 groups included 12 cases of healthy accidental death with myocardial autopsy and 23 healthy blood donors. The mouse model of viral myocarditis (VMC) was established by Coxsackie virus B3 infection and myocardial tissues and skeletal muscle were collected. Mitochondrial DNA (mtDNA) deletion rate was quantitatively determined using polymerase chain reaction. Results There was significantly difference of myocardial mitochondrial DNA deletion rate between VMD or VMC group and control group (P<0.05). Moreover, the loss of mitochondrial membrane phospholipids was significantly different between VMD or VMC group and control group. In VMC mice, there were negative correlations between myocardial mtDNA3867 deletion rate and left ventricular peak systolic pressure (LVPSP) (r = −0.66, P<0.05), and between myocardial mtDNA3867 deletion rate and +dp/dtmax (r = −0.79, P<0.05), while there was positive correlation between myocardial mtDNA3867 deletion rate and −dp/dtmax (r = 0.80, P<0.05). Conclusion Mitochondrial damage is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction. The mitochondrial damage in the skeletal muscle and lymphocytes reflect a “window” of myocardial mitochondrial damage.

Mitochondria are the main energy engines in the cells. The abnormality of myocardial energy metabolism is one of the important mechanisms of the development of cardiomyocyte hypertrophy and heart failure [9][10][11]. The existence of multiple deletions and mutations of mitochondrial deoxyribonucleic acid (mtDNA) in patients with dilated cardiomyopathy has been demonstrated by Arbustini and other investigators [12]. But it has been unclear yet if these changes are the cause or the sequence of myocardial injury. The present study was designed to provide more evidence for the roles of the mitochondrial damage in the pathogenesis of viral myocardial disease. Moreover, given the limitations of clinical application of myocardial biopsy, we also studied the mitochondrial damages in skeletal muscle, peripheral blood samples, and myocardium and analyzed their correlations in order to obtain a ''window'' in peripheral cells which reflects mitochondrial damage in myocardium.

Viral myocardial disease patients and control group
Data was collected from 83 patients with viral myocardial disease in the inpatients units of the Department of Cardiology, Affiliated Hospital of Xi'an Jiaotong University, collected from January 1997-July 2000. These patients included 50 patients with viral myocarditis (blood coxsackievirus B-IgM was positive for all of these 50 patients) and 33 patients with dilated cardiomyopathy (the clinical data of patients in Table 1). The diagnosis of viral myocarditis was based on the criteria established by the Chinese Heart Association in 1999 [13]. The diagnosis of dilated cardiomyopathy was based on the criteria established by the World Health Organization/International Federation of Society of Cardiology (WHO/ISFC) in 1996 [14]. All the DCM patients included in this study had the history of viral myocarditis. Myocardial biopsy has been performed via right jugular vein catheterization in 20 cases of viral myocarditis and 12 patients of dilated cardiomyopathy. Myocardial tissues were collected at 2-4 sites on the right side of interventricular septum using Cordis biopsy forceps and immediately stored in liquid nitrogen. Lymphocytes were isolated from 10 ml of blood of patients and saved at 280˚C for the later analysis. Control groups included 12 cases of healthy accidental death (10 males and 2 females) for the myocardial autopsy in the same sites of myocardium and 23 healthy blood donors (11 males and 12 females) for the normal subject control. Although only 32 out of 83 patients agreed to have cardiac biopsy, we received blood samples from all the cases participated in this study. There were no age differences among groups of total 50 patients of viral myocarditis, 20 patients of viral myocarditis with cardiac biopsy, total 33 patients of dilated cardiomyopathy, 12 patients of dilated cardiomyopathy with biopsy, 12 cases of healthy accidental death, and 23 healthy blood donors. Written consent was obtained from all of the participants or family members of the healthy accidental death in the study and the study protocol was approved by Institutional Review Board of Xi'an Jiaotong University School of Medicine.

Viral myocarditis mice model
Establishment of the mouse model Mouse model of viral myocarditis was established as described previously [15]. Forty of 6-8 weeks old male BALB/c mice with body weight of 18-22 g were obtained from Xi'an Jiaotong University School of Medicine Laboratory Animal Center, and were randomly divided into 2 groups. All animal study protocols were approved by the Institutional Animal Research and Ethics Committee of Xi'an Jiaotong University (SCXK2007-001). The experimental group with 30 mice were intraperitoneally injected with 0.1 ml of Eagle solution containing Coxsackie virus B 3 [CVB 3, The 50% infectious dose (TCID 50 )510 8 ] (provided by the laboratory of viral heart disease at Fudan University Zhongshan Hospital). Ten mice in control group were intraperitoneally injected with the equal volume of Eagle medium without CVB 3. We found that the mice injected with CVB 3 began to show towering hair, curling, reduced activity, reduced body weight in 3 days after injection, mild systemic cyanosis in 4 days, progressive hind limb paralysis, ear and tail ischemia, necrosis, and scab in 6 days after injection. 3 mice died in 7,15 days after injection and no more death afterward.

Determination of heart function
At day 3, 11, and 24 after viral injection, heart functions were measured using multi-channel polygraph system (Pawerlab, USA) through carotid artery catheterization under the anesthesia of 20% urethane (0.01 ml/g body weight, intraperitoneal injection).

Sample collection
Myocardium from heart apex and skeletal muscle from forelimb were collected after the measurement of heart functions. Each tissue was cut into two 1 mm 3 pieces for the following experiments: the first fixed using improved Demer's method, serially dehydrated in acetone, embedded in Epson 812 and sectioned with a microtome. The sections were observed directly under a transmission electron microscope without uranium staining [16]. The second homogenized in 1000 mL of Tris-EDTA (TE) buffer. The tissues were digested with 1% trypsin (final concentration was 1-2.5 mg/mL) at 37˚C for 24 h. Phenol/Chloroform extraction followed by ethanol precipitation was used to isolate total DNA. The quality and quantity of DNA were measured by ultraviolet spectrophotometry [17].

Quantitative analysis of mitochondrial DNA deletion
Quantitative analysis of mitochondrial DNA 4977 and mtDNA 7436 deletion rate of myocardium and lymphocytes in VMD patients by polymerase chain reaction (PCR) The common mtDNA deletion types, mtDNA 4977 and mtDNA 7436 deletion, were compared in VMC and DCM patients. Three primers were needed for PCR analysis [18][19][20][21][22]: Primers P1/P2 (located in mtDNA L8282-8305 and H13832-13852) were used to amplify a fragment of 594 base pairs (bp) representing mtDNA 4977 deletion. Primers P3/P4 (located in mtDNA L8150-8166 and H16142-16159) were used to amplify a fragment of 574 bp representing mtDNA 7436 deletion. Primers P5/P6 were designed in the highly conserved region of the mtDNA molecule 12S rRNA gene region, located in the L1077-1098 and H1552-1572 position. For either wild type or mtDNA deletion, P5/P6 could amplify a fragment of 496 bp which was used as an internal reference in order to calculate the mtDNA 4977 and mtDNA 7436 deletion ratio. The PCR reaction system included 0.4 mg of DNA, 200 mmol/L of 46dNTP including a-32 P-dATP (3000 Ci/mmol), 0.5 mmol/L of each primer, MgCl 2 2 mmol/L, TaqDNA enzyme (Promega) 2U. The conditions of PCR were as follows: pre-denaturing at 94˚C for 7 min, denaturing at 94˚C for 60 s, annealing at 55˚C for 60 s, extension at 72˚C for 105 s, 35 cycles, final extension at 72˚C for 8 min. The PCR products from primers P1/P2 and P3/P4 were digested with restriction enzyme DdeI and XbaI to confirm the specificity of the PCR products. For quantitative analysis of PCR products, the PCR products were electrophoresed on a 1.2% agarose gel and observed under ultraviolet light. Specific bands were cut and put in the scintillation vial with 0.4 ml of 30% H 2 O 2. After the gel was dissolved at 70˚C in a water bath, 5 ml of scintillation fluid was added and put in the dark for overnight followed by the liquid scintillation counting. To eliminate the quenching effect of the gel, exactly 10 mg of the gel was cut and used for quench correction. Finally, an amplification curve was made with the number of PCR cycles and the amount of PCR products (cpm number) as the horizontal and vertical axis, respectively.
To have the best amplification efficiency, our pilot studies have shown that the best amount of DNA template is 0.4 mg, and the best number of PCR cycles is 35 cycles. The ratio of product from P1/P2 over the product from P5/P6 reflected the rate of mtDNA 4977 deletion over the total mtDNA. The ratio of product from P3/ P4 over the product from P5/P6 reflected the rate of mtDNA 7436 deletion over the total mtDNA.
Quantitative analysis of mitochondrial DNA 3867 deletion rate (mtDNA 3867 )