*2.1.4. mPTP opening*

the sarcoplasmic reticulum (SR), which binds to troponin C, and allows for the myosin and actin filaments to interact [10]. During diastole, the Ca2+ either goes back into the SR or is

by β-adrenergic stimulation, increases the number of Ca2+ transients as well as the size of the transients, leading to stronger cardiac contractions [14]. Additionally, mitochondria can also indirectly contribute to Ca2+ regulation by inducing changes in the concentration of ATP, NAD(P)H, pyruvate, and ROS, which in turn regulate other Ca2+ signaling machinery components [15]. This associated Ca2+ signaling is involved in the Ca2+ buffering, the Ca2+ release from internal stores and the influx from the extracellular solution, the Ca2+ uptake into cellular

Calcium signaling in the mitochondria also contributes to the regulation of cellular energy metabolism. ATP is hydrolyzed to ADP in order to power energy-requiring processes and is shuttled into the mitochondria to be reconverted into ATP as a final step in respiration. This enhances the

is transferred into the mitochondria through the mitochondrial Ca2+ uniporter (MCU), activating the enzymes of the Krebs cycle to adjust NADH regeneration to match its oxidation [14]. In addition, excessive mitochondrial Ca2+ uptake and Ca2+ accumulation, irreversible ΔΨ collapse,

Type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s) are Ca2+ release channels found on cardiac SR. Recent studies have demonstrated that leaky RyR2 channels, but not IP3R2, contribute to mitochondrial Ca2+ overload and dysfunction in heart failure (HF) [11]. NO signaling and its downstream effectors such as *S*-nitrosylation have also been shown to be key processes in regulating calcium signaling. The neuronal nitric oxide synthase (nNOS or NOS1) has been linked to the reduction of calcium influx through the L-type Ca2+ channel [5, 18]. This decrease in Ca2+ influx may be responsible for the cardioprotection induced by NO. Furthermore, decreased *S*-nitrosylation of key SR Ca2+ handling proteins such as the RyR2s due to impaired NOS1 can result in increased Ca2+-

*S*-nitrosylation of the SR Ca2+ ATPase (SERCA) has been associated with lower Ca2+ uptake in

While substantial efforts were undertaken to characterize the kinetic properties of mitochondrial calcium cycling, the experimental approaches and techniques have not been able to reach explicit conclusions on cardiac mitochondrial responses to cytosolic Ca2+ oscillations during each heartbeat. However, it is widely accepted that Ca2+ is a second messenger for the regulation of mitochondrial tasks and represents a crucial link for the role of mitochondria for

Mitochondria are also a large cellular source of ROS. ROS includes the superoxide anion radi-

enzymatic and nonenzymatic mechanisms. The most abundant form of ROS in the body is O2

OH), as well as nonradical oxidants, such as hydrogen per-

O2

) [21]. They can be converted from one to the other by

organelles, and the extrusion by plasma membrane Ca2+ pumps [16].

electron flux within the ETC, resulting in the oxidation of NADH to NAD<sup>+</sup>

ATP depletion, and oxidative stress contribute to the opening of the mPTP [17].

mediated ventricular arrhythmia in the setting of elevated myocardia [Ca2+]

excitation-metabolism and excitation-contraction coupling in the heart.

O2

the SR and impaired myocardial relaxation [20].

*2.1.3. Generation of ROS*

O2

·−) and hydroxyl radical (·

) and singlet oxygen (1

which is enzymatically or spontaneously dismutated to H2

cal (O2

oxide (H2

/Ca2+ exchanger [13]. An increase in workload, as triggered

. Concurrently, Ca2+

i

. In the human body, there are

[19]. Inhibition of

·−,

exported out of the cell via the Na+

34 Mitochondrial Diseases

Mitochondria can mediate cell death through the opening or activation of the mPTP [25]. The mPTP is a high conductance channel that generates a sudden increase in inner mitochondrial membrane (IMM) permeability to ions and small solutes when opened [26, 27]. The pore is regulated by the concentration of Ca2+, ADP, NADH, and ROS. Regulation of the mPTP opening is a key essential mechanism for cardiomyocyte survival and function.

Intense research efforts have been focused on elucidating the molecular components of the mPTP. The original mPTP model hypothesized that the channel comprised these principal proteins: cyclophilin D (CyPD), located in the mitochondrial matrix; the ANT, found in the inner membrane; the voltage-dependent anion channel (VDAC) in the outer membrane [28]; and other interacting mitochondrial molecules such as the phosphate carrier, BH3 proteins, and p53 [29]. However, genetic ablation of the proposed components revealed that only the deletion of the CyPD gene resulted in impaired opening of the mPTP, suggesting that the other proposed components are not a necessary part of the pore [30, 31].

One of the major changes during the cardiac development is the use of energy fuels to generate ATP in cardiomyocyte. In the fetal heart, glucose and lactate are the predominant substrates used in the generation of ATP [43, 44]. The fetal heart boasts of a large endogenous glycogen supply, which is a significant source of the glucose on which the heart relies. Glycogenolysis is also particularly important in conditions of oxygen deprivation, allowing the fetal heart to resist the effects of hypoxia and ischemia better than the adult heart [43]. Fetal hearts have less mitochondria and therefore lower levels of respiratory and TCA cycle activities [2]. Notably, circulating levels of fatty acids are low, reducing the role of FAO in the generation of ATP. FAO is further inhibited by the high lactate levels present in the fetal heart [2]. Postnatally, an important switch occurs as fatty acids replace glucose and lactate as the primary energy substrates in the developing heart [43]. Consequently, the activity of the proteins of the carnitine shuttle, particularly the M isoform of the mitochondrial carnitine palmitoyltransferase I (CPT I) and mitochondrial carnitine palmitoyltransferase II (CPT II), is markedly increased during the early postnatal period [45]. Other key proteins that have been associated with the uptake of fatty acids into cardiac muscle cells also exhibit increased mRNA expression during matu-

Mitochondria and Heart Disease

37

http://dx.doi.org/10.5772/intechopen.72611

ration of the heart, reflecting increased fatty acid uptake and metabolism [46].

as mitoCK allows maximal activation of the processes of OXPHOS [47].

**2.3. Cardiac mitochondria during aging**

activities, may occur with aging.

mitochondria and cellular signaling are changed.

In addition, there is a change in the transfer and use of the energy currency in the mitochondria during the cardiac development. MitoCK is responsible for the production of high-energy phosphates in adult heart. In the fetal heart, mitoCK levels are undetectable, with its expression starting between weeks 1 and 2 in the Wistar rat pup and rising to adult levels after 6 weeks [47]. MitoCK expression was associated with creatine-activated respiration and the affinity of OXPHOS to ADP. Importantly, there is a change in the organization of the cardiac mitochondria from a random arrangement from day 1 in a rat to a fine network of myofibrils by week 3,

Aging is a major risk factor for cardiovascular diseases. During aging, mitochondrial oxidative stress responses, mitochondrial damage, and biogenesis as well as the cross-talk between

Aging may induce changes to the shape and size of mitochondria in the heart [48]. In aged mice, mitochondria appeared more rounded and less spherical [49]. It was further noted that aged mitochondria exhibit a lower total area of inner membrane per mitochondria, suggesting a reduced capacity for OXPHOS [50]. Reciprocally, increased levels of large-scale deletions and point mutations in cardiac mtDNA, as well as reduced levels of mitochondrial enzymatic

Additionally, the multiple metabolic changes that occur in cardiac muscle with advancing age include increasing levels of saturated fatty acids and reduced levels of polyunsaturated fatty acids and cardiolipin [51]. Cardiolipin is a key cellular phospholipid and an important constituent of the mitochondrial inner membrane. Reduced cardiolipin influences cardiac mitochondrial membrane transport function, fluidity, and stability of the membrane and facilitates optimal energy generation [51]. Significant reduction in carnitine and acetyl carnitine levels has also been reported in older subjects, suggesting lowered ability to transfer fatty acids into the mitochondria to be metabolized [52]. In addition, the effect of aging on cardiac OXPHOS

Recent studies indicated that the ATP synthase is a major component of the mPTP [32]. There are two working proposals about the mechanism for ATP synthase in the mPTP formation. The first one suggests that the pore forms at the interface of two dimers of ATP synthase [33]. It has been showed that the current that was observed from reconstituted lipid bilayers with purified dimers of the ATP synthase was electrophysiologically equivalent to that of the mPTP. Additionally, genetic ablation of two specific subunits of the F0 subcomplex that are necessary for dimerization did not result in opening of the pore, which further underscores the importance of dimerization for the formation of the mPTP [34]. The second hypothesis focuses on the c-subunit ring of the F0 subcomplex [35]. In purified ATP synthase extracts in yeast, the ring structure produced by the c-subunits exhibited a current that was equivalent to that of the mPTP [34], and the currents were inhibited by regulators of the mPTP, suggesting that the ring and the mPTP are the same. While debate continues about the precise components and mechanism of the mPTP, its importance in physiology and pathology is clear and its regulation is paramount to cell survival.

While a short-term opening of the mPTP appears to act as a normal calcium-release mechanism that is required for proper metabolic regulation [29, 36–38], irreversible formation and consequent opening of the mPTP are key factors in mitochondrial dysfunction and mitochondria-driven cell death [32, 39, 40]. When the mitochondria are exposed to high concentrations of calcium, they undergo a massive and permanent swelling that leads to an abrupt increase in permeability to small solutes of the IMM, abolishing the chemiosmotic gradient across the IMM [29], which subsequently uncouples OXPHOS, leading to a decrease in ATP production and an increase in ROS formation [25]. Further rupture of the outer mitochondrial membrane results in the extrusion of cytochrome c, a key step in the initiation of apoptosis [41]. The mPTP may also play a role in the regulation of energy production due to the dual role of the ATP synthase in both ATP production and mPTP formation [25].

Interestingly, small increases in *O*-GlcNAcylation were correlated with improved ability of cardiac mitochondria to sequester Ca2+ as well as resistance to mPTP opening. Key regulatory proteins in the mPTP, the VDAC and ANT, were also found to be able to be *O*-GlcNAcylated. The ATP synthase, the key molecule that forms the mPTP, has also been shown to be able to be *O*-GlcNAcylated [6]. Since mPTP opening is influenced by the loss of the mitochondrial potential as well as calcium overload, any change in mitochondrial potential or calcium dynamics may have adverse effects in the mitochondria. Key calcium signaling participants of mPTP regulation include the pore of the outer membrane, VDAC, the pore of the IMM calcium uniporter, and a key regulator of the mPTP, cyclophilin D [42]. Our most recent study also showed that overexpression of VCP protects against stress-induced mPTP opening in cardiomyocyte through an iNOS-dependent mechanism [9].
