Because mitochondrial bioenergetics has been extensibly discussed in Chap. 5, it suffices to point out here that energy production by the organelle is depending on both nuclear- and mtDNA-encoded genetic factors—which modulate normal mitochondrial function, including enzyme activity and cofactor availability—and on environmental factors such as substrate availability of sugars, fats, proteins, and oxygen. Several interacting bioenergetic pathways contribute to mitochondrial energy metabolism including pyruvate oxidation, the tricarboxylic acid (TCA) cycle, fatty acid β-oxidation (FAO), and the common final pathway of oxidative phosphorylation (OXPHOS), which generates most of the cellular ATP. OXPHOS is performed by complexes of proteins located at the mitochondrial inner membrane [1] including the electron transport chain (ETC)/respiratory complexes I–IV, the ATP synthase (complex V), and the adenine nucleotide translocator (ANT) Fig. 8.1. In order to be effectively utilized for bioenergetic production via mitochondrial FAO, fatty acids need to be transported into the cell and subsequently into the mitochondria, a process requiring several transport proteins including the carnitine shuttle (composed of carnitine acyltransferase and two carnitine palmitoyl transferases as well as carnitine). FAO and carbohydrate oxidation, via the TCA cycle, generates the majority of intramitochondrial NADH and FADH2, the direct source of electrons for the ETC.

Since mitochondrial biogenesis has been comprehensively discussed in Chap. 3, here it will suffice to mention that organelle biogenesis increases in specific cell types (e.g., cardiac and skeletal muscle) during cell hypertrophy and treatment with a variety of agents, such as thyroid hormone (TH), exercise, glucocorticoids, xenobiotics, and electrical stimulation [2–8]. While it is widely accepted that mtDNA copy number is highly regulated within a given cell or tissue type [9], the mechanisms that control specific mtDNA levels and overall mitochondrial copy number have not been established. On the other hand, it is well known that the nuclear genome encodes the full complement of proteins involved in mtDNA replication and transcription, the protein components of mitochondrial ribosomes, the multiple structural and transport proteins of the mitochondrial membranes, the remaining peptide subunits of the respiratory complexes (other than the 13 mtDNA-encoded peptide subunits), and the mitochondrial enzymes involved in mitochondrial lipid metabolism and the TCA cycle [10]. These nuclear-encoded proteins, synthesized on cytosolic ribosomes, are targeted to mitochondria and translocated to the organelle by an elaborated process, which involves signal peptide recognition, membrane receptors, proteases, and an arrangement of molecular chaperones [11] Fig. 8.1. Regulation of many of the nuclear-encoded OXPHOS proteins is mediated by changing gene expression, which is affected by an array of physiological (e.g., hypoxia) and developmental stimuli [12]. Tissue-specific isoforms for specific peptides (e.g., cardiac/skeletal muscle-specific isoforms exist for genes encoding cytochrome c oxidase subunits VIa, VIIa, and VIII) show entirely different patterns of gene expression in adult compared to the fetal stages of development [12, 13].

Since a comprehensive discussion on the role of ROS in cell signaling, including their positive and negative effects, has been presented in Chap. 10, here it will suffice to note that ROS are critical by-products of mitochondrial bioenergetic activity and that side reactions of mitochondrial ETC enzymes with oxygen directly generate superoxide anion radical. Significantly, the primary sites for mitochondrial ROS generation, as a by-product of normal metabolism, are at complex I, II, and III of the respiratory chain, and either excessive or diminished electron flux at these sites can favor the auto-oxidation of flavins and quinones (including coenzyme Q) producing superoxide radicals [14].

Whereas a large number of receptors have been identified in different tissues and cell types, few well-characterized heart mitochondrial receptors have been hitherto detected. Interestingly, the thyroid hormone (TH) receptor Erb-Aa, which has been identified as an “orphan” receptor interacting with mtDNA during targeted hormonal stimulation (Fig. 8.1) [15], heretofore has not been found in cardiac tissue, notwithstanding the significant effect of TH in heart mitochondria. Moreover, a large number of nuclear transcription factors including p53, NF-κB, PPAR-α, RXR-a, and TR3 (an “orphan receptor” of the steroid-thyroid hormone-retinoid receptor superfamily of transcription factors), which have been characterized in many tissues/cell types as translocating to mitochondria, hitherto have not been found in heart mitochondria.

Although TNF-α can be induced in cardiomyocyte mitochondria [16], no specific mitochondrial receptor binding TNF-α or to various cytokines known to effect cardiac mitochondrial function have been found. Also, in contrast with other noncardiac tissues, characterization in heart mitochondria of anchoring proteins that bind and concentrate protein kinases has been rather limited. Further research on mitochondrial receptors and on the proteins associated with ­apoptosis and mitochondrial removal may increase our understanding of the role that these receptors and mitophagy play in normal physiological processes and in cardiac diseases [17]. For a comprehensive discussion on mitophagy, see Chap. 10.

A unifying concept regarding mitochondrial signaling is the stimuli-generated translocation of specific cytosolic proteins into the organelle; the increasing list of translocated proteins includes many of the proapoptotic proteins (e.g., Bax, Bid) as well as protein kinases. Many of these kinases appear to target specific proteins on the outer mitochondria membrane, and others, imported as preproteins, are recognized by a small set of specific receptors (so-called translocases) on the mitochondrial outer membrane (TOM). The import of proteins into mitochondria is often mediated by heat shock proteins (e.g., HSP60 and HSP70), which specifically interact with a complex mitochondrial protein import apparatus, which includes matrix proteases. Physiological stimuli and stresses, including temperature changes and hormone treatment (e.g., thyroid hormone [TH]), may modulate the cardiac mitochondrial import apparatus pathways [18–20].

The number of proteins translocated from cytosol to the mitochondria, which include several of the proapoptotic ­proteins (e.g., Bax, Bid) and some protein kinases, seems to be increasing.

Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus that impacts many cellular activities under both normal and pathophysiological conditions. In both yeast and animal cells, retrograde signaling is linked to the mammalian (or mechanistic) target of rapamycin (mTOR) signaling, but its precise connections in cardiomyocytes have not yet been established. It has been suggested that inhibition of mTOR by rapamycin suppresses myocardial hypertrophy although its mechanism has not been found [21]. Song et al. [22] have recently studied the effect of mTOR on pathological hypertrophy and whether cardiac mTOR regulates the inflammatory response. Transgenic mice with cardiac-specific overexpression of wild-type mTOR (mTOR-Tg) were used. These mice were protected against the cardiac dysfunction induced by left ventricular pressure overload—aortic constriction (AC)—and showed lower levels of interstitial fibrosis than littermate controls (WT) at 4-week post-AC. In contrast, AC caused cardiac dysfunction in the WT. At 1-week post-AC, the proinflammatory cytokines interleukin (IL)-1β and IL-6 were significantly increased in WT mice but not in mTOR-Tg mice. This study further characterized the effects of mTOR activation, by exposing HL-1 cardiomyocytes transfected with mTOR to lipopolysaccharide (LPS). mTOR overexpression suppressed LPS-induced secretion of IL-6, and mTOR inhibitors rapamycin and PP242 abolished this inhibitory effect. Furthermore, mTOR overexpression reduced NF-κB-regulated transcription in HL-1 cells. Taken together, these findings suggest that mTOR protects against pressure-overload changes and its cardioprotective effect appears to be mediated by regulation of the inflammatory reaction [22]. The mTOR plays a critical role in the regulation of cell growth and in the response to energy state changes. Zhang et al. [23] have shown that ablation of Mtor in the adult mouse myocardium results in fatal, dilated cardiomyopathy (DCM) that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an mTOR-containing multiprotein complex-1 (mTORC1) substrate that inhibits translation initiation. Mtor-ablated mice subjected to pressure overload showed impaired hypertrophic response and accelerated progression of HF. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvement was observed in apoptosis, heart function, and survival. These data demonstrate a role for the mTORC1 signaling network in myocardial response to stress and in particular highlight the role of 4E-BP1 in the regulation of cardiomyocyte viability and HF. Because the effects of reduced mTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy to improve cardiac function in HF.

In mammalian cells, mitochondrial dysfunction sets off signaling cascades through altered Ca²⁺ dynamics including calcineurin activation, which activate several protein kinase pathways (e.g., PKC and MAPK) and transcription factors such as NF-κB, calcineurin-dependent NFAT, CREB, and ATF leading to stress protein expression (e.g., chaperone proteins) and activities [24]. This can result in both changes in cell morphology and phenotype including proliferative growth, apoptotic signaling, and glucose metabolism. Thus, calcium appears to be an important link between the relay of extracellular signals to the nucleus and the bidirectional communication between nucleus and mitochondria. Furthermore, this concept is strengthened by the observation that CREB is found in its transcriptionally active, ­phosphorylated state in cells exhibiting mitochondrial ­dysfunction resulting either from mtDNA loss or by pathological mutation in mtDNA [25], and this seems to happen simultaneously with activation of CaMKIV, leading to CREB phosphorylation in response to calcium. Nevertheless, whether calcium signaling through CREB, and the PGC-1 family coactivators, represents a physiologically significant pathway of retrograde regulation in vertebrate cells is not known [26].

The endoplasmic reticulum (ER) is a multifunctional signaling organelle that contributes to the regulation of cellular processes such as the entry and release of Ca²⁺, sterol biosynthesis, apoptosis, and release of arachidonic acid [27]. One of its primary roles is to function as a source of the Ca²⁺ signals that are released through either IP3 or ryanodine receptors (RyRs), which are themselves Ca²⁺-sensitive. Another significant function of the cardiomyocyte ER is to regulate apoptosis by operating in tandem with mitochondria. In cells undergoing activation or excitation, calcium is released from the endoplasmic/sarcoplasmic reticulum to activate calcium-dependent kinases and phosphatases, thereby regulating numerous cellular processes, for example, apoptosis and autophagy [28]. Antiapoptotic regulators of apoptosis such as Bcl-2 may act by reducing the ebb and flow of Ca²⁺ through ER/mitochondrial cross talk [29]. Because both organelles are in close proximity, calcium is rapidly taken up by mitochondria via the calcium uniporter on the inner mitochondrial membrane [30–32]. Bcl-2 and Bcl-xL inhibit calcium redistribution to the mitochondria, thereby limiting calcium uptake [29, 33]. Also, it has been suggested that Bcl-2 inhibits calcium release through L-type channels, thereby preventing mitochondrial calcium uptake [34].

The capability of the ER in spreading signals throughout the cell, mediated by a process of Ca²⁺-induced Ca²⁺ release, is particularly important in the control of cardiomyocyte function. The role of ER as an internal reservoir of Ca²⁺ is coordinated with its role in protein synthesis since a constant luminal level of Ca²⁺ is essential for protein folding. In order to achieve this regulation, the ER also contains several stress signaling pathways that can activate transcriptional cascades to regulate the luminal content of the Ca²⁺-dependent chaperones responsible for the folding and packaging of secretory proteins.

Nuclear-mitochondrial interactions depend on the interplay between numerous transcription factors (NRF-1, NRF-2, PPARα, ERRα, Sp1, and others) and members of the PGC-1 family of regulated coactivators (PGC-1α, PGC-1β, and PRC) [26]. These nuclear transcription modulators govern the expression of a wide array of mitochondrial proteins in response to diverse cellular stimuli and signals. For instance, nuclear transcription factors such as nuclear respiratory factors (NRFs) 1 and 2 are implicated in the activation of mitochondrial biogenesis [35, 36, 38]. These factors exert a direct effect on the synthesis of specific nuclear-encoded subunits of the mitochondrial respiratory enzymes as well as indirectly by upregulating levels of TFAM, involved in both mtDNA replication and transcription (see Fig. 8.1). In addition, a master transcription coactivator (PGC-1) activates directly or indirectly the expression of transcription factors NRF1 and NRF2 [37]. These transcriptional models serve as a framework to understand the integration of cell-signaling events with mitochondrial biogenesis and function [26].

The nucleus also contains global regulatory transcription factors such as PPARs and their transcriptional coactivators (i.e., PPAR, RXR) which also play a pivotal regulatory role in the expression of mitochondrial protein components of FAO pathway, integral to bioenergetic metabolism [38–40]. Transcriptional regulation by these activators is affected by hypoxia, ischemia, and HF [41, 42]. Expression of PGC-1 and mitochondrial biogenesis are modulated by activation of a calcium-/calmodulin-dependent protein kinase, indicating that the calcium-regulated signaling pathway plays a critical role in transcriptional activation of genes responsible for mitochondrial biogenesis [43]. Furthermore, these nuclear activators are modulated during cardiac development [44, 45]; activation of the nuclear-factor-activated T cell gene has been shown to be crucial in early cardiac development and is required to maintain myocardial mitochondrial oxidative function. Targeted cardiac gene disruption of nuclear-factor-activated T cell genes results in cardiomyocyte mitochondria ETC dysfunction, reduced ventricular size, and aberrant cardiomyocyte structure in mice embryo [45]. These nuclear transcriptional factors modulate the cardiac phenotype in transgenic animals exhibiting either mutations in specific transcriptional factors or overexpressed genes [37–45]. Also, it has been shown that specific nuclear transcription factors are essential for normal cardiac phenotype and mitochondrial function. For instance, cardiac-specific PGC-1 overexpression in transgenic mice results in uncontrolled mitochondrial proliferation and extensive loss of sarcomeric structure leading to dilated cardiomyopathy (DCM) [37]. Myocardial overexpression of PPAR may lead to severe cardiomyopathy with both increased myocardial fatty acid uptake and mitochondrial FAO [46]. In a similar manner, mutations targeting TFAM produce inactivation of myocardial mitochondrial gene expression and ETC dysfunction resulting in DCM and atrioventricular conduction defects [47].

Indications that mitochondria contain multiple phosphoprotein substrates for protein kinases and that a number of protein kinases are translocated into heart mitochondria strongly suggest that protein phosphorylation within the mitochondria is a critical element of mitochondrial signaling pathways [48]. However, it is important to note that detection of a protein in a phosphorylated state does not mean that such phosphorylation plays a regulatory role. Many proteins can be phosphorylated in vitro by protein kinases yet show no changes in activity. Up to this point, protein kinases identified in heart mitochondria include PDH kinase, branched-chain keto acid dehydrogenase kinase, PKA, PKC isoforms, and c-Jun N-terminal kinase [49–54], and their characterization has provided new insights into the fundamental mechanisms regulating mitochondrial responses to diverse physiological stimuli and stresses.

In cardiomyocytes, PKC after translocation to the mitochondria forms a signaling module by complexing with specific mitogen-activated protein kinases (e.g., extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase), resulting in phosphorylation of the proapoptotic protein Bad [53]. Also, PKC forms physical interaction with components of the cardiac mitochondrial permeability transition pore (MPTP) (in particular, VDAC and ANT) [55]. This interaction may inhibit the pathological opening of the pore (including Ca²⁺-induced opening and subsequent mitochondrial swelling) contributing to PKC-induced cardioprotection (CP). While PKC activation in CP likely precedes mitoK_ATP channel opening, its direct interaction with the mitoK_ATP channels has not been demonstrated. Following treatment with diazoxide, PKC is also translocated to cardiac mitochondria [52]; however, several observations have shown that PKC does not play a contributory role in the CP provided by ischemic preconditioning [56, 57].

A mitochondrial PKA (mtPKA) and its protein substrates have been localized to the matrix side of the inner mitochondrial membrane [51]. In cardiomyocytes the NDUFS4 is phosphorylated by mtPKA, and increased levels of cAMP promote NDUFS4 phosphorylation, enhancing both complex I activity and NAD-linked mitochondrial respiration [51, 58, 59]. These posttranslational changes can be reversed by dephosphorylation via a mitochondria-localized phosphatase. Phosphorylation of several subunits of COX, including COXI, III, and Vb, occurs at serine residues by mtPKA, modulates COX activity [60], and has been considered to be a critical element of respiratory control. This cAMP-dependent phosphorylation occurs with high ATP/ADP ratios and results in allosteric inhibition of COX activity; at the same time this regulatory control can result in reduced membrane potential and more efficient energy transduction in the resting state. On the other hand, increases in mitochondrial phosphatase (Ca²⁺-induced) reverse allosteric COX inhibition/respiratory control resulting in increased membrane potential and ROS formation; interestingly, various stress stimuli leading to increased Ca²⁺ flux result in increased potential and mitochondrial ROS formation. Newly developed kinase inhibitor assays and proteomic analysis have allowed identification of a variety of mitochondrial phosphoprotein targets for these kinases. A set of proteins has been found in the mitochondrial phosphoprotein proteome of bovine heart as protein targets of kinase-mediated phosphorylation [61]. The majority of these phosphoproteins were involved in mitochondrial bioenergetic function either in the TCA cycle (e.g., aconitase, isocitrate, and pyruvate dehydrogenase) as respiratory complex subunits (e.g., NDUFA 10 of complex I, succinate dehydrogenase flavoprotein subunit of complex II, core I and core III subunits of complex III, and subunits of complex V) or as essential players in the homeostasis of mitochondrial bioenergetics (e.g., creatine kinase, ANT). Interestingly, phosphorylation of the elongation factor Tu, a key regulatory protein of the cardiac mitochondrial protein translation apparatus, is modulated during myocardial ischemia [62].

The import of Ca²⁺ from cytosol into cardiac mitochondria is an important regulatory event in cell signaling. The organelle couples the cellular metabolic state with Ca²⁺ transport processes not only modulating their own intraorganelle Ca²⁺ but also influencing the entire cellular network of cellular Ca²⁺ signaling, including the ER, the plasma membrane, and the nucleus [63]. These interactions are critical in Ca²⁺ homeostasis and signaling in the cardiomyocyte and in the numerous cellular functions regulated by the cation [64, 65]. Bidirectional signaling mediated by Ca²⁺ provides a framework by which mitochondrial biogenesis, structure, and metabolic activity will dictate a number of adaptive mechanisms during cell proliferation and cellular stress. PGC-1α selectively reduced mitochondrial Ca²⁺ responses to cell stimulation by reducing the efficacy of mitochondrial Ca²⁺ uptake sites and increasing organelle volume. In turn, this affected ER Ca²⁺ release and cytosolic responses in HeLa cells. Above all, the modulation of mitochondrial Ca²⁺ uptake significantly reduced cellular sensitivity to the Ca²⁺-mediated proapoptotic effect of C₂ ceramide [66, 67]. Mitochondrial calcium flux, particularly in cultured cardiomyocytes, has become detectable using advanced cell imaging techniques with fluorescent dyes, confocal microscopy, and recombinantly derived Ca²⁺-sensitive photoprobes [68, 69]. Mitochondrial calcium influx is primarily provided by a Ca²⁺ pump uniporter (see Fig. 8.2) located in the inner membrane and driven by the mitochondrial membrane potential as well as by low matrix Ca²⁺ levels and can be blocked by ruthenium red [70].

Mitochondrial Ca²⁺ uptake is significantly and rapidly elevated in cardiomyocytes during physiological Ca²⁺ signaling and is often accompanied by a highly localized transient mitochondrial depolarization [69]. Efflux of Ca²⁺ from cardiomyocyte mitochondria is mediated by a Na⁺/Ca²⁺ exchanger (NCE) linked to ETC proton pumping, although calcium efflux also occurs with MPTP opening. Activation of the MPTP and mitochondrial Ca²⁺ flux also occur in early myocardial apoptosis and IRI and are involved in the generation of a calcium wave delivering system between adjacent mitochondria [71]. A major consequence of increased mitochondrial Ca²⁺ uptake is the upregulation of energy metabolism and stimulation of mitochondrial OXPHOS. Elevated mitochondrial Ca²⁺ levels allosterically stimulate the activity of three TCA cycle enzymes including pyruvate, isocitrate, and 2-oxoglutarate dehydrogenases [72, 73]. Activation of these enzymes by Ca²⁺ results in increased NADH/NAD⁺ ratios and ultimately leads to increased mitochondrial ATP synthesis. A thermokinetic model of cardiac bioenergetics showed calcium-dependent activation of the dehydrogenases as the rate-limiting determinant of respiratory flux regulating myocardial oxygen consumption, proton efflux, and NADH and ATP synthesis [74]. In cardiomyocytes mitochondrial ATP synthase activity can be directly modulated by increased mitochondrial Ca²⁺ levels [75, 76].

Many cellular processes require cooperation between the plasma membrane, the nucleus, and the subcellular vesicular/tubular networks such as the mitochondria and endoplasmic reticulum. Contacts between these cellular structures are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation, and apoptosis. Contacts between smooth subdomains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure [63]. The small distance between the organelle membranes as measured by electron microscopy allows contact formation by proteins present on their surfaces, permitting and regulating their interactions. Intracompartment Ca²⁺ signaling is recognized as a key mode of signal transduction and amplification in mitochondria [68, 69]. Using product of phosphatidylinositol 4,5-bisphosphate hydrolysis, such as inositol trisphosphate (IP3), as second messengers, a variety of cell-surface hormones and neurotransmitters signal the release of Ca²⁺ from endoplasmic reticulum (ER) and Golgi apparatus to the cytosol. The proximity of mitochondria to ER membranes appears to be a significant factor for ER Ca²⁺ release and mitochondrial Ca²⁺ uptake [77]. This dramatic increase in mitochondrial Ca²⁺ is rapidly mobilized from the ER-IP3 receptor channels (Fig. 8.2) when in close contact to mitochondria, albeit the precise molecular mechanism of this transfer has not been fully established. Similarly, the sarcoplasmic reticulum ryanodine receptors are also located near the cardiomyocyte mitochondria undergoing calcium release [78]. Proposed mechanisms for the rapid calcium mitochondrial import include the involvement of diffusible cytosolic factors that stimulate the Ca²⁺ uniporter, activation of rapid mode of uptake (RaM), and enhanced uptake by mitochondrial analogues of ryanodine receptors residing in the inner membrane [79–82]. A voltage-dependent anion carrier has also been identified as a component in Ca²⁺ transport from ER through the outer mitochondrial membrane [83].

Besides their sarcolemmal location, K_ATP channels present in the inner membrane of mitochondria (mitoK_ATP) Fig. 8.2. These cardioprotective K_ATP channels consist of pore-forming (Kir6.1 and/or Kir6.2) and sulphonylurea-binding modulatory subunits [sulfonylurea receptor (SUR) 1, 2A, or 2B]. While K_ATP channels consisting of Kir6.2/SUR2A subunits present in heart muscle and Kir6.1/SUR2B subunit-containing K_ATP channels present in smooth muscle, the SUR1-containing channels have been found to participate in pancreatic insulin release. However, the SUR1 subunits have also been found recently in mouse cardiac tissue and show protection from myocardial IRI in SUR1-null mice.

The molecular identification and functional characterization of a mitochondrial sulfonylurea receptor 2 splice variant generated by intraexonic have been reported by Ye et al. [84] They found that isolation of transcripts encoding the 55-kDa forms showed a nonconventional intraexonic splicing (IES) event, which occurred in the SUR2 mRNA to generate such variants. Further investigation suggested these modified channels formed by IES variants generated a distinct ATP-sensitive current. This same group of investigators also reported that SUR2 knockout (SUR2KO) male mice are protected against IRI and that a 55-kDa intraexonic splice variant of SUR2A (SUR2A-55), unique to mitochondrial membrane fractions, forms functional K_ATP channels and is more abundant in mitochondria of SUR2KO mice than in wild type. Interestingly, overexpression of SUR2A-55 seems to reduce infarct size and maintains cardiac function with improved maintenance of the mitochondria membrane potential [85].

ATP-sensitive potassium channels of the inner mitochondrial membrane (mitoK_ATP) are blocked by ATP and have been implicated as potential mediators of cardioprotective mechanisms such as IPC [86]. This cardioprotective effect is partially mediated by attenuating Ca²⁺ overloading in the mitochondrial matrix and by increased ROS generation during preconditioning, further leading to protein kinase activation and decreased ROS levels generated during reperfusion [87]. The mitoK_ATP channel is also regulated by a variety of ligands (e.g., adenosine, opioids, bradykinin, acetylcholine), which bind sarcolemmal G protein-coupled receptors, with subsequent activation of calcium flux, tyrosine protein kinases, and phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) pathway [88, 89]. Marked changes in mitochondrial matrix volume associated with mitoK_ATP channel opening may play a contributory role in the CP process [89], although this has been questioned [90]. MitoK_ATP channel opening is specifically activated by drugs such as diazoxide and nicorandil and can also inhibit H₂O₂-induced apoptotic progression in cardiomyocytes, suggesting that mitoK_ATP channels may also play a significant role in mediating OS signals in the mitochondrial apoptotic pathway [91, 92]. Another ion channel (i.e., the calcium-activated K⁺ channel) has been identified on the mitochondrial inner membrane and has been shown to have a cardioprotective effect [93]. Nevertheless, the precise temporal order of events in the mitochondrial CP cascade and the exact molecular nature of the mitoK_ATP channel remain to be defined [94].

The MPTP is a protein pore formed in the mitochondria membranes under certain pathological conditions. The opening of this megachannel, located at contact sites between the inner and outer membranes, has been suggested to cause a number of important changes in mitochondrial structure and metabolism, including increased mitochondrial matrix volume (leading to mitochondrial swelling); release of matrix Ca²⁺; altered cristae; cessation of ATP production, primarily due to uncoupling of the ETC; and dissipation of the mitochondrial membrane potential [95]. The MPTP may be a site where the organelle integrates multiple cell-signaling stimuli as well as metabolic responses, and the induction of the MPTP can lead to cell death and play a critical role in apoptosis. Interestingly, MPTP opening during apoptosis is transient, allowing mitochondria to maintain the ATP levels necessary for fueling the downstream apoptotic responses. It should not be surprising that early apoptotic events involve the modulation of ATP levels, given the close proximity of the MPTP to the respiratory complexes in the mitochondrial inner membrane as well as its involvement in the mitochondrial loss of cytochrome c, a critical molecule in ETC ­function. Present at the MPTP site are a number of energy-associated mitochondrial molecules including the ANT, the glycolytic enzyme hexokinase, the outer membrane voltage-dependent anion channel protein (VDAC or porin), the mitochondrial creatine kinase, and the phospholipid cardiolipin [96]. While ample data exist on MPTP subunit composition, genetic knockout experiments suggest that the identity of the core factors of the MPTP is still unresolved, and gathered evidence suggests a much more complex composition of this protein complex than anticipated [97].

Following an episode of myocardial ischemia, at the onset of reperfusion, opening of this nonspecific pore is a critical determinant of myocyte death. Besides its role in the mitochondrial pathway of apoptosis, the opening of the MPTP, if unrestrained, leads to the loss of ionic homeostasis and ultimately to necrotic cell death [98]. Fatty acids, high matrix Ca²⁺ levels, pro-oxidants, metabolic uncouplers, NO, and excessive mitochondrial ROS production (primarily from respiratory complex I and III) promote the opening of the MPTP.

Moreover, the MPTP may be also an important target of CP. It has been shown that suppressing MPTP opening at the onset of reoxygenation can protect human myocardium against lethal hypoxia-reoxygenation injury [99] and that the inhibition of MPTP opening can be mediated either directly by cyclosporin A (CsA) and sanglifehrin A (SfA) or indirectly by decreasing calcium loading and ROS levels (Fig. 8.3).

Recently, Penna et al. [100] reported that very low concentrations (pmol/L) of platelet-activating factor (PAF) infused before ischemia induce CP similar to those afforded by ischemic preconditioning (IP) and that endogenous PAF production participates in the induction of IP itself. This IP-like action exerted by low concentrations of PAF is due to the activation/phosphorylation of kinases included in the reperfusion injury salvage kinase (RISK) pathway, such as protein kinase C, Akt/PKB, and nitric oxide synthase. Together with the activation of mitochondrial K_ATP channels, these events may allow prevention of MPTP opening at ­reperfusion. In addition, the nitric oxide-dependent S-nitrosylation of L-type Ca²⁺ channels induced by PAF reduces intracellular Ca²⁺ overload.

The PI3K/Akt pathway promotes cell survival primarily by intervening in the mitochondrial apoptosis cascade at events before cytochrome c release and caspase activation occur (Fig. 8.4). Akt activation inhibits changes in the inner mitochondrial membrane potential that occur in apoptosis (suppressing apoptotic progression and cytochrome c release induced by several proapoptotic proteins). While Akt also contributes to the phosphorylation and inactivation of the proapoptogenic protein Bad, it remains unclear whether Bad phosphorylation is the mechanism by which Akt ensures cell survival and mitochondrial integrity since other mitochondrial targets of Akt remain to be identified [111]. Interestingly, microarray analysis has demonstrated that once unnoticed Akt regulation of genes could modulate the influence of Akt on cardiomyocyte survival, metabolism, and growth [112].

Deprivation of nutrients (e.g., amino acids), glucose, and growth factor, which can lead to cardiomyocyte apoptosis [104], has been found to signal via the mitochondria-associated mTOR protein (Fig. 8.4) [113]. Moreover, both the Akt pathway and the downstream mTOR protein impact cardiomyocyte survival and cell size largely through increased cytoplasmic protein synthesis by mediating activation of translational initiation factors and ribosomal proteins. In addition, serotonin binding to the serotonin 2B receptor protects cardiomyocytes against serum deprivation-induced apoptosis via the PI3K pathway impacting ANT and Bax expression. Transgenic mice harboring serotonin 2B receptor null mutations manifest pronounced myocardial mitochondrial defects in addition to altered mitochondrial ETC activities (complex II and IV), ANT-1, and Bax expression [114].

Finally, Akt signaling also provides CP against ischemic injury in response to diverse treatments including cardiotrophin-1, acetylcholine, adenosine, and bradykinin-mediated preconditioning [115–117], although the precise target of Akt action in mitochondria-based cardioprotection remains undetermined since Akt does not associate directly with mitoK_ATP channels.

Stresses in cardiac hypertrophy (e.g., mechanical) and ischemia/hypoxia (e.g., oxidative) elicit a variety of adaptive responses at the tissue, cellular, and molecular levels. A model showing the cardiac physiological response to hypoxia reveals that mitochondria function as O₂ sensors both by increasing ROS generation during hypoxia and via their abundant heme-containing proteins (e.g., COX), which reversibly bind oxygen [118]. Oxidant signals such as ROS act as second messengers initiating signaling cascades and are prominent features in both adaptive responses to hypoxia and mechanically stressed heart. Downregulation of COX activity contributes to the increased ROS generation and signaling observed in cardiomyocytes during hypoxia [119]. Also, hypoxia stimulates NO synthesis in cardiomyocytes [120], and NO downregulates COX activity with subsequent mitochondrial H₂O₂ production. This event has been proposed to provide a mitochondria-generated signal for further regulating redox-sensitive signaling pathways, including apoptosis, and can proceed even in the absence of marked changes in ATP levels [108]. Interestingly, nitric oxide synthase has been identified in heart mitochondria although its role in regulating OXPHOS is not clear [121, 122]. Mitochondrial ROS has also been shown to activate p38 kinase in hypoxic cardiomyocytes [123]. Longer-term responses to hypoxia have been shown to involve increased gene expression of hypoxia-induced factors and the activation of transcription factors such as NF-κB which has also been implicated in the complex regulation of cardiac hypertrophy and inflammatory cytokines (TNF-α, interleukin-1). While increased ROS has been shown to be an important element in NF-κB gene activation, there is recent evidence showing that cardiomyocyte hypoxia-induced factor gene activation can also occur in the absence of ROS [124].