**4. NADH-ubiquinone oxidoreductase (complex I)**

and organ storage, or hypotension in septic shock. Functional deficits caused by ischemia in major organs result in significant morbidity, disability, and mortality. Mitochondria are double-membrane-bound, dynamic organelles present in most eukaryotic cell types. They require large amounts of oxygen to generate ~90–95% of the total energy in oxidative phosphorylation occurring in a majority of cells [1]. Therefore, mitochondria are the key subcellular targets of ischemia, which undergo pathological changes that trigger cellular and tissue damage when oxygen is unavailable. Mitochondria perform several major cellular functions including energy and intermediary metabolisms, several biosyntheses, regulation of calcium storage, and redox homeostasis [1]. They are also involved in signaling, cell cycle, growth,

Proteins of the mitochondrial electron transport chain and oxidative phosphorylation are among the primary targets of ischemia and oxidative stress during reperfusion [3]. Decreases in activities of adenine nucleotide translocase (ANT) and ATP synthase are among the earliest events after the onset of cardiac ischemia [4–7]. Inhibition of NADH:ubiquinone dehydrogenase (complex I) and reduced cytochrome c content occur early during ischemia [5, 8], whereas the damage to ubiquinol:cytochrome c oxidoreductase (complex III) and cytochrome oxidase (complex IV) occurs in prolonged ischemia [5, 8]. Reduced activity of complex I is primarily caused by decreases in NADH dehydrogenase activity [5, 9] due to, in part, oxidative damage to the flavin mononucleotide (FMN) prosthetic groups, which results in electron leakage, superoxide production, and the generation of reactive oxygen species (ROS) in ischemic tissues [5, 10]. Ischemia decreases the activity of complex III by inactivating the iron-sulfur center, which contributes to electron leakage and superoxide production, and exacerbates oxidative stress originating from complex I [11]. The voltage-dependent anion channel (VDAC) and proteins of the mitochondrial permeability transition (MPT) pore are also targeted by I/R, which disrupts the transport of ions and solutes and the membrane

Mitochondria are dynamic organelles that regularly undergo fission (fragmentation) and fusion (formation of a network of mitochondria) [14]. Fission is mediated by the dynaminrelated protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) [14]. Fusion is regulated by the mitofusins (Mfn1 and Mfn2) and the optic atrophy protein 1 (Opa1) [14]. Drp1 and Opa1 are rapidly activated and translocated to mitochondria after ischemia in major organs. This promotes fission, the permeabilization of the mitochondrial outer membrane, and the release of proteins that initiate the intrinsic cascade of apoptosis [15, 16]. Inhibition of Drp1 inhibits fission and reduces cardiomyocyte death, the size of myocardial infarct, and acute kidney injury after ischemia [17]. Mitochondria also regulate the intrinsic cascade of apoptosis, which is activated after the release of mitochondrial proteins. Therefore, preservation of mitochondrial integrity and function is crucial for organ protection against

differentiation, and cell death by apoptosis [2].

184 Mitochondrial Diseases

potential for ATP synthesis [12, 13].

I/R injury.

**2. Major mitochondrial targets of ischemia/reperfusion (I/R)**

Dysfunction of complex I is the most common disorder of oxidative phosphorylation in humans. It is often due to defects of the subunit assembly to form the mature complex I. Complex I is a major mitochondrial target of I/R. Its activity decreases as early as 10 minutes into cardiac ischemia [24]. Our studies demonstrated that the activity of complex I in renal cortical mitochondria is decreased after renal ischemia [25]. Changes in the activity of complex I during I/R are also regulated by phosphorylation [4, 26]. Phosphorylations occur on several subunits of complex I. The NDUFA10 subunit is phosphorylated on S59 and S95 [27]. The NDUFS4 and NDUFA10 subunits are phosphorylated by mitochondrial protein kinase A (PKA), which stimulates the activity of complex I [27–29]. Phosphorylations of subunit ESSS on S20 and subunit MWFE (NDUFA1) on S55 regulate complex I assembly. Blocking these phosphorylations inhibits assembly of subunits to form a mature complex I and reduces its activity [26, 30]. Tyrosine phosphorylation of the NDUFB10 subunit by Src kinases also increases the activity of complex I, possibly by increasing its affinity toward NADH or increasing assembly of subunits into the fully active complex I [31, 32]. The reduced assembly of subunits leads to decreased levels of complex I [32]. This adaptation shifts fuel utilization from fuels that generate primarily NADH (carbohydrates) to fuels generating more FADH2 (fatty acids) oxidized by complex II. This shift may occur during nutritional restriction or after ischemic injury [32]. Further, complex I is primarily assembled into mitochondrial super-complexes, which increases O2 consumption and reduces ROS production [33]. Cardiac I/R induces disintegration of mitochondrial super-complexes, which reduces activity of the electron transport chain [34]. Thus, phosphorylation controls the formation, stability, and function of complex I and its assembly into super-complexes.

c-Src kinase activates complex IV [41, 42]. The latter event is required for the normal function of cells, which are dependent on the efficient production of ATP to maintain their functions [42]. Interestingly, phosphorylation of the same subunit by the receptor tyrosine kinase ERBb2 decreases the activity of complex IV and mitochondrial respiration [43, 44]. Complex IV activity is inhibited in a time-dependent manner after myocardial ischemia, which stimulates multiple phosphorylations of complex IV: (1) subunit I on S115 and S116, (2) subunit IVi1 on T52, and (3) subunit Vb on S40 in the heart [38, 45]. These inhibitory phosphorylations are mediated by PKA and inhibition of PKA reduces I/R injury to the myocardial tissue [45]. Phosphorylation of subunit IV-1 on S58 by PKA increases the activity of complex IV by preventing allosteric inhibition of complex IV by ATP [46]. It was proposed that phosphorylation of S58 switches mitochondrial metabolism from energy utilization to energy storage in

Protein Kinases and Regulation of Mitochondrial Function in Ischemia/Reperfusion Injury

http://dx.doi.org/10.5772/intechopen.71094

187

Phosphorylations of serine, threonine, and 2 tyrosine residues have been mapped on cytochrome c [39, 47]. Phosphorylation of T28 results in a partial inhibition of the electron transport chain and respiration [48]. It was suggested that the other phosphorylations regulate the mobility of cytochrome c between complexes, its binding to cardiolipin, and the interaction

Multiple and differential phosphorylations of ATP synthase have been reported in different

alytic β-subunit is extensively phosphorylated on S106, T107, T262/S263, T312, and T368 in mammalian cardiomyocytes, whereas in yeast, the β-subunit is phosphorylated on T58, S213,

ATP synthase activity [51]. They occur in cardiac preconditioning, which offers protection

downregulates its levels *in vivo* [6, 51]. We have shown serine phosphorylations on the α and/or β- and γ-subunits of ATP synthase in RPTC [52]. These phosphorylations are PKC-α-

cells [52]. Phosphoproteomics demonstrated phosphorylation of S146 on the γ-subunit [52]. Phosphorylation of the γ-subunit is associated with the formation of ATP synthase dimers [52].

ANT, an antiporter embedded in the inner mitochondrial membrane that facilitates the exchange of ADP and ATP, is one of the most abundant mitochondrial proteins and a primary

against ischemia [51]. Phosphorylation of T213 on the β-subunit of skeletal muscle F<sup>o</sup>


domain are mediated by Src kinase [49, 50]. The α and e sub-

F1

domain) and

domain and reduce

F1



organisms and tissues. Tyrosine phosphorylations of the ε-subunit (in the F<sup>o</sup>

T262, and T318 [51]. These phosphorylations affect assembly of the F<sup>1</sup>

dependent and preserve the levels of the γ-subunit and F<sup>o</sup>

**10. Adenine nucleotide translocase (ANT)**

pathological conditions including I/R-induced injury [46].

**8. Cytochrome c**

with Apaf-1 during apoptosis [47].

**F1**

**-ATPase)**

**9. ATP synthase (Fo**

F1

units of Fo

the α- and δ-subunits of the F<sup>1</sup>
