Cardioprotection by Pre- and Post-conditioning: Implications for the Role of Mitochondria

The mitochondrion has evolved as an important organelle in determining cell survival and cell death. It is involved in a plethora of processes in mammalian cells including ATP production, steroid synthesis, and cell division and cell death. Indeed, mitochondrial dysfunction is associated with numerous human maladies including heart disease. Mitochondrial diseases have traditionally been attributed to defects in the electron transport chain (ETC), the major source of mitochondrial reactive oxygen species (ROS), a byproduct of mitochondrial respiration. Mitochondrial cation channels and exchangers function to maintain matrix homeostasis and are likely involved in modulating mitochondrial function in part by regulating O2 •generation. Insofar as mitochondria are involved in oxidative damage that leads to apoptosis, antioxidants and other therapeutic strategies that target the organelle appear to be a novel approach to alleviate some cardiovascular diseases. This novel approach has gained unprecedented attention recently with a significant potential for future therapeutic purpose. Whether mitochondria are targets or end effectors of cardiac preand post-conditioning remain unresolved. This brief review will provide the latest information gleaned from the literature on the role of mitochondria in preand post-conditioning during cardiac ischemia and reperfusion. AAC 8 (1) 1-5 ( 2011) 8 consumption by functioning mitochondria to produce energy with minimal electron leak to generate the superoxide (O2 •-) anion (15,71). Mitochondria are therefore vital for normal cellular function including intracellular metabolic activities and signal transduction of various cellular pathways during normal and abnormal states. Cell signaling pathways induced physiologically by reactive oxygen species (ROS) include effects on thiol groups and disulfide linkages, which post-translationally modifies protein structure to activate/inactivate specific kinase/phosphatase pathways (15). Similarly, mitochondrial proteins can be targets of extra-matrix signaling molecules with concomitant changes in mitochondrial bioenergetics and cellular protection against oxidative stress. Mitochondrial constituents such as proteins, lipids, and mitochondrial DNA are themselves targets of oxidative/nitrosative stress, and the mitochondrial control of oxidative stress has consequences both for cellular energy metabolism and for the processes that control the onset and progression of the cell death response (15). A protected electron transport chain (ETC) during preand post-conditioning is therefore critically important for the recovery of the post-ischemic heart. An understanding of mitochondrial function in normal and pathological states is essential to ameliorate or prevent mitochondria related cardiac dysfunction, like I/R injury. Thus, the recent spotlight on mitochondria is attributed to its role in cell death, in which excess ROS (O2 •and its products) and dysfunction in the energy production process are underlying factors. O2 •-, which is generated at several sites within the ETC and the matrix, is mostly converted to H2O2 both inside and outside the mitochondrial matrix by O2 •dismutases. H2O2 is a major, highly diffusible, chemical messenger that, in low amounts, physiologically modulates cell function (15). Diseases of the mitochondria appear to cause most damage to cells that are metabolically active, like the heart, brain, liver, kidneys and skeletal muscle. In a recent comprehensive review (15), we discussed several mitochondrial related diseases, which will not be discussed here. The objective of this brief review is to provide the role of mitochondria in I/R injury and to note the potential therapeutic approaches to mitigate mitochondria-related dysfunction, specifically preand post-conditioning. Overview of Mitochondrial Anatomy and Function: Implications for Ischemia and Reperfusion Injury and Cytoprotection Mitochondria are the key cellular organelles charged with synthesizing ATP via oxidative phosphorylation (OXPHOS) (Figure 1). They have major roles in cellular physiology beyond ETC and OXPHOS. Mitochondria are also involved in intracellular Ca2+ homeostasis, ROS production, and apoptosis. Mitochondrial compartments include the outer and inner mitochondrial membrane (OMM and IMM, respectively) (Figure 1), the inter-membrane space (IMS), and the matrix. The membranes and the IMS are intimately involved in control of cell function and cell death. The OMM plays an important role in controlling mitochondrial activity. The OMM and IMS limit the passage of proteins and small solutes that regulate many cellular processes and initiate or inhibit apoptotic pathways. The OMM is a relatively simple phospholipid structure that contains many channel proteins. OMM permeabilization is considered the ‘point of no return’ as this event is responsible for initiating the apoptotic cascade in numerous cell death pathways (22). The release of, for example, cytochrome c triggers formation of an “apoptosome” that leads to activation of the executioner caspases (22). It is possible that under normal conditions changes in mitochondrial dynamics also modulate OMM permeabilization to induce apoptosis (17,18). The most abundant protein on the OMM is the VDAC. It acts as a conduit for translocating ions and a variety of metabolites such as ATP and ADP across the OMM. As a major gateway in and out of the mitochondrion, VDAC mediates a delicate balance between metabolism and death in cells (15). However, following I/R, a 4-fold increase in VDAC phosphorylation has been reported (66) and the cardioprotective effect of PD169316, an antagonist of p38 mitogen-activated protein kinase (MAPK) resulted in a significant reduction in ischemia-induced phosphorylation of VDAC, specifically a reduction of tyrosine phosphorylation (66). The VDAC is also a receptor for hexokinases (HK) and the VDAC-HK binding has been implicated in cell survival and cell death. HK II binding to VDAC is believed to maintain mitochondrial membrane integrity and to prevent the Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) (80). The mPTP is a mega-pore that connects IMM and OMM proteins with the result that mitochondrial permeability increases and cell demise ensues. Exposure to apoptotic stimuli causes a decline in mitochondrial HK II activity (31). In the heart, it has been suggested that the mitochondria-HK interaction may indeed be an integral part of cardioprotection, including ischemic and pharmacologic preconditioning (IPC and PPC, respectively) (85). In addition to mCa2+, mPTP opening appears to be instigated by high levels of ROS, increased Pi and a high matrix pH (15). Since matrix pH likely becomes more acidic during ischemia, pore opening will not be favored. Therefore, matrix acidification during I/R has AAC 8 (1) 1-5 ( 2011) 9 been shown to be a beneficial strategy in attenuating I/R damage (15). In contrast, matrix alkalinization on reperfusion may induce pore activation, so continued acidification during reperfusion or inhibition of Na2+/ Ca2+-exchanger (NCE) to minimize cytosolic and mitochondrial Ca2+ overload would be strategically beneficial during this period to reduce cell death. Indeed, acidification has been shown to be effective in postconditioning protection as demonstrated by inhibited pore opening (15). We and other investigators have also assessed myocardial mPTP opening in the intact heart, prior to the onset of contractile dysfunction during I/R, and in isolated mitochondria (2,10,33). We showed that I/R increases the vulnerability of isolated mitochondria to increased extra-matrix Ca2+ challenges (2). In the cardiac dystrophied mdx mouse model, evaluation of mPTP in the intact isolated heart model using the 3H-DOG method showed increased mPTP opening as evidenced by release of cytochrome c when hearts underwent I/R (10). Mitochondria from isolated hypertrophied rat hearts exhibited an increased susceptibility to opening of mPTP in response to physiological stressors (i.e., Ca2+ overload and anoxia and reoxygenation) and H2O2 (10,15). In the IMM, a large DYm (negative inside) is formed across the IMM by outward proton pumping by the respiratory enzymes of the ETC. The IMM charge separation and transmembrane cation fluxes via specialized cation channels/uniporters, symporters, and exchangers are essential for mitochondrial respiration and function (15). Activation of the exchangers allows extrusion of cations entering the matrix down their concentration gradient, thus preventing volume expansion. The IMM also contains the adenine nucleotide translocator (ANT), which is considered a crucial component of the mPTP. The ANT has been shown to be a target for oxidative and nitrosative stress and therefore is an important component in I/R injury. In cardiac myocytes, connexin 43 (Cx43), the predominant protein in gap junctions in ventricular myocardium, is also localized in the IMM of cardiomyocyte mitochondria (15) and has been implicated recently as a factor in preand post-conditioning (65). There is increasing evidence that mitochondria, which have a large capacity to take up Ca2+, play a critical role in maintaining intracellular Ca2+ homeostasis in I/R (15). During I/R, increased cytosolic Ca2+ may lead to mCa2+ overload via the mitochondrial Ca2+ uniporter (Figure 1), and in the presence of ROS, may lead to mPTP opening and cell death. Therefore, preventing Ca2+ uptake at times of increased cytosolic Ca2+ due to oxidative stress has proven to be a viable strategy to mitigate cellular damage during I/R. In a recent study it was reported that melatonin, a pineal gland hormone protect against brain I/R injury in part by preventing