ROS production in mitochondria is regulated by a number of thermodynamic and kinetic factors [45]. The diverse, and even contrary, effects of mKATP channels opening on ROS production in mitochondria are difficult to evaluate because mitochondrial ROS production depends on a wide variety of conditions, which include mitochondrial energy state (quantitatively represented by Δ*μ*H), redox potential of the main sites of ROS formation in the respiratory chain [69–71], the source of the electron supply to the respiratory chain, the rate of respiration [70] and, at last, the concentration of oxygen [3, 4], which is the end electron acceptor in the

Standard redox potential of one-electron oxygen reduction to superoxide constitutes −160 mV, and on this basis, the respiratory chain in highly energized mitochondria comprises multiple putative sites of ROS formation [45, 69]. At complex I, ROS formation largely occurs in the course of thermodynamically unfavorable reverse electron transport, which requires high Δ*μ*H and critically depends on both ΔΨm and ΔpH [72, 73]. This mechanism of ROS formation is one best studied "classical" example of thermodynamically regulated ROS production in mitochondria. Unlike this, ROS production at complex III is dependent on both thermodynamic (such as the redox state of the ubiquinone pool) and kinetic factors [45, 69–71], such as the quantity and the life span of free radical intermediates of the redox reactions, which are regulated by the rate of respiration and the relations between the rates of ROS formation and the removal of these species. Q-cycle is supposed to be the main source of ROS in complex III [45], and ROS formation at this site exhibits a bell-shaped dependence on the redox state of Q-cycle [69]. Partially oxidized Q-cycle was shown to be most favorable for ROS production at complex III [74], which implies its dependence both on mitochondrial energy state and the

dependent on cell type, are largely affected by the effects of ATP-sensitive K+

*3.1.1. The impact of mKATP channels opening on ROS production in mitochondria*

production [21, 50, 64]), the effects of ATP-sensitive K+

nels opening on ROS production in mitochondria.

redox reactions in the respiratory chain.

rate of respiration.

uptake dissipates Δ*μ*H, largely at the cost of ΔΨm. Thus,

Mitochondrial KATP Channel Function under Hypoxia http://dx.doi.org/10.5772/intechopen.80323

transport on ROS production appear to

/

25

transport on

of K+

of oxygen consumption due to K+

ΔΨm and the rate of respiration.

H+

#### **3.1. Direct bioenergetic consequences of mKATP channels opening**

In energized mitochondria, potential-dependent potassium transport directed to the matrix space takes place at the cost of proton-motive force (Δ*μ*H), a free energy generated by the electron transport chain. As ΔΨm is the main part of Δ*μ*H, K+ uptake, accompanied by the obligatory electroneutral water uptake [24], occurs at the cost of ΔΨm and thus results in depolarization. Because of its dramatic effect on ΔΨm and matrix swelling, K+ uptake would be detrimental for mitochondria, if there was not the work of respiratory chain and K+ /H+ exchange. Thus, the loss of ΔΨm is opposed by the "compensatory" work of respiratory chain [62], which increases oxygen consumption proportional to the rate of K+ transport in order to restore ΔΨm; on the other hand, matrix swelling is opposed by potassium extrusion *via* K+ /H+ -exchanger, which is accompanied by the matrix contraction [24]. Concurrent work of K+ channels and K+ /H+ exchanger constitutes mitochondrial K+ cycle [24], of which potential-dependent component (K+ uptake) dissipates Δ*μ*H and in this way uncouples mitochondria and affects potentialdependent mitochondrial functions: ATP synthesis, Ca2+ transport and ROS production.

The impact of mKATP channels opening on mitochondrial bioenergetics greatly depends on the channels' activity and their abundance in mitochondrial membrane, which is responsible for the effects of mKATP channels opening on mitochondrial energy state and decides for cell specificity of mKATP channel functions [63]. Thus, higher density of mKATP channel distribution in brain results in slight depolarization, which was observed in the literature and in our studies [63, 64], while lower amount of the channels in the heart and liver was of no effect on ΔΨm even at full activation [24, 65, 66]. Elevated expression of mKATP channel and the channel activation that were observed under hypoxia [20, 21, 41, 59] increase the "weight" of ATP-sensitive K+ transport in the regulation of mitochondrial functions and metabolism. This is still more visible in malignant cells functioning in hypoxic environment, in which overexpression of mKATP channel was shown [53].

Unlike protonophoric uncoupling that reduces transmembrane pH (ΔpH), uncoupling of the respiratory chain by mKATP channel opening is accompanied by the elevated ΔpH because of K+ uptake into matrix occurring in exchange for protons. However, the activation of K+ / H+ -exchanger reduces this minor gain in ΔpH, and besides, simultaneous increase in the rate of oxygen consumption due to K+ uptake dissipates Δ*μ*H, largely at the cost of ΔΨm. Thus, the regulation of ROS production and other potential-dependent functions of mitochondria, dependent on cell type, are largely affected by the effects of ATP-sensitive K+ transport on ΔΨm and the rate of respiration.

#### *3.1.1. The impact of mKATP channels opening on ROS production in mitochondria*

In the literature, it was supposed that protection of tissues against the impairments caused by

transport and ROS production is largely dependent on the abundance of the channel in mitochondrial membrane. Oxygen deficiency affects functional consequences of mKATP channels opening by modulating channels expression and activity [20, 53, 59]. As it was shown in cardiomyocytes, the activation of cardiac mKATP channels under hypoxia was mediated by the interactions of conducting subunit Kir6.2 with heat shock protein 90, HSP90 [25] and Kir6.1 with gap junction protein connexin 43 and PKCε [26]. Silencing or pharmacological inhibition of HSP90 and connexin 43 abolished protective effects afforded by mKATP channels opening [25, 26]. At present, there are scarce data on such interactions, which could contribute to cell specificity of molecular mechanisms regulating mKATP channels opening and its functional consequences under hypoxia. To assess how mKATP channels can be involved in mitochondrial response to oxygen deprivation, direct bioenergetic and functional effects of mKATP

In energized mitochondria, potential-dependent potassium transport directed to the matrix space takes place at the cost of proton-motive force (Δ*μ*H), a free energy generated by the elec-

electroneutral water uptake [24], occurs at the cost of ΔΨm and thus results in depolarization.

loss of ΔΨm is opposed by the "compensatory" work of respiratory chain [62], which increases

uptake) dissipates Δ*μ*H and in this way uncouples mitochondria and affects potential-

The impact of mKATP channels opening on mitochondrial bioenergetics greatly depends on the channels' activity and their abundance in mitochondrial membrane, which is responsible for the effects of mKATP channels opening on mitochondrial energy state and decides for cell specificity of mKATP channel functions [63]. Thus, higher density of mKATP channel distribution in brain results in slight depolarization, which was observed in the literature and in our studies [63, 64], while lower amount of the channels in the heart and liver was of no effect on ΔΨm even at full activation [24, 65, 66]. Elevated expression of mKATP channel and the channel activation that were observed under hypoxia [20, 21, 41, 59] increase the "weight"

This is still more visible in malignant cells functioning in hypoxic environment, in which

transport in the regulation of mitochondrial functions and metabolism.

dependent mitochondrial functions: ATP synthesis, Ca2+ transport and ROS production.

transport on the oxygen consumption, membrane potential, ATP synthesis, Ca2+

transport is largely based on bioenergetic effects of K+

channels opening [16, 41, 50, 53]. The impact of ATP-

uptake, accompanied by the obligatory

/H+

transport in order to restore ΔΨm; on the

/H+

cycle [24], of which potential-dependent component

uptake would be detrimental

exchange. Thus, the

/H+

channels and K+

hypoxia afforded by mitochondrial K+

transport and signaling triggered by K+

channels opening need to be considered.

**3.1. Direct bioenergetic consequences of mKATP channels opening**

tron transport chain. As ΔΨm is the main part of Δ*μ*H, K+

oxygen consumption proportional to the rate of K+

overexpression of mKATP channel was shown [53].

exchanger constitutes mitochondrial K+

(K+

of ATP-sensitive K+

Because of its dramatic effect on ΔΨm and matrix swelling, K+

for mitochondria, if there was not the work of respiratory chain and K+

other hand, matrix swelling is opposed by potassium extrusion *via* K+

is accompanied by the matrix contraction [24]. Concurrent work of K+

sensitive K+

24 Hypoxia and Anoxia

Generally, it is supposed that cytoprotective effects of mKATP channels opening are primarily based on the modulation of Ca2+ transport and mitochondrial ROS production, which prevent Ca2+ overload [54, 56, 57] and trigger ROS-dependent signaling, thereby preventing the opening of cyclosporine-sensitive pore (mPTP) [50]. However, of all functional effects produced by mKATP channels opening (the modulation of mitochondrial morphology [9, 10], respiration [63, 65], Ca2+ transport [54–56], potassium cycle [24, 66], ATP synthesis [20, 67, 68] and ROS production [21, 50, 64]), the effects of ATP-sensitive K+ transport on ROS production appear to be the most controversial. This diversity needs to consider the direct effects of mKATP channels opening on ROS production in mitochondria.

ROS production in mitochondria is regulated by a number of thermodynamic and kinetic factors [45]. The diverse, and even contrary, effects of mKATP channels opening on ROS production in mitochondria are difficult to evaluate because mitochondrial ROS production depends on a wide variety of conditions, which include mitochondrial energy state (quantitatively represented by Δ*μ*H), redox potential of the main sites of ROS formation in the respiratory chain [69–71], the source of the electron supply to the respiratory chain, the rate of respiration [70] and, at last, the concentration of oxygen [3, 4], which is the end electron acceptor in the redox reactions in the respiratory chain.

Standard redox potential of one-electron oxygen reduction to superoxide constitutes −160 mV, and on this basis, the respiratory chain in highly energized mitochondria comprises multiple putative sites of ROS formation [45, 69]. At complex I, ROS formation largely occurs in the course of thermodynamically unfavorable reverse electron transport, which requires high Δ*μ*H and critically depends on both ΔΨm and ΔpH [72, 73]. This mechanism of ROS formation is one best studied "classical" example of thermodynamically regulated ROS production in mitochondria. Unlike this, ROS production at complex III is dependent on both thermodynamic (such as the redox state of the ubiquinone pool) and kinetic factors [45, 69–71], such as the quantity and the life span of free radical intermediates of the redox reactions, which are regulated by the rate of respiration and the relations between the rates of ROS formation and the removal of these species. Q-cycle is supposed to be the main source of ROS in complex III [45], and ROS formation at this site exhibits a bell-shaped dependence on the redox state of Q-cycle [69]. Partially oxidized Q-cycle was shown to be most favorable for ROS production at complex III [74], which implies its dependence both on mitochondrial energy state and the rate of respiration.

The share of ATP-sensitive K+ transport in the total K+ transport in brain and liver mitochondria by our estimations, which agreed with literary data [24], was about ~30–35% [64, 66]. However, in spite of the well-defined characteristics of ATP-sensitive K+ transport obtained in mitochondria of different cell types, the effects of mKATP channels opening on ROS production are difficult to quantify because of their dependence on several mutually dependent parameters. Overlay of the moderate alterations in mitochondrial functions caused by ATP-sensitive K+ transport with closely interrelated thermodynamic and kinetic factors regulating ROS formation in mitochondria could explain apparently contradictory effects of mKATP channel opening on ROS production reported in the literature. Interestingly, both the elevation [16, 41] and suppression [21, 40] of ROS production were reported to improve cardiac and cardiomyocyte functions after the exposure to hypoxia in a way dependent on mKATP channel opening. Based on the published data, it is tempting to hypothesize that bidirectional regulation of ROS production by potassium transport observed in the literature could represent a flexible mechanism of the fast response to the elevation of ROS levels generally observed under hypoxia and that, dependent on conditions, could prevent ROS overproduction [57, 75] or trigger ROS-dependent signaling [16, 41, 50, 53], which makes this function of mKATP channel of especial importance under limited oxygen availability.

its physiological meaning appears to be more evident and needs to be considered. In agreement with the literature, we suppose that it is the regulation of cellular levels of ATP [67, 77], but what is still more important, the regulation of the oxygen consumption by mitochondria.

The functional effects of mKATP channels opening under hypoxia are similar to those observed in normoxic cells. Thus, under oxygen deprivation, mKATP channel activation reduced mitochondrial Ca2+ loading [57, 79], preserved ATP levels [67, 77] and increased cell survival [16, 40, 41, 80] by suppression of apoptosis *via* targeting glycogen synthase kinase 3β (GSK3β), an enzyme involved in triggering cell death by promotion of the opening of mitochondrial permeability transition pore, mPTP [81]. Suppression of cell death pathways resulted in stabilization of membrane potential [16, 59, 80] and the restoration of ATP synthesis [82]. Increased expression of both Kir6.2 [59] and SUR2A [37], similar to pharmacological mKATP channels opening, too was shown to improve the viability and the resistance of cardiomyocytes to hypoxia.

As one can see from the above examples, cell response to hypoxia was essentially dependent on the bioenergetic effects of mKATP channels opening. This allows assume that cytoprotection afforded by mKATP channels opening is largely based on a synergistic action of bioenergetic effects of mKATP channel functioning (primarily ROS production and ATP synthesis [20, 40, 41, 67, 79]), and the redox signaling critically dependent on ROS formation caused by

In the literature, it was obtained rather unambiguous evidence of the suppression of the OxPhos by mKATP channels opening [67, 76, 77]. However, it seems to be surprising that under hypoxia, similar to other metabolic stress conditions, cytoprotection was afforded by contrary effects of mKATP channels opening on free radical formation, and both the reduction [40, 80] and the elevation [16, 41, 50] of ROS production were shown to afford cytoprotective effects. To smooth this apparent contradiction, we recently proposed [83] that, dependent

channels opening could afford protection at least in two ways: either directly, by the direct reduction of ROS formation under certain conditions [40, 75, 80, 84] or indirectly, by the elevation of ROS production and triggering of ROS-dependent signaling shown to be cytoprotective under oxygen deprivation (ischemia and hypoxia) [16, 41, 50]. To shed more light on physiological role(s) of mKATP channels under hypoxia, functional consequences of mKATP channels opening on ROS production and ATP synthesis should be considered in more detail.

With reference to hypoxia, it is generally supposed that mitochondria respond to oxygen deprivation by the generation of ROS and activation of ROS-dependent signaling pathways [3–5, 14]. mKATP channel was shown to be involved in ROS signaling triggered both upstream (by the activation of kinases PI3K/Akt, PKCε [16, 80]) and downstream (p38MAPK

*3.2.1. Triggering of ROS-dependent signaling and controlling of ROS production in*

transport on mitochondrial bioenergetics, mKATP

Mitochondrial KATP Channel Function under Hypoxia http://dx.doi.org/10.5772/intechopen.80323 27

**3.2. Functional consequences and molecular targets of mKATP channel opening**

**under hypoxia**

mKATP channels opening [16, 50].

on the direct impact of ATP-sensitive K+

*mitochondria*

#### *3.1.2. Direct effects of mKATP channel opening on F0 F1 ATP synthase activity*

In several works, including our own studies, an inhibition of both ATP synthesis and hydrolysis, ensuing from mKATP channels opening, was reported [67, 68, 76, 77]. Biochemical mechanism of this effect is not well understood, but, based on the published data, its physiological relevance can be considered.

As we have observed in our work on liver mitochondria, even full activation of mKATP channel by diazoxide moderately increased the rate of state 4 respiration and resulted in slight mitochondrial uncoupling not accompanied by depolarization [64]. However, these moderate changes in mitochondrial functions apparently suppressed phosphorylation, which could not be explained by the mild uncoupling effect. This was reflected in the decreased rates of state 3 respiration and phosphorylation, which were proved by measuring respective rates of proton transport after ADP addition [68]. It is worth mentioning that mKATP channel opening essentially reduced oxygen consumption in the course of phosphorylation and increased apparent P/O ratio [68]. These effects were coincident with concurrent activation of K+ cycling, which was the cause of stimulation of state 4 respiration [66]. Based on the literature [78], we assumed that activation of K+ cycling could be the plausible cause for inhibition of F0 F1 ATP synthase functioning, not explained by respiratory uncoupling caused by ATP-sensitive K+ transport.

Considering that ATP synthesis and hydrolysis are coupled to proton translocation across mitochondrial membrane, we supposed that concurrent K+ cycling could disturb the molecular mechanism of F0 F1 ATP synthase both at the stage of ATP synthesis and hydrolysis. Possibly, close mechanism of such molecular uncoupling called "decoupling" was observed in the literature under the action of K+ /H+ -ionophore gramicidin, which occurred without apparent changes in Δ*μ*Н [78]. While the biochemical mechanism of such decoupling is not quite clear, its physiological meaning appears to be more evident and needs to be considered. In agreement with the literature, we suppose that it is the regulation of cellular levels of ATP [67, 77], but what is still more important, the regulation of the oxygen consumption by mitochondria.

#### **3.2. Functional consequences and molecular targets of mKATP channel opening under hypoxia**

The share of ATP-sensitive K+

*3.1.2. Direct effects of mKATP channel opening on F0*

relevance can be considered.

assumed that activation of K+

F1

erature under the action of K+

mitochondrial membrane, we supposed that concurrent K+

/H+

transport.

mechanism of F0

by ATP-sensitive K+

26 Hypoxia and Anoxia

transport in the total K+

However, in spite of the well-defined characteristics of ATP-sensitive K+

dria by our estimations, which agreed with literary data [24], was about ~30–35% [64, 66].

in mitochondria of different cell types, the effects of mKATP channels opening on ROS production are difficult to quantify because of their dependence on several mutually dependent parameters. Overlay of the moderate alterations in mitochondrial functions caused

regulating ROS formation in mitochondria could explain apparently contradictory effects of mKATP channel opening on ROS production reported in the literature. Interestingly, both the elevation [16, 41] and suppression [21, 40] of ROS production were reported to improve cardiac and cardiomyocyte functions after the exposure to hypoxia in a way dependent on mKATP channel opening. Based on the published data, it is tempting to hypothesize that bidirectional regulation of ROS production by potassium transport observed in the literature could represent a flexible mechanism of the fast response to the elevation of ROS levels generally observed under hypoxia and that, dependent on conditions, could prevent ROS overproduction [57, 75] or trigger ROS-dependent signaling [16, 41, 50, 53], which makes this function of mKATP channel of especial importance under limited oxygen availability.

*F1*

In several works, including our own studies, an inhibition of both ATP synthesis and hydrolysis, ensuing from mKATP channels opening, was reported [67, 68, 76, 77]. Biochemical mechanism of this effect is not well understood, but, based on the published data, its physiological

As we have observed in our work on liver mitochondria, even full activation of mKATP channel by diazoxide moderately increased the rate of state 4 respiration and resulted in slight mitochondrial uncoupling not accompanied by depolarization [64]. However, these moderate changes in mitochondrial functions apparently suppressed phosphorylation, which could not be explained by the mild uncoupling effect. This was reflected in the decreased rates of state 3 respiration and phosphorylation, which were proved by measuring respective rates of proton transport after ADP addition [68]. It is worth mentioning that mKATP channel opening essentially reduced oxygen consumption in the course of phosphorylation and increased

apparent P/O ratio [68]. These effects were coincident with concurrent activation of K+

which was the cause of stimulation of state 4 respiration [66]. Based on the literature [78], we

synthase functioning, not explained by respiratory uncoupling caused by ATP-sensitive K+

Considering that ATP synthesis and hydrolysis are coupled to proton translocation across

close mechanism of such molecular uncoupling called "decoupling" was observed in the lit-

changes in Δ*μ*Н [78]. While the biochemical mechanism of such decoupling is not quite clear,

cycling could be the plausible cause for inhibition of F0

ATP synthase both at the stage of ATP synthesis and hydrolysis. Possibly,

 *ATP synthase activity*

transport with closely interrelated thermodynamic and kinetic factors

transport in brain and liver mitochon-

transport obtained

cycling,

F1 ATP

cycling could disturb the molecular

The functional effects of mKATP channels opening under hypoxia are similar to those observed in normoxic cells. Thus, under oxygen deprivation, mKATP channel activation reduced mitochondrial Ca2+ loading [57, 79], preserved ATP levels [67, 77] and increased cell survival [16, 40, 41, 80] by suppression of apoptosis *via* targeting glycogen synthase kinase 3β (GSK3β), an enzyme involved in triggering cell death by promotion of the opening of mitochondrial permeability transition pore, mPTP [81]. Suppression of cell death pathways resulted in stabilization of membrane potential [16, 59, 80] and the restoration of ATP synthesis [82]. Increased expression of both Kir6.2 [59] and SUR2A [37], similar to pharmacological mKATP channels opening, too was shown to improve the viability and the resistance of cardiomyocytes to hypoxia.

As one can see from the above examples, cell response to hypoxia was essentially dependent on the bioenergetic effects of mKATP channels opening. This allows assume that cytoprotection afforded by mKATP channels opening is largely based on a synergistic action of bioenergetic effects of mKATP channel functioning (primarily ROS production and ATP synthesis [20, 40, 41, 67, 79]), and the redox signaling critically dependent on ROS formation caused by mKATP channels opening [16, 50].

In the literature, it was obtained rather unambiguous evidence of the suppression of the OxPhos by mKATP channels opening [67, 76, 77]. However, it seems to be surprising that under hypoxia, similar to other metabolic stress conditions, cytoprotection was afforded by contrary effects of mKATP channels opening on free radical formation, and both the reduction [40, 80] and the elevation [16, 41, 50] of ROS production were shown to afford cytoprotective effects. To smooth this apparent contradiction, we recently proposed [83] that, dependent on the direct impact of ATP-sensitive K+ transport on mitochondrial bioenergetics, mKATP channels opening could afford protection at least in two ways: either directly, by the direct reduction of ROS formation under certain conditions [40, 75, 80, 84] or indirectly, by the elevation of ROS production and triggering of ROS-dependent signaling shown to be cytoprotective under oxygen deprivation (ischemia and hypoxia) [16, 41, 50]. To shed more light on physiological role(s) of mKATP channels under hypoxia, functional consequences of mKATP channels opening on ROS production and ATP synthesis should be considered in more detail.

#### *3.2.1. Triggering of ROS-dependent signaling and controlling of ROS production in mitochondria*

With reference to hypoxia, it is generally supposed that mitochondria respond to oxygen deprivation by the generation of ROS and activation of ROS-dependent signaling pathways [3–5, 14]. mKATP channel was shown to be involved in ROS signaling triggered both upstream (by the activation of kinases PI3K/Akt, PKCε [16, 80]) and downstream (p38MAPK [52], PKCε, Akt [16, 41]) of mKATP channels opening. This implies the ability of mKATP channel to sense and convey ROS signals, which agrees with the function of the channel as a "ROS sensor" proposed in the literature [75, 84]. The ability of mKATP channel to accept and convey ROS signals is well illustrated by the fast response to hypoxia exposure by NOX/ ROS-dependent activation of PKCε *via* mKATP channel opening and feedback ROS/PKCεdependent activation of NOX [16, 42, 47], PI3K/Akt and PKC activation upstream and feedback PKCε activation downstream of mKATP channel opening *via* increase in ROS formation [51] and ROS-dependent Akt and PKCε activation downstream of mKATP channel opening [41], which exerted anti-apoptotic effect by the inhibition of GSK3β and mPTP opening. The ability of ATP-sensitive K+ transport to trigger cytoprotective signaling based on the modulation of ROS production has adverse effects in tumors functioning under limited oxygen supply and known to exhibit high mKATP channel activity. Thus, radioresistance of malignant glioma cells overexpressing mKATP channel was shown to be dependent on mKATP channel opening, increasing mitochondrial ROS emission and triggering of MAPK/ERK signaling, which also resulted in suppression of mPTP opening and prevention of tumor cell death [53].

after application of pharmacological mKATP channel openers under pathophysiological conditions [67, 77]. This assumption was supported by the data showing that inhibition

cellular ATP levels under ischemic conditions [67]. Possibly, under hypoxia, suppression of ATP hydrolysis would be helpful in saving ATP available from the glycolytic pathway. Besides, the dramatic fall of the total level of ATP under hypoxia and suppression of ATP

state in order to maintain other physiological functions of the channel. However, we suppose that inhibition of ATP synthesis could be of particular significance under oxygen

Under hypoxia, controlling of cellular oxygen level becomes important for cell survival [3–5, 46]. Mitochondria consume most part (up to 90%) of cellular oxygen. With reference to hypoxia, it needs to be considered that ATP synthesis, which continually occurs in a living cell, is a highly oxygen-consuming process. Thus, it is reasonable to suppose that controlling of oxygen consumption by controlling the rate of ATP synthesis and the reduction of oxygen expenses for oxidative phosphorylation is one vitally important function of mKATP channel under hypoxia. This is in line with other mechanisms suppressing mitochondrial respiration and OxPhos reported in the literature: the activation of AMPK and glycolysis, S-nitrosation of

and save oxygen for oxygen-dependent processes by suppression of the oxidative ATP synthesis could move into first place under hypoxia. Concomitant suppression of ATP hydrolysis should prevent excess ATP consumption, which was confirmed by the data showing a preser-

Mitochondria respond to hypoxia by triggering ROS signaling, HIFs activation, controlling of oxygen consumption, ROS production and the level of cellular ATP. Potassium channels of mitochondria and plasma membrane were shown to be both oxygen sensors and ROS sensors and thus are first to respond to the changes of ROS and oxygen levels in the cell. Several published data discussed in this review allow us suppose that activation of potassium transport in mitochondria and controlling the above processes *via* mKATP channel opening could be

As it was supposed by many authors, mitochondria, being the main oxygen consumers, deprive the rest of the cell of oxygen. Under these conditions, ATP synthesis *via* OxPhos becomes too oxygen expensive function of mitochondria. So, phosphorylation should be down-regulated in order to rescue the whole cell from severe oxygen deficiency. Thus, under hypoxia, several mechanisms are brought into action in order to reduce oxygen consumption by mitochondria, i.e. by downregulation and nitrosylation of respiratory complexes, by

F1

ATP synthase by mKATP channel openers helped to preserve

Mitochondrial KATP Channel Function under Hypoxia http://dx.doi.org/10.5772/intechopen.80323 29

transport should keep mKATP channel in functionally active

ATP synthase. Possibly, this function

transport on the whole) to reduce oxygen consumption

of hydrolytic activity of F0

synthesis by ATP-sensitive K+

deprivation.

of ATP-sensitive K+

**functions under hypoxia**

F1

the respiratory complexes and downregulation of F0

transport (and K+

vation of cellular ATP ensuing from the mKATP channel opening.

**4. Conclusions: physiological relevance of mKATP channel**

one of the key events in the adaptive responses of the organelles to hypoxia.

As shown in the above examples, a hypothesis of mKATP channels acting as ROS sensors [75, 84] could be useful in the appraisal of physiological functions of mKATP channel under hypoxia. It is well known that mKATP channel can be activated by ROS [85], and elevated channel activity in response to excess ROS formation could serve to regulate mitochondrial metabolism and prevent ROS overproduction [57, 79]. This enables us to consider mKATP channel as the trigger of both ROS-dependent signaling and "ROS sensor" involved in the regulation of mitochondrial ROS production *via* modulation of mitochondrial bioenergetics. Oxidative modification of mKATP channel can represent a feedback mechanism for the regulation of mKATP channel activity. Being at one time a subject of an oxidative modification and a regulator of ROS formation, mKATP channel could be an effective tool in controlling of mitochondrial ROS production under hypoxia.

The impact of mKATP channel opening on mitochondrial energy state, dependent on the channel activity, could serve as a regulatory mechanism directed either on triggering of redox signaling or prevention of ROS overproduction. Apparently, controversial data on the regulation of ROS production by mKATP channel opening possibly reflect one integrated mechanism regulating fast response of mitochondria to the changes of ROS levels in the mitochondrial environment.

#### *3.2.2. ATP-sensitive K+ transport in the regulation of oxidative phosphorylation*

Physiological role of mKATP channel functioning under hypoxia is not limited to the regulation of ROS production and antiapoptotic effects. In our recent work [68], we proposed that F0 F1 ATP synthase can be one of the principal targets of mKATP channels opening. The modulation of ATP synthesis by ATP-sensitive K+ transport can play particularly important role under hypoxia, which is the regulation of cellular ATP and controlling of oxygen consumption:

Generally, it is supposed that suppression of ATP hydrolysis by mKATP channels opening is a plausible explanation for the preservation of cellular ATP of excess depletion observed after application of pharmacological mKATP channel openers under pathophysiological conditions [67, 77]. This assumption was supported by the data showing that inhibition of hydrolytic activity of F0 F1 ATP synthase by mKATP channel openers helped to preserve cellular ATP levels under ischemic conditions [67]. Possibly, under hypoxia, suppression of ATP hydrolysis would be helpful in saving ATP available from the glycolytic pathway. Besides, the dramatic fall of the total level of ATP under hypoxia and suppression of ATP synthesis by ATP-sensitive K+ transport should keep mKATP channel in functionally active state in order to maintain other physiological functions of the channel. However, we suppose that inhibition of ATP synthesis could be of particular significance under oxygen deprivation.

[52], PKCε, Akt [16, 41]) of mKATP channels opening. This implies the ability of mKATP channel to sense and convey ROS signals, which agrees with the function of the channel as a "ROS sensor" proposed in the literature [75, 84]. The ability of mKATP channel to accept and convey ROS signals is well illustrated by the fast response to hypoxia exposure by NOX/ ROS-dependent activation of PKCε *via* mKATP channel opening and feedback ROS/PKCεdependent activation of NOX [16, 42, 47], PI3K/Akt and PKC activation upstream and feedback PKCε activation downstream of mKATP channel opening *via* increase in ROS formation [51] and ROS-dependent Akt and PKCε activation downstream of mKATP channel opening [41], which exerted anti-apoptotic effect by the inhibition of GSK3β and mPTP opening. The

lation of ROS production has adverse effects in tumors functioning under limited oxygen supply and known to exhibit high mKATP channel activity. Thus, radioresistance of malignant glioma cells overexpressing mKATP channel was shown to be dependent on mKATP channel opening, increasing mitochondrial ROS emission and triggering of MAPK/ERK signaling, which also resulted in suppression of mPTP opening and prevention of tumor

As shown in the above examples, a hypothesis of mKATP channels acting as ROS sensors [75, 84] could be useful in the appraisal of physiological functions of mKATP channel under hypoxia. It is well known that mKATP channel can be activated by ROS [85], and elevated channel activity in response to excess ROS formation could serve to regulate mitochondrial metabolism and prevent ROS overproduction [57, 79]. This enables us to consider mKATP channel as the trigger of both ROS-dependent signaling and "ROS sensor" involved in the regulation of mitochondrial ROS production *via* modulation of mitochondrial bioenergetics. Oxidative modification of mKATP channel can represent a feedback mechanism for the regulation of mKATP channel activity. Being at one time a subject of an oxidative modification and a regulator of ROS formation, mKATP channel could be an effective tool in controlling of

The impact of mKATP channel opening on mitochondrial energy state, dependent on the channel activity, could serve as a regulatory mechanism directed either on triggering of redox signaling or prevention of ROS overproduction. Apparently, controversial data on the regulation of ROS production by mKATP channel opening possibly reflect one integrated mechanism regulating fast response of mitochondria to the changes of ROS levels in the mito-

 *transport in the regulation of oxidative phosphorylation*

Physiological role of mKATP channel functioning under hypoxia is not limited to the regulation of ROS production and antiapoptotic effects. In our recent work [68], we proposed that

under hypoxia, which is the regulation of cellular ATP and controlling of oxygen consumption: Generally, it is supposed that suppression of ATP hydrolysis by mKATP channels opening is a plausible explanation for the preservation of cellular ATP of excess depletion observed

ATP synthase can be one of the principal targets of mKATP channels opening. The modu-

transport can play particularly important role

transport to trigger cytoprotective signaling based on the modu-

ability of ATP-sensitive K+

mitochondrial ROS production under hypoxia.

lation of ATP synthesis by ATP-sensitive K+

cell death [53].

28 Hypoxia and Anoxia

chondrial environment.

*3.2.2. ATP-sensitive K+*

F0 F1 Under hypoxia, controlling of cellular oxygen level becomes important for cell survival [3–5, 46]. Mitochondria consume most part (up to 90%) of cellular oxygen. With reference to hypoxia, it needs to be considered that ATP synthesis, which continually occurs in a living cell, is a highly oxygen-consuming process. Thus, it is reasonable to suppose that controlling of oxygen consumption by controlling the rate of ATP synthesis and the reduction of oxygen expenses for oxidative phosphorylation is one vitally important function of mKATP channel under hypoxia. This is in line with other mechanisms suppressing mitochondrial respiration and OxPhos reported in the literature: the activation of AMPK and glycolysis, S-nitrosation of the respiratory complexes and downregulation of F0 F1 ATP synthase. Possibly, this function of ATP-sensitive K+ transport (and K+ transport on the whole) to reduce oxygen consumption and save oxygen for oxygen-dependent processes by suppression of the oxidative ATP synthesis could move into first place under hypoxia. Concomitant suppression of ATP hydrolysis should prevent excess ATP consumption, which was confirmed by the data showing a preservation of cellular ATP ensuing from the mKATP channel opening.

### **4. Conclusions: physiological relevance of mKATP channel functions under hypoxia**

Mitochondria respond to hypoxia by triggering ROS signaling, HIFs activation, controlling of oxygen consumption, ROS production and the level of cellular ATP. Potassium channels of mitochondria and plasma membrane were shown to be both oxygen sensors and ROS sensors and thus are first to respond to the changes of ROS and oxygen levels in the cell. Several published data discussed in this review allow us suppose that activation of potassium transport in mitochondria and controlling the above processes *via* mKATP channel opening could be one of the key events in the adaptive responses of the organelles to hypoxia.

As it was supposed by many authors, mitochondria, being the main oxygen consumers, deprive the rest of the cell of oxygen. Under these conditions, ATP synthesis *via* OxPhos becomes too oxygen expensive function of mitochondria. So, phosphorylation should be down-regulated in order to rescue the whole cell from severe oxygen deficiency. Thus, under hypoxia, several mechanisms are brought into action in order to reduce oxygen consumption by mitochondria, i.e. by downregulation and nitrosylation of respiratory complexes, by producing H2 S as electron donor to the respiratory chain, by downregulation of the OxPhos and by the activation of mitochondrial ATP-sensitive K+ transport to reduce ATP synthesis and oxygen expenses for one of the most oxygen-consuming mitochondrial functions. Thus, hypoxia upregulates glycolysis in order to save oxygen and preserve cellular ATP needed for energy-consuming processes, such as maintenance of membrane potentials, metabolism, protein synthesis and other cell functions. Inhibition of ATP hydrolysis by potassium transport helps to save ATP obtained by glycolytic pathway.

Being important for the understanding of physiological role of mKATP channel, these aspects of mKATP channel functions, largely based on bioenergetic effects of ATP-sensitive K+

port, cannot yet help in appraisal of the specificity of mKATP channel, as compared to other potassium channels present in mitochondria. Possibly, novel concepts of physiological role(s) of mKATP channels based on the molecular and cellular mechanisms regulating mKATP channel functioning are required to extend our understanding of physiological relevance and

the mechanisms regulating mKATP channel functions under hypoxia.

AA Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine,

[1] Navarrete-Opazo A, Mitchell GS. Therapeutic potential of intermittent hypoxia: A matter of dose. American Journal of Physiology. Regulatory, Integrative and Comparative

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Academy of Sciences of the United States of America. 1996;**93**(3):1215-1220

sensing, mitochondria and ROS signaling:

Address all correspondence to<EMAIL_ADDRESS>
Physiology. 2014;**307**(10):R1181-R1197

[5] Waypa GB, Smith KA, Schumacker PT. O2

2006;**91**(5):807-819

ascent of Everest. The FASEB Journal. 2012;**26**(4):1431-1441

HIF. Cell Death and Differentiation. 2008;**15**(4):660-666

The fog is lifting. Molecular Aspects of Medicine. 2016;**47-48**:76-89

**Author details**

Olga V. Akopova

Kiev, Ukraine

**References**

trans-

31

Mitochondrial KATP Channel Function under Hypoxia http://dx.doi.org/10.5772/intechopen.80323

The activation of mitochondrial potassium transport is a ubiquitous phenomenon under the limited oxygen availability. The above brief survey of the literature enables us to propose the following important functions of mKATP channels relevant to hypoxia: (1) ability to accept and convey ROS signals, triggering of ROS signaling specific for hypoxia; (2) controlling of mitochondrial ROS production and preventing overproduction; (3) controlling the level of cellular oxygen by oxygen-saving control of OxPhos and ATP production and (4) saving cellular ATP (obtained from both oxidation and glycolysis) by suppression of ATP hydrolysis. Multiple mKATP channel functions under hypoxia discussed in this work can be summarized in the following scheme.

**Scheme.** mKATP channels functions under hypoxia.

Being important for the understanding of physiological role of mKATP channel, these aspects of mKATP channel functions, largely based on bioenergetic effects of ATP-sensitive K+ transport, cannot yet help in appraisal of the specificity of mKATP channel, as compared to other potassium channels present in mitochondria. Possibly, novel concepts of physiological role(s) of mKATP channels based on the molecular and cellular mechanisms regulating mKATP channel functioning are required to extend our understanding of physiological relevance and the mechanisms regulating mKATP channel functions under hypoxia.

#### **Author details**

producing H2

30 Hypoxia and Anoxia

following scheme.

and by the activation of mitochondrial ATP-sensitive K+

helps to save ATP obtained by glycolytic pathway.

**Scheme.** mKATP channels functions under hypoxia.

S as electron donor to the respiratory chain, by downregulation of the OxPhos

and oxygen expenses for one of the most oxygen-consuming mitochondrial functions. Thus, hypoxia upregulates glycolysis in order to save oxygen and preserve cellular ATP needed for energy-consuming processes, such as maintenance of membrane potentials, metabolism, protein synthesis and other cell functions. Inhibition of ATP hydrolysis by potassium transport

The activation of mitochondrial potassium transport is a ubiquitous phenomenon under the limited oxygen availability. The above brief survey of the literature enables us to propose the following important functions of mKATP channels relevant to hypoxia: (1) ability to accept and convey ROS signals, triggering of ROS signaling specific for hypoxia; (2) controlling of mitochondrial ROS production and preventing overproduction; (3) controlling the level of cellular oxygen by oxygen-saving control of OxPhos and ATP production and (4) saving cellular ATP (obtained from both oxidation and glycolysis) by suppression of ATP hydrolysis. Multiple mKATP channel functions under hypoxia discussed in this work can be summarized in the

transport to reduce ATP synthesis

Olga V. Akopova

Address all correspondence to<EMAIL_ADDRESS>
AA Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kiev, Ukraine

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**Chapter 3**

**Provisional chapter**

**Hypoxia Signaling in Cardiovascular Diseases**

**Hypoxia Signaling in Cardiovascular Diseases**

DOI: 10.5772/intechopen.80456

Cardiovascular diseases such as stroke, coronary artery disease, and thrombosis remain a global health burden. Understanding the mechanism of these diseases paves the way for development of prophylactics/therapeutics. It is well known at cellular levels; the pathophysiology of most of the cardiovascular disease involves a complicated yet coordinated signaling networks triggered in response to either cellular or tissue levels of hypoxic milieu. Information related to types of hypoxia and signaling mechanism associated to such complications if complied and presented in a comprehensive manner shall prove relevant in proposing common therapeutic targets for wide array of cardiovascular complications. The relative functional roles of hypoxia-triggered signaling pathways are also an area of current research. Based upon these facts, this chapter discusses the types of hypoxia and role of hypoxia-mediated signaling pathways in various types of commonly

**Keywords:** hypoxia, signaling, cardiovascular disorders, thrombosis, therapeutics

Oxygen concentration below the tissue specific physiological levels is termed as 'Hypoxia'. Depending upon the cause of oxygen scarcity, hypoxia can be classified into Hypoxic hypoxia (occurs due to deficiency in oxygen exchange in lungs or arises due to reduced partial pressure of oxygen in air), Anemic hypoxia (arises when the transport of oxygen is affected), stagnant hypoxia (due to delayed blood renewal, or insufficient blood flow) or histotoxic hypoxia (body is not able to use the available oxygen) [1]. Among its various types, stagnant hypoxia and hypoxic hypoxia are most common types associated with pathophysiology of a variety of cardiovascular disorders (CVDs) such as hypoxic milieu developing in veins due to reduced blood flow promotes thrombus formation [2, 3], whereas, environmental hypoxia at

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Neha Gupta and Mohammad Zahid Ashraf

Neha Gupta and Mohammad Zahid Ashraf

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80456

occurring cardiovascular disorders.

**Abstract**

**1. Introduction**

#### **Hypoxia Signaling in Cardiovascular Diseases Hypoxia Signaling in Cardiovascular Diseases**

DOI: 10.5772/intechopen.80456

Neha Gupta and Mohammad Zahid Ashraf Neha Gupta and Mohammad Zahid Ashraf

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80456

#### **Abstract**

Cardiovascular diseases such as stroke, coronary artery disease, and thrombosis remain a global health burden. Understanding the mechanism of these diseases paves the way for development of prophylactics/therapeutics. It is well known at cellular levels; the pathophysiology of most of the cardiovascular disease involves a complicated yet coordinated signaling networks triggered in response to either cellular or tissue levels of hypoxic milieu. Information related to types of hypoxia and signaling mechanism associated to such complications if complied and presented in a comprehensive manner shall prove relevant in proposing common therapeutic targets for wide array of cardiovascular complications. The relative functional roles of hypoxia-triggered signaling pathways are also an area of current research. Based upon these facts, this chapter discusses the types of hypoxia and role of hypoxia-mediated signaling pathways in various types of commonly occurring cardiovascular disorders.

**Keywords:** hypoxia, signaling, cardiovascular disorders, thrombosis, therapeutics

#### **1. Introduction**

Oxygen concentration below the tissue specific physiological levels is termed as 'Hypoxia'. Depending upon the cause of oxygen scarcity, hypoxia can be classified into Hypoxic hypoxia (occurs due to deficiency in oxygen exchange in lungs or arises due to reduced partial pressure of oxygen in air), Anemic hypoxia (arises when the transport of oxygen is affected), stagnant hypoxia (due to delayed blood renewal, or insufficient blood flow) or histotoxic hypoxia (body is not able to use the available oxygen) [1]. Among its various types, stagnant hypoxia and hypoxic hypoxia are most common types associated with pathophysiology of a variety of cardiovascular disorders (CVDs) such as hypoxic milieu developing in veins due to reduced blood flow promotes thrombus formation [2, 3], whereas, environmental hypoxia at

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

high altitude exposures also promotes a prothrombotic tendency [4, 5]. Physiological alterations are ultimately orchestrated as a myriad of changes at both cellular and molecular levels. These changes involving the activation of transcription factors (Hypoxia-inducible Factors-1, NF-kB), their downstream signaling pathways, generation of reactive oxygen species and many other molecular adaptive responses in cells, also contribute toward the development of diseased phenotype. A better understanding of these signaling pathways would lead to the identification of putative targets for development of therapeutics and prophylactics to reduce the burden of CVDs. The current chapter discusses the hypoxia-associated pathophysiological changes toward disease progression and major transcription factors playing a role in hypoxic conditions, the signaling and molecular events involved in commonly occurring CVDs. Expanding the understanding of the hypoxia-associated molecular-signaling pathways and cross-talk between them will provide new avenues of therapeutic opportunity of the disease.

the major regulator of oxygen homeostasis, for adaptation to hypoxia involving increasing tissue reperfusion, and oxygenation, thereby, overcoming initial hypoxic insult [13]. Even under normoxic conditions also HIF-1 regulates the shift to increased glycolysis and anaerobic metabolism at low oxygen tensions [13]. HIF-1 regulates a number of target genes (such VEGF-1, EPO). Furthermore, mammalian HIF-1α has three isoforms viz. HIF-1α, 2α, and 3α.

Although, NF-kβ and HIF-1 has independent roles in gene regulation, their cross-talk plays equally important role in pathophysiology of a number of diseases. Structurally, there lies an active NF-kβ binding site, in the proximal promoter site of HIF-1 gene and NF-kB, regulates the basal levels of HIF-1 gene expression [16]. Even under hypoxic conditions, HIF-1 transcription is upregulated through NF-kβ dependent mechanism [17]. Reports are there to show that hypoxia-induced transcription of NF-kβ depends upon the presence of HIF-1 and HIF-1 also directly, regulates neutrophil survival in hypoxia via NF-kβ modulation [18]. Higher expressions of HIF-1 are related to increased NF-kβ activity and an enhanced inflammatory response [18, 19]. Some of the common gene products are shared by HIF-1α such as eNOS, a potent vasodilator whose bioavailability is increased by coordinated action of HIF-1 and

OSAS have been recognized as a major health problem affecting developed countries. The disorder is characterized by obstruction of upper airways during sleep resulting in sleep fragmentation and excessive day time sleepiness. OSAS has shown to have a causal relationship with CVDs [21–23]. Although pathogenesis of CVDs in OSAS is multifactorial, the proposed mechanism by which OSAS predisposes to CVD includes sympathetic excitation, vascular endothelial dysfunction, metabolic dysregulation as well as oxidative stress and inflammation induced by cyclical intermittent hypoxia [24]. Evidences are there to show that inflammatory pathways mediate the pathobiology of cardiovascular complications in OSAS. The pattern of intermittent hypoxia in patients of OSAS can be either repetitive cycles of hypoxia/ reoxygenation or it can be with prolonged periods of sustained hypoxia allowing for development of an adaptive response, associated with increased tissue perfusion and oxygenation whereas shorter intermittent hypoxic exposures may also activate inflammatory pathways [25, 26]. Intermittent hypoxia directly promotes the production of cytokines and inflammatory cells in OSAS patients. The inflammatory response in OSAS is regulated by NF-kβ and HIF-1α. A rise in NF-kβ activity and its downstream product TNF-α has been observed in OSAS. Levels of TNF-α has also been found to be higher in serum samples of OSAS patients as compared to age and sex matched controls. OSAS patients also show elevated monocyte

condi-

41

levels [14, 15].

Hypoxia Signaling in Cardiovascular Diseases http://dx.doi.org/10.5772/intechopen.80456

HIF-1α accumulation is the key regulatory subunit for assembly of HIF under low O2

tions. Regulation of HIF-1α occurs post-translationally in response to low O2

NF-kB. In OSAS even crosstalk of NF-kβ and HIF-1 also play a central role [20].

**2.3. Interaction between NF-kβ and HIF-1 pathways**

**3. Hypoxia in various CVDs**

**3.1. Hypoxia in obstructive sleep apnea syndrome**

## **2. Master regulators of hypoxia responsive factors**

#### **2.1. Transcription factors**

#### *2.1.1. Nuclear factor (NF)-kβ*

Nuclear factor (NF)-kβ is a eukaryotic transcription factor that mediates inflammatory processes through Rel family of proteins and was originally described as a nuclear factor required for immunoglobulin k light-chain transcription in B cells [6]. Normally, in most of the cells, NF-kβ lies in its inactive state by binding to the inhibitor IkB and is retained in cytoplasm. Upon sensing an inflammatory stimulus, IkB undergoes ubiquitin-mediated degradation and NF- kβ translocation takes place to the nucleus [7]. Inside the nucleus, NF-kB regulates the transcription of a number of genes. NF-kβ plays a central role in inflammatory processes by orchestrating the expression of numerous factors (cytokines, adhesion molecules and enzymes) [8, 9].

Activation of NF-kβ also occurs in cardiovascular tissue with a concomitant increase in expression of *i*NOS (inducible nitric oxide synthase) protein. Increased NF-kβ activity in circulating neutrophils and raised plasma levels of NF-kβ, controlled gene products, soluble E-selectin and soluble vascular cell-adhesion molecule-1(VCAM-1) are a response to hypoxia in patients of obstructive sleep apnea syndrome (OSAS) [10].

#### **2.2. Hypoxia-inducible factor-1 (HIF-1)**

HIF-1 is a heterodimeric transcription factor consisting of a constitutively expressed-β subunit and an α-subunit containing an oxygen dependent degradation (ODD) domain [11]. Under normoxic conditions, hydroxylation of ODD occurs in an oxygen dependent manner rendering α-subunit vulnerable to proteasomal degradation [12]. Therefore, HIF-1 is suppressed in normoxia, whereas under hypoxic conditions, HIF-1 is stable and active, capable to bind to the regulatory regions of its target genes and inducing their expression. HIF-1 is the major regulator of oxygen homeostasis, for adaptation to hypoxia involving increasing tissue reperfusion, and oxygenation, thereby, overcoming initial hypoxic insult [13]. Even under normoxic conditions also HIF-1 regulates the shift to increased glycolysis and anaerobic metabolism at low oxygen tensions [13]. HIF-1 regulates a number of target genes (such VEGF-1, EPO). Furthermore, mammalian HIF-1α has three isoforms viz. HIF-1α, 2α, and 3α. HIF-1α accumulation is the key regulatory subunit for assembly of HIF under low O2 conditions. Regulation of HIF-1α occurs post-translationally in response to low O2 levels [14, 15].

#### **2.3. Interaction between NF-kβ and HIF-1 pathways**

high altitude exposures also promotes a prothrombotic tendency [4, 5]. Physiological alterations are ultimately orchestrated as a myriad of changes at both cellular and molecular levels. These changes involving the activation of transcription factors (Hypoxia-inducible Factors-1, NF-kB), their downstream signaling pathways, generation of reactive oxygen species and many other molecular adaptive responses in cells, also contribute toward the development of diseased phenotype. A better understanding of these signaling pathways would lead to the identification of putative targets for development of therapeutics and prophylactics to reduce the burden of CVDs. The current chapter discusses the hypoxia-associated pathophysiological changes toward disease progression and major transcription factors playing a role in hypoxic conditions, the signaling and molecular events involved in commonly occurring CVDs. Expanding the understanding of the hypoxia-associated molecular-signaling pathways and cross-talk between them will provide new avenues of therapeutic opportunity

Nuclear factor (NF)-kβ is a eukaryotic transcription factor that mediates inflammatory processes through Rel family of proteins and was originally described as a nuclear factor required for immunoglobulin k light-chain transcription in B cells [6]. Normally, in most of the cells, NF-kβ lies in its inactive state by binding to the inhibitor IkB and is retained in cytoplasm. Upon sensing an inflammatory stimulus, IkB undergoes ubiquitin-mediated degradation and NF- kβ translocation takes place to the nucleus [7]. Inside the nucleus, NF-kB regulates the transcription of a number of genes. NF-kβ plays a central role in inflammatory processes by orchestrating the expression of numerous factors (cytokines, adhesion molecules and

Activation of NF-kβ also occurs in cardiovascular tissue with a concomitant increase in expression of *i*NOS (inducible nitric oxide synthase) protein. Increased NF-kβ activity in circulating neutrophils and raised plasma levels of NF-kβ, controlled gene products, soluble E-selectin and soluble vascular cell-adhesion molecule-1(VCAM-1) are a response to hypoxia in patients

HIF-1 is a heterodimeric transcription factor consisting of a constitutively expressed-β subunit and an α-subunit containing an oxygen dependent degradation (ODD) domain [11]. Under normoxic conditions, hydroxylation of ODD occurs in an oxygen dependent manner rendering α-subunit vulnerable to proteasomal degradation [12]. Therefore, HIF-1 is suppressed in normoxia, whereas under hypoxic conditions, HIF-1 is stable and active, capable to bind to the regulatory regions of its target genes and inducing their expression. HIF-1 is

**2. Master regulators of hypoxia responsive factors**

of obstructive sleep apnea syndrome (OSAS) [10].

**2.2. Hypoxia-inducible factor-1 (HIF-1)**

of the disease.

40 Hypoxia and Anoxia

enzymes) [8, 9].

**2.1. Transcription factors**

*2.1.1. Nuclear factor (NF)-kβ*

Although, NF-kβ and HIF-1 has independent roles in gene regulation, their cross-talk plays equally important role in pathophysiology of a number of diseases. Structurally, there lies an active NF-kβ binding site, in the proximal promoter site of HIF-1 gene and NF-kB, regulates the basal levels of HIF-1 gene expression [16]. Even under hypoxic conditions, HIF-1 transcription is upregulated through NF-kβ dependent mechanism [17]. Reports are there to show that hypoxia-induced transcription of NF-kβ depends upon the presence of HIF-1 and HIF-1 also directly, regulates neutrophil survival in hypoxia via NF-kβ modulation [18]. Higher expressions of HIF-1 are related to increased NF-kβ activity and an enhanced inflammatory response [18, 19]. Some of the common gene products are shared by HIF-1α such as eNOS, a potent vasodilator whose bioavailability is increased by coordinated action of HIF-1 and NF-kB. In OSAS even crosstalk of NF-kβ and HIF-1 also play a central role [20].

### **3. Hypoxia in various CVDs**

#### **3.1. Hypoxia in obstructive sleep apnea syndrome**

OSAS have been recognized as a major health problem affecting developed countries. The disorder is characterized by obstruction of upper airways during sleep resulting in sleep fragmentation and excessive day time sleepiness. OSAS has shown to have a causal relationship with CVDs [21–23]. Although pathogenesis of CVDs in OSAS is multifactorial, the proposed mechanism by which OSAS predisposes to CVD includes sympathetic excitation, vascular endothelial dysfunction, metabolic dysregulation as well as oxidative stress and inflammation induced by cyclical intermittent hypoxia [24]. Evidences are there to show that inflammatory pathways mediate the pathobiology of cardiovascular complications in OSAS. The pattern of intermittent hypoxia in patients of OSAS can be either repetitive cycles of hypoxia/ reoxygenation or it can be with prolonged periods of sustained hypoxia allowing for development of an adaptive response, associated with increased tissue perfusion and oxygenation whereas shorter intermittent hypoxic exposures may also activate inflammatory pathways [25, 26]. Intermittent hypoxia directly promotes the production of cytokines and inflammatory cells in OSAS patients. The inflammatory response in OSAS is regulated by NF-kβ and HIF-1α. A rise in NF-kβ activity and its downstream product TNF-α has been observed in OSAS. Levels of TNF-α has also been found to be higher in serum samples of OSAS patients as compared to age and sex matched controls. OSAS patients also show elevated monocyte NF-kβactivity [25]. In OSAS, patients with several nocturnal hypoxemia, HIF-1, can be viewed as a pro-inflammatory contributor to hypoxic response by promoting inflammatory cell survival [27, 28].

egr-1 has also been validated in *in vivo* model system where egr-1 null animals when exposed to hypoxia showed a minimal rise in TF mRNA levels with no change in antigen levels in comparison to normoxia exposed controls [37]. Earlier evidences indicate that oxygen deprivation promotes egr-1 synthesis due to binding of ternary complex factor to serum response element (SRE) sites in egr-1 promoter region [38]. Egr-1 also plays a central role in monocyte expression of TF under hypoxia. Expression of egr-1 initiated mechanism in pathologic changes associated with hypoxia point toward novel strategies to prevent these events, that is, target egr-1 rather than directly targeting coagulation mechanism. These series of events producing exposure of TF in hypoxemic vasculature especially in mononuclear phagocytes and smooth muscle cells provide a new biologic context to consider mechanisms underlying and possible

**3.4. Hypoxia signaling in inflammation and tissue regeneration, with role in chronic**

Most of the *in vitro* studies have been complemented by *in vivo* model systems to obtain a more physiologically relevant setting to understand the inter-relationship of hypoxia and disease. Rodents are most widely used. Mice and rats are highly amenable to manipulation and are small enough to fit into hypoxic chambers for longer periods of time. With advent of technology and in present era, Zebra fish are also used as a new whole-organism model of disease to understand the complex physiology involved [39]. One of the primary reasons is that zebrafish have optically transparent larvae; an opportunity to visualize disease processes *in vivo* using fluorescence microscopy. In addition, high-throughput drug screening can be done easily by addition of small molecule compounds to the embryo in water [40]. An injury to blood vessel is often associated with tissue hypoxia when blood flow is restricted in localized milieu. Even, inflammation can occur and recruitment of immune cells at the injury site occurs and clearance of damaged cells takes place to prevent infection [41]. Innate immune cells (leucocytes) are the first one to respond to injury and sense change in local oxygen levels. This inflammatory response is highly regulated by HIF, signaling, contributing to the regulation of immune activity and lifespan of leucocytes, as timely resolution of inflammation is also necessary otherwise failure to timely resolution may result in inflammatory diseases such as COPD [42, 43]. Once inflammation is resolved, tissues thus regenerate and homeostasis is restored. In zebrafish models, cardiomyocytes regeneration is dependent upon HIF-1α signaling by virtue of which cardiomyocytes can survive and regenerate with an injury of 20% of

**obstructive pulmonary disease (COPD) (studies with zebrafish models)**

heart tissue thereby identifying HIF-1α as a potential therapeutic target [44, 45].

Myocardial gene expression highly depends upon the levels of O2

**3.5. Hypoxia in heart and cardiac dysfunction: role of reactive oxygen species (ROS)**

ing isolated hypoxia or ischemia-associated hypoxia, as a result gene expression patterns are altered. In experiments, conducted with myocardial infarction-induced mice, HIF-1α stability was found to reduce infarct size and decrease the number of apoptotic cells [46]. The possible explanation is an upregulation of cardiotrophin-1 (member of IL-6 family) by HIF-1α in hypoxic environment. Further, impaired cardiac muscle contractility due to reduced calcium

. O2

levels change either dur-

Hypoxia Signaling in Cardiovascular Diseases http://dx.doi.org/10.5772/intechopen.80456 43

interventions to prevent hypoxia-induced thrombosis.

#### **3.2. Venous thrombosis: role of valvular stasis-associated hypoxia**

Venous Thrombosis involves the formation of a thrombus inside deep veins usually in legs. Such thrombus can break off and travels in circulation and may lodge at pulmonary vasculature leading to Pulmonary Embolism, which may cause death. As per current understanding the luminal thrombus in veins develops in the presence of increased stasis and hypoxia resulting from the outgrowth of a progressively occlusive thrombus extending from valve to lumen [2, 29]. Evidences for the role of stasis (reduced blood flow), include clinical scenario like long term immobilization due to hospitalization. Pressure of stasis in venous valves is supported by the observation that contrast media used in venography lingers in the veins for up to 60 min after the procedure in the elderly with a clear gradient of increasing stasis with age [30]. Further, pO2 measurements in sinuses of dogs by Hamer et al. have established that prolonged stasis leads to severe hypoxia at venous valvular sinus. A steeply declining pO2 gradient from 5 to 1 kPa was observed after 2 h of stasis [31]. However, the anatomical location of the severe hypoxia and thrombus initiation were same site [29]. Changes in blood flow pattern are attributed to generation of hypoxia. Role of HIF-1α is venous thrombus is contradictory and interplayed. An earlier study revealed that HIF-1α stimulates, vein recanalization and thrombus resolution [32] however, study by Gupta et al. suggest that HIF-1α plays a role in thrombus development [33].

#### **3.3. Pathways associated with ischemia-associated thrombosis**

Reduced blood flow in veins (also called as stasis), is associated with reduced intravascular O2 tension and thrombus progression. However, only reduced levels of O2 have not been found sufficient to trigger fibrin clot formation. Although interplay of hypoxia with different cell types, majorly mononuclear phagocytes and polymorphonuclear leucocytes, can contribute, the association between hypoxia and hypoxemia has remained strong, despite extensive mechanistic explanations [3, 34]. In thrombotic episodes, hypoxemia is found severe in proximity to venous valve cusps and nascent thrombi develop on apparently intact endothelial surface at the parietal aspect of valve cusps during hypoxemia [3]. In addition, *in vivo* exposures to intermittent hypoxia/reoxygenation are also associated with thrombus formation. Under such settings, hypoxia/hypoxemia is sufficient to cause venous thrombosis. Stasis leads to ischemia that is associated with a myriad of changes in vascular microenvironment, increased vascular permeability [3].

In a mice model of hypoxia-induced thrombosis, administration of blocking Ab to tissue factor (TF) suppressed fibrin deposition [35]. This observation was also supported by the evidence generated when TF expression was analyzed in hypoxic murine lungs. Hypoxic exposure produced ~20 fold rise in TF transcripts in hypoxic lungs in comparison to normoxic ones [36]. In such cases, early growth receptor-1 (egr-1) has been identified as the primary driving motif for hypoxia-induced TF transcriptional upregulation. The biological importance of egr-1 has also been validated in *in vivo* model system where egr-1 null animals when exposed to hypoxia showed a minimal rise in TF mRNA levels with no change in antigen levels in comparison to normoxia exposed controls [37]. Earlier evidences indicate that oxygen deprivation promotes egr-1 synthesis due to binding of ternary complex factor to serum response element (SRE) sites in egr-1 promoter region [38]. Egr-1 also plays a central role in monocyte expression of TF under hypoxia. Expression of egr-1 initiated mechanism in pathologic changes associated with hypoxia point toward novel strategies to prevent these events, that is, target egr-1 rather than directly targeting coagulation mechanism. These series of events producing exposure of TF in hypoxemic vasculature especially in mononuclear phagocytes and smooth muscle cells provide a new biologic context to consider mechanisms underlying and possible interventions to prevent hypoxia-induced thrombosis.

NF-kβactivity [25]. In OSAS, patients with several nocturnal hypoxemia, HIF-1, can be viewed as a pro-inflammatory contributor to hypoxic response by promoting inflammatory cell sur-

Venous Thrombosis involves the formation of a thrombus inside deep veins usually in legs. Such thrombus can break off and travels in circulation and may lodge at pulmonary vasculature leading to Pulmonary Embolism, which may cause death. As per current understanding the luminal thrombus in veins develops in the presence of increased stasis and hypoxia resulting from the outgrowth of a progressively occlusive thrombus extending from valve to lumen [2, 29]. Evidences for the role of stasis (reduced blood flow), include clinical scenario like long term immobilization due to hospitalization. Pressure of stasis in venous valves is supported by the observation that contrast media used in venography lingers in the veins for up to 60 min after the procedure in the elderly with a clear gradient of increasing stasis with

prolonged stasis leads to severe hypoxia at venous valvular sinus. A steeply declining pO2 gradient from 5 to 1 kPa was observed after 2 h of stasis [31]. However, the anatomical location of the severe hypoxia and thrombus initiation were same site [29]. Changes in blood flow pattern are attributed to generation of hypoxia. Role of HIF-1α is venous thrombus is contradictory and interplayed. An earlier study revealed that HIF-1α stimulates, vein recanalization and thrombus resolution [32] however, study by Gupta et al. suggest that HIF-1α plays a role

Reduced blood flow in veins (also called as stasis), is associated with reduced intravascular

found sufficient to trigger fibrin clot formation. Although interplay of hypoxia with different cell types, majorly mononuclear phagocytes and polymorphonuclear leucocytes, can contribute, the association between hypoxia and hypoxemia has remained strong, despite extensive mechanistic explanations [3, 34]. In thrombotic episodes, hypoxemia is found severe in proximity to venous valve cusps and nascent thrombi develop on apparently intact endothelial surface at the parietal aspect of valve cusps during hypoxemia [3]. In addition, *in vivo* exposures to intermittent hypoxia/reoxygenation are also associated with thrombus formation. Under such settings, hypoxia/hypoxemia is sufficient to cause venous thrombosis. Stasis leads to ischemia that is associated with a myriad of changes in vascular microenvironment,

In a mice model of hypoxia-induced thrombosis, administration of blocking Ab to tissue factor (TF) suppressed fibrin deposition [35]. This observation was also supported by the evidence generated when TF expression was analyzed in hypoxic murine lungs. Hypoxic exposure produced ~20 fold rise in TF transcripts in hypoxic lungs in comparison to normoxic ones [36]. In such cases, early growth receptor-1 (egr-1) has been identified as the primary driving motif for hypoxia-induced TF transcriptional upregulation. The biological importance of

tension and thrombus progression. However, only reduced levels of O2

measurements in sinuses of dogs by Hamer et al. have established that

have not been

**3.2. Venous thrombosis: role of valvular stasis-associated hypoxia**

**3.3. Pathways associated with ischemia-associated thrombosis**

vival [27, 28].

42 Hypoxia and Anoxia

age [30]. Further, pO2

O2

in thrombus development [33].

increased vascular permeability [3].

#### **3.4. Hypoxia signaling in inflammation and tissue regeneration, with role in chronic obstructive pulmonary disease (COPD) (studies with zebrafish models)**

Most of the *in vitro* studies have been complemented by *in vivo* model systems to obtain a more physiologically relevant setting to understand the inter-relationship of hypoxia and disease. Rodents are most widely used. Mice and rats are highly amenable to manipulation and are small enough to fit into hypoxic chambers for longer periods of time. With advent of technology and in present era, Zebra fish are also used as a new whole-organism model of disease to understand the complex physiology involved [39]. One of the primary reasons is that zebrafish have optically transparent larvae; an opportunity to visualize disease processes *in vivo* using fluorescence microscopy. In addition, high-throughput drug screening can be done easily by addition of small molecule compounds to the embryo in water [40]. An injury to blood vessel is often associated with tissue hypoxia when blood flow is restricted in localized milieu. Even, inflammation can occur and recruitment of immune cells at the injury site occurs and clearance of damaged cells takes place to prevent infection [41]. Innate immune cells (leucocytes) are the first one to respond to injury and sense change in local oxygen levels. This inflammatory response is highly regulated by HIF, signaling, contributing to the regulation of immune activity and lifespan of leucocytes, as timely resolution of inflammation is also necessary otherwise failure to timely resolution may result in inflammatory diseases such as COPD [42, 43]. Once inflammation is resolved, tissues thus regenerate and homeostasis is restored. In zebrafish models, cardiomyocytes regeneration is dependent upon HIF-1α signaling by virtue of which cardiomyocytes can survive and regenerate with an injury of 20% of heart tissue thereby identifying HIF-1α as a potential therapeutic target [44, 45].

#### **3.5. Hypoxia in heart and cardiac dysfunction: role of reactive oxygen species (ROS)**

Myocardial gene expression highly depends upon the levels of O2 . O2 levels change either during isolated hypoxia or ischemia-associated hypoxia, as a result gene expression patterns are altered. In experiments, conducted with myocardial infarction-induced mice, HIF-1α stability was found to reduce infarct size and decrease the number of apoptotic cells [46]. The possible explanation is an upregulation of cardiotrophin-1 (member of IL-6 family) by HIF-1α in hypoxic environment. Further, impaired cardiac muscle contractility due to reduced calcium ion uptake by cardiomyocytes along with certain amount of dilation in muscles was the additional factor involved [47–49]. This elevated HIf-1α levels may lead to better cardiomyocyte survival under hypoxia.

In addition to the regulatory role of transcription factors, formation of ROS is another major event occurring under oxygen regulation conditions. ROS participates as a benevolent molecule in cell signaling processes and can induce irreversible cellular damage. Formation of ROS in heart or other tissues may occur by several mechanisms either by xanthine oxidase (XO), NAD(P)H, oxidases, and cytochrome P450, or by auto-oxidation of catecholamines and by uncoupling of NO synthase (NOS) [50–52]. Presence of unpaired e- on NO facilitates its reaction with O2 - to form peroxynitrites (ONOO−), an oxidant. Further, Formation of ROS is also induced by cytokine stimulus, growth factors such as angiotensin II (ATII), PDGF and TNF-α [50, 53]. As an adaptive response, production of ROS is counterbalanced by several enzymatic (such as superoxide dismutase (SOD), Catalase, Thioredoxin) and non-enzymatic mechanisms (intracellular oxidants such as vitamins E, C, β-carotene, ubiquinone lipoic acid and glutathione) [54–56]. Deletion of Thioredoxin reductase leads to cardiac abnormalities and even cardiac death, secondary to severe dilated cardiomyopathy [57]. Activation of ROS occurs in response to various stressors and in failing heart as well (**Table 1**).

#### **3.6. Hypoxia-mediated inflammation in atherosclerosis**

Inflammation and hypoxia are integral parts in development of atherosclerosis. Data from recent reports suggest that HIF-1α is involved in the pathogenesis of atherosclerosis. Smooth muscle cells extracted from coronary arteries showed that HIF-1α increased activity was related to increased VEGF expression required for proliferation of smooth muscle cells (SMC) [70]. Moreover, hypoxia also produces HIF-1α dependent increase in macrophage migration inhibitory factor (MIF)-required for escalation of migration increased proliferation of vascular SMCs during progression of atherosclerosis [71]. In developing atherosclerosis chronic inflammation and various types of cells (SMC, EC monocytes/macrophages, and T lymphocytes) are involved in plaque formation [72]. Even, the oxygen supply from the luminal blood strongly affects the cells of blood vessel wall [73]. In developed atherosclerosis, tissue hypoxia occurs at the plaque lesion and HIF-1α expression occurs at the macrophage rich center of plaque [72, 74].

heart serves as a signal to regulate the expansion of CPC and cardiac morphogenesis. The response is also mediated by HIF-1α, where HIF-1α forms a complex with notch effector HES family bHLH transcription factor 1 (HES 1) and protein deacetylase sirtuin1 (SIRT1) at the ISL1 gene (islet gene) where ISL1 repression occurs in hypoxic heart tube or as a response to ectopic hypoxic response and prevents CHDs. Thus this is an example where spatial difference in physiological hypoxia maintains the homeostasis for CPCs and provides mechanistic

**Disease ROS related mechanism References**

Myocardial infarction ROS plays a role in necrosis and reperfusion, injury. Overexpression

post MI cardiac function.

via oxidation of cysteine residues.

and occlusion

ROS contributes to the formation of oxidized LDL, the key molecular player in progression of atherosclerosis. Even the activation of MMPs by ROS contributes to plaque rupture initiating coronary thrombosis

of SOD (an antioxidant molecule), reduces the infarct size. Evidences that ROS play an important role in myocardial infarction (MI). Around 20% of patients suffering from MI, often develop heart failure which is also determined by the healing and remodeling patterns of ventricles. The latter highly depends upon ROS. Even inhibition of XO with allopurinol diminishes ROS production in myocardium and attenuates maladaptive LV remodeling, leading to

ROS activates either directly or indirectly many extracellular factors as well as downstream signaling pathways that mediate hypertrophic growth response to these factors. Molecules such as PKC, MAPKs, p38, JNK, ERK1/2, Akt, Tyrosine kinase, NF-kB. For instance, AgIIinduced hypertrophy is mediated by induction of extracellular signal, whereas direct activation involves ROS-mediated activation of PKC

[58–60]

45

Hypoxia Signaling in Cardiovascular Diseases http://dx.doi.org/10.5772/intechopen.80456

[61–65]

[66–69]

PAH is clinically manifested as elevated BP in pulmonary artery with resulting right ventricular heart failure [79, 80]. Hypoxia is known to elicit pulmonary vasoconstriction and arterial remodeling [81, 82]. Hypoxic exposures are the commonly used murine models of PAH. Recently, pulmonary endothelial specific HIF-2α deficient mice showed tolerance to hypoxia-induced PAH as compared to HIF-1α deficient or control mice [83]. As a molecular mechanism HIF-2α regulates NO production in pulmonary vasculature via induction of arginase-1, thereby indicating that HIF-2α-Arginase-1 axes may be used as a therapeutic target to

An imbalance between tissue demand and actual oxygen supply also develops due to environmental hypoxia or reduced oxygen content in ambient air [4]. Such episodes are commonly

explanation for non- congenital CHDs [78].

**Table 1.** Different diseases associated to ROS signaling.

Heart failure function (In ischemic syndrome, heart failure is a sequelae of myocardial ischemia and necrosis is a major cause of death worldwide)

Cardiac hypertrophy (cardiac hypertrophy often serves as a maladaptive precursor to

heart failure)

**3.8. Pulmonary arterial hypertension (PAH)**

improve NO availability in PAH [83].

**3.9. High-altitude hypoxia and thrombosis**

HIF-1α also upregulates the expression of low density lipoprotein receptor related protein-1 (LRP1) associated with cholesterol independent progression of atherosclerosis [75]. In fact, bone marrow transplantation of muscle specific HIF-1α deficient mice reduced the plaque burden in aorta of Ldlr−/− mice. Furthermore, expression of inflammatory genes (M1 macrophage accumulation) was also suppressed in HIF-1α deficient mice [76]. It is also known that tissue hypoxia in plaque lesion is not a consequence of increased plaque burden but a consequence of HIF-1α signaling-mediated M1 macrophage activation [72, 77].

#### **3.7. Congenital heart diseases (CHD): role of spatially differential hypoxia**

CHDs are the major inborn abnormality with major role of environmental factors. The role of non-physiological hypoxia during early pregnancy also induces CHD. Reports are there to show that cells in the mouse heart tube are hypoxic while cardiac progenitor cells (CPCs) in the secondary heart field are normoxic. This spatial difference in the oxygenation of developing

**Table 1.** Different diseases associated to ROS signaling.

ion uptake by cardiomyocytes along with certain amount of dilation in muscles was the additional factor involved [47–49]. This elevated HIf-1α levels may lead to better cardiomyocyte

In addition to the regulatory role of transcription factors, formation of ROS is another major event occurring under oxygen regulation conditions. ROS participates as a benevolent molecule in cell signaling processes and can induce irreversible cellular damage. Formation of ROS in heart or other tissues may occur by several mechanisms either by xanthine oxidase (XO), NAD(P)H, oxidases, and cytochrome P450, or by auto-oxidation of catecholamines and by uncoupling of NO synthase (NOS) [50–52]. Presence of unpaired e- on NO facilitates its

also induced by cytokine stimulus, growth factors such as angiotensin II (ATII), PDGF and TNF-α [50, 53]. As an adaptive response, production of ROS is counterbalanced by several enzymatic (such as superoxide dismutase (SOD), Catalase, Thioredoxin) and non-enzymatic mechanisms (intracellular oxidants such as vitamins E, C, β-carotene, ubiquinone lipoic acid and glutathione) [54–56]. Deletion of Thioredoxin reductase leads to cardiac abnormalities and even cardiac death, secondary to severe dilated cardiomyopathy [57]. Activation of ROS

Inflammation and hypoxia are integral parts in development of atherosclerosis. Data from recent reports suggest that HIF-1α is involved in the pathogenesis of atherosclerosis. Smooth muscle cells extracted from coronary arteries showed that HIF-1α increased activity was related to increased VEGF expression required for proliferation of smooth muscle cells (SMC) [70]. Moreover, hypoxia also produces HIF-1α dependent increase in macrophage migration inhibitory factor (MIF)-required for escalation of migration increased proliferation of vascular SMCs during progression of atherosclerosis [71]. In developing atherosclerosis chronic inflammation and various types of cells (SMC, EC monocytes/macrophages, and T lymphocytes) are involved in plaque formation [72]. Even, the oxygen supply from the luminal blood strongly affects the cells of blood vessel wall [73]. In developed atherosclerosis, tissue hypoxia occurs at the plaque

lesion and HIF-1α expression occurs at the macrophage rich center of plaque [72, 74].

**3.7. Congenital heart diseases (CHD): role of spatially differential hypoxia**

HIF-1α also upregulates the expression of low density lipoprotein receptor related protein-1 (LRP1) associated with cholesterol independent progression of atherosclerosis [75]. In fact, bone marrow transplantation of muscle specific HIF-1α deficient mice reduced the plaque burden in aorta of Ldlr−/− mice. Furthermore, expression of inflammatory genes (M1 macrophage accumulation) was also suppressed in HIF-1α deficient mice [76]. It is also known that tissue hypoxia in plaque lesion is not a consequence of increased plaque burden but a consequence of HIF-1α signaling-mediated M1 macrophage activation [72, 77].

CHDs are the major inborn abnormality with major role of environmental factors. The role of non-physiological hypoxia during early pregnancy also induces CHD. Reports are there to show that cells in the mouse heart tube are hypoxic while cardiac progenitor cells (CPCs) in the secondary heart field are normoxic. This spatial difference in the oxygenation of developing

occurs in response to various stressors and in failing heart as well (**Table 1**).

**3.6. Hypoxia-mediated inflammation in atherosclerosis**

survival under hypoxia.

44 Hypoxia and Anoxia

reaction with O2

heart serves as a signal to regulate the expansion of CPC and cardiac morphogenesis. The response is also mediated by HIF-1α, where HIF-1α forms a complex with notch effector HES family bHLH transcription factor 1 (HES 1) and protein deacetylase sirtuin1 (SIRT1) at the ISL1 gene (islet gene) where ISL1 repression occurs in hypoxic heart tube or as a response to ectopic hypoxic response and prevents CHDs. Thus this is an example where spatial difference in physiological hypoxia maintains the homeostasis for CPCs and provides mechanistic explanation for non- congenital CHDs [78].

#### **3.8. Pulmonary arterial hypertension (PAH)**

PAH is clinically manifested as elevated BP in pulmonary artery with resulting right ventricular heart failure [79, 80]. Hypoxia is known to elicit pulmonary vasoconstriction and arterial remodeling [81, 82]. Hypoxic exposures are the commonly used murine models of PAH. Recently, pulmonary endothelial specific HIF-2α deficient mice showed tolerance to hypoxia-induced PAH as compared to HIF-1α deficient or control mice [83]. As a molecular mechanism HIF-2α regulates NO production in pulmonary vasculature via induction of arginase-1, thereby indicating that HIF-2α-Arginase-1 axes may be used as a therapeutic target to improve NO availability in PAH [83].

#### **3.9. High-altitude hypoxia and thrombosis**

An imbalance between tissue demand and actual oxygen supply also develops due to environmental hypoxia or reduced oxygen content in ambient air [4]. Such episodes are commonly found on exposure to high altitude, mountain climbing or while traveling through air travel, (commercial flights). In commercial flights, when cabin pressure reduces and becomes equivalent to an altitude of 1.5–2.5 km hypoxia is often followed by reoxygenation in majority of such cases, serves as an exacerbating factor for thrombus development in veins [84, 85]. In an earlier study, such observations have been recorded with simulated mouse models where hypoxiareoxygenation is known to promote thrombosis in mouse model of DVT thereby validating incidence of DVT under H/R conditions. The mechanistic explanation given is that hypoxia promotes the secretion of Weibel-Palade bodies, thereby initiating thrombosis in stenosis model [5]. In addition, a recent report also elucidated the possible early factors for hypoxia-induced venous thrombosis; however, in these cases, animals were exposed to hypoxia/normoxia postthrombus induction (by ligation method) and the study has reported the role of novel regulators NLRP3-Caspase-1-IL-1β signaling axis under the transcriptional regulation of HIF-1. Using the pre-clinical rat model for hypoxia-induced thrombosis, the investigators have clearly demonstrated that under hypoxic environments (as found at high altitude), NLRP3-Caspase-1- IL-1α signaling axis could serve as therapeutic target to prevent thrombogenesis under hypoxic settings. Nonetheless, the translational potential of these pre-clinical observations were also made evident in patients of altitude induce venous thrombosis obtained from army soldiers posted at regions of High altitude [33]. In another parallel study, aimed to investigate the role of hypoxia-induced platelet hyper-reactivity, platelet specific novel regulator protein 'calpain' was found to be involved in promoting prothrombotic tendency on ascension to high altitude [86]. A genome wide expression analysis of genes in patients of high altitude-induced venous thrombosis revealed that the progression of venous thrombus formation is attributed to the differential expression of hypoxia responsive genes in response to environmental hypoxia [87].

In the cell types of pulmonary artery, adventitial fibroblasts proliferation occurs under reduced

interesting effects on mTORC1 pathway. In experimental models of ischemia/reperfusion cells showed failure in response with mTORC1 inhibition. Additionally, reperfusion also resulted in increased mTORC1 signaling with increased P70S6K and 4E-BP1 phosphorylation. mTORC1 signaling during ischemia imparts/contributes to withstand the associated stresses

Activation of hypoxia-induced signaling mechanisms form an integral component in development of widely known CVDs (**Figure 1**). These mechanisms are activated as an adaptive response toward hypoxia, and involve a coordinated action of Transcription

**Figure 1.** Commonly known CVDs with pathophysiology as a function of hypoxia signaling mechanisms.

 conditions [93]. Signaling pathways include MAPK, PKC. In fact, hypoxic exposure leads to mTORC1 activation as in aortic SMC [92]. Along with mTORC1 activation, P70S6K activity and 4E-BP1 phosphorylation increase, thus affecting the rate of protein synthesis changing

followed by O2

availability that produce

Hypoxia Signaling in Cardiovascular Diseases http://dx.doi.org/10.5772/intechopen.80456 47

O2

with hypoxic gradient [94].

**5. Conclusion**

Ischemia is characterized by exposure of cells to O2

and help in recovery following ischemic insult [95].

#### **4. HIF-1–independent responses**

These responses become functional to promote ATP conservation by limiting energy consuming processes such as ribosome biogenesis ion channel activity. Such types of responses include mTORC1 & UPR pathways-mediated regulation of mRNA translation [88]. Responses include inhibition of protein synthesis by affecting the assembly of active eukaryotic initiation factor (eIF) 4F & eIF2-GTP-met-tRNA ternary complex. mTOR is a highly conserved serine/threonine kinase which integrates environmental stimuli to regulate metabolism, translation & structural organization in cell in response to growth factors and O2 availability [89, 90].

mTOR occurs in two distinct complexes. mTORC1 (comprising of raptor and GBL/mLST8) and mTORC2 (raptor and GBL/mLST8). mTORC1 plays a role in ribosome biogenesis, mRNA translation, and nutrient import. mTORC2 regulates Akt catalysis and actin organization. Hypoxia regulates the translational activity via involvement of mTORC1 and C2-mediated action. The activity of mTORC1 is regulated by different types of kinases (upstream such as PI3K/Akt/MAPK) by phosphorylation of tuberous sclerosis complex (TSC) [90].

#### **4.1. Biological manifestation of mTORC1 pathways in cardiovascular cells**

Cells of cardiovascular system respond to hypoxic environments by exhibiting increased growth and program of vascular remodeling operating in tissues and cells (SMC, EC, fibroblasts), involving signaling pathways (mTORC1 mediated and its downstream targets) [91, 92].

In the cell types of pulmonary artery, adventitial fibroblasts proliferation occurs under reduced O2 conditions [93]. Signaling pathways include MAPK, PKC. In fact, hypoxic exposure leads to mTORC1 activation as in aortic SMC [92]. Along with mTORC1 activation, P70S6K activity and 4E-BP1 phosphorylation increase, thus affecting the rate of protein synthesis changing with hypoxic gradient [94].

Ischemia is characterized by exposure of cells to O2 followed by O2 availability that produce interesting effects on mTORC1 pathway. In experimental models of ischemia/reperfusion cells showed failure in response with mTORC1 inhibition. Additionally, reperfusion also resulted in increased mTORC1 signaling with increased P70S6K and 4E-BP1 phosphorylation. mTORC1 signaling during ischemia imparts/contributes to withstand the associated stresses and help in recovery following ischemic insult [95].

#### **5. Conclusion**

found on exposure to high altitude, mountain climbing or while traveling through air travel, (commercial flights). In commercial flights, when cabin pressure reduces and becomes equivalent to an altitude of 1.5–2.5 km hypoxia is often followed by reoxygenation in majority of such cases, serves as an exacerbating factor for thrombus development in veins [84, 85]. In an earlier study, such observations have been recorded with simulated mouse models where hypoxiareoxygenation is known to promote thrombosis in mouse model of DVT thereby validating incidence of DVT under H/R conditions. The mechanistic explanation given is that hypoxia promotes the secretion of Weibel-Palade bodies, thereby initiating thrombosis in stenosis model [5]. In addition, a recent report also elucidated the possible early factors for hypoxia-induced venous thrombosis; however, in these cases, animals were exposed to hypoxia/normoxia postthrombus induction (by ligation method) and the study has reported the role of novel regulators NLRP3-Caspase-1-IL-1β signaling axis under the transcriptional regulation of HIF-1. Using the pre-clinical rat model for hypoxia-induced thrombosis, the investigators have clearly demonstrated that under hypoxic environments (as found at high altitude), NLRP3-Caspase-1- IL-1α signaling axis could serve as therapeutic target to prevent thrombogenesis under hypoxic settings. Nonetheless, the translational potential of these pre-clinical observations were also made evident in patients of altitude induce venous thrombosis obtained from army soldiers posted at regions of High altitude [33]. In another parallel study, aimed to investigate the role of hypoxia-induced platelet hyper-reactivity, platelet specific novel regulator protein 'calpain' was found to be involved in promoting prothrombotic tendency on ascension to high altitude [86]. A genome wide expression analysis of genes in patients of high altitude-induced venous thrombosis revealed that the progression of venous thrombus formation is attributed to the differential expression of hypoxia responsive genes in response to environmental hypoxia [87].

These responses become functional to promote ATP conservation by limiting energy consuming processes such as ribosome biogenesis ion channel activity. Such types of responses include mTORC1 & UPR pathways-mediated regulation of mRNA translation [88]. Responses include inhibition of protein synthesis by affecting the assembly of active eukaryotic initiation factor (eIF) 4F & eIF2-GTP-met-tRNA ternary complex. mTOR is a highly conserved serine/threonine kinase which integrates environmental stimuli to regulate metabolism, translation & structural

mTOR occurs in two distinct complexes. mTORC1 (comprising of raptor and GBL/mLST8) and mTORC2 (raptor and GBL/mLST8). mTORC1 plays a role in ribosome biogenesis, mRNA translation, and nutrient import. mTORC2 regulates Akt catalysis and actin organization. Hypoxia regulates the translational activity via involvement of mTORC1 and C2-mediated action. The activity of mTORC1 is regulated by different types of kinases (upstream such as

Cells of cardiovascular system respond to hypoxic environments by exhibiting increased growth and program of vascular remodeling operating in tissues and cells (SMC, EC, fibroblasts), involving signaling pathways (mTORC1 mediated and its downstream targets) [91, 92].

PI3K/Akt/MAPK) by phosphorylation of tuberous sclerosis complex (TSC) [90].

**4.1. Biological manifestation of mTORC1 pathways in cardiovascular cells**

availability [89, 90].

**4. HIF-1–independent responses**

46 Hypoxia and Anoxia

organization in cell in response to growth factors and O2

Activation of hypoxia-induced signaling mechanisms form an integral component in development of widely known CVDs (**Figure 1**). These mechanisms are activated as an adaptive response toward hypoxia, and involve a coordinated action of Transcription

**Figure 1.** Commonly known CVDs with pathophysiology as a function of hypoxia signaling mechanisms.

factors (HIF-1, NF-kβ), reactive oxygen species and downstream effector molecules, which can serve as therapeutic targets to control the development of the related disease.

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#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Neha Gupta1 \* and Mohammad Zahid Ashraf2 \*

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
1 Department of Biosciences, Jamia Millia Islamia, New Delhi, India

2 Department of Biotechnology, Jamia Millia Islamia, New Delhi, India

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**Conflict of interest**

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Neha Gupta1

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