Myocardial ischemia and reperfusion injury

An imbalance between the supply and demand of oxygen in the heart tissue due to restricted coronary blood flow results in myocardial ischemia (134). The absence of oxygen and metabolic substrates decreases the energy available to cardiomyocytes and leads to the tissue injury (infarction). The extent of the injury is determined by various factors including the duration of ischemia, the severity of ischemia (zero-flow versus low-blood flow) and the accompanying inflammatory response. It is also apparent that heart reperfusion, which is a requisite for cardiac tissue survival, can also increase injury over and above that sustained during ischemia, and independently lead to myocardial cell death (6, 15, 28, 94, 301, 317).

This phenomenon is called ischemia-reperfusion injury and is a major cause of morbidity and mortality in western nations. Fortunately, it has become clear that the cardiac response to ischemia-reperfusion insult can be manipulated to delay or decrease the severity of the injury, which has motivated intense studies on the mechanisms of cardioprotection.

Cell death as a result of ischemia-reperfusion injury has been reported to have features of both apoptosis and necrosis (201). The exact contribution of both in myocardial cell injury is still unclear. Pharmacological inhibition of pro-apoptotic signaling pathways during reperfusion is able to reduce both apoptotic and necrotic components of cell death (330). Therefore, it seems that they both share the common mechanisms in the early stages of cell death.

Necrosis is characterized by cell swelling leading to plasma membrane disruption with release of cytosolic components that in turn trigger an inflammatory response (90). Rupture of the plasma membrane can be facilitated by proteolytic degradation of crucial cytoskeletal and membrane proteins, such as calpain-mediated cleavage of ankyrin and fodrin, anchor proteins for Na⁺/K⁺-ATPase (138). Calpains are Ca²⁺-dependent proteases, and the increase in cytosolic free Ca²⁺ concentration has been consistently observed in ischemia and reperfusion (284).

Apoptosis is programmed cell death. It results in chromatin condensation, DNA fragmentation, cell shrinkage, and plasma membrane budding with release of apoptotic bodies, which are phagocytized by other cells in the organism (147). The inflammatory response is not involved in apoptosis due to the preserved cell membrane integrity. Apoptosis already occurs during ischemia and is accelerated in reperfusion (28) or it can be triggered at the onset of the reperfusion (94). In severely ischemic tissue, apoptosis progresses to necrosis as a result of ATP loss, which inhibits energy-dependent processes (e.g. functioning of ion pumps) resulting in swelling and rupturing of the cell membrane (284). Transduction of apoptotic signals can be divided into two pathways. The extrinsic pathway is a receptor-mediated cascade triggered by tumor necrosis factor-α (TNF-α) and Fas receptors and orchestrated by activation of caspase-8 and caspase-3 (215) The intrinsic pathway is mediated through mitochondria and activated by various stimuli such as ischemia and reperfusion or hypoxia (322). The pro-apoptotic stimuli of the intrinsic pathway induce increased permeability of the outer mitochondrial membrane (106). This phenomenon is associated with the opening of mitochondrial permeability transition pore (MPTP), which is a protein complex located at the junctions of both mitochondrial membranes (65). It has been reported to contain the voltage-dependent anion channel (VDAC, porin) in the OMM, the adenine nucleotide translocase (ANT) in the IMM and cyclophilin-D in the matrix, although the exact structure of MPTP is still unknown (325). MPTP opening occurs at the onset of and during the reperfusion phase as a result of calcium overload, depletion of adenine nucleotides, increase in phosphate levels, augmented ROS generation and mitochondrial depolarization (106).

In contrast, low pH inhibits pore opening. During cardiac ischemia, electron transport chain and proton pumping ceases leading to the reduction of mitochondrial membrane potential (ΔΨm). In order to maintain ΔΨm ATP synthase starts to work in a reverse mode to hydrolize ATP generated from glycolysis (98). The calcium uptake into the mitochondria depends on ΔΨm (212, 213). However, with the onset of reperfusion, ETC functioning and membrane potential are restored. Unfortunately, this leads to the additional accumulation of Ca²⁺ in the mitochondria, likely to predispose the MPT pore opening (213). Therefore, any agents that are able to inhibit calcium uptake (ruthenium red) (205) or mildly dissipate the ΔΨm (diazoxide) (126) may be considered as cardioprotective.

Ischemia-mediated permeabilization of outer mitochondrial membrane results in the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, endonuclease G (EndoG) and apoptosis-inducing factor (AIF) into the cytosol (102). All of these proteins play a crucial role in apoptosis signaling (325). In cytosol, cytochrome c binds to the Apaf-1 protein and activates caspase-9, which in turn activates caspase-3, the ‘executioner’

of the apoptotic pathway (28). Apoptosis is an energy-dependent process, thus the presence of ATP is necessary to execute the apoptotic program. Therefore, severe ischemia with substantial ATP depletion limits caspase activation and leads to the necrosis. Smac/DIABLO and HtrA2/Omi neutralize caspase-inhibitory proteins, which results in activation of caspases (289, 308). EndoG and AIF translocate to the nucleus where they orchestrate the DNA fragmentation (41, 180).

Apoptosis is highly regulated by the Bcl-2 family of proteins (101). Its members exhibit both, the anti-apoptotic (Bcl-2 and Bcl-xL), and pro-apoptotic (Bak, Bax, BAD, Bid, Bim) function. It has been reported that Bcl-2 family proteins (Bcl-xL) can bind directly to the components of MPTP controlling the pore opening and closing (276). Additionally, pro-apoptotic members of Bcl-2 family, Bak and Bax, can oligomerize into channel structures in the outer mitochondrial membrane (mitochondrial outer membrane permeabilization, MOMP) independently of the MPTP (152), and thus mediate the cytochrome c release. This process can be regulated by anti-apoptotic peptides, for instance it is known that Bcl-2 sequesters inactivated, monomeric form of Bax, preventing its oligomerization in OMM (96).

Moreover, Bcl-2 family proteins seem to converge the intrinsic and extrinsic apoptotic pathways. For instance, after Bid is cleaved by death receptor-dependent caspase-8, it translocates to the mitochondria and induces MPT pore opening (324). The Bcl-2 family proteins can be regulated through different mechanisms. Phosphorylation of Bim results in its degradation by proteasomes (179), while phosphorylation of BAD by kinases leads to its inactivation (19), all resulting in the inhibition of pro-apoptotic signaling.

Any manipulations that inhibit MPTP opening or enhance pore closure (106), influence cytosolic free Ca²⁺ concentration (205, 216) and regulate the pro- and anti-apoptotic proteins (124, 136, 185) should provide cardioprotection against ischemia-reperfusion injury. This concept is a basis of the cardioprotective strategy of pre- and postconditioning (Figure 1.2).

Figure 1.2. Schematic presentation of pre- and postconditioning as the means of cardioprotection against ischemia and reperfusion injury. ROS, reactive oxygen species; Jak, Janus kinase; Stat, signal transducer and activator of transcription; SAFE, survivor activating factor enhancement pathway; PI3K, phosphatidylinositol 3-kinase; Akt, serine-threonine Akt kinase; MAPKs, mitogen-activated protein kinases; ERK, extracellular signal-regulated kinase; RISK, reperfusion injury salvage kinase pathway; PKCε, protein kinase C isoform ε; p70S6K, serine-threonine kinase of S6 ribosomal protein; GSK-3β, glycogen synthase kinase-3β; mTOR, mammalian target of rapamycin;

eNOS, endothelial nitric oxide synthase; mito K_ATP, mitochondrial ATP-sensitive potassium channel;

Bcl-2, B-cell lymphoma 2 protein; Bax, Bcl-2-associated X protein; MPT, mitochondrial permeability transition; MOMP, mitochondrial outer membrane permeabilization. Figure adapted from Liem et. al (182).

1.6. Preconditioning and postconditioning in myocardial ischemia and