The large conductance calcium and voltage-activated potassium channels (MaxiK, BK_Ca, K_Ca1.1) encoded by Kcnma1 gene are ubiquitously expressed in excitable and non-excitable cells (1, 2). The functional channel is comprised of four pore-forming α-subunits, each with seven transmembrane domains where S4 serves as a voltage sensor and C-terminus contains Ca²⁺-sensing RCK1 and RCK2 domains (3). Ca²⁺ and voltage sensing allow activation of BK_Ca (4), resulting in its physiological involvement in neurotransmitter release and secretion (2). Increasing evidence indicates that BK_Ca channels are located in intracellular organelles in addition to the plasma membrane, extending their functional roles in cellular physiology from organelle to organ level (1, 2, 5–10).

Studies involving activation (10–15) and inactivation (11, 16) with pharmacological and genetic tools, including global (10), and tissue-specific knockouts (17), have implicated BK_Ca channels in cardiac function, neuroprotection (18), and cardioprotection from ischemia-reperfusion (IR) injury, in addition to IR-induced inflammation and mucosal barrier disruption in the small intestine (19). Further, it was shown that BK_Ca is present in the mitochondria of adult cardiomyocytes (10, 20). Tissue-specific knockouts in which BK_Ca was ablated in adult cardiomyocytes showed that expression of mitochondrial BK_Ca is responsible for its cardioprotective effect (17). It has been shown that agonists or antagonists have no effect on global (10) and cardiomyocytes-specific (17) knockouts. However, mice expressing activated BK_Ca have not been tested for cardioprotection from IR injury (8). Genetically modifying BK_Ca in mice by introducing a mutation responsible for its constitutive activation (8), independent of pharmacological agents, can further support the role of BK_Ca in cardioprotection from IR injury.

One of the possible outcomes of pharmacological activation or inactivation of BK_Ca is decrease/increase in the production of reactive oxygen species (ROS) (21–24). The reduction in the levels of ROS accompanied by “mild” mitochondrial uncoupling (25) by BK_Ca agonists is assigned as a possible mechanism for organ and cellular protection from IR injury (26). As stated earlier, all of these studies rely on the use of pharmacological tools with possible non-specific effects. To understand the role of activation of BK_Ca and its influence on mitochondrial ROS generation, studies need to be performed independent of the pharmacological agents. Non-specific and off-target effects of pharmacological tools have generated reservations (12) on the role of BK_Ca in modulating levels of mitochondrial ROS as well as cardioprotection from IR injury.

In the current study, we have used genetically-activated mice where BK_Ca is constitutively active due to incorporation of a gain of function mutation (Tg-BK^R207Q or Tg-BK_Ca) (8) to test the role of BK_Ca activation in mitochondrial ROS generation and cardioprotection from IR injury. We have established that the activation of BK_Ca is vital for a cardioprotective effect in both IR as well as IPC using an ex vivo isolated perfused heart model. We have further shown that activation of BK_Ca, attenuates ROS from complex I and complex II/III of mitochondria only after IR injury. Our results presented here further corroborate the role of BK_Ca in cardioprotection.

All of the experiments on mice were approved by the Institutional Animal Care and Use Committee at the Drexel University and the Ohio State University. Animals were housed in the vivarium with food and water available ad libitum. Experiments were carried out on 3 month-old male and female. Experimentalists were blinded for the genotype of mice used.

Overall, our results demonstrate that expression and activation of BK_Ca are vital for cardioprotection from IR injury as well as IPC-mediated cardioprotection. Cardioprotection mediated by BK_Ca is possibly modulated by mitochondrial ROS production.

BK_Ca channels are modulated by calcium, voltage, and several other cellular components, making them key pathophysiological targets. Auxiliary proteins, β and γ subunits also directly regulate the activity of BK_Ca channels, as well as, their sensitivity toward channel activators and blockers. One of the major breakthroughs in BK_Ca-centric research over the past two decades is the availability of small molecular openers for BK_Ca channels. These small molecules and pharmacological agents have provided ample opportunities to decipher the role of BK_Ca channels in physiological functions. The use of several pharmacological agents including, NS1619 and NS11021, to activate BK_Ca has been shown to play a vital role in cardioprotection and neuroprotection from IR injury. However, due to non-specific and off-target effects of drugs (40–44), clinical application of BK_Ca agonists is not yet initiated. Preclinical and basic research provides sufficient evidence for a translational aspect of BK_Ca activators but due to limitations related to specificity and the lack of potency (40–44), concerns have been raised on the role of activation of BK_Ca in pathological conditions (45). The need to revisit the issue related to the role of activation of BK_Ca in a pathological condition such as IR injury is critical in the light of recent findings that expression of BK_Ca is vital for cardioprotection using global (10, 32) and cardiac-specific (17) knockout mice.

In this study, we have used transgenic mice expressing the BKCaR207Q mutant subunit driven by Per1 locus. Per1 is ubiquitously expressed in a wide variety of tissues and in a previous study, it was shown that Tg-BKCaR207Q mice displayed overexpression of constitutively-activated BK_Ca in several different tissues including aorta. In adult cardiomyocytes and cardiac mitochondria isolated from Tg-BK_Ca mice, there were no differences observed in the localization of BK_Ca, implying that overexpression of the BKCaR207Q mutant does not interfere with its cellular or organelle localization. We have not observed any abnormal phenotype or behavior in Tg-BK_Ca mice. Cardiovascular analysis using echocardiography also did not demonstrate abnormal cardiac function, in comparison to the wild-type mice. Hence, Tg-BK_Ca mice provide an appropriate tool to test whether overexpression and activation of BK_Ca play a role in cardioprotection as shown by global (10) and cardiomyocytes-specific (17) null mutant mice.

In the last two decades, several studies using pharmacological tools have suggested a protective role of BK_Ca from IR injury and in IPC-mediated cardioprotection and are summarized in recent reviews (1, 5). The first study to implicate BK_Ca in cardioprotection (i.e., improved LVDP and reduced infarction) from global IR injury was conducted using the agonist NS1619, the effects of which were blocked by paxilline (20). Improved LVDP by preconditioning with 3 μM NS1619 was confirmed by Stowe et al. (38) possibly by modulating mitochondrial ROS and Ca²⁺ concentrations. The same group has proposed that opening of BK_Ca elevates the level of K⁺ in the matrix of mitochondria, which is exchanged for H⁺ ion by a K⁺/H⁺ exchanger (46). Increase in the level of H⁺ in the mitochondrial matrix stabilizes mitochondrial membrane potential (ΔΨ_m) so that unutilized electrons combine with oxygen to generate a small amount of mitochondrial oxygen radicals and other ROS, which then protects the cardiomyocytes by stimulating downstream protective signaling pathways. In addition to NS1619, studies involving other BK_Ca channel openers, NS11021 (14) and naringenin (47), have also demonstrated that cellular and cardioprotection from IR injury further provide strong evidence for the role of BK_Ca in cardioprotection. However, in primary rat cortical neurons, NS1619-preconditioning caused mitochondrial depolarization which could not be prevented by paxilline (48).

Pharmacological data using agonists and antagonists provides strong evidence for the role of BK_Ca in cardioprotection. However, the same set of drugs have resulted in non-specific effects in cells and organs. Paxilline, although is a specific BK_Ca-blocker, abolished isoflurane-mediated cardioprotection in wild-type as well as Kcnma1^−/− mice¹². NS1619 (>10 μM) is known to inhibit SERCA (49), mitochondrial respiratory chain (44, 50), and H⁺/K⁺ leak (46). These issues have largely been addressed by using low concentrations (≤10 μM) of NS1619 in conjunction with global (10) and cardiomyocytes specific (17) BK_Ca knockout mice. Our results using activated BK_Ca reiterated and further supported the studies involving global (10, 12, 32) and cardiomyocytes specific (17) knockout mice.

Interestingly, we observed an increase in the LVDP amongst a few hearts from Tg-BK_Ca mice exposed to IPC (4 out of 7) beyond 100% during the reperfusion stage, in comparison to the baseline. We used a constant flow approach for this experiment. We anticipate that in the presence of some degree of infarction, the remaining cardiomyocytes would contract more and beat faster to balance the pressure overload arising due to the constant flow of buffer solution to the isolated heart. An increase in LVDP and + dp/dt was also observed by Okazaki et al. after IR-injury (51). Similarly, Papanicolaou et al., also observed an increase in LDVP up to 150% in mitofusin-2 deleted hearts after 10 min of ischemia and 20 min of reperfusion ex vivo, using Langendorff model (52). Moreover, an increase in the LVDP is associated with an increase in the flow rate (51) in ex vivo IR study and BK_Ca activator, rottlerin, is known to increase the flow rate post ex vivo IR (24).

As others have shown, we too provide evidence that BK_Ca is exclusively present in mitochondria of adult cardiomyocytes and modulate mitochondrial function. In a recently published study, the notion of BK_Ca in adult cardiomyocytes playing a role in cardioprotection was confirmed, and cardioprotective role via BK_Ca in smooth muscle cells was ruled out by using tissue-specific knockouts (17). However, an argument regarding the role or necessity of activation of BK_Ca to provide cardioprotection from IR injury is still valid. Our study involving mice expressing activated BK_Ca without any pharmacological tools support the notion that in addition to the expression of BK_Ca, activation of BK_Ca is also important in cardioprotection from IR injury. Expression of BKCaR207Q protected the heart from IR injury, as well, as improved the recovery of LVDP after IPC and IR injury. Further, we also noticed a remarkable decrease in myocardial infarction in hearts isolated from Tg-BK_Ca mice as compared to wild-type mice. IPC as anticipated, reduced myocardial infarction in wild-type mice which was further augmented in Tg-BK_Ca mice, implying that activation of BK_Ca can further enhance the IPC-mediated cardioprotection from IR injury. Soltysinka et al. (32) demonstrated a cause-effect relationship between “IPC-mediated cardioprotection” and “BK_Ca” using global BK_Ca knockout mice. Cardioprotective property of IPC was lost in the hearts collected from BK_Ca knockout mice (31). Our results along with other studies using global or tissue-specific knockout mice imply that expression and activation of BK_Ca play an important role in cardioprotection from IR injury.

In IR injury, ROS is well-characterized to be the major player as a second messenger involved in preconditioning (53). Complex III of the electron transport chain (ETC) is the main site for ROS production, and the ROS produced is directed away from the antioxidant defenses of the mitochondrial matrix (54). However, complex I mediated ROS products are released in the mitochondrial matrix in the proximity of defense enzyme systems and is known to change the redox state of proteins present in the mitochondrial matrix, which causes a deleterious impact on cellular physiology (55). Association of K⁺ channels to mitochondrial ROS has been well-established (56, 57). Specifically, K_ATP channels mediate influx of K⁺ into the mitochondrial matrix resulting in a small augmentation of ROS production to induce cardioprotection (56, 57). In contrast, pharmacological tools aimed at BK_Ca channels indicate that the opening of BK_Ca reduces IR-induced large-scale ROS production whereas closing the BK_Ca channel increases deleterious ROS production (22, 24, 58). Recently, using cardiac-specific BK_Ca knockout mice, it was shown that the absence of BK_Ca increases ROS (17) which has been proposed earlier to regulate endoplasmic calcium release (59). Increase in ROS in the knockout mice is independent of change in expression of ROS degrading enzymes such as CuZnSOD (SOD1) and MnSOD (SOD2) (17). We have observed that activation of BK_Ca does not affect the ROS production at the basal levels, however, after IR injury, activation of BK_Ca results in a reduction in ROS production. The decline was observed for both complexes of the ETC in isolated mitochondria from Tg-BK_Ca mice. The other factor to be taken into account is the amount in addition to the site of production of ROS available in the cell (55). IPC reduced ROS generated by complexe I in wild-type mice which indicate that reducing the total amount of ROS can also protect the cardiac tissue.

Activation of BK_Ca is also known to increase the Ca²⁺ retention capacity of mitochondria (10). Our recent findings on Ca²⁺ (10), and ROS in this study, and studies from other groups have indicated that BK_Ca-mediated cardioprotection involves an interplay between ROS, Ca²⁺ and mitochondrial permeability transition pore (mPTP) (1, 5, 22, 31, 38, 46, 60–62). One possible mechanism is an increase in Ca²⁺ retention capacity possibly by modulating a mitochondrial Ca²⁺ pump on activation of BK_Ca, hence allowing more Ca²⁺ uptake during ischemia-reperfusion. Also, blocking BK_Ca channels either pharmacologically or genetically enhances ROS production, hence increasing myocardial infarction. The precise manner of how Ca²⁺ modulates ROS generation is not well-understood. However, three-dimensional conformational changes induced by Ca²⁺ in the ETC complexes, such as changes in complex IV have been reported (63, 64). Direct addition of BK_Ca channel activator in isolated mitochondrial preparation results in depolarization (5, 46) of mitochondrial membrane potential which made it difficult to study mitochondrial BK_Ca channels in isolated preparations. However, the use of genetic approaches clearly indicates that partial activation (Tg-BK_Ca) does not change mitochondrial ROS generation under physiological conditions. During stress, the increase in Ca²⁺ influx could further increase the open probability of the channel which could result in a reduction of ROS as observed in our studies. Since the decrease in ROS levels has been associated with cardioprotection from IR injury (65, 66), we anticipate that activation of BK_Ca reduces deleterious ROS production, hence decreases Ca²⁺ release from endoplasmic reticulum and reduction in an influx of Ca²⁺ to the matrix and prevents Ca²⁺ overload in mitochondria (59). This complex interplay between Ca²⁺ and ROS is known to result in apoptosis, possibly by opening the mPTP. Hence, activation of BK_Ca can be linked to delay in the formation and/or closing of mPTP.

In summary, our study implicates overexpression of activated BK_Ca in cardioprotection against IR injury, and cardioprotection is mediated, in part, by decreasing deleterious mitochondrial ROS generation.

Even though, our study does not involve pharmacological tools, there are several limitations which should be mentioned. The Tg-BK_Ca mice are not homozygous but are generated in the background of wild-type mice as the homozygous mouse is embryonic lethal (8). The animals are phenotypically normal. This finding is important as usage of heterozygous mice suggests that partial activation of BK_Ca is sufficient for cardioprotection and reduction of mitochondrial ROS. The gain-of-function BK_Ca is present in all the cells under Per1 locus. Since Per1 is present in most cells types, the effect we observed could also arise from non-cardiomyocytes. Non-cardiomyocyte cells such as cardiac neurons are known to play a role in cardioprotection as well as cellular protection as reported earlier (12). Our study does not rule out the role of non-cardiomyocyte BK_Ca in cardioprotection from IR injury. Our ex vivo approach using the Langendorff method partially rules out non-cardiac BK_Ca as the heart is excised and isolated from other organs during the experiment. Since hearts were not paced at a constant rate, ± dp/dt may not be a good index of cardiac function. Therefore, we did not report ± dp/dt. We further isolated mitochondria and measured ROS levels with and without IR. Our results also indicate that BK_Ca channel activation can modulate mitochondrial ROS levels. We have observed statistical significance in between WT and Tg-BK_Ca IR for LVDP, infarction, and ROS production but not in WT IR vs. IPC when one-way ANOVA followed by Tukey's multiple comparison tests was used. This could be due to the shorter duration of reperfusion (30 min).

Tg-BK_Ca construct is generated on DEC splice variant (8) which is known to facilitate localization of BK_Ca to mitochondria (10), and we did not observe any change in localization of BK_Ca in cardiomyocytes or isolated mitochondria. Therefore, we corroborate earlier findings involving pharmacological and global, as well as cardiac-specific null mutant mice, in addition to the expression of BK_Ca, activation of BK_Ca plays an important role in cardioprotection from IR injury.

The study was carried out in accordance with the recommendations from National Institute of Health. All protocols involving animals were approved by the Drexel University College of Medicine and the Ohio State University IACUC.

SKG, DP, ATH, KS, PK, SGR, MK, and HS performed the research, analyzed data and wrote the manuscript. ALM generated Tg-BK and BK mutant mice.