Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in developed countries (1). The most common cause of cardiac injury is ischemic heart disease secondary to progressive coronary occlusion and is complicated frequently by complications such as ventricular arrhythmias and congestive heart failure (2). The most effective therapy, thus, far for limiting ischemic injury is early reperfusion, but reperfusion in itself has risks (3–5) to the extent that acute coronary occlusion and therapeutic intervention are collectively considered to produce an aggregate phenomenon recognized as ischemia-reperfusion (IR) injury (6–9).

Continuing research is aimed to explore the therapeutic interventions against IR injury. Although numerous pharmacological and preconditioning approaches to cardioprotection have been explored, regular exercise participation is recognized as an important, cost-effective, and safer lifestyle intervention in the prevention and treatment of IR injury (10–14). Redundant protective effects are evident in the exercised heart, namely, increased levels of heat shock proteins (15), altered nitric oxide (NO) signaling (16–18), enhanced Ca²⁺ handling proteins (19), improved ATP-sensitive potassium channels (20), and enhanced endogenous antioxidant (13, 21).

As an important site for ATP production via oxidative phosphorylation, mitochondria are critical in regulating normal cardiac metabolism and play a key role in the susceptibility of the heart to IR injury (22–28). The heart is an organ with high metabolic demand. Providing the myocardium with adequate coronary perfusion and oxygen delivery is crucial, but enhanced mitochondrial capacity also is essential. Mitochondrial respiratory rate and enzyme activities are the major elements that drive the oxidative phosphorylation process and structural integrity of the mitochondria. Myocardial IR can cause severe effects on mitochondrial homeostasis which dramatically affects mitochondrial function and survival (29–32). In addition to the detrimental effects of impaired mitochondrial energy production, mitochondrial ionic imbalance, and cell stress signaling can cause mitochondrial-mediated cell death (33–37).

Myocardial IR injury is an important cause of impaired heart function in the early postoperative period after cardiac surgery and acute myocardial ischemia. Growing evidence has become available supporting a crucial role of mitochondrial dysfunction in myocardial IR injury. Mitochondrial dysfunction during ischemia is a major mechanism that contributes to cardiomyocytes damage during IR (24, 26–31). Increased reactive oxygen species (ROS) generation, defects in electron transport chain activity and oxidative phosphorylation process, impaired respiratory chain complexes activity, opening of the mitochondrial permeability transition pore (mPTP), and release of cytochrome care considered contributing factors in mitochondrial dysfunction associated with heart IR (30–36).

Stunned (reversibly injured and nonfunctioning) myocardium displays relatively excess oxygen consumption for a specified rate of contractile work and, therefore, has a declined mechanical efficiency, which may be due to a rapid recovery of the intracellular pH during reperfusion (6). Once perfusion is restored, the intracellular increased accumulation of H⁺ during ischemia is transported into the extracellular space to normalize the pH in exchange for Na⁺ via Na⁺/H⁺ exchanger, while ATP depletion inactivates Na⁺/K⁺-ATPase. The combined effect results in an increase in intracellular Na⁺ which, in turn, activates the sarcolemmal 2Na⁺/Ca²⁺ exchanger, resulting in the exchange of intracellular Na⁺ with extracellular Ca²⁺. A high rate of 2Na⁺/Ca²⁺exchange can finally lead to Ca²⁺ overload which, in turn, induce arrhythmogenesis, myocardial stunning, contracture, and ultimately apoptotic or autophagic cell death. Fluctuations in Ca²⁺from the sarcoplasmic reticulum during reperfusion stimulate the opening of the mPTP (29, 30). Opening of the mPTP leads to rapid dissipation of the membrane potential gradient which is essential for the synthesis of ATP, water enters through the open pore causing mitochondrial swelling and lysis triggering apoptosis and cell death (28–30).

The proposed mechanisms underlying exercise-induced cardioprotection in IR are numerous. Some are systemic (12), some are vascular (12–14, 21), some are neural (38, 39), some are structural (9), and some are energetic/metabolic (40, 41) including expression of selected mitochondrial proteins resulting in a mitochondrial phenotype that is resistant to IR-induced injury (13, 19). While these studies generally support exercise-induced adaptations that produce resistance to injury, few address the mitochondrial functional consequences following an injury.

Although regular exercise training is recognized as an important lifestyle intervention in the prevention and treatment of CVD and IR injury, not all the individuals experience the same benefits from participating in the exercise. There is a paradox that some individuals with many modifiable risk factors (hypertension, diabetes, and obesity), and who do not exercise, also do not get CVD, while others who have no risk factors, and exercise regularly, and still experience adverse cardiac outcomes, suggesting a significant intrinsic component to the exercise effect. It has been estimated that up to 70% of the variation in exercise capacity is due to the intrinsic genetic component (42). Thus, studying the differential impacts of intrinsic aerobic exercise capacity after cardiac IR injury using intrinsic aerobic phenotype rats bred for low and high aerobic running capacity would provide a better platform for understanding the influences of intrinsic aerobic capacity on cardiac metabolic capacity and mitochondrial adaptive response pre- and post-IR injury in these phenotypes.

Koch and Britton used phenotypic selection based on treadmill running time at 11 weeks of age in an outbred rat strain (NIN:N) to create divergent strains that have become known as high capacity aerobic running (HCR) and low capacity aerobic running (LCR) rats (43–47). The HCR animals generally are characterized as “disease resistant,” while the LCR animals are characterized as disease prone. Interestingly, the HCR/LCR strains demonstrate many of the same traits related to CVD that had been previously associated with active exercise (48–52) including some mitochondrial/metabolic effects (53, 54). Still, cardioprotection in the HCRs is not always observed (55–57), and while LCR did associate with a higher incidence of pump failure, it did not associate with multiorgan system failure in hemorrhagic shock (58). We have previously reported that cardioprotection in HCRs was present but limited, was likely intrinsic to the tissue, and could be overwhelmed by IR severity (57). Given the consistent metabolic differences that characterized HCR and LCR, the current studies were designed to identify the relative influence of aerobic phenotype on the impact of IR on subsequent mitochondrial function.

The results of this study showed that while there was increased mitochondrial capacity in HCRs compared to LCRs, the increased cellular respiratory capacity did not translate to reduced injury from ischemic arrest and reperfusion even with the benefit of supplemental cardioprotection by using established cardioplegia regimens. Moreover, there was accelerated relative loss of mitochondrial respiratory capacity in the HCRs compared to the LCRs, even though coronary blood flow was better preserved during reperfusion in the HCRs compared to the LCRs. The lack of benefit despite better flow was a bit unexpected, as dynamic exercise has generally associated cardioprotection with improved perfusion. A decrease in infarct size after training was seen in rats subjected to a swim training regimen after permanent occlusion which the authors attributed to increased myocardial vascularity (59). In a much-cited paper, Brown et al. demonstrated that prolonged endurance training confers a cardioprotective effect against infarction in myocardium subjected to severe ischemia and subsequent reperfusion (21). In addition, they observed that during severe ischemia, coronary flow to regions of the myocardium outside the ischemic area at risk was better maintained in hearts isolated from endurance-trained rats. Furthermore, on reperfusion of the area at risk, the increase in flow to the previously ischemic region of the heart was markedly higher in hearts isolated from trained rats (21).

We have demonstrated that the cardioprotection associated with the HCR phenotype may be limited (57). We did not have sufficient animals available to complete a preliminary dose response study to determine the optimal cold storage time, and it is possible that despite the cardioplegia, the ischemic time was simply too long and overwhelmed and benefit the HCR phenotype might have conferred. However, the results are consistent with those reported by others working with the model (55, 56). Still, there are important differences worth noting. In particular, active exercise training programs produce improvements to ischemic tolerance via a host of mechanisms (5, 6, 9, 10, 14, 15), notably including increased mitochondrial functional capacity (19, 24, 59). Gallego-Selles et al. described experiments in humans subjects indicating that, at least in skeletal muscle, nrf2 is activated rapidly by exercise to exhaustion and can dynamically modulate ROS and mitochondrial-sourced proteins such as catalase (61). Flockhart demonstrated, also in human subjects, that participating in an intense, active exercise program produced functional impairment in the mitochondria that also was associated with at least a transient decrease in glucose tolerance (62). In our hands, despite an increased resting mitochondrial capacity in the HCRs, there was no protection against the ischemic insult. Together, it seems clear that there can be substantial differences in the role of mitochondria in the response to ischemia that can be influenced by the nature and duration of the exercise training and confounded by differences dictated by the intrinsic capacity for exercise, independent of actual active exercise. The importance and role of pathways such as nrf2/keap1/catalase and others in bridging the components of an exercise effect (intrinsic vs. active) remain to be determined.

Several previous experimental studies of myocardial ischemia generally have indicated a good correlation between disruption of complex I through a variety of mechanisms (16–18, 33, 41), as well as opening of the permeability transition pore, with outcomes in cardiac ischemia and reperfusion (23, 26, 28–30). Some have suggested that complex I is considered the main site of damage to the respiratory chain in IR, while downstream electron transport chains are relatively resistant to IR injury (60, 63, 64). Furthermore, Veitch and his colleagues (63) found a major decline in complex I activity in perfused rat hearts subjected to 20 min of global ischemia, and Cairns and his group (64) demonstrated that this damage was exacerbated by reperfusion.

In contrast to these studies, our results suggest a more generalized loss of mitochondrial respiratory capacity following IR. Perhaps that should not be surprising. Animals that have been selectively inbred using aerobic running capacity as the selection criteria create a strain optimized for performance when aerobic conditions exist. Ischemia certainly is not an aerobic condition. While it is possible that the HCR phenotype augments several factors that could delay the onset of ischemia and which would be protective (57), they may be maladapted if the transition to anaerobic conditions still occurs. Structural adaptations like increased capillary density or increased vasodilator capacity, which might have been beneficial in delaying the onset of ischemia, would also be factors that could aggravate the potential for reperfusion injury once perfusion was reestablished after an acute ischemic episode. When ample oxygen is present, cardiac mitochondria have greater substrate flexibility than many other tissues, meaning that cardiac mitochondria can respire effectively using a wide variety of substrates (67). Tolerance for IR may not be related so much to the capacity to respire using a given substrate, but more to the ability to switch from one substrate to another. With the newest approaches that have been developed (68), and the energy hypothesis that is being advanced by the HCR LCR work (46, 53), the HCR LCR phenotypes could provide interesting and valuable perspective on factors contributing to substrate preference. To the extent that HCR complex I respiratory capacity was impacted strongly by IR, and that there was a similarity between PTP function and infraction size in HCR and LCR, suggest that a more complex assessment of specific mitochondria adaptations in these strains might be warranted.

Mitochondria comprise a large fraction of the heart mass and are critical for the normal mechanical and electrophysiological function of the cardiomyocyte, playing roles that extend beyond bioenergetics and metabolism. Proper function is required to meet the high energetic demand of the cardiomyocyte and playing an essential role in controlling oxidative stress and Ca²⁺ handling (34). Ischemia-reperfusion injury increases the production of ROS and induces calcium overload into mitochondria (35) which can interact together to induce opening of the mPTP and, therefore, triggering apoptosis by promoting the release of proapoptotic proteins (i.e., cytochromec) and subsequent activation of the programmed cell (65, 66).

Our results indicate that intrinsic aerobic capacity must be tied to mitochondrial performance at a subcellular level. It also is clear that the dynamic role of mitochondria in cardiac ischemia, perfusion, and heart failure is only now beginning to be appreciated, but remains confused, perhaps in part because the underlying intrinsic elements have not been adequately considered in the assessment of the active exercise response. Models such as these HCR and LCR phenotypes should provide a unique window on how that background influences the extent to which inducible adaptive responses can be accomplished best using an active exercise regimen.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) East Carolina University.

RL participated in designing, conducting, analyzing, and writing. MA and MZ participated in conducting, analyzing, and writing. KF-W participated in designing, conducting, and analyzing. LCK participated in conducting and analyzing. LGK and SB provided animals and insight on study design. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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