O`Brief et al [71] reported ten individuals who developed severe congestive heart failure while on imatinib. They showed that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib showed mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome *c* into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib. [71]

In turn, international and randomized phase III study involving 1106 patients with newly diagnosed chronic myeloid leukemia with Philadelphia chromosome, who are in chronic phase, severe cardiac failure and left ventricular dysfunction was reported in 0.7% of patients receiving imatinib, compared to 0,9% of patients treated with interferon alpha (IFN) with cytosine arabinoside (Ara-C). However, in the IRIS study, after imatinib treatment of heart failure incidence was 1% of patients. [72]

Cardiac toxicity of imatinib may be due to inhibition by this drug tyrosine kinase c-Abl. It was confirmed that by the action of imatinib in response to oxidative stress occur:

#### **3.1.4 Dasatinib**

#### **<IP_ADDRESS> Mechanism of antitumor action of dasatinib**

Dasatinib is a potent inhibitor of the tyrosine kinase Bcr-Abl. Also it blocks a number of other tyrosine kinases, including compounds belonging to the families: scr, c-kit and PDGFR eEPHA 2.

#### **<IP_ADDRESS> Clinical application of dasatinib**

 This drug is used in the treatment of CML and Ph + ALL patients who had developed resistance to imatinib.

#### **<IP_ADDRESS> Mechanism of cardiotoxicity of dasatinib**

There have been reports of congestive heart failure and cardiac arrhythmias in patients during dasatinib therapy. This medicine may also cause QT prolongation. Treatment with dasatinib may increase the risk of pulmonary arterial hypertension. Patients who reported the occurrence of pulmonary hypertension during treatment with dasatinib usually received other cardiotoxic drugs or suffered from other chronic diseases. In these patients improved hemodynamic and clinical parameters after discontinuation of treatment dasatinib was reported. [73]

Cardiac Complications of Cancer Treatment 51

and asymptomatic decreased LVEF among 1674 breast cancer patients was 1.3% and was also 1.3% among 1453 patients with non- breast malignancies. Lapatinib- associated LVEF decrease was symptomatic in 0.1%, generally reversible/nonprogressive. Average duration of LVEF decrease was 40 days. The other studies conducted so far showed that the cardiotoxicity associated with lapatinib is not severe, is reversible, and usually patients can

Recent case reports provide alarming signals that treatment with bortezomib might be

It is used in treatment of patients suffering from multiple myeloma or mantle cell lymphoma.

Nowis et al. [74] reported that bortezomib treatment leads to left ventricular contractile dysfunction in rats manifested by a significant drop in left ventricle ejection fraction. It is important that in this study rats were not treated with other drugs. Dramatic ultrastructular abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. [74] Bortezomib induced cardiac effects seem to be reversible. The risk of cardiotoxicity after bortezomib is higher in patients which have previously cardiac problems or being concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. In a recent phase III clinical study of bortezomib was used in combination with pegylated liposomal doxorubicin, LVEF ejection fraction was reported to decrease in 7% of patients treated with bortezomib. [75] In another clinical study, grade 3 to 4 cardiac heart failure was reported in

2 cases (one fatal) receiving bortezomib in combination with doxorubicin. [76]

presence is associated with poor prognosis and shorter overall survival time. [22]

**3.3 Monoclonal antibodies directed against the receptor or tyrosine kinase ligands**

Trastuzumab is a monoclonal antibody selectively connecting to the extracellular domain of HER-2 receptor, which belongs to the family of epidermal growth factor receptors. Overexpression of this receptor is found in approximately 20-25% of breast cancers. It is

Trastuzumab improves response rate and survival in women with metastatic breast cancer with known overexpression of the HER-2 receptor. Also the use of trastuzumab

continue lapatinib therapy, when symptomatic treatment has been started. [54]

**3.2 Bortezomib**

**3.3.1 Trastuzumab**

associated with cardiac events.

**3.2.1 Mechanism of antitumor action of bortezomib**

Bortezomib is a reversible proteasome inhibitor.

**3.2.3 Mechanism of cardiotoxicity of bortezomib**

**<IP_ADDRESS> Mechanism of antitumor action of trastuzumab**

**<IP_ADDRESS> Clinical application of trastuzumab**

**3.2.2 Clinical application of bortezomib**

#### **3.1.5 Lapatinib (GW572016)**

#### **<IP_ADDRESS> Mechanism of antitumor action of lapatinib**

Lapatinib is an oral dual tyrosine kinase inhibitor selective for inhibition of EGFR/ErbB1 and HER2/ErbB2.

#### **<IP_ADDRESS> Clinical application of lapatinib.**

This small molecule represents one of the most promising target therapies in breast cancer that overexpressed HER2. It is used in trastuzumab-refractory breast cancer.

#### **<IP_ADDRESS> Mechanism of cardiotoxicity of lapatinib**

Perez et al. reviewed the cardiotoxicity data of lapatinib in 3558 patients, including 1674 breast cancer patients, already treated with the drug alone or in combination with other agents. A total of 1090 patients had >6 months exposure to lapatinib. Evaluation of cardiac left ventricular ejection fraction (LVEF) was done every 8 weeks while patients were receiving therapy, in addition to follow-up for any cardiac clinical events. A preliminary analysis of patients treated with lapatinib to date revealed that incidence of symptomatic

Table 4. Tyrosine kinase inhibitors- SUMMARY

and asymptomatic decreased LVEF among 1674 breast cancer patients was 1.3% and was also 1.3% among 1453 patients with non- breast malignancies. Lapatinib- associated LVEF decrease was symptomatic in 0.1%, generally reversible/nonprogressive. Average duration of LVEF decrease was 40 days. The other studies conducted so far showed that the cardiotoxicity associated with lapatinib is not severe, is reversible, and usually patients can continue lapatinib therapy, when symptomatic treatment has been started. [54]

#### **3.2 Bortezomib**

50 Cardiotoxicity of Oncologic Treatments

Lapatinib is an oral dual tyrosine kinase inhibitor selective for inhibition of EGFR/ErbB1

This small molecule represents one of the most promising target therapies in breast cancer

Perez et al. reviewed the cardiotoxicity data of lapatinib in 3558 patients, including 1674 breast cancer patients, already treated with the drug alone or in combination with other agents. A total of 1090 patients had >6 months exposure to lapatinib. Evaluation of cardiac left ventricular ejection fraction (LVEF) was done every 8 weeks while patients were receiving therapy, in addition to follow-up for any cardiac clinical events. A preliminary analysis of patients treated with lapatinib to date revealed that incidence of symptomatic

 Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of several malignancies, converting lethal diseases into manageable, if not curable, chronic

The goal of tumor-cell killing by TKIs must be balanced against cardiotoxicity,

function, either as a primary or secondary effect. Therefore, alterations in

 Very few clinical trials have examined cardiotoxicities of TKIs in a prospective fashion with predefined cardiac endpoints, including left ventricular function. Therefore, there is a wide gap in our knowledge regarding the types, and risk of,

tumour cells but that are involved in cardiomyocyte survival.

but cardiotoxicity as an 'off-target' effect should be minimized.

 Cardiomyocytes are contractile and have an extremely high demand for ATP. As a result, they might be particularly susceptible to agents that perturb mitochondrial

mitochondrial function could have a role in the cardiotoxicities of some currently

 Some current kinase targets in cancer are not expressed in cardiomyocytes. Therefore have little or no direct role in cardiomyocyte survival. The current generation of TKIs is inherently non-selective, and the purposeful design of multitargeted TKIs might allow a single agent to be more effective, and to be used in more types of cancer, but with this comes an increased risk of cardiotoxicity. In some cases this will probably be due to inhibition of 'bystander' targets that are not essential for the killing of

 Identification of the kinase responsible for cardiotoxicity of an agent is important for future drug design. Future kinases should avoid this kinase. Thus, greater selectivity of individual agents may require the use of more agents to treat a particular cancer,

because in some instances tumor cell death and preservation of cardiomyocyte health

diseases. This is essential to limit toxicities of these agents.

that overexpressed HER2. It is used in trastuzumab-refractory breast cancer.

**3.1.5 Lapatinib (GW572016)**

and HER2/ErbB2.

**<IP_ADDRESS> Mechanism of antitumor action of lapatinib**

**<IP_ADDRESS> Mechanism of cardiotoxicity of lapatinib**

**<IP_ADDRESS> Clinical application of lapatinib.**

may be mutually exclusive.

cardiotoxicity for most of these agents.

Table 4. Tyrosine kinase inhibitors- SUMMARY

approved agents.

Recent case reports provide alarming signals that treatment with bortezomib might be associated with cardiac events.

#### **3.2.1 Mechanism of antitumor action of bortezomib**

Bortezomib is a reversible proteasome inhibitor.

#### **3.2.2 Clinical application of bortezomib**

It is used in treatment of patients suffering from multiple myeloma or mantle cell lymphoma.

#### **3.2.3 Mechanism of cardiotoxicity of bortezomib**

Nowis et al. [74] reported that bortezomib treatment leads to left ventricular contractile dysfunction in rats manifested by a significant drop in left ventricle ejection fraction. It is important that in this study rats were not treated with other drugs. Dramatic ultrastructular abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. [74] Bortezomib induced cardiac effects seem to be reversible. The risk of cardiotoxicity after bortezomib is higher in patients which have previously cardiac problems or being concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. In a recent phase III clinical study of bortezomib was used in combination with pegylated liposomal doxorubicin, LVEF ejection fraction was reported to decrease in 7% of patients treated with bortezomib. [75] In another clinical study, grade 3 to 4 cardiac heart failure was reported in 2 cases (one fatal) receiving bortezomib in combination with doxorubicin. [76]

#### **3.3 Monoclonal antibodies directed against the receptor or tyrosine kinase ligands**

#### **3.3.1 Trastuzumab**

#### **<IP_ADDRESS> Mechanism of antitumor action of trastuzumab**

Trastuzumab is a monoclonal antibody selectively connecting to the extracellular domain of HER-2 receptor, which belongs to the family of epidermal growth factor receptors. Overexpression of this receptor is found in approximately 20-25% of breast cancers. It is presence is associated with poor prognosis and shorter overall survival time. [22]

#### **<IP_ADDRESS> Clinical application of trastuzumab**

Trastuzumab improves response rate and survival in women with metastatic breast cancer with known overexpression of the HER-2 receptor. Also the use of trastuzumab

Cardiac Complications of Cancer Treatment 53

27% of patients who received trastuzumab with anthracyclines and cyclophosphamide compared to 8% of patients after treatment with chemotherapy alone (anthracycline and cyclophosphamide without trastuzumab). In 13% of patients treated with paclitaxel with trastuzumab cardiac complications occurred. Continuation of trastuzumab did not affect the severity of dysfunction of the cardiovascular system in most patients. The symptomatic

Further analysis concerned three trials [338 patients] where trastuzumab monotherapy was used. It was estimated that the risk of cardiotoxicity associated with trastuzumab was 4%. 3% of patients had grade 3 and 4 toxicity according to Common Toxicity Criteria (CTC). [78] Mechanism of cardiotoxicity induced by trastuzumab is still not fully clear. But role of inhibition of normal cardiac repair pathways by this drug seem to be probable. Her-2 heterodimerizes to Her-4, leading to autophosphorylation of the Her-2 tyrosine kinase domain. [79] This complex, which is the antitumor target of trastuzumab, is also active in cardiac repair. The complex is activated by neuregulin 1, which is secreted in paracrine fashion by cardiac endothelial cells that are under stress. [79] Activation of the complex leads to multiple downstream effects. In turn they lead to hypertrophy of cardiac myocytes in vivo. In mice, deletion of Her-2 results in a dilated cardiomyopathy. [80] Chien proposed a model in which various types of cardiac stress such as mechanical strain, anthracyclines, or hypoxia trigger two competing pathways of cardiac myocyte survival (mediated by neuregulin-1 or gp 130 cytokines) or apoptosis. The clinical outcome depends on which process prevails. In this model, treatment with trastuzumab blocks the survival pathway by preventing Her-2/Her-4 heterodimerization, thus shifting the balance to apoptosis. The

treatment was effective. [78]

result is decreased cardiac contractility and CHF. [81]

 induced apoptosis of cardiomyocytes, cased hypertrophy of cardiac myocytes,

**<IP_ADDRESS> Clinical application of bevacizumab**

**3.3.2 Bevacizumab**

renal and breast cancer.

Table 5. Mechanism of cardiotoxicity of trastuzumab

**<IP_ADDRESS> Mechanism of antitumor action of bevacizumab**

**<IP_ADDRESS> Mechanism of cardiotoxicity of bevacizumab**

receptor and thereby inhibits the process of neoangiogenesis.

by the presence of HER-2 receptor on cardiomyocytes,

Mechanism for inducing cardiotoxicity by trastuzumab is presented in Table nr 5.

blocks the cardiac survival pathway by preventing Her-2/Her-4 heterodimerization,

Bevacizumab is a recombinant humanised IgG monoclonal antibody specifically binds to vascular endothelial growth factor (VEGF) and blocking its connection to the membrane

Bevacizumab is adjunct to cytotoxic drug combination. It is approved for use in colon, lung,

Based on the research phase I and II found that the possible side effects of bevacizumab are:

concurrently with adjuvant chemotherapy significantly improves the relapse-free survival and overall survival in women with overexpressing HER-2 receptor in localized form of breast cancer. [22]

#### **<IP_ADDRESS> Risk factors for cardiotoxicity after treatment with trastuzumab**

The risk factors associated with trastuzumab cardiotoxicity include:

It was proven that the risk of cardiomyopathy after trastuzumab (as opposed to anthracycline antibiotics) does not depend on the total dose of drug. Contrast, cardiac dysfunction associated with trastuzumab therapy is completely reversible after discontinuation of the drug and after symptomatic treatment and leaves without residual effects in the structure of cardiac muscle. [77]

#### **<IP_ADDRESS> Mechanism of cardiotoxicity of trastuzumab**

Cardiotoxicity associated with trastuzumab was analyzed only in the phase III trials, sporadic cases relate to assessment of phase II trials, because nobody knows whether this drug may cause clinically significant cardiac complications. No patients were monitored in terms of efficiency of the cardiovascular system during trastuzumab therapy.

Cardiac complications, grade IV NYHA (New York Heart Association) was observed in 2% of patients treated with trastuzumab in the first line, in 4% of patients treated with trastuzumab because of resistance to previous treatment, in 2% of patients treated with concomitant paclitaxel and trastuzumab (compared to 1% of patients the group receiving paclitaxel alone) and in 16% of patients receiving chemotherapy: adriamycin with cyclophosphamide (AC) plus trastuzumab (compared to 4% in the group receiving chemotherapy alone AC). Symptoms of heart failure occurred in 75.5% of patients (83 in the group of 110 patients). In 79% of cases symptomatic treatment of heart failure has proved effective. [77]

Based on 7 studies: phase II and III, Cardiac Review and Evaluation Committee (CREC) conducted a retrospective analysis of cardiac events associated with trastuzumab therapy. One analysis was based on international study 222 women who received trastuzumab in the second or third line treatment of metastatic breast cancer. Patients were previously treated with anthracyclines (94% women), taxanes (67% women), radiotherapy (71% women), hormonal therapy (57% women). Cardiac complications were seen in 10 patients (4.7%), including 3 patients after treatment with trastuzumab. 9 patient out of all who develop complication (n=10) had been treated with anthracylines previously. Most of the cardiac complications in this study were clinically significant. There was one death following a ventricular arrhythmia, in a patient after previous treatment with anthracyclines with reduced baseline left ventricular ejection fraction. [78]

Another study conducted by CREC concerned randomized, international phase III study comparing trastuzumab in combination with chemotherapy to chemotherapy alone in 469 patients with metastatic breast cancer. The symptoms of cardiotoxicity were observed in

concurrently with adjuvant chemotherapy significantly improves the relapse-free survival and overall survival in women with overexpressing HER-2 receptor in localized form of

It was proven that the risk of cardiomyopathy after trastuzumab (as opposed to anthracycline antibiotics) does not depend on the total dose of drug. Contrast, cardiac dysfunction associated with trastuzumab therapy is completely reversible after discontinuation of the drug and after symptomatic treatment and leaves without residual

Cardiotoxicity associated with trastuzumab was analyzed only in the phase III trials, sporadic cases relate to assessment of phase II trials, because nobody knows whether this drug may cause clinically significant cardiac complications. No patients were monitored in

Cardiac complications, grade IV NYHA (New York Heart Association) was observed in 2% of patients treated with trastuzumab in the first line, in 4% of patients treated with trastuzumab because of resistance to previous treatment, in 2% of patients treated with concomitant paclitaxel and trastuzumab (compared to 1% of patients the group receiving paclitaxel alone) and in 16% of patients receiving chemotherapy: adriamycin with cyclophosphamide (AC) plus trastuzumab (compared to 4% in the group receiving chemotherapy alone AC). Symptoms of heart failure occurred in 75.5% of patients (83 in the group of 110 patients). In 79% of cases

Based on 7 studies: phase II and III, Cardiac Review and Evaluation Committee (CREC) conducted a retrospective analysis of cardiac events associated with trastuzumab therapy. One analysis was based on international study 222 women who received trastuzumab in the second or third line treatment of metastatic breast cancer. Patients were previously treated with anthracyclines (94% women), taxanes (67% women), radiotherapy (71% women), hormonal therapy (57% women). Cardiac complications were seen in 10 patients (4.7%), including 3 patients after treatment with trastuzumab. 9 patient out of all who develop complication (n=10) had been treated with anthracylines previously. Most of the cardiac complications in this study were clinically significant. There was one death following a ventricular arrhythmia, in a patient after previous treatment with anthracyclines with

Another study conducted by CREC concerned randomized, international phase III study comparing trastuzumab in combination with chemotherapy to chemotherapy alone in 469 patients with metastatic breast cancer. The symptoms of cardiotoxicity were observed in

terms of efficiency of the cardiovascular system during trastuzumab therapy.

symptomatic treatment of heart failure has proved effective. [77]

reduced baseline left ventricular ejection fraction. [78]

**<IP_ADDRESS> Risk factors for cardiotoxicity after treatment with trastuzumab**

The risk factors associated with trastuzumab cardiotoxicity include:

past radiotherapy prior therapy with anthracyclines.

effects in the structure of cardiac muscle. [77]

**<IP_ADDRESS> Mechanism of cardiotoxicity of trastuzumab**

breast cancer. [22]

 diabetes, obesity, older age,

history of hypertension,

27% of patients who received trastuzumab with anthracyclines and cyclophosphamide compared to 8% of patients after treatment with chemotherapy alone (anthracycline and cyclophosphamide without trastuzumab). In 13% of patients treated with paclitaxel with trastuzumab cardiac complications occurred. Continuation of trastuzumab did not affect the severity of dysfunction of the cardiovascular system in most patients. The symptomatic treatment was effective. [78]

Further analysis concerned three trials [338 patients] where trastuzumab monotherapy was used. It was estimated that the risk of cardiotoxicity associated with trastuzumab was 4%. 3% of patients had grade 3 and 4 toxicity according to Common Toxicity Criteria (CTC). [78]

Mechanism of cardiotoxicity induced by trastuzumab is still not fully clear. But role of inhibition of normal cardiac repair pathways by this drug seem to be probable. Her-2 heterodimerizes to Her-4, leading to autophosphorylation of the Her-2 tyrosine kinase domain. [79] This complex, which is the antitumor target of trastuzumab, is also active in cardiac repair. The complex is activated by neuregulin 1, which is secreted in paracrine fashion by cardiac endothelial cells that are under stress. [79] Activation of the complex leads to multiple downstream effects. In turn they lead to hypertrophy of cardiac myocytes in vivo. In mice, deletion of Her-2 results in a dilated cardiomyopathy. [80] Chien proposed a model in which various types of cardiac stress such as mechanical strain, anthracyclines, or hypoxia trigger two competing pathways of cardiac myocyte survival (mediated by neuregulin-1 or gp 130 cytokines) or apoptosis. The clinical outcome depends on which process prevails. In this model, treatment with trastuzumab blocks the survival pathway by preventing Her-2/Her-4 heterodimerization, thus shifting the balance to apoptosis. The result is decreased cardiac contractility and CHF. [81]

Mechanism for inducing cardiotoxicity by trastuzumab is presented in Table nr 5.

Table 5. Mechanism of cardiotoxicity of trastuzumab

#### **3.3.2 Bevacizumab**

#### **<IP_ADDRESS> Mechanism of antitumor action of bevacizumab**

Bevacizumab is a recombinant humanised IgG monoclonal antibody specifically binds to vascular endothelial growth factor (VEGF) and blocking its connection to the membrane receptor and thereby inhibits the process of neoangiogenesis.

#### **<IP_ADDRESS> Clinical application of bevacizumab**

Bevacizumab is adjunct to cytotoxic drug combination. It is approved for use in colon, lung, renal and breast cancer.

#### **<IP_ADDRESS> Mechanism of cardiotoxicity of bevacizumab**

Based on the research phase I and II found that the possible side effects of bevacizumab are:

Cardiac Complications of Cancer Treatment 55

Cardiotoxicity of anagrelide also confirmed case of 50-year-old man diagnosed with essential thrombocythaemia. Due to the increasing resistance to first-line treatment the patient was treated with anagrelide in a dose gradually increasing up to 2.5 mg twice a day. The patient developed clinical features of congestive heart failure, NYHA IV. LVEF was 18%. ECG findings were typical for myocardial ischemia but troponin T level was normal. Coronary angiography was performed revealing normal in which the coronary arteries. Treatment of anagrelide was stopped, hydroxyurea therapy was started again. Patients received symptomatic therapy of heart failure: diuretics and ACE-inhibitors with good response. Hydroxyurea therapy once again proved to be ineffective. Doctors were decided to join hydroxyurea of anagrelide at a reduced dose 0.5 mg twice a day. Modification of treatment was effective, the level of platelets decreased to the normal limits, the symptoms of heart failure was absent. ECHO, made by 2 and 8 months after starting treatment with anagrelide [low doses] and hydroxyurea, showed increasing LVEF from 40 to 50%. [83]

These observations confirm that the use of anagrelide is not absolutely safe to the cardiovascular system. Cardiomyopathy, during anagrelide treatment is rare but clinically significant. Therefore, patients with a history of heart disease should be treated with anagrelide with caution, when potential benefits exceed risk of this kind therapy. [82,83] Mechanism of cardiotoxicity associated with the action of anagrelide is showen in Table nr 6.

Hematopoietic stem cell transplantation (HSCT) has now become the treatment of choice for large number of malignant and non-malignant diseases. Cardiac complications may result from high-dose chemotherapy or irradiation administered during the conditioning phase of bone marrow and blood stem cell transplantation (BMT). Cardiac complications of high-dose cyclophosphamide and total body irradiation (TBI) or other intensive conditioning regimens administered prior to bone marrow and stem cell transplantation (BMT) have been well documented. Clinically, patients present with congestive heart failure and pancarditis. Hemorrhagic perimyocarditis with endothelial damage and microthrombi in capillaries as well as acute fibrinous pericarditis. Several other cytotoxic drugs such as busulfan, carmustine or cytarabine used in pre-BMT regimens may cause significant cardiotoxicity. Acute myocardial infarction and various cardiac arrhythmias including cardiac arrest as a consequence of infusion of cryopreserved marrow have been reported during the acute phase of BMT. Pretreatment with anti-tumor antibiotics including anthracyclines and prior mediastinal irradiation may increase the risks of adverse cardiac sequelae after BMT, but identification of the risk factors and effective strategies for cardiologic prescreening have not been well established. Although cardiac complications associated with BMT have been documented in several series, the incidence of reported cardiotoxicity has varied among investigators, probably reflecting patient

by positive inotropic effects,

Table 6. The mechanism of cardiotoxicity of anagrelide

**Hematopoietic Stem Cell Transplantation (HSCT)**

**5. Cardiotoxicity of High Dose Chemotherapy (HDC) followed by**

 by vasodilatation, by tachyarrythmias,

The presence of hypertension was reported in 5-7% of patients participating in clinical trials. It was also observed [in rare cases] development of encephalopathy associated with hypertension and subarachnoid haemorrhage. Patients after treatment with anthracyclines and radiotherapy are at increased risk for cardiac complications during or after treatment with bevacizumab. In patients previously treated with anthracyclines followed by mediastinal radiotherapy, the incidence of left heart failure was about 4%. When anthracyclines and radiotherapy were used at the same time heart failure was observed in 14% of patients. [54]

In a randomized study breast cancer patients receiving paclitaxel with bevacizumab compared to patients receiving paclitaxel alone the risk of hypertension 3 and 4 level rose to about 15%, thromboembolic events up to about 2.5%, proteinuria to about 3.1% and cardiac function the left ventricle to 1.4%. One patient receiving bevacizumab with paclitaxel died of a myocardial infarction. [54]

#### **4. Cardiotoxicity of anagrelide**

1. Mechanism of antitumor action of anagrelide.

Anagrelide works by inhibiting the maturation of platelets from megakaryocytes. The exact mechanism of action is unclear, although it is known to be a phosphodiesterase inhibitor. It is a potent inhibitor of phosphodiesterase-II. It inhibits PDE-3 and phospholipase A2.

2. Clinical application of anagrelide.

This medicine is a standard treatment of thrombocythemia in chronic myeloproliferative disorders: essential thrombocythaemia (ET), polycythemia vera (PV) and bone marrow fibrosis.

3. The mechanism of cardiotoxicity of anagrelide.

The mechanism of cardiotoxicity of this drug is not fully known. It may be due to medicine`s positive inotropic effect or consequence of vasodilatation and tachyarrhythmia induced by anagrelide. During the treatment following can occur: palpitations and tachycardia, less common- congestive heart failure, hypertension, arrhythmia, atrial fibrillation and the occasional- angina, myocardial infarction, cardiomegaly, cardiomyopathy, pericardial effusion, and orthostatic hypotension. [82]

Anagrelide was found as a cause of cardiomyopathy in 2000 at the Mayo Clinic [Rochester, USA]. The data were collected from 434 patients suffered from essential thrombocythaemia and polycythemia vera. Investigators confirmed by echocardiography the development of idiopathic cardiomyopathy during treatment of these chronic myeloproliferative disorders with anagrelide in 11 patients. The decrease in LVEF was significant from 35% (two persons) to 10% (one person). Discontinuation of treatment with anagrelide resulted in an increased LVEF. [82]

The presence of hypertension was reported in 5-7% of patients participating in clinical trials. It was also observed [in rare cases] development of encephalopathy associated with hypertension and subarachnoid haemorrhage. Patients after treatment with anthracyclines and radiotherapy are at increased risk for cardiac complications during or after treatment with bevacizumab. In patients previously treated with anthracyclines followed by mediastinal radiotherapy, the incidence of left heart failure was about 4%. When anthracyclines and radiotherapy were used at the same time heart failure was observed in 14% of patients. [54]

In a randomized study breast cancer patients receiving paclitaxel with bevacizumab compared to patients receiving paclitaxel alone the risk of hypertension 3 and 4 level rose to about 15%, thromboembolic events up to about 2.5%, proteinuria to about 3.1% and cardiac function the left ventricle to 1.4%. One patient receiving bevacizumab with paclitaxel died of a myocardial

Anagrelide works by inhibiting the maturation of platelets from megakaryocytes. The exact mechanism of action is unclear, although it is known to be a phosphodiesterase inhibitor. It

This medicine is a standard treatment of thrombocythemia in chronic myeloproliferative disorders: essential thrombocythaemia (ET), polycythemia vera (PV) and bone marrow

The mechanism of cardiotoxicity of this drug is not fully known. It may be due to medicine`s positive inotropic effect or consequence of vasodilatation and tachyarrhythmia induced by anagrelide. During the treatment following can occur: palpitations and tachycardia, less common- congestive heart failure, hypertension, arrhythmia, atrial fibrillation and the occasional- angina, myocardial infarction, cardiomegaly, cardiomyopathy, pericardial

Anagrelide was found as a cause of cardiomyopathy in 2000 at the Mayo Clinic [Rochester, USA]. The data were collected from 434 patients suffered from essential thrombocythaemia and polycythemia vera. Investigators confirmed by echocardiography the development of idiopathic cardiomyopathy during treatment of these chronic myeloproliferative disorders with anagrelide in 11 patients. The decrease in LVEF was significant from 35% (two persons) to 10% (one person). Discontinuation of treatment with anagrelide resulted in an increased

is a potent inhibitor of phosphodiesterase-II. It inhibits PDE-3 and phospholipase A2.

bleeding,

infarction. [54]

fibrosis.

LVEF. [82]

**4. Cardiotoxicity of anagrelide**

2. Clinical application of anagrelide.

effusion, and orthostatic hypotension. [82]

1. Mechanism of antitumor action of anagrelide.

3. The mechanism of cardiotoxicity of anagrelide.

 thromboembolism, proteinuria, hypertension.

Cardiotoxicity of anagrelide also confirmed case of 50-year-old man diagnosed with essential thrombocythaemia. Due to the increasing resistance to first-line treatment the patient was treated with anagrelide in a dose gradually increasing up to 2.5 mg twice a day. The patient developed clinical features of congestive heart failure, NYHA IV. LVEF was 18%. ECG findings were typical for myocardial ischemia but troponin T level was normal. Coronary angiography was performed revealing normal in which the coronary arteries. Treatment of anagrelide was stopped, hydroxyurea therapy was started again. Patients received symptomatic therapy of heart failure: diuretics and ACE-inhibitors with good response. Hydroxyurea therapy once again proved to be ineffective. Doctors were decided to join hydroxyurea of anagrelide at a reduced dose 0.5 mg twice a day. Modification of treatment was effective, the level of platelets decreased to the normal limits, the symptoms of heart failure was absent. ECHO, made by 2 and 8 months after starting treatment with anagrelide [low doses] and hydroxyurea, showed increasing LVEF from 40 to 50%. [83]

These observations confirm that the use of anagrelide is not absolutely safe to the cardiovascular system. Cardiomyopathy, during anagrelide treatment is rare but clinically significant. Therefore, patients with a history of heart disease should be treated with anagrelide with caution, when potential benefits exceed risk of this kind therapy. [82,83]

Mechanism of cardiotoxicity associated with the action of anagrelide is showen in Table nr 6.

Table 6. The mechanism of cardiotoxicity of anagrelide

#### **5. Cardiotoxicity of High Dose Chemotherapy (HDC) followed by Hematopoietic Stem Cell Transplantation (HSCT)**

Hematopoietic stem cell transplantation (HSCT) has now become the treatment of choice for large number of malignant and non-malignant diseases. Cardiac complications may result from high-dose chemotherapy or irradiation administered during the conditioning phase of bone marrow and blood stem cell transplantation (BMT). Cardiac complications of high-dose cyclophosphamide and total body irradiation (TBI) or other intensive conditioning regimens administered prior to bone marrow and stem cell transplantation (BMT) have been well documented. Clinically, patients present with congestive heart failure and pancarditis. Hemorrhagic perimyocarditis with endothelial damage and microthrombi in capillaries as well as acute fibrinous pericarditis. Several other cytotoxic drugs such as busulfan, carmustine or cytarabine used in pre-BMT regimens may cause significant cardiotoxicity. Acute myocardial infarction and various cardiac arrhythmias including cardiac arrest as a consequence of infusion of cryopreserved marrow have been reported during the acute phase of BMT. Pretreatment with anti-tumor antibiotics including anthracyclines and prior mediastinal irradiation may increase the risks of adverse cardiac sequelae after BMT, but identification of the risk factors and effective strategies for cardiologic prescreening have not been well established. Although cardiac complications associated with BMT have been documented in several series, the incidence of reported cardiotoxicity has varied among investigators, probably reflecting patient

Cardiac Complications of Cancer Treatment 57

years (range, 16.6 to 68.6 years); median time to CHF was 3.0 years (range, 1.03 to 18.9 years); 68% received autologous HCT. Median ejection fraction was 36.9% (range, 15% to 53%). Compared with matched controls (n = 166), patients with late CHF received more cycles of pre-HCT chemotherapy (8.6 v 4.9 cycles; P= 0.01), had greater body mass index at HCT (28.4 v 26.2 kg/m2; P= 0.01), greater lifetime anthracycline exposure (285.3 v 175.6 mg/m2; P= 0.01), and were more likely to have multiple chronic comorbidities (30.0% v 13.9%; P= 0.01). Multivariable analysis revealed number of pre-HCT chemotherapy cycles (odds ratio [OR] 1.2; P= 0.01), anthracycline dose 250 mg/m2 (OR 3.2; P= 0.05), and two or more chronic comorbidities (OR 4.3; P= 0.01) to be independently associated with late CHF. Pre-HCT exposure to anthracyclines and presence of comorbidities are primarily risk associated with late CHF after HCT. Conditioning-related therapeutic exposure does not contribute significantly to the risk. These results form the basis for identifying high-risk individuals for targeted surveillance, as well as developing preventive strategies in the

Considering all of the above the important factors affecting the occurrence of cardiotoxic high dose chemotherapy complications include exposure to high doses of cyclophosphamide, total body irradiation, prior anthracycline therapy, the presence of dimethyl sulfoxide (DMSO) in intravenous products, infectious complications associated with neutropenia. The risk of cardiac complications after HSCT is also influenced by patient

Dimethyl sulphoxide (DMSO) is cells protective factor, preventing the crystallization of water and damage to cell membranes collected stem cells, which are subjected to freezing in liquid nitrogen at temperatures below -120 degrees C. Cardiotoxicity associated with DMSO may result in hypotension, requiring fluid infusion or even inotropic drugs. Ones also reported cardiac arrhythmias such as atrial fibrillation, or bradycardia, and incidents of acute coronary syndromes. Cardiotoxicity associated with DMSO is rare. Wether DMSO is responsible for these has been questioned. [84,85] Recent reports of similar acute cardiac events despite DMSO depletion have led to the suggestion that perhaps the complications seen following infusion may be more a product of the amount of infused granulocytes,

Infectious complications during early post-transplant neutropenia or prolonged immunosuppression for GVHD prophylaxis are common problems following HSCT. Overwhelming sepsis can lead to cardiopulmonary failure, necessitating prolonged intubation and cardiac inotropic support, potentially causing subclinical cardiotoxicity or other end-organ compromise that may not be evident until years following HSCT. [84]

The Tichellis study [84] provided preliminary evidence for an association between GVHD and the development of arterial disease. There are emerging data to suggest that chronic GVHD could play a role in the development of cardiovascular disease. Cardiac side effects of chronic GVHD, while rare, likely occur as a result of direct organ lymphocytic infiltration. Increased levels of circulating tumor necrosis factor alfa [TNF alfa] may impair muscle electrical activity and compromise myocardial contractility. Furthermore, increased amounts of inflammatory markers, such as TNF alfa and interleukin-6, could perpetuate endothelial injury, contributing to premature arterial events in long-term survivors after allogeneic HSCT. Treatment of GVHD is not without cardiovascular consequences.

form of aggressive management of comorbidities. [87]

rather than DMSO. [84]

age, type of therapy applied and previous medical history. [84]

selection, differences in BMT preparative regimens and lack of universal grading system for cardiotoxic events. [84-87]

Cardiac complications associated with exposure to the conditioning factors can be acute, with a relatively short or long period of latency after HSCT. [84-87]

To assess the frequency of clinically serious cardiac toxicity related to the acute phase of bone marrow transplantation (BMT), investigators from University of Minnesota retrospectively examined life-threatening or fatal cardiotoxicity identified using the complications records of their transplant center clinical database. All serious cardiac toxicity events within 100 days of BMT except those attributable to septic shock, pneumonitis or multi-organ failure were reviewed. During the first 100 days after transplantation 628 cardiac complications experienced and divided by four-level scale cardiac toxicity [86]:

Of 2821 BMT patients at the University of Minnesota between 1977 and 1997, 26 were identified as having suffered major or fatal (n = 13) cardiotoxicity (0.9%, 19 adults and seven children). Rapidly progressive heart failure resulted in death of 11 patients, one patient had fatal pericardial tamponade, and one had an acute ventricular fibrillation arrest. The remaining 13 patients (50%) had life-threatening cardiotoxicity including four patients with pericardial tamponade and nine patients with cardiac arrhythmias. Overall, we observed that acute, major cardiotoxic events attributable to BMT are uncommon, occurring with a frequency of 1%. These data suggest that with appropriate pre-transplant clinical evaluation, high-dose cyclophosphamide and irradiation in the BMT preparative phase does not result in frequent, clinically relevant short-term cardiac toxicity. [86]

The most common late complications of HSCT include valvular dysfunction, conduction disturbances, pericarditis and cardiomyopathies. Are also observed vascular complications, which include: coronary heart disease, cerebral vascular dysfunction and disease. [84-87]

Investigators from City of Hope National Medical Center [87] examined the independent roles of pre-hematopoietic cell transplantation (HCT) therapeutic exposures, transplantation-related conditioning, and comorbidities (pre- and post-HCT) in the development of late congestive heart failure (CHF) after HCT. This was a nested case-control design. Individuals with late CHF (diagnosed 1 year after HCT) were identified from a cohort of 2,938 1 year survivors who underwent transplantation at City of Hope National Medical Center, Duarte, CA. This cohort formed the sampling frame for selecting controls (without CHF) matched for age and year of HCT, donor source (allogeneic v autologous), and length of follow-up. Sixty patients with late CHF were identified; median age at HCT was 45.3

selection, differences in BMT preparative regimens and lack of universal grading system

Cardiac complications associated with exposure to the conditioning factors can be acute,

To assess the frequency of clinically serious cardiac toxicity related to the acute phase of bone marrow transplantation (BMT), investigators from University of Minnesota retrospectively examined life-threatening or fatal cardiotoxicity identified using the complications records of their transplant center clinical database. All serious cardiac toxicity events within 100 days of BMT except those attributable to septic shock, pneumonitis or multi-organ failure were reviewed. During the first 100 days after transplantation 628 cardiac complications experienced and divided by four-level scale cardiac toxicity [86]:

Stage I- asymptomatic cardiomegaly, mild ECG changes, not requiring treatment,

 Stage II- moderate changes in the ECG, which require and well respond to routine treatment, congestive heart failure responsive to afterload reduction in the treatment of

 Stage III: severe abnormalities in the ECG with no or partial response to medical intervention, congestive heart failure, requiring drugs with isotropic, cardiogenic shock,

Of 2821 BMT patients at the University of Minnesota between 1977 and 1997, 26 were identified as having suffered major or fatal (n = 13) cardiotoxicity (0.9%, 19 adults and seven children). Rapidly progressive heart failure resulted in death of 11 patients, one patient had fatal pericardial tamponade, and one had an acute ventricular fibrillation arrest. The remaining 13 patients (50%) had life-threatening cardiotoxicity including four patients with pericardial tamponade and nine patients with cardiac arrhythmias. Overall, we observed that acute, major cardiotoxic events attributable to BMT are uncommon, occurring with a frequency of 1%. These data suggest that with appropriate pre-transplant clinical evaluation, high-dose cyclophosphamide and irradiation in the BMT preparative phase does not result in frequent, clinically relevant

The most common late complications of HSCT include valvular dysfunction, conduction disturbances, pericarditis and cardiomyopathies. Are also observed vascular complications, which include: coronary heart disease, cerebral vascular dysfunction and disease. [84-87]

Investigators from City of Hope National Medical Center [87] examined the independent roles of pre-hematopoietic cell transplantation (HCT) therapeutic exposures, transplantation-related conditioning, and comorbidities (pre- and post-HCT) in the development of late congestive heart failure (CHF) after HCT. This was a nested case-control design. Individuals with late CHF (diagnosed 1 year after HCT) were identified from a cohort of 2,938 1 year survivors who underwent transplantation at City of Hope National Medical Center, Duarte, CA. This cohort formed the sampling frame for selecting controls (without CHF) matched for age and year of HCT, donor source (allogeneic v autologous), and length of follow-up. Sixty patients with late CHF were identified; median age at HCT was 45.3

reducing the voltage wave QRS&> 50%, pericardial tamponade.

with a relatively short or long period of latency after HSCT. [84-87]

asymptomatic stroke in the pericardium,

diuretics and digitalis, pericarditis,

Stage IV- death following cardiac toxicity.

short-term cardiac toxicity. [86]

for cardiotoxic events. [84-87]

years (range, 16.6 to 68.6 years); median time to CHF was 3.0 years (range, 1.03 to 18.9 years); 68% received autologous HCT. Median ejection fraction was 36.9% (range, 15% to 53%). Compared with matched controls (n = 166), patients with late CHF received more cycles of pre-HCT chemotherapy (8.6 v 4.9 cycles; P= 0.01), had greater body mass index at HCT (28.4 v 26.2 kg/m2; P= 0.01), greater lifetime anthracycline exposure (285.3 v 175.6 mg/m2; P= 0.01), and were more likely to have multiple chronic comorbidities (30.0% v 13.9%; P= 0.01). Multivariable analysis revealed number of pre-HCT chemotherapy cycles (odds ratio [OR] 1.2; P= 0.01), anthracycline dose 250 mg/m2 (OR 3.2; P= 0.05), and two or more chronic comorbidities (OR 4.3; P= 0.01) to be independently associated with late CHF. Pre-HCT exposure to anthracyclines and presence of comorbidities are primarily risk associated with late CHF after HCT. Conditioning-related therapeutic exposure does not contribute significantly to the risk. These results form the basis for identifying high-risk individuals for targeted surveillance, as well as developing preventive strategies in the form of aggressive management of comorbidities. [87]

Considering all of the above the important factors affecting the occurrence of cardiotoxic high dose chemotherapy complications include exposure to high doses of cyclophosphamide, total body irradiation, prior anthracycline therapy, the presence of dimethyl sulfoxide (DMSO) in intravenous products, infectious complications associated with neutropenia. The risk of cardiac complications after HSCT is also influenced by patient age, type of therapy applied and previous medical history. [84]

Dimethyl sulphoxide (DMSO) is cells protective factor, preventing the crystallization of water and damage to cell membranes collected stem cells, which are subjected to freezing in liquid nitrogen at temperatures below -120 degrees C. Cardiotoxicity associated with DMSO may result in hypotension, requiring fluid infusion or even inotropic drugs. Ones also reported cardiac arrhythmias such as atrial fibrillation, or bradycardia, and incidents of acute coronary syndromes. Cardiotoxicity associated with DMSO is rare. Wether DMSO is responsible for these has been questioned. [84,85] Recent reports of similar acute cardiac events despite DMSO depletion have led to the suggestion that perhaps the complications seen following infusion may be more a product of the amount of infused granulocytes, rather than DMSO. [84]

Infectious complications during early post-transplant neutropenia or prolonged immunosuppression for GVHD prophylaxis are common problems following HSCT. Overwhelming sepsis can lead to cardiopulmonary failure, necessitating prolonged intubation and cardiac inotropic support, potentially causing subclinical cardiotoxicity or other end-organ compromise that may not be evident until years following HSCT. [84]

The Tichellis study [84] provided preliminary evidence for an association between GVHD and the development of arterial disease. There are emerging data to suggest that chronic GVHD could play a role in the development of cardiovascular disease. Cardiac side effects of chronic GVHD, while rare, likely occur as a result of direct organ lymphocytic infiltration. Increased levels of circulating tumor necrosis factor alfa [TNF alfa] may impair muscle electrical activity and compromise myocardial contractility. Furthermore, increased amounts of inflammatory markers, such as TNF alfa and interleukin-6, could perpetuate endothelial injury, contributing to premature arterial events in long-term survivors after allogeneic HSCT. Treatment of GVHD is not without cardiovascular consequences.

Cardiac Complications of Cancer Treatment 59

receptors located on nerve endings centripetal parasympathetic system. Parasympathetic system also innervates the heart muscle, which explains the cause of the cardiac

Results of the review of clinical trials with MEDLINE database from the period 1963-2002

ECG changes (PR, QRS, QT, QTc, JT) were small, reversible, clinically insignificant and

 ECG changes occurred most frequently between 1 and 2 hours after administration of dolasetron, ondansetron and granisetron and returned to normal within 24 hours, no

Based on these data it was concluded that the benefits of this class of drugs greatly outweigh

Considering the high risk of cardiac complications due to oncological treatment, it is important to establish standards of conduct with patients undergoing cancer therapy

[1] Ewer MS, Von Hoff DD, Benjamin RS. A historical perspective of anthracycline

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[7] Armenian Saro H., Bhatia S. Cardiovascular disease after hematopoietic cell transplantation- lessons learned. Haematologica 2003; 93: 1132-1136. [8] Singal PK, Li T, Kumar D, et al. Adriamycin-induced heart failure: mechanizm and

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transcription factor: implication in the regulation of cardiac myocyte apoptosis.

Anthracyclines: selected New developments. Curr Med Chem Anticancer Agents.

induced symptomatic and asymptomatic cardiotoxicity duriong and after

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complications associated with this class of drugs. [89]

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their adverse effects on the cardiovascular system. [91]

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old song? FASEB J 1999; 13: 199-212.

Mol Pharmacol. 2003; 63: 368-377.

proven to cardiac toxicity.

2001; 1: 113-130.

**8. References**

for cardiotoxicity antagonists 5-HT3 receptor were as follows [89-91]:

evidence of acute, clinically significant cardiac complications.

cardiotoxicity. Heart Fail Clin 2011; 17: 363-372.

modulation. Mol Cell Biochem. 2000; 207: 77-86.

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Prolonged treatment with calcineurin inhibitors and steroids can lead to myocardial hypertrophy, as well as increase the likehood of cardiovascular disease risk factors such as hypertension, diabetes, and renal insufficiency. [84]

#### **6. Carditoxicity of radiotherapy**

In anticancer treatment, radiotherapy plays an important role. It can also damage the heart muscle. Due to the location of the heart in human body, mediastinal radiotherapy poses the highest risk of cardiac complications. This form of treatment is commonly used in lymphoma, breast, esophageal and lung cancers. In connection with the expected long period of survival of patients with Hodgkin lymphoma and breast cancer the risk of complications from cardiovascular diseases is the highest. The maximum potential safe dose of ionizing radiation on the heart area are as follows [88]:

Early cardiac complication after radiation therapy is acute pericarditis, which usually occurs within a few weeks after treatment. The most common late effect of radiotherapy on cardiovascular system is coronary artery disease. It usually appears within 10-15 years after radiation. Ones have been shown that ionizing radiation can initiate or accelerate the atherosclerotic process, this phenomenon refers mainly to people with other risk factors for cardiovascular disease. On others cardiac structures radiotherapy also has a negative effect, but to a lesser extent. It may lead to development of restrictive cardiomyopathy, cardiac diastolic dysfunction, impaired contractile function [that occurs after mated treatment with anthracyclines], aortic stenosis, QT prolongation, persistent tachycardia [damage of autonomic nervous system]. [88]

Risk factors for acute or chronic cardiac complications after radiation therapy include [88]:

#### **7. Cardiotoxicity of antagonists of 5-HT3 receptor**

Potentially cardiotoxic drugs, inevitably associated with oncology therapy, are also antagonists of 5-HT3 receptor. Chemotherapy, especially given in high doses causes of nausea and vomiting, which are very burdensome for the patient with neoplastic disease. The mechanism of vomiting is the release of large amount of serotonin, stimulating 5-HT3

Prolonged treatment with calcineurin inhibitors and steroids can lead to myocardial hypertrophy, as well as increase the likehood of cardiovascular disease risk factors such as

In anticancer treatment, radiotherapy plays an important role. It can also damage the heart muscle. Due to the location of the heart in human body, mediastinal radiotherapy poses the highest risk of cardiac complications. This form of treatment is commonly used in lymphoma, breast, esophageal and lung cancers. In connection with the expected long period of survival of patients with Hodgkin lymphoma and breast cancer the risk of complications from cardiovascular diseases is the highest. The maximum potential safe dose

about 45 Gy, while 65% of the volume of the heart is irradiated with a standard

Early cardiac complication after radiation therapy is acute pericarditis, which usually occurs within a few weeks after treatment. The most common late effect of radiotherapy on cardiovascular system is coronary artery disease. It usually appears within 10-15 years after radiation. Ones have been shown that ionizing radiation can initiate or accelerate the atherosclerotic process, this phenomenon refers mainly to people with other risk factors for cardiovascular disease. On others cardiac structures radiotherapy also has a negative effect, but to a lesser extent. It may lead to development of restrictive cardiomyopathy, cardiac diastolic dysfunction, impaired contractile function [that occurs after mated treatment with anthracyclines], aortic stenosis, QT prolongation, persistent tachycardia [damage of

Risk factors for acute or chronic cardiac complications after radiation therapy include [88]: irradiated volume of the heart (cardiac risk is proportional to the irradiated volume of

patient age (younger age at the time of exposure to ionizing radiation is a risk factor for

time of exposure (the majority of published reports that the need for a minimum of 10

 dose and technique of application of ionizing radiation (it is considered that the total radiation dose in the case of mediastinal radiotherapy, increasing the risk of cardiovascular disease is> 35-40 Gy, whereas a fractional dose> 2 Gy per day) and

Potentially cardiotoxic drugs, inevitably associated with oncology therapy, are also antagonists of 5-HT3 receptor. Chemotherapy, especially given in high doses causes of nausea and vomiting, which are very burdensome for the patient with neoplastic disease. The mechanism of vomiting is the release of large amount of serotonin, stimulating 5-HT3

years to the risk of death from heart attack increased above 10%),

prior chemotherapy (especially anthracycline-based regimens).

**7. Cardiotoxicity of antagonists of 5-HT3 receptor**

hypertension, diabetes, and renal insufficiency. [84]

of ionizing radiation on the heart area are as follows [88]:

approximately 60 Gy when 25% or less heart volume is irradiated,

**6. Carditoxicity of radiotherapy**

fractionation at 2 Gy per day.

autonomic nervous system]. [88]

the heart),

vascular disease),

receptors located on nerve endings centripetal parasympathetic system. Parasympathetic system also innervates the heart muscle, which explains the cause of the cardiac complications associated with this class of drugs. [89]

Results of the review of clinical trials with MEDLINE database from the period 1963-2002 for cardiotoxicity antagonists 5-HT3 receptor were as follows [89-91]:

Based on these data it was concluded that the benefits of this class of drugs greatly outweigh their adverse effects on the cardiovascular system. [91]

Considering the high risk of cardiac complications due to oncological treatment, it is important to establish standards of conduct with patients undergoing cancer therapy proven to cardiac toxicity.

#### **8. References**

Cardiac Complications of Cancer Treatment 61

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2508.

**4**

*USA*

*Cardiovascular Medicine,*

 *St. Elizabeth's Medical Center and*

*Tufts University School of Medicine, Boston,*

**Neuregulin1-ErbB Signaling in**

**Doxorubicin-Induced Cardiotoxicity**

David Goukassian, James P. Morgan and Xinhua Yan\*

This chapter will review basic and clinical findings regarding the cardioprotective role of Neuregulin1-ErbB signaling against the cardiotoxicity of doxorubicin, a widely used chemotherapeutic agent. In 2001, *The New England Journal of Medicine* published the results from clinical trials in breast cancer patients using Trastuzumab, a monoclonal antibody that blocks the ErbB2 receptor. These studies showed that the incidence of New York Heart Association (NYHA) class III/IV heart failure was 16% in patients who were concurrently treated with doxorubicin and Trastuzumab compared to 3% and 2% respectively in patients who were treated with doxorubicin or Trastuzumab alone (Slamon et al., 2001). These results for the first time suggest that the ErbB signaling has cardioprotective effects against doxorubicin-induced cardiotoxicity. Since then, a significant amount of basic and clinical research has been conducted to investigate the mechanisms of the ErbB signaling pathway in protecting the heart from doxorubicin-induced toxicity. At the same time, studies have been performed searching for factors that stimulate ErbB signaling to protect the heart from doxorubicin. Although ErbB receptors have at least 13 ligands, Neuregulin1 proteins have become the focus of this search (Yarden and Sliwkowski, 2001a). Studies from cardiomyocyte culture, animal models and clinical trials have demonstrated that Neuregulin1 may be effective for preventing or treating doxorubicin-induced cardiotoxicity.

The anthracycline drug doxorubicin was discovered about 40 years ago and continues to be used as a first line antineoplastic drug (Outomuro et al., 2007a; Moretti et al., 2009). Doxorubicin is effective for the treatment of a wide variety of cancers in children and adults, such as leukemia, lymphoma, breast, lung and colon cancers (Outomuro et al., 2007a; Anderson and Sawyer, 2008). Doxorubicin has significantly improved survival of childhood cancer patients, in which the survival rate is now approaching 75% (Curry et al., 2006). There are an estimated 300,000 childhood cancer survivors in the United States (Jemal et al.,

**1. Introduction**

**2.1 Background**

\* Corresponding Author

**2. Doxorubicin-induced cardiotoxicity**

### **Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity**

David Goukassian, James P. Morgan and Xinhua Yan\* *Cardiovascular Medicine, St. Elizabeth's Medical Center and Tufts University School of Medicine, Boston, USA*

#### **1. Introduction**

64 Cardiotoxicity of Oncologic Treatments

[90] Goodin S.,Cunningham R. 5-HT3 receptor antagonists for the treatment of nausea and vomiting. A reappraisal of their side-effect profile. Oncologist 2002; 7: 424-436. [91] Navari R.M., Koeller J.M. Electrocardiographic and cardiovascular effects of the 5-HT3

receptor antagonists. Ann Pharmacother 2003; 37: 1276-1286.

This chapter will review basic and clinical findings regarding the cardioprotective role of Neuregulin1-ErbB signaling against the cardiotoxicity of doxorubicin, a widely used chemotherapeutic agent. In 2001, *The New England Journal of Medicine* published the results from clinical trials in breast cancer patients using Trastuzumab, a monoclonal antibody that blocks the ErbB2 receptor. These studies showed that the incidence of New York Heart Association (NYHA) class III/IV heart failure was 16% in patients who were concurrently treated with doxorubicin and Trastuzumab compared to 3% and 2% respectively in patients who were treated with doxorubicin or Trastuzumab alone (Slamon et al., 2001). These results for the first time suggest that the ErbB signaling has cardioprotective effects against doxorubicin-induced cardiotoxicity. Since then, a significant amount of basic and clinical research has been conducted to investigate the mechanisms of the ErbB signaling pathway in protecting the heart from doxorubicin-induced toxicity. At the same time, studies have been performed searching for factors that stimulate ErbB signaling to protect the heart from doxorubicin. Although ErbB receptors have at least 13 ligands, Neuregulin1 proteins have become the focus of this search (Yarden and Sliwkowski, 2001a). Studies from cardiomyocyte culture, animal models and clinical trials have demonstrated that Neuregulin1 may be effective for preventing or treating doxorubicin-induced cardiotoxicity.

#### **2. Doxorubicin-induced cardiotoxicity**

#### **2.1 Background**

The anthracycline drug doxorubicin was discovered about 40 years ago and continues to be used as a first line antineoplastic drug (Outomuro et al., 2007a; Moretti et al., 2009). Doxorubicin is effective for the treatment of a wide variety of cancers in children and adults, such as leukemia, lymphoma, breast, lung and colon cancers (Outomuro et al., 2007a; Anderson and Sawyer, 2008). Doxorubicin has significantly improved survival of childhood cancer patients, in which the survival rate is now approaching 75% (Curry et al., 2006). There are an estimated 300,000 childhood cancer survivors in the United States (Jemal et al.,

<sup>\*</sup> Corresponding Author

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 67

(3) inhibition of cardiac specific muscle gene transcription and translation, in combination with an increase in myofibril protein degradation, leading to loss of myofibrils (Lewis and Gonzalez, 1987; Ito et al., 1990; Kurabayashi et al., 1994; Toyoda et al., 1998; d'Anglemont de Tassigny et al., 2004; Lim et al., 2004b), and (4) disturbance of intracellular calcium homeostasis (De Beer et al., 2001; Wallace, 2003). The mechanism of doxorubicin-induced free radical generation and oxidative stress has been reviewed in other chapters of this book as well as comprehensive reviews in the field (Singal et al., 2000; Berthiaume and Wallace, 2007; Simunek et al., 2009). In this chapter, we will focus on the mechanisms that connect the

Studies have been conducted to investigate doxorubicin-induced cardiomyocyte apoptosis in H9c2 rat embryonic cardiomyocytes, neonatal and adult rat cardiomyocytes, as well as in hearts from rats and mice treated with doxorubicin (Childs et al., 2002a; Fukazawa et al.,

Doxorubicin induces apoptosis via both intrinsic and extrinsic pathways. Doxorubicin alters the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins, including Bcl-2, Bad, Bim, Bax, Bak and Bik (Aries et al., 2004; Rohrbach et al., 2005b; Kobayashi et al., 2006); it also causes DNA damage and p53 activation (Liu et al., 2004; L'Ecuyer et al., 2006). All these changes can cause loss of mitochondrial integrity, the leakage of cytochrome c and the activation of caspase 9 (Hengartner, 2000; Green and Leeuwenburgh, 2002). Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis in cardiomyocytes (Green and Leeuwenburgh, 2002). Within the extrinsic pathway, doxorubicin increases Fas and FasL, followed by the activation of caspase 8 (Nakamura et al., 2000; Kalivendi et al., 2005b). The activation of caspase 9 and/or caspase 8 eventually leads to the activation of

Doxorubicin selectively down-regulates cardiac specific muscle gene expressions. These may involve decreases in cardiac muscle gene transcription and translation, as well as

Studies have shown that doxorubicin decreases the expression of α-sarcomeric actin, cardiac troponin I (cTnI), and myosin light chain 2 (MLC 2) (Ito et al., 1990). Doxorubicin treatment decreases cardiac troponins in left ventricular tissues of mice and in cultured rat neonatal cardiomyocytes (Bian et al., 2009). Down-regulations of cardiac troponins by doxorubicin are caused by decreased transcription and translation as well as increased caspase and proteasome degradation of these proteins (Bian et al., 2009). Caspase 3, 5, 6 or 10 directly cleaves cardiac troponins, while caspase 9 or 13 may indirectly cause degradation of these

Other reports also have shown that doxorubicin inhibits the expression of transcription factors or cofactors that are important for regulation of cardiac-specific gene transcription. These include GATA4, MEF2C, dHAND, Nkx2.5 and p300 (Poizat et al., 2000; Aries et al., 2004). In addition to cardiac genes, GATA4 may also regulate genes that are involved in the process of apoptosis. Overexpression of GATA4 in cardiomyocytes or mouse hearts

increases in selective proteasome degradation of these proteins (Poizat et al., 2000).

2003; Liu et al., 2005; Youn et al., 2005; Fu and Arcasoy, 2007; Bian et al., 2009).

caspase 3, cleavage of genomic DNA and apoptosis (Hengartner, 2000).

**2.2.2 Doxorubicin-induced myofibril loss in cardiomyocytes**

proteins (Bian et al., 2009).

Neuregulin1-ErbB signaling to doxorubicin cardiotoxicity.

**2.2.1 Doxorubicin-induced apoptosis in the cardiomyocyte**

2006; Oeffinger et al., 2006). Nearly 60% of them have been treated with doxorubicin or its analogs (van Dalen et al., 2006; Bryant et al., 2007; LoPiccolo et al., 2008). In adult patients, such as breast cancer patients, it is estimated that 30% of them have been treated with doxorubicin (Paik et al., 2000; Pritchard et al., 2006). However, dose-related cardiotoxicity has limited the use of doxorubicin at all in a subset of cancer patients with pre-existing cardiovascular conditions and in cancer patients in general, due to significant risk of developing irreversible heart failure months and years after doxorubicin treatment (Barry et al., 2007a; Outomuro et al., 2007a; Anderson and Sawyer, 2008).

The incidence of doxorubicin-induced cardiotoxicity has been reported to be in a variable range of 0.4 - 41% (Outomuro et al., 2007a). The risk of doxorubicin-induced cardiotoxicity mainly depends on the cumulative dose of the drug administered. The incidence of doxorubicin-induced heart failure is about 3% at a cumulative dose of 400 mg/m2 , 7.5% at a cumulative dose of 550 mg/m2 and 18% at 700 mg/m2 (Von Hoff et al., 1979; Swain et al., 2003; Outomuro et al., 2007a). Other factors, such as age (very young or elderly patients), pre-existing cardiovascular disease, and previous or concurrent use of other anti-cancer cytotoxic or targeted therapies, can increase the risk of doxorubicin-induced heart failure (Von Hoff et al., 1979; Safra, 2003; Outomuro et al., 2007b). Trastuzumab, a monoclonal antibody that blocks the HER2 receptor, is the first drug approved by the US Food and Drug Administration (FDA) for targeted cancer therapy. When Trastuzumab and doxorubicin were concurrently used in breast cancer patients, the incidence of New York Heart Association type III and IV heart failure rose from 2-3% to 16% over a period of 50 months of observation (Slamon et al., 2001).

Doxorubicin can cause acute, subacute and late cardiotoxicity. The acute doxorubicin cardiotoxicity starts within 24 hours of drug infusion, and may present as cardiac arrhythmia, myocarditis and pericarditis (Appelbaum et al., 1976). The long-term prognosis of acute doxorubicin cardiotoxicity is relatively good. Subacute doxorubicin cardiotoxicity occurs weeks and months after the treatment, while late cardiotoxicity can occur 4-20 years after the cessation of the treatment. The onset of chronic doxorubicin cardiotoxicity is insidious; the disease, however, progressively develops to severe and irreversible heart failure (Simsir et al., 2005). Therefore, doxorubicin cardiotoxicity is a Type I chemotherapyrelated cardiac dysfunction (CRCD) (Ewer and Lippman, 2005). It is irreversible and presents a life-long threat for cancer survivors (Ewer and Lippman, 2005).

The pathological changes in chronic doxorubicin cardiotoxicity include cardiac hypertrophy, which may be followed with thinning of the ventricular wall (dilated cardiomyopathy), interstitial fibrosis, vascular and mitochondrial degeneration.  Nearly 60% of them have been treated with doxorubicin or its analogs (van Dalen et al., 2006; Bryant et al., 2007; LoPiccolo et al., 2008). In adult patients, such as breast cancer patients, it is estimated that 30% of them have been treated with doxorubicin (Paik et al., 2000; Pritchard et al., 2006). However, dose-related cardiotoxicity has limited the use of doxorubicin at all in a subset of cancer patients with pre-existing cardiovascular conditions and in cancer patients in general, due to significant risk of developing irreversible heart failure months and years after doxorubicin treatment (Barry et al., 2007a;

The incidence of doxorubicin-induced cardiotoxicity has been reported to be in a variable range of 0.4 - 41% (Outomuro et al., 2007a). The risk of doxorubicin-induced cardiotoxicity mainly depends on the cumulative dose of the drug administered. The incidence of doxorubicin-induced heart failure is about 3% at a cumulative dose of 400 mg/m2 , 7.5% at a cumulative dose of 550 mg/m2 and 18% at 700 mg/m2 (Von Hoff et al., 1979; Swain et al., 2003; Outomuro et al., 2007a). Other factors, such as age (very young or elderly patients), pre-existing cardiovascular disease, and previous or concurrent use of other anti-cancer cytotoxic or targeted therapies, can increase the risk of doxorubicin-induced heart failure (Von Hoff et al., 1979; Safra, 2003; Outomuro et al., 2007b). Trastuzumab, a monoclonal antibody that blocks the HER2 receptor, is the first drug approved by the US Food and Drug Administration (FDA) for targeted cancer therapy. When Trastuzumab and doxorubicin were concurrently used in breast cancer patients, the incidence of New York Heart Association type III and IV heart failure rose from 2-3% to 16% over a period of 50 months of

Doxorubicin can cause acute, subacute and late cardiotoxicity. The acute doxorubicin cardiotoxicity starts within 24 hours of drug infusion, and may present as cardiac arrhythmia, myocarditis and pericarditis (Appelbaum et al., 1976). The long-term prognosis of acute doxorubicin cardiotoxicity is relatively good. Subacute doxorubicin cardiotoxicity occurs weeks and months after the treatment, while late cardiotoxicity can occur 4-20 years after the cessation of the treatment. The onset of chronic doxorubicin cardiotoxicity is insidious; the disease, however, progressively develops to severe and irreversible heart failure (Simsir et al., 2005). Therefore, doxorubicin cardiotoxicity is a Type I chemotherapyrelated cardiac dysfunction (CRCD) (Ewer and Lippman, 2005). It is irreversible and

The pathological changes in chronic doxorubicin cardiotoxicity include cardiac hypertrophy, which may be followed with thinning of the ventricular wall (dilated cardiomyopathy), interstitial fibrosis, vascular and mitochondrial degeneration.

Lipshultz et al., 2005; Barry et al., 2007b).

(3) inhibition of cardiac specific muscle gene transcription and translation, in combination with an increase in myofibril protein degradation, leading to loss of myofibrils (Lewis and Gonzalez, 1987; Ito et al., 1990; Kurabayashi et al., 1994; Toyoda et al., 1998; d'Anglemont de Tassigny et al., 2004; Lim et al., 2004b), and (4) disturbance of intracellular calcium homeostasis (De Beer et al., 2001; Wallace, 2003). The mechanism of doxorubicin-induced free radical generation and oxidative stress has been reviewed in other chapters of this book as well as comprehensive reviews in the field (Singal et al., 2000; Berthiaume and Wallace, 2007; Simunek et al., 2009). In this chapter, we will focus on the mechanisms that connect the Neuregulin1-ErbB signaling to doxorubicin cardiotoxicity.

#### **2.2.1 Doxorubicin-induced apoptosis in the cardiomyocyte**

Studies have been conducted to investigate doxorubicin-induced cardiomyocyte apoptosis in H9c2 rat embryonic cardiomyocytes, neonatal and adult rat cardiomyocytes, as well as in hearts from rats and mice treated with doxorubicin (Childs et al., 2002a; Fukazawa et al., 2003; Liu et al., 2005; Youn et al., 2005; Fu and Arcasoy, 2007; Bian et al., 2009).

Doxorubicin induces apoptosis via both intrinsic and extrinsic pathways. Doxorubicin alters the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins, including Bcl-2, Bad, Bim, Bax, Bak and Bik (Aries et al., 2004; Rohrbach et al., 2005b; Kobayashi et al., 2006); it also causes DNA damage and p53 activation (Liu et al., 2004; L'Ecuyer et al., 2006). All these changes can cause loss of mitochondrial integrity, the leakage of cytochrome c and the activation of caspase 9 (Hengartner, 2000; Green and Leeuwenburgh, 2002). Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis in cardiomyocytes (Green and Leeuwenburgh, 2002). Within the extrinsic pathway, doxorubicin increases Fas and FasL, followed by the activation of caspase 8 (Nakamura et al., 2000; Kalivendi et al., 2005b). The activation of caspase 9 and/or caspase 8 eventually leads to the activation of caspase 3, cleavage of genomic DNA and apoptosis (Hengartner, 2000).

#### **2.2.2 Doxorubicin-induced myofibril loss in cardiomyocytes**

Doxorubicin selectively down-regulates cardiac specific muscle gene expressions. These may involve decreases in cardiac muscle gene transcription and translation, as well as increases in selective proteasome degradation of these proteins (Poizat et al., 2000).

Studies have shown that doxorubicin decreases the expression of α-sarcomeric actin, cardiac troponin I (cTnI), and myosin light chain 2 (MLC 2) (Ito et al., 1990). Doxorubicin treatment decreases cardiac troponins in left ventricular tissues of mice and in cultured rat neonatal cardiomyocytes (Bian et al., 2009). Down-regulations of cardiac troponins by doxorubicin are caused by decreased transcription and translation as well as increased caspase and proteasome degradation of these proteins (Bian et al., 2009). Caspase 3, 5, 6 or 10 directly cleaves cardiac troponins, while caspase 9 or 13 may indirectly cause degradation of these proteins (Bian et al., 2009).

Other reports also have shown that doxorubicin inhibits the expression of transcription factors or cofactors that are important for regulation of cardiac-specific gene transcription. These include GATA4, MEF2C, dHAND, Nkx2.5 and p300 (Poizat et al., 2000; Aries et al., 2004). In addition to cardiac genes, GATA4 may also regulate genes that are involved in the process of apoptosis. Overexpression of GATA4 in cardiomyocytes or mouse hearts

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 69

the adverse effects of doxorubicin on Ca2+ homeostasis, thereby exacerbating the disease. On the other hand, activation of SERCA2a at a later stage of the disease may be beneficial.

The Neuregulin1-ErbB signaling pathway is an evolutionally conserved signaling pathway. It is pivotal for the development of various organ systems including the heart. It is also important for maintaining normal physiological functions of these organs. During the past two decades, studies using genetically modified mouse models, and most recently systems biology approaches, have revealed that ErbB receptors and their ligands form a complex signaling network, which includes an input layer, signal-processing layers and an output layer. This signaling system regulates a wide range of functions of the cell. In the heart, a

The ErbB receptors, also known as HER receptors, are epidermal growth factor (EGF) receptor tyrosine kinases (RTKs). Worm *C. elegans* contains one ErbB receptor and one ligand (Aroian et al., 1990). In Drosophila, there are one ErbB receptor and four ligands (Freeman, 1998). In humans, there are four members of this RTK family, which include ErbB1 (also known as EGFR, HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) (Citri and Yarden, 2006). There are 13 polypeptide extracellular ErbB ligands, which include Neuregulin proteins, epidermal growth factor (EGF), epiregulin, betacellulin and others (Yarden and Sliwkowski, 2001b). All ErbB ligands contain a conserved epidermal growth factor (EGF)-like domain (Yarden and Sliwkowski, 2001b; Citri and Yarden, 2006). This multilayered and various combinations of ErbB receptors and ligands suggest an apparently more sophisticated and fine-tuned NRG1-ErbB axis-dependent regulation of signal

A unique feature of the ErbB family is that there is no known ligand for the ErbB2 receptor (Klapper et al., 1999), while ErbB3 lacks intrinsic kinase activity (Guy et al., 1994). However, ErbB2 and ErbB3 can form heterodimers to generate potent cellular signals (Citri et al., 2003). The ErbB2 receptor is a preferred heterodimeric partner of the other three ErbB receptors (Graus-Porta et al., 1997). Heterodimers which contain ErbB2 have higher affinity

The ErbB receptors are expressed in various types of cells, including epithelial, mesenchymal, neuronal and cardiomyocytes. The ErbB receptors play important roles in organ development and maintaining the normal physiological function of adult tissues. In cancer cells, ErbB receptors are aberrantly expressed and constitutively activated. Therefore, they are major drug targets for cancer therapy (Alimandi et al., 1995; Moasser, 2007). In the developing heart, the ErbB2 and ErbB4 receptors are detected in the cardiac myocardium and endocardium (Erickson et al., 1997; Meyer et al., 1997; Zhao et al., 1998; Fuller et al., 2008; Pentassuglia and Sawyer, 2009; De Keulenaer et al., 2010), while the ErbB3 receptor is expressed in the cardiac endocardium and mesenchyme (Erickson et al., 1997; Camenisch et

**3. The Neuregulin1-ErbB signaling and its physiological functions in the**

significant number of studies have been performed in this area.

**3.1 The ErbB receptor tyrosine kinases 3.1.1 Members of the ErbB receptor family**

transduction and biological responses in humans.

and broader specificity for ligands.

**heart**

attenuates doxorubicin-induced apoptosis. On the other hand, GATA4 null mice are more susceptible to doxorubicin cardiotoxicity (Aries et al., 2004).

In addition to myofibril loss, doxorubicin also induces myofibril disarray in cardiomyocytes (Sawyer et al., 2002). Degradation of titin, a myofilament protein, may contribute to this effect of doxorubicin. Titin is a scaffold protein that assembles myofilament proteins into sarcomeres. It regulates cardiomyocyte contractile function via length-dependent activation in stretched sarcomeres during the transition from diastole to systole (Helmes et al., 2003). Doxorubicin activates calcium-dependent proteases calpains which in turn cause titin degradation (Lim et al., 2004a).

#### **2.2.3 Doxorubicin disturbs calcium homeostasis in cardiomyocytes**

The sarcoplasmic reticulum Ca2+ pump (SERCA2a) plays a pivotal role in intracellular calcium mobilization and thus myocardial contractility. The sarcoplasmic reticulum (SR) orchestrates the movement of calcium during both contraction and relaxation of the heart. Excitation leads to the opening of voltage gated L-type calcium channels, allowing the entry of calcium, which then stimulates the release of a much larger amount of calcium from SR and subsequent contraction. During relaxation, calcium is re-sequestered into SR by SERCA2a and extruded to the extracellular fluid by the sarcolemmal sodium-calcium exchanger (NCX) (del Monte et al., 1999; Wehrens and Marks, 2004). A decrease in SR Ca2+ ATPase activity and Ca2+ uptake is responsible for the abnormal Ca2+ homeostasis in human cardiomyocytes from failing hearts (Schmidt et al., 1998; Schmidt et al., 1999).

Studies have shown that doxorubicin can either increase or decrease cardiomyocyte contractility. The discrepancy of these findings may be caused by different animal and cell culture models, the dosage of doxorubicin, the duration of the treatment and especially the developmental stage of the disease. In the early stage of the disease, doxorubicin tends to induce Ca2+ release from SR and increase cardiomyocyte contractility (Brown et al., 1989b; Kim et al., 1989; Ondrias et al., 1990; Kapelko et al., 1996). In the late stage of the disease, doxorubicin inhibits Ca2+ regulatory proteins and reduces cardiomyocyte contractility (Ondrias et al., 1990; Dodd et al., 1993; Maeda et al., 1998; Boucek et al., 1999; Chugun et al., 2000; Gambliel et al., 2002; Timolati et al., 2006). In a subacute doxorubicin mouse model, doxorubicin induces an increase in cardiac contractile function as measured by dP/dtmax and dP/dtmin during the first few days after the doxorubicin injection; however, cardiac function declines later on (our unpublished data). These results are consistent with the findings in doxorubicin-treated patients (Brown et al., 1989b; Barry et al., 2007a).

Doxorubicin-induced reduction of cardiomyocyte contractility is often associated with decreased expression of SERCA2a (Dodd et al., 1993; Boucek et al., 1999; Gambliel et al., 2002), suggesting that impaired SERCA2a function may contribute to doxorubicin-induced cardiomyocyte contractile dysfunction.

Studies in mice with cardiomyocyte-specific overexpression of SERCA2a, however, showed that SERCA2a overexpression exacerbated doxorubicin-induced mortality and morphological damage to cardiac tissue (Burke et al., 2003). These results may be caused by constitutive activation of SERCA2a, especially during the early stage of doxorubicin cardiac injury. Increase of SERCA2a activities at the early stage of the disease may further aggravate

attenuates doxorubicin-induced apoptosis. On the other hand, GATA4 null mice are more

In addition to myofibril loss, doxorubicin also induces myofibril disarray in cardiomyocytes (Sawyer et al., 2002). Degradation of titin, a myofilament protein, may contribute to this effect of doxorubicin. Titin is a scaffold protein that assembles myofilament proteins into sarcomeres. It regulates cardiomyocyte contractile function via length-dependent activation in stretched sarcomeres during the transition from diastole to systole (Helmes et al., 2003). Doxorubicin activates calcium-dependent proteases calpains which in turn cause titin

The sarcoplasmic reticulum Ca2+ pump (SERCA2a) plays a pivotal role in intracellular calcium mobilization and thus myocardial contractility. The sarcoplasmic reticulum (SR) orchestrates the movement of calcium during both contraction and relaxation of the heart. Excitation leads to the opening of voltage gated L-type calcium channels, allowing the entry of calcium, which then stimulates the release of a much larger amount of calcium from SR and subsequent contraction. During relaxation, calcium is re-sequestered into SR by SERCA2a and extruded to the extracellular fluid by the sarcolemmal sodium-calcium exchanger (NCX) (del Monte et al., 1999; Wehrens and Marks, 2004). A decrease in SR Ca2+ ATPase activity and Ca2+ uptake is responsible for the abnormal Ca2+ homeostasis in human

Studies have shown that doxorubicin can either increase or decrease cardiomyocyte contractility. The discrepancy of these findings may be caused by different animal and cell culture models, the dosage of doxorubicin, the duration of the treatment and especially the developmental stage of the disease. In the early stage of the disease, doxorubicin tends to induce Ca2+ release from SR and increase cardiomyocyte contractility (Brown et al., 1989b; Kim et al., 1989; Ondrias et al., 1990; Kapelko et al., 1996). In the late stage of the disease, doxorubicin inhibits Ca2+ regulatory proteins and reduces cardiomyocyte contractility (Ondrias et al., 1990; Dodd et al., 1993; Maeda et al., 1998; Boucek et al., 1999; Chugun et al., 2000; Gambliel et al., 2002; Timolati et al., 2006). In a subacute doxorubicin mouse model, doxorubicin induces an increase in cardiac contractile function as measured by dP/dtmax and dP/dtmin during the first few days after the doxorubicin injection; however, cardiac function declines later on (our unpublished data). These results are consistent with the

susceptible to doxorubicin cardiotoxicity (Aries et al., 2004).

**2.2.3 Doxorubicin disturbs calcium homeostasis in cardiomyocytes**

cardiomyocytes from failing hearts (Schmidt et al., 1998; Schmidt et al., 1999).

findings in doxorubicin-treated patients (Brown et al., 1989b; Barry et al., 2007a).

Doxorubicin-induced reduction of cardiomyocyte contractility is often associated with decreased expression of SERCA2a (Dodd et al., 1993; Boucek et al., 1999; Gambliel et al., 2002), suggesting that impaired SERCA2a function may contribute to doxorubicin-induced

Studies in mice with cardiomyocyte-specific overexpression of SERCA2a, however, showed that SERCA2a overexpression exacerbated doxorubicin-induced mortality and morphological damage to cardiac tissue (Burke et al., 2003). These results may be caused by constitutive activation of SERCA2a, especially during the early stage of doxorubicin cardiac injury. Increase of SERCA2a activities at the early stage of the disease may further aggravate

degradation (Lim et al., 2004a).

cardiomyocyte contractile dysfunction.

the adverse effects of doxorubicin on Ca2+ homeostasis, thereby exacerbating the disease. On the other hand, activation of SERCA2a at a later stage of the disease may be beneficial.

#### **3. The Neuregulin1-ErbB signaling and its physiological functions in the heart**

The Neuregulin1-ErbB signaling pathway is an evolutionally conserved signaling pathway. It is pivotal for the development of various organ systems including the heart. It is also important for maintaining normal physiological functions of these organs. During the past two decades, studies using genetically modified mouse models, and most recently systems biology approaches, have revealed that ErbB receptors and their ligands form a complex signaling network, which includes an input layer, signal-processing layers and an output layer. This signaling system regulates a wide range of functions of the cell. In the heart, a significant number of studies have been performed in this area.

#### **3.1 The ErbB receptor tyrosine kinases**

#### **3.1.1 Members of the ErbB receptor family**

The ErbB receptors, also known as HER receptors, are epidermal growth factor (EGF) receptor tyrosine kinases (RTKs). Worm *C. elegans* contains one ErbB receptor and one ligand (Aroian et al., 1990). In Drosophila, there are one ErbB receptor and four ligands (Freeman, 1998). In humans, there are four members of this RTK family, which include ErbB1 (also known as EGFR, HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) (Citri and Yarden, 2006). There are 13 polypeptide extracellular ErbB ligands, which include Neuregulin proteins, epidermal growth factor (EGF), epiregulin, betacellulin and others (Yarden and Sliwkowski, 2001b). All ErbB ligands contain a conserved epidermal growth factor (EGF)-like domain (Yarden and Sliwkowski, 2001b; Citri and Yarden, 2006). This multilayered and various combinations of ErbB receptors and ligands suggest an apparently more sophisticated and fine-tuned NRG1-ErbB axis-dependent regulation of signal transduction and biological responses in humans.

A unique feature of the ErbB family is that there is no known ligand for the ErbB2 receptor (Klapper et al., 1999), while ErbB3 lacks intrinsic kinase activity (Guy et al., 1994). However, ErbB2 and ErbB3 can form heterodimers to generate potent cellular signals (Citri et al., 2003). The ErbB2 receptor is a preferred heterodimeric partner of the other three ErbB receptors (Graus-Porta et al., 1997). Heterodimers which contain ErbB2 have higher affinity and broader specificity for ligands.

The ErbB receptors are expressed in various types of cells, including epithelial, mesenchymal, neuronal and cardiomyocytes. The ErbB receptors play important roles in organ development and maintaining the normal physiological function of adult tissues. In cancer cells, ErbB receptors are aberrantly expressed and constitutively activated. Therefore, they are major drug targets for cancer therapy (Alimandi et al., 1995; Moasser, 2007). In the developing heart, the ErbB2 and ErbB4 receptors are detected in the cardiac myocardium and endocardium (Erickson et al., 1997; Meyer et al., 1997; Zhao et al., 1998; Fuller et al., 2008; Pentassuglia and Sawyer, 2009; De Keulenaer et al., 2010), while the ErbB3 receptor is expressed in the cardiac endocardium and mesenchyme (Erickson et al., 1997; Camenisch et

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 71

et al., 1993). Subsequently, it was found that all these factors were encoded by the same gene, which is the Neuregulin1 gene (Burden and Yarden, 1997). Therefore, Neuregulin1 proteins were also called Neu differentiation factor (NDF), heregulins, glial growth factor (GGF), acetylcholine receptor inducing activity (ARIA). Subsequently, three other Neuregulin genes were discovered, which are Neuregulin2, Neuregulin3 and Neuregulin4 (Carraway et al., 1997; Zhang et al., 1997; Harari et al., 1999). However, limited information is available regarding the biological functions of Neuregulin2, 3 and 4 gene encoded proteins. Neureuglin1 proteins bind directly to the ErbB3 and the ErbB4 receptors. The ErbB2 receptor does not bind directly to Neureuglin1; rather it serves as a co-receptor and forms heterodimers with the

ErbB3 or the ErbB4 receptor (Falls et al., 1993; Burden and Yarden, 1997).

Fig. 1. The ErbB Signaling Network. ErbB receptors and their ligands form a complex signaling network which regulates a wide variety of cell functions. There are four ErbB receptors (ErbB1-ErbB4). The ErbB2 receptor does not bind to any ligand, but is a preferred partner of other ErbB receptors. The ErbB3 receptor is devoid of kinase activity. ErbB2 and ErbB3 can form functional and potent heterodimers. There are at least 13 known ligands for the ErbB receptors, including Neuregulin1 proteins. Ligand binding with the ErbB receptors activates various signaling molecules in the cell. The PI3K and MAPK pathways are among the most studied pathways. Multiple feedback loops exist within and outside of this signaling network. The activation and integration of these pathways lead to the regulation of cell survival, growth, proliferation, migration and differentiation. NRG, Neuregulin; EGF,

epidermal growth factor; LPA, lysophosphatidic acid; ET, endothelin; GAP, GTPase

PLC, phospholipase C; Shp2, Src homology domain-2-containing protein tyrosine phosphatase 2; Stat, signal transducer and activator of transcription. Reprinted by

permission from *Nat Rev Mol Cell Biol; 2, 127-137, 2001*.

activating protein; HB-EGF, heparin-binding EGF; Jak, Janus kinase; PKC, protein kinase C;

al., 2002). ErbB2 and ErbB4 are expressed in adult cardiomyocytes (Zhao et al., 1998). Further more, recent studies have shown that adult cardiomyocytes also express the ErbB3 receptor (Camprecios et al., 2011).

#### **3.1.2 The signaling network activated by the ErbB receptors**

The ErbB receptors contain an extracellular ligand binding domain, a single transmembrane domain and intracellular kinase domain. Upon ligand binding, ErbB receptors form homoor heterodimers, which trigger auto- or trans-phosphorylation on tyrosine residues of the receptors. These residues then serve as docking sites for recruiting signaling molecules from the cytosol to the cell membrane, which then activate various signaling pathways.

The dimerization of ErbB receptors induces activation of a wide range of signaling molecules. The PI3K and MAPK pathways are among the most studied. In addition, the ErbB receptors activate other pathways, such as STATs, PLCs and JNK. These molecules further regulate the activities of key transcriptional factors, such as Jun, fos and Myc, leading to the modulation of various aspects of cell function including survival, proliferation, and migration.

Systems biology studies have revealed that this signaling network is highly organized and precisely regulated by multiple layers of regulatory and control mechanisms, which include activation of specific pathways, positive and negative feedback loops and horizontal control by parallel signaling networks.

Phosphoproteomics studies have shown that each ErbB receptor has a specific and preferred binding pattern with signaling molecules (Schulze et al., 2005). The ErbB2 receptor has few binding partners, among which, Shc is the most common partner. The ErbB3 receptor has multiple binding sites for the PI3K subunit p85. ErbB1 and ErbB4 receptors show a diversity of interaction partners including STAT5 and Grb2.

Feedback loops in and outside of this network help maintain homeostasis. For example, ErbB-mediated activation of the MAPK pathway induces the transcription of TGFα and HB-EGF (Schulze et al., 2001), which in turn further activate the ErbB receptors. ErbB activation can also lead to transcription of proteins that inhibit further activation of a particular pathway. For example, ErbB1 receptor activation by EGF causes expression of the suppressor of cytokine signaling (SOCS), which in turn promotes ErbB1 degradation (Kario et al., 2005). In addition, the ErbB signaling can be regulated by G-protein coupled receptors (GPCRs). Studies have shown that thrombin and endothelin have positive effects on the ErbB signaling via activation of matrix metalloproteinases (MMPs), subsequent activation of ErbB ligands, or activation of Src or Pyk2 which phosphorylate ErbB receptors (Dikic et al., 1996; Yarden and Sliwkowski, 2001b; Negro et al., 2006).

#### **3.2 Neuregulin1 proteins**

Neuregulin1 proteins were discovered during 1992-1993 by four independent research groups. At the time, two of the groups were searching for a ligand for the oncogene ErbB2 (Holmes et al., 1992; Peles et al., 1992; Wen et al., 1992). One group was searching for a factor that stimulated the proliferation of Schwann cells (Marchionni et al., 1993), and the third was searching for a factor that stimulated the synthesis of muscle receptors for acetylcholine (Falls

al., 2002). ErbB2 and ErbB4 are expressed in adult cardiomyocytes (Zhao et al., 1998). Further more, recent studies have shown that adult cardiomyocytes also express the ErbB3

The ErbB receptors contain an extracellular ligand binding domain, a single transmembrane domain and intracellular kinase domain. Upon ligand binding, ErbB receptors form homoor heterodimers, which trigger auto- or trans-phosphorylation on tyrosine residues of the receptors. These residues then serve as docking sites for recruiting signaling molecules from

The dimerization of ErbB receptors induces activation of a wide range of signaling molecules. The PI3K and MAPK pathways are among the most studied. In addition, the ErbB receptors activate other pathways, such as STATs, PLCs and JNK. These molecules further regulate the activities of key transcriptional factors, such as Jun, fos and Myc, leading to the modulation of various aspects of cell function including survival,

Systems biology studies have revealed that this signaling network is highly organized and precisely regulated by multiple layers of regulatory and control mechanisms, which include activation of specific pathways, positive and negative feedback loops and horizontal control

Phosphoproteomics studies have shown that each ErbB receptor has a specific and preferred binding pattern with signaling molecules (Schulze et al., 2005). The ErbB2 receptor has few binding partners, among which, Shc is the most common partner. The ErbB3 receptor has multiple binding sites for the PI3K subunit p85. ErbB1 and ErbB4 receptors show a diversity

Feedback loops in and outside of this network help maintain homeostasis. For example, ErbB-mediated activation of the MAPK pathway induces the transcription of TGFα and HB-EGF (Schulze et al., 2001), which in turn further activate the ErbB receptors. ErbB activation can also lead to transcription of proteins that inhibit further activation of a particular pathway. For example, ErbB1 receptor activation by EGF causes expression of the suppressor of cytokine signaling (SOCS), which in turn promotes ErbB1 degradation (Kario et al., 2005). In addition, the ErbB signaling can be regulated by G-protein coupled receptors (GPCRs). Studies have shown that thrombin and endothelin have positive effects on the ErbB signaling via activation of matrix metalloproteinases (MMPs), subsequent activation of ErbB ligands, or activation of Src or Pyk2 which phosphorylate ErbB receptors (Dikic et al.,

Neuregulin1 proteins were discovered during 1992-1993 by four independent research groups. At the time, two of the groups were searching for a ligand for the oncogene ErbB2 (Holmes et al., 1992; Peles et al., 1992; Wen et al., 1992). One group was searching for a factor that stimulated the proliferation of Schwann cells (Marchionni et al., 1993), and the third was searching for a factor that stimulated the synthesis of muscle receptors for acetylcholine (Falls

the cytosol to the cell membrane, which then activate various signaling pathways.

receptor (Camprecios et al., 2011).

proliferation, and migration.

by parallel signaling networks.

**3.2 Neuregulin1 proteins**

of interaction partners including STAT5 and Grb2.

1996; Yarden and Sliwkowski, 2001b; Negro et al., 2006).

**3.1.2 The signaling network activated by the ErbB receptors**

et al., 1993). Subsequently, it was found that all these factors were encoded by the same gene, which is the Neuregulin1 gene (Burden and Yarden, 1997). Therefore, Neuregulin1 proteins were also called Neu differentiation factor (NDF), heregulins, glial growth factor (GGF), acetylcholine receptor inducing activity (ARIA). Subsequently, three other Neuregulin genes were discovered, which are Neuregulin2, Neuregulin3 and Neuregulin4 (Carraway et al., 1997; Zhang et al., 1997; Harari et al., 1999). However, limited information is available regarding the biological functions of Neuregulin2, 3 and 4 gene encoded proteins. Neureuglin1 proteins bind directly to the ErbB3 and the ErbB4 receptors. The ErbB2 receptor does not bind directly to Neureuglin1; rather it serves as a co-receptor and forms heterodimers with the ErbB3 or the ErbB4 receptor (Falls et al., 1993; Burden and Yarden, 1997).

Fig. 1. The ErbB Signaling Network. ErbB receptors and their ligands form a complex signaling network which regulates a wide variety of cell functions. There are four ErbB receptors (ErbB1-ErbB4). The ErbB2 receptor does not bind to any ligand, but is a preferred partner of other ErbB receptors. The ErbB3 receptor is devoid of kinase activity. ErbB2 and ErbB3 can form functional and potent heterodimers. There are at least 13 known ligands for the ErbB receptors, including Neuregulin1 proteins. Ligand binding with the ErbB receptors activates various signaling molecules in the cell. The PI3K and MAPK pathways are among the most studied pathways. Multiple feedback loops exist within and outside of this signaling network. The activation and integration of these pathways lead to the regulation of cell survival, growth, proliferation, migration and differentiation. NRG, Neuregulin; EGF, epidermal growth factor; LPA, lysophosphatidic acid; ET, endothelin; GAP, GTPase activating protein; HB-EGF, heparin-binding EGF; Jak, Janus kinase; PKC, protein kinase C; PLC, phospholipase C; Shp2, Src homology domain-2-containing protein tyrosine phosphatase 2; Stat, signal transducer and activator of transcription. Reprinted by permission from *Nat Rev Mol Cell Biol; 2, 127-137, 2001*.

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 73

A common phenotype of these transgenic mice is that all homozygous mice die around E10.5 during embryogenesis with the absence or underdevelopment of ventricular trabeculae. EGF-like domain disruption also causes cardiac endocardial cushion defects (Meyer and Birchmeier, 1995). Hearts around E10.5 display depressed contractility, dilatation of the common ventricle, decreased emptying of the ventricle and slow irregular

In mice with EGF-like domain disruption, the activity of all Neureuglin1s is abolished; while in mice with Ig-like domain deletion, Type III Neuregulin1s are still functional; in mice with cytoplasmic tail disruption, secretive Neuregulin1 proteins are not affected. The Ig-like domain interacts with the extracellular matrix (Kramer et al., 1996a), while the cytoplasmic tail regulates the proteolytic release of the Neuregulin1 extracellular domains (Liu et al., 1998b). Therefore, these studies suggest that Ig-like Neuregulin1s and the release of membrane anchored Neuregulin1s are necessary for cardiac trabeculae formation. They also suggest that Type III Neuregulin1s may not be pivotal for cardiac development. In mice with CRD-Neureuglin1 knockout, no cardiac defects were reported (Wolpowitz et al., 2000). Recently, Hedhli and colleagues tested whether endothelial-derived Neuregulin1s are important for protecting the heart from ischemia-reperfusion injury (Hedhli et al., 2011). They generated mice with tamoxifen-inducible and endothelium-selective Neuregulin1 gene knockout by cross-breeding mice with VE-cadherin promoter driven Cre-ER (Monvoisin et al., 2006) and mice carrying homozygously floxed alleles of the Neuregulin1 gene (Yang et al., 2001). At the baseline, hearts from these knockout mice showed normal wall thickness, left ventricular chamber size, and systolic function. Cardiac morphology, including capillary density was not different from non-transgenic mice. However, after ischemia-reperfusion, the infarct area, the number of TUNEL positive cells, and the number of infiltrating leukocytes were significantly increased in knockout mice compared with controls. The activation of ErbB4 receptors was decreased in ischemia-reperfusion treated knockout mouse hearts. In addition, injection of a recombinant Neuregulin1 (EGF-like domain) reversed the adverse effects observed in the knockout mice. These results suggested that loss of endothelium-derived Neuregulin1 was the cause of worsening ischemia-reperfusion cardiac injury. This study has identified that Neuregulin1 is one of the protective factors

heart rate (Kramer et al., 1996a; Liu et al., 1998b).

derived from the cardiac endothelium.

**3.3.2 Transgenic mouse models with mutations of the ErbB receptors**

To understand the physiological roles of the ErbB receptors, transgenic mice with deletion of the ErbB2, ErbB3 or ErbB4 gene were generated (Gassmann et al., 1995; Lee et al., 1995; Erickson et al., 1997; Crone et al., 2002; Ozcelik et al., 2002). Deletion of the ErbB2 or the ErbB4 gene caused a similar cardiac phenotype as that observed in pan-Neuregulin1 knockout mice, which was the underdevelopment of cardiac ventricular trabeculae (Gassmann et al., 1995; Lee et al., 1995; Meyer and Birchmeier, 1995). ErbB2 or ErbB4 knockout mice died around E10.5 due to cardiac defects. Cardiac specific overexpression of the ErbB2 receptor in ErbB2 knockout mice restored normal ventricular trabeculation (Morris et al., 1999). These data suggest that the effects of Neuregulin1 proteins on cardiac development depend on both ErbB2 and ErbB4 receptors. They also suggest that ErbB2 and ErbB4 heterodimers are needed for propagating Neuregulin1 signals in the developing hearts. In mice with the ErbB3 gene knockout, however, the defect of ventricular trabeculation was not observed. Instead, cardiac cushions lacked mesenchyme and cardiac

The Neuregulin1 gene is located on the short arm of the human chromosome 8 (Stefansson et al., 2002). At least 15 Neuregulin1 isoforms are produced by the Neuregulin1 gene as a result of alternative splicing and multiple promoters (Falls, 2003; Hayes and Gullick, 2008). All Neuregulin1 proteins contain the epidermal growth factor (EGF)-like domain, which binds to ligands and also is sufficient for activation of the ErbB receptors. According to the N-terminal structural differences, Neuregulin1 proteins are divided into three types (type I, II and III). Type I Neuregulin1 proteins contain an immunoglobulin (Ig)-like domain, an EGF-like domain, a proteolysis site, a hydrophobic transmembrane domain and a cytoplasmic tail. Like type I Neuregulin1, Type II Neuregulin1 isoforms contain an Ig-like domain and EGF-like domain. In addition, type II isoforms contain a signal peptide, a kringle-like sequence in their N-terminal. Type III isoforms do not have an Ig-like domain; instead, they contain a cysteine-rich domain in the N-terminal part of the protein. Therefore, Type I and II Neuregulin1 proteins are known as Ig-Neuregulin1s. The Type III Neuregulin1 proteins are known as CRD-Neuregulin1s. Other factors that differentiate

Neuregulin1 proteins are the type of EGF-like domain (α, β) and whether the isoform is initially synthesized as a transmembrane or non-membrane protein (Falls et al., 1993).

Type I and II Neuregulin1 proteins are synthesized as secreted proteins or single-pass transmembrane proteins. Proteolytic cleavage of the Ig-Neuregulin1s releases the Nterminal fragment that contains the EGF-like domain to the extracellular space, which may, in turn, bind to the ErbB receptors in a paracrine or autocrine manner. Studies have shown that metalloproteases, such as ADAM17 and ADAM19, are capable of mediating the shedding of Neuregulin1 proteins from cells (Kuramochi et al., 2004; Kalinowski et al., 2010). Type III Neuregulin1 proteins are synthesized as two-pass transmembrane proteins, with a transmembrane domain located at the C-terminal of the EGF domain and a transmembrane domain within the CRD-domain. Cleavage of the type III Neuregulin1 proteins exposes the EGF-like domain of the protein, which may interact with ErbB receptors in a juxtacrine mechanism (Falls et al., 1993).

#### **3.3 Neuregulin-ErbB signaling in developing and adult hearts: Findings in transgenic mouse models**

The physiological function of Neuregulin1 proteins and their ErbB receptors have been intensively studied during the past two decades, mainly in transgenic mouse models, as well as in mice treated with recombinant Neuregulin1s and in cell culture. Neuregulin1 proteins are localized in the cardiac endocardium and the endothelium of the cardiac microvasculature (Meyer and Birchmeier, 1995; Lemmens et al., 2006). The ErbB2 and ErbB4 receptors are expressed in cardiac myocardium (Gassmann et al., 1995; Lee et al., 1995) and ErbB3 proteins are expressed in endocardial cushion mesenchyme (Erickson et al., 1997). Neuregulin1s activate ErbB receptors in a paracrine manner (Marchionni, 1995). Studies have demonstrated that Neureuglin-ErbB signaling is essential for cardiac development in embryos and pivotal for protecting adult hearts from stress.

#### **3.3.1 Transgenic mouse models with mutations of the Neuregulin1 gene**

Several transgenic mouse models are generated with deletion/disruption of the EGF-like domain, Ig-like domain or the cytoplasmic tail of the Neureuglin1 proteins (Meyer and Birchmeier, 1995; Kramer et al., 1996b; Liu et al., 1998a).

The Neuregulin1 gene is located on the short arm of the human chromosome 8 (Stefansson et al., 2002). At least 15 Neuregulin1 isoforms are produced by the Neuregulin1 gene as a result of alternative splicing and multiple promoters (Falls, 2003; Hayes and Gullick, 2008). All Neuregulin1 proteins contain the epidermal growth factor (EGF)-like domain, which binds to ligands and also is sufficient for activation of the ErbB receptors. According to the N-terminal structural differences, Neuregulin1 proteins are divided into three types (type I, II and III). Type I Neuregulin1 proteins contain an immunoglobulin (Ig)-like domain, an EGF-like domain, a proteolysis site, a hydrophobic transmembrane domain and a cytoplasmic tail. Like type I Neuregulin1, Type II Neuregulin1 isoforms contain an Ig-like domain and EGF-like domain. In addition, type II isoforms contain a signal peptide, a kringle-like sequence in their N-terminal. Type III isoforms do not have an Ig-like domain; instead, they contain a cysteine-rich domain in the N-terminal part of the protein. Therefore, Type I and II Neuregulin1 proteins are known as Ig-Neuregulin1s. The Type III Neuregulin1

Neuregulin1 proteins are the type of EGF-like domain (α, β) and whether the isoform is initially synthesized as a transmembrane or non-membrane protein (Falls et al., 1993).

Type I and II Neuregulin1 proteins are synthesized as secreted proteins or single-pass transmembrane proteins. Proteolytic cleavage of the Ig-Neuregulin1s releases the Nterminal fragment that contains the EGF-like domain to the extracellular space, which may, in turn, bind to the ErbB receptors in a paracrine or autocrine manner. Studies have shown that metalloproteases, such as ADAM17 and ADAM19, are capable of mediating the shedding of Neuregulin1 proteins from cells (Kuramochi et al., 2004; Kalinowski et al., 2010). Type III Neuregulin1 proteins are synthesized as two-pass transmembrane proteins, with a transmembrane domain located at the C-terminal of the EGF domain and a transmembrane domain within the CRD-domain. Cleavage of the type III Neuregulin1 proteins exposes the EGF-like domain of the protein, which may interact with ErbB

**3.3 Neuregulin-ErbB signaling in developing and adult hearts: Findings in transgenic**

The physiological function of Neuregulin1 proteins and their ErbB receptors have been intensively studied during the past two decades, mainly in transgenic mouse models, as well as in mice treated with recombinant Neuregulin1s and in cell culture. Neuregulin1 proteins are localized in the cardiac endocardium and the endothelium of the cardiac microvasculature (Meyer and Birchmeier, 1995; Lemmens et al., 2006). The ErbB2 and ErbB4 receptors are expressed in cardiac myocardium (Gassmann et al., 1995; Lee et al., 1995) and ErbB3 proteins are expressed in endocardial cushion mesenchyme (Erickson et al., 1997). Neuregulin1s activate ErbB receptors in a paracrine manner (Marchionni, 1995). Studies have demonstrated that Neureuglin-ErbB signaling is essential for cardiac development in

Several transgenic mouse models are generated with deletion/disruption of the EGF-like domain, Ig-like domain or the cytoplasmic tail of the Neureuglin1 proteins (Meyer and

proteins are known as CRD-Neuregulin1s. Other factors that differentiate

receptors in a juxtacrine mechanism (Falls et al., 1993).

embryos and pivotal for protecting adult hearts from stress.

Birchmeier, 1995; Kramer et al., 1996b; Liu et al., 1998a).

**3.3.1 Transgenic mouse models with mutations of the Neuregulin1 gene**

**mouse models**

A common phenotype of these transgenic mice is that all homozygous mice die around E10.5 during embryogenesis with the absence or underdevelopment of ventricular trabeculae. EGF-like domain disruption also causes cardiac endocardial cushion defects (Meyer and Birchmeier, 1995). Hearts around E10.5 display depressed contractility, dilatation of the common ventricle, decreased emptying of the ventricle and slow irregular heart rate (Kramer et al., 1996a; Liu et al., 1998b).

In mice with EGF-like domain disruption, the activity of all Neureuglin1s is abolished; while in mice with Ig-like domain deletion, Type III Neuregulin1s are still functional; in mice with cytoplasmic tail disruption, secretive Neuregulin1 proteins are not affected. The Ig-like domain interacts with the extracellular matrix (Kramer et al., 1996a), while the cytoplasmic tail regulates the proteolytic release of the Neuregulin1 extracellular domains (Liu et al., 1998b). Therefore, these studies suggest that Ig-like Neuregulin1s and the release of membrane anchored Neuregulin1s are necessary for cardiac trabeculae formation. They also suggest that Type III Neuregulin1s may not be pivotal for cardiac development. In mice with CRD-Neureuglin1 knockout, no cardiac defects were reported (Wolpowitz et al., 2000).