Sarcolemmal Atp-Sensitive Potassium Channels and Their Influence on Exercise Training-Mediated Cardioprotection

Introduction

The western world is adversely affected by coronary artery disease, which manifests as myocardial damage resulting from ischemia-reperfusion (IR) injury. The IR-induced myocardial injury has various levels ranging from myocardial damage from minor injuries to myocyte death resulting from serious injuries. This paper seeks to identify the need to provide a strategy for cardioprotection against IR-induced damage due to the increasing global prevalence of coronary artery disease and the associated IR-induced cardiac injury. There have been a variety of approaches to attain cardioprotection against IR injury that have been investigated. Presently, the only practical and sustainable countermeasure capable of providing cardioprotection is regular bouts of endurance exercise (Powers, Quindry, & Kavazis, 2008).

According to studies on human epidemiological, frequent exercise minimizes the chances of fatalities during myocardial IR insult. In addition to this, animal studies confirm that regular bouts of aerobic exercise (i.e., running or swimming) protect the heart from IR-induced injury. This paper looks at a variety of material from investigations conducted on the effects of muscular exercise in providing cardioprotection against IR-induced injury. The first step involves an evaluation of the different levels of IR injury, followed by an assessment of the events that result in IR-induced myocardial injury with an emphasis on the role that reactive oxygen species (ROS) play in this injurious process. Next, the paper looks at the proof provided to verify that exercise is cardioprotective, followed by an assessment of the putative mechanisms responsible for exercise-induced cardioprotection (Powers, Quindry, & Kavazis, 2008).

One of the hypotheses is that the increased sarcKATP channel expression would be sustained following months of training, and that pharmacological blockade of this channel population would abolish the protection from infarction afforded by training. Evidence from the literature indicated that delayed PC induced by a variety of stimuli is comprehensively abolished by 5-hydroxydecanoic acid (5HD), a putative mitochondrial ATP-sensitive K+ (mitoKATP) channel antagonist, administered minutes before index ischemia (Powers, Quindry, & Kavazis, 2008).

These experiments provide compelling evidence for the role of the mitoKATP channel as a distal mediator of protection from infarction. As such, our interest in exercise-induced PC led us to hypothesize that mitoKATP channels may also be integrally involved in delayed protection following exercise training, and that administration of 5HD would abrogate training-induced reductions in myocardial infarct size following I–R. Therefore, the study sought to determine if blockade of either the sarcolemmal and/or mitochondrial isoforms of the KATP channel abolished the protection afforded by chronic exercise training (Powers, Quindry, & Kavazis, 2008).

Background

The cardiac function has previously been analyzed based on physical activity. This is because the ability of the heart to increase the delivery of oxygen and metabolic fuels relies on an array of adaptive responses that are vital in the matching of bodily demand. At the same time, they avoid exhaustion of cardiac energy and functional resources. The most abundant cardiac membrane protein complex is the ATP-sensitive potassium (KATP) channel, which is capable of regulating membrane excitability in response to changes in ATP and ADP levels.

Levels of IR injury

There are three levels of IR-induced cardiac injury, which are dependent on the duration of ischemia. The first detectable level of injury is the generation of reperfusion-induced cardiac arrhythmias. Ventricular tachycardia or fibrillation without cell death or a deficit in ventricular contractile performance, are some of the possibilities of reperfusion after 1–5 min of ischemia.

The second level of myocardial injury or “myocardial stunning” occurs when reperfusion occurs after an ischemic period of 5–20 min. This level of injury has distinct aspects including a deficit in myocardial contractility that occurs without myocardial cell death. The injury causes ventricular contractile deficits that last for 24–72 hours, after the IR event.

The third and highest level of IR injury occurs when ischemia is extended beyond 20 min. In these circumstances, cardiac myocytes become irreversibly damaged, resulting in cell death; myocardial infarction. This kind of death results from apoptosis and necrosis. It is also important to note that mitochondrial injury plays a major role in both forms of cell death.

Events leading to myocardial ischemia-reperfusion injury

The factors leading to IR injury include a decrease in cellular ATP levels; production of Reactive oxygen species (ROS), accumulation of hydrogen ions; generation of reactive nitrogen species (RNS); calcium overload; calpain activation; and leukocyte activation. These factors collectively enhance cellular injury, followed by cardiac myocyte death (Powers, Quindry, & Kavazis, 2008).

Evidence for exercise-induced cardioprotection

Previously conducted research indicates that endurance exercise training (ExTr) improves myocardial tolerance to IR in both male and female animals as well as young and old animals. ExTr protects cardiac myocytes against IR-induced oxidative stress. In addition to this, ExTr also protects mitochondria against IR-induced damage. The ExTr-mediated cardioprotection is observed in both short-to moderate-duration ischemia (i.e., 5–20 min) and moderate to severe (i.e., 20–60 min) ischemic insults.

Collectively, these studies reveal that ExTr protects the heart against IR-induced arrhythmias, myocardial stunning, and myocardial cell death. Interestingly, only a few days of consecutive exercise are required to achieve the maximal benefit of extra-induced cardioprotection. It has been observed that short-term exercise training (i.e., 3–5 consecutive days) provides the same level of cardiac protection as that observed after weeks to months of regular exercise. Exercise-induced cardioprotection is lost within 18 days after the cessation of exercise training (National Health and Nutrition Examination Survey III, 2000).

Exercise-induced cardioprotection: potential mechanisms

The exact mechanism(s) responsible for exercise-induced myocardial protection against IR injury is not quite clear; though, numerous putative mechanisms of ExTr-induced cardioprotection have been proposed and investigated. These include anatomic and physiological changes in the coronary arteries (collateral circulation), induction of myocardial heat shock proteins (HSPs), increased myocardial cyclooxygenase-2 (COX-2) activity, elevated endoplasmic reticulum (ER) stress proteins, enhanced function of sarcolemmal ATP-sensitive potassium (sarcoKATP) channels, elevated levels of mitochondrial ATP-sensitive potassium (mitoKATP) channels, and increased myocardial antioxidant capacity (Herlitz, Bengtson, Hjalmarson, & Karlson, 1988).

Sarcolemmal ATP-sensitive potassium channels

Sarcolemmal ATP-sensitive K+ channels located in the sarcolemma of cardiac myocytes were discovered in 1983. They get their name from the inhibitory effect of ATP, which acts from the cytosolic side of the plasma membrane to reduce potassium channel opening. Structurally, sarcoKATP channels comprise two distinct proteins, an inwardly rectifying potassium channel pore and a sulfonylurea receptor subunit. The latter plays a regulatory role in modulating the channel opening in response to activating factors such as transient ischemia and increases in adenosine and MgADP along with activation of protein kinase C (PKC).

Researchers have shown that the pharmacological opening of sarcoKATP permits K+ outflow from the myocyte and provides cardioprotection during an IR insult. Opening sarcoKATP channels are postulated to protect against IR injury by shortening the cardiac action potential duration by accelerating phase 3 repolarization. Shortening the cardiac action potential would inhibit Ca2+ entry into the cell via L-type Ca2+ channels and prevent Ca2+ overload.

Importantly, the slowing of depolarization could also reduce Ca2+ entry by preventing the reversal of the Na+/Ca2+exchanger. These actions collectively protect the cardiac myocyte during IR by reducing the cytosolic Ca2 overload. Several lines of evidence indicate that sarcoKATP channels are involved in ischemic preconditioning-induced protection against an IR insult. According to Brown et al., ExTr increases the expression of sarcoKATP channels in the cardiac myocyte. In addition to this, the use of Langendorf isolated and perfused heart model of IR, these researchers indicated that pharmacological blockage of the sarcoKATP channel impairs the ExTr induced protective benefits against IR-induced myocardial infarction. This is a vital observation and additional research on the role that sarcoKATP channels play in ExTr induced cardioprotection is warranted.

Gaps in Exercise-induced cardioprotection

There are a couple of unanswered questions despite the recent progress that has been made in delineating the mechanisms responsible for ExTr-induced cardioprotection. For instance, although it appears that myocardial levels of both MnSOD and sarcoKATP are increased in the heart after ExTr, it is unknown if these two cardioprotective mediators act independently or interact to contribute to ExTr-induced cardioprotection.

Whereas ExTr-induced increase in mitochondrial levels of MnSOD would accelerate the removal of superoxide radicals, an increase in MnSOD activity would also increase hydrogen peroxide (H2O2) production. Hence, it would seem logical that ExTr increases in MnSOD activity would be associated with an increased ability to remove H2O2 from the mitochondria and/or cytosol. However, at present, it is unknown if ExTr results in an increased ability of cardiac myocytes to remove H2O2, requiring further research in order to determine if ExTr promotes an increase in an endogenous antioxidant to remove H2O2.

It is clear that mitochondria play an important role in IR-induced cardiac injury. Mitochondria have been referred to as the arbitrators of life and death for the cardiac myocyte exposed to an IR insult. New evidence suggests that ExTr protects against anoxia–reoxygenation-induced damage in mitochondria isolated from the hearts of ExTr animals. Some investigations have indicated that mitochondria isolated from the hearts of ExTr animals experience a lower rate of H2O2 production in both State 3 and State 4 respiration. Collectively, these findings indicate that ExTr enhances protective changes in the mitochondria that may contribute to ExTr-induced cardioprotection.

A problem lies in the fact that mechanisms responsible for these ExTr-induced changes in mitochondria are unknown. It is also unclear if ExTr results in a protection against anoxia–reoxygenation-induced injury in both subsarcolemmal and intermyofibrillar mitochondria (Yamashita, Hoshida, Otsu, Asahi, Kuzuya, & Hori, 1999).

A recent report revealed that male and female animals differ in the time course of exercise-induced cardioprotection. However, the mechanisms responsible for this gender difference in the time course of ExTr-induced cardioprotection are unknown. In this regard, investigation of gender differences in ExTr-induced cardioprotection could provide a useful experimental paradigm to improve our understanding of which cellular mediators are required to achieve the maximal benefit of ExTr-induced cardioprotection.

There is overwhelming evidence in support of the positive effects of endurance exercise training in promoting cardioprotection. In spite of this, it is unclear whether other forms of exercise, such as high-intensity anaerobic exercise, also promote cardioprotection. To date, there is an absence of published studies on the impact of resistance exercise training (weight training) on cardioprotection.

On the other hand, it was observed, from a recent study, that high-intensity (sprint) training protects the heart against IR-induced diastolic dysfunction; though, additional studies are necessary to determine if this form of high-intensity exercise training will result in cardioprotection against IR-induced cell death.

The part played by sarcoKATP channels in ExTr-induced cardioprotection remains relatively uninvestigated and to date, only one report exists regarding the role that sarcoKATP channels play in ExTr induced protection against myocardial infarction. There exists a close relationship between sarcKATP protein expression and protection from I–R injury, though a pharmacological blockade of this channel population to confirm the postulated channel-mediated cardioprotection needs to be performed.

Preliminary studies

The phenomenon of myocardial preconditioning has been extensively studied since its discovery by Murry and colleagues (Murry et al. 1986). Cardiac preconditioning (PC) can be triggered by several different stimuli and has been shown to reduce infarct size and defend against myocardial stunning. The protection afforded by PC appears to be biphasic, with an initial window of protection (‘classic PC’) lasting 1–2 hours after the PC stimulus, and the second window of protection (‘late’ or ‘delayed PC’), which occurs 24–72 hrs following the PC stimulus (Yellon & Downey, 2003).

While many factors appear to be potent in reducing myocardial infarct size when administered acutely, repetitive administration of the preconditioning stimuli such as ischemic PC ( (Cohen, Yang, & Downey, 1994) or adenosine receptor agonist (Tsuchida et al., 1994) results in a loss of efficacy, indicative of a tachyphylaxis response. Therefore, the clinical applicability of these stimuli as infarct-sparing strategies continues to be in question (Yellon & Downey, 2003).

A single bout of exercise training has been shown to protect the myocardium against ischemia-reperfusion (I–R) damage in a biphasic manner (Yamashita et al., 1999) consistent in amplitude and temporal onset as that afforded by other PC stimuli (Bolli, 2000).

Other than chronic moderate ethanol consumption (Kehl et al. 2003), exercise training is the only stimulus demonstrated to sustain myocardial protection against infarction over such an extended period. This finding continues to be extremely relevant from a clinical standpoint, as myocardial infarction affects over 1 million Americans annually (National Health and Nutrition Examination Survey III, 2000), and the size of infarction correlates inversely with a chance of both short-term (Miller et al. 1995) and long-term survival (Herlitz et al. 1988).

The role of cardiac ATP-sensitive potassium (KATP) channels in preconditioning has received much attention over the last 10 years, with investigations exploring the protective role of both the sarcolemmal (sarcKATP) and the mitochondrial (mitoKATP) isoforms of this channel. Several investigations have implicated a distal role for the KATP channel in delayed protection afforded by a variety of stimuli (Bolli, 2000); however, only one study has examined the role of the KATP channels in exercise-induced protection from infarction. According to previously conducted research, short-term exercise was identified to lead to reduced infarct size following I–R, with the delayed protection correlating closely with increased expression of the sarcKATP channel in both males and females (Herlitz, Bengtson, Hjalmarson, & Karlson, 1988).

Methods

Animal model

Adult female Sprague–Dawley rats (Harlan; n =103 total animals). Experiments are to be conducted with prior approval from the Institutional Animal Care and Use Committee at the institution, and in accordance with guidelines established by the American Physiological Society.

I–R protocol

Animals will be trained for not less than 12 weeks, as suggested by Brown et al (2003). At the time of death, animals will be anesthetized with sodium pentobarbital (35 mg kg−1; i.p. injection) and hearts excised and rapidly hung by the aorta to a cannula of a modified Langendorff apparatus. Both left and right adrenal glands will be removed and weighed, and plantaris muscle will be dissected for analysis of citrate synthase activity (Brown et al. 2003).

Retrograde perfusion will be initiated using an established buffer, and a 3-F pressure-transducing catheter (Millar) will be simultaneously inserted into the left ventricle (LV) via the aortic valve for the collection of left-ventricular developed pressure (LVDP) waveforms. After a 10 min equilibration period, baseline measurements of LVDP will be obtained. Following the baseline measurements, hearts from trained and sedentary animals will be divided further into a total of six groups. The concentrations of HMR 1098 and 5HD will be useful in abolishing the infarct-sparing effects of classic ischemic preconditioning.

A separate group of hearts should undergo three bouts of 5 min alternating regional I–R before index ischemia, and the reduction in infarct size following ischemic preconditioning (5.4±2.6% of the zone at risk) will be abolished when hearts receive 5HD during the ischemic preconditioning (IPC) stimulus (22.6±2.7% of the zone at risk; P <0.05). LVDP measurements will be taken after five additional minutes of exposure to KATP channel blockers (or control buffer) before regional ischemia is initiated.

Non-ischemic time controls

A separate group of hearts is will be subjected to the 3 h perfusion protocol in the absence of ischemia to determine the effect of the crystalloid perfusate and pharmacological agents on hemodynamic parameters and infarct size.

Western blotting

A separate group of Tr and Sed animals (n =9 Tr and 9 Sed) will be anesthetized and hearts excised. LV

free wall will be isolated and rinsed in saline (4◦C), and homogenized and probed for Kir6.2, SUR2a, Akt, p-Akt, glycogen synthase kinase (GSK)-3β, and p-GSK-3β (Brown et al. 2003).

Intracellular Ca2+ content

A separate group of hearts fromTr and Sed animals (n =36 total) will be exposed to a truncated I–R protocol (30 min each) in the presence or absence of the experimental drugs, and subsequently analyzed for intracellular Ca2+ content (Alto&Dhalla, 1979).

Exclusion criteria

Data can be omitted from analysis if one of the following criteria is met: the unclear resolution of heart slice images precluded analysis of infarction (n =1); coronary flow does not decrease at the onset of ischemia or increase at the onset of reperfusion (indicative of inefficient suture placement; n =14); or hearts do not complete the I–R protocol due to excessive fibrillation or technical difficulty (n =8).

Statistical analyses

Data will be presented as means±standard error. All statistical analyses will be performed using SPSS Software, and α-level can be predetermined to be P <0.05. A priori confirmatory comparison of infarct size and LVDP between Sed and Tr groups will be performed with a two-tailed Student’s t-test. All other infarct size comparisons will be performed with a 2×3 ANOVA (training group × drug). The LV, body, spleen, and adrenal gland weights, and citrate synthase activity data, will be pooled according to training status and analyzed with a two-tailed Student’s t-test (all Tr versus all Sed). A repeated-measure ANOVA will be employed for analysis of LVDP during ischemia and again following the onset of reperfusion. The main effects of the drug will be analyzed at each time point using a one-way ANOVA with a Bonferroni correction for post hoc comparisons.

Possible conclusions

The precise mechanisms responsible for exercise-induced cardioprotection are unclear, though there are numerous cardioprotection candidates that exist including improvements in the coronary circulation, increased ER stress proteins, elevated COX-2 activity, increased myocardial levels of HSP72, increased mitoKATP channels, elevated levels of sarcoKATP channels, and/or improvements in cardiac antioxidant capacity. Research has indicated that elevated myocardial MnSOD activity and increased expression of sarcoKATP channels are both important contributors to exercise-induced cardioprotection. In addition to this, it is possible that other cardioprotective mediators exist and may contribute to ExTr induced cardioprotection. Delineating the mechanisms responsible for exercise-induced protection against IR injury is important and could lead to the development of pharmacological or molecular approaches to the prevention of IR-induced myocardial injury.

References

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