Bangladesh Journal of Pharmacology Research Article Hydrogen sulfide-mediated cardiopro- tection against ischemia reperfusion is linked to KATP channel for mitochondrial preservation but not for its distinct preference on interfibrillar mitochondria BJP Introduction Ischemic heart diseases are the leading causes of morta- lity all over the world (Judith et al., 2013) and the strategies to restore blood flow in the ischemic areas of the heart often encounter ischemia reperfusion injury. Hydrogen sulfide, one of the well-known cardioprotec- tive gasotransmitters (Wang, 2004), is reported to attenu -ate the ischemia reperfusion injury by preserving the mitochondrial functional activities. Miotchondrial ATP- sensitive potassium channel (mitoKATP) channel play a crucial role in regulating the mitochondrial functional activity by changing the mitochondrial matrix volume (Kaasik et al., 2007), mitochondrial respiration rate and modulating membrane potential. Among the four pota- ssium channels in the inner mitochondrial membrane, mitoKATP and mitoBKCa channels are known to interact with hydrogen sulfide (Walewska et al., 2018). Eviden- ces from the literature support a direct interaction of hydrogen sulfide on mitoKATP, where it sulphydrates the Cys43 in the Kir6.1 subunit (Mustafa et al., 2011). Many drugs like nicorandil, bimakalin, pinacidil and levosimendan, that specifically target mitoKATP are known to be effective in the management of cardio- vascular diseases especially ischemia reperfusion (Pollesello and Mebazaa, 2004). Moreover, gas signaling molecules like H2S, NO and CO are reported to provide cytoprotection via modulating the opening of mito- chondrial potassium channels. This in turn regulates different cardioprotective signaling pathways, repor- ted to have converging targets in mitochondria (O’Rourke, 2004). But recent studies have reported the presence of Abstract Hydrogen sulfide has been shown to protect myocardium against ischemia- reperfusion injury by preserving interfibrillar mitochondria functional activi- ties than subsarcolemmal mitochondria. In this study, the role of the KATP channel in modulating the mitochondrial subpopulations during the cardioprotection mediated by NaSH (H2S donor) was investigated. Isolated rat hearts were treated with mitochondrial KATP channel closer glibenclamide (10 μM)/opener diazoxide (0.8 mM) via Langendorff perfusion apparatus before ischemia-reperfusion. The results showed that NaSH pre-conditioning in presence of glibenclamide significantly improved cardiac recovery without any significant difference between interfibrillar mitochondria and subsarcole- mmal mitochondria. In conclusion, targeting KATP channel may not be good option to target interfibrillar mitochondria/subsarcolemmal mitochondria against ischemia-reperfusion injury. Article Info Received: 21 January 2019 Accepted: 1 June 2019 Available Online: 9 June 2019 DOI: 10.3329/bjp.v14i2.39890 Cite this article: Chandrasekaran P, Ravindran S, Boovarahan SR, Kurian GA. Hydro- gen sulfide-mediated cardioprotection against ischemia reperfusion is linked to KATP channel for mitochondrial preservation but not for its distinct preference on interfibrillar mitochon- dria. Bangladesh J Pharmacol. 2019; 14: 107-15. Hydrogen sulfide-mediated cardioprotection against ischemia reperfusion is linked to KATP channel for mitochondrial preservation but not for its distinct preference on interfibrillar mitochondria Priyadharshini Chandrasekaran, Sriram Ravindran, Sri Rahavi Boovarahan and Gino A. Kurian Vascular Biology Lab, School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur, Tamilnadu 613401, India. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2019; 14: 107-115 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 heterogeneous mitochondria in the myocardium that not only differ with respect to the spatial location but also with respect to biochemical activities and morpho- logy (Kuznetsov and Margreiter, 2009). Our lab has shown that mitochondria present in the myofibril named interfibrillar mitochondria exhibits resistance to ischemia reperfusion injury (Banu et al., 2016) than those present near to the membrane named subsarco- lemmal mitochondria. Moreover, there is distinct res- ponse of interfibrillar mitochondria and subsarcolem- mal mitochondria toward diazoxide, an opener of ATP- sensitive K+ channels (Holmuhamedov et al., 2012). Despite the higher preclinical success rate against ische- mia reperfusion for hydrogen sulfide, it’s potential to ameliorate revascularization injury in clinical subjects were moderate, indicating the existence of dearth in knowledge with regards to hydrogen sulfide mode of action. This brings the need to address the overlooked area of hydrogen sulfide research on ischemia reperfu- sion injury that focus on its effect on the mitochondrial sub population. In the present study, the role of KATP channel in interfibrillar mitochondria and subsarco- lemmal mitochondria in determining the efficacy of hydrogen sulfide in the management of ischemia reper- fusion injury was evaluated. Materials and Methods Animals and experimental design The experiments were carried out with 250-300 g Wistar male rats. The rats were kept under a standard condition with regular diet and water ad libitum. Perfusion protocol The rats were anesthetized with 60 mg/kg sodium thiopentone and the hearts were excised and mounted in the Langendorff apparatus (AD Instruments, Aus- tralia) as per the previous procedure with slight modifi- cation (Chevion et al., 1993). The heart was stabilized for 20 min with Krebs-Henseleit buffer (NaCl 118.0 mM, KCl 4.7 mM, CaCl2 1.9 mM, MgSO4 1.2 mM, NaHCO3 25.0 mM, KH2PO4 1.2 mM, glucose 10.1 mM) perfusion, maintained at 37°C and pH of 7.4. The buffer was oxygenated (95% O2 + 5% CO2) throughout the experiment. The perfusion was carried out according to the animal groups. Ischemia was given for 30 min by switching off the buffer flow, followed by reperfusion (reflowing the buffer into the heart) for 60 min. Preconditioning was given for 15 min in three cycles following 10 min stabilization. Each cycle consisted of 2 min ischemia followed by 3 min reperfusion. Experimental groups Rats were divided into 7 groups (n=6/group) randomly namely a) normal (to establish baseline parameters for the effects caused by subsequent manipulations, normal group hearts were subjected to continuous perfusion for 115 min with Krebs-Henseleit buffer); b) ischemia- reperfusion (ischemia-reperfusion challenged isolated rat hearts were obtained by arresting the flow of Krebs- Henseleit buffer for 30 min followed by reperfusing the heart for 60 min after 25 min of stabilization with Krebs -Henseleit buffer); c) hydrogen sulfide preconditioning (hydrogen sulfide preconditioning hearts were perfused with NaSH (20 µM) after 10 min of stabilization for 15 min followed by ischemia for 30 min and reperfusion for 60 min); d) glibenclamide ischemia- reperfusion control (treated with glibenclamide (10 µM) prior to hydrogen sulfide preconditioning protocol); e) diazoxide ischemia-reperfusion control (treated with diazoxide (0.8 mM) respectively prior to hydrogen sulfide preconditioning protocol); f) glibenclamide plus hydrogen sulfide preconditioning (treated with glibenclamide and diazoxide respectively followed by ischemia-reperfusion protocol described above), g) diazoxide plus hydrogen sulfide preconditioning (treated with diazoxide followed by ischemia-reperfusion protocol described above). The subsequent hemodynamic parameters like left ventricular end diastolic pressure in mmHg, developed pressure in mmHg, heart rate in beats per min, and rate pressure products were evaluated using LabChart pro of AD Instruments, Australia. Rate pressure product (RPP=HR*DP) was calculated using heart rate and developed pressure. Mitochondrial isolation Mitochondrial subpopulation were isolated from the heart by the method described previously (Palmer et al., 1977). Briefly, tissue homogenate was centrifuged at 800 x g for 5 min and the resulting supernatant was centri- fuged at 9,000 x g for another 10 min. The resultant pellet was centrifuged at 8000 x g twice to yield the subsarcolemmal mitochondria fraction. The pellet obtained in the initial step (800 x g, 5 min) was treated with nagarase enzyme (0.5 mg/g tissue) and subjected to differential centrifugation procedure similar to sub- sarcolemmal mitochondria isolation to yield the inter- fibrillar mitochondria fraction. All procedures were carried out at 4°C. Estimation of lactate dehydrogenase and creatine kinase The cardiac injury markers lactate dehydrogenase and creatine kinase were estimated in the heart homogenate spectrophotometrically using the previously described method (Kurian et al., 2005). Determination of infarct size Myocardial infarct size was measured after staining the heart sections with 1.5% triphenyl tetrazolium chloride at 37°C for 10 min. Images were taken using zoom stereomicroscope (Nikon SMZ1270) having high-defini- 108 Bangladesh J Pharmacol 2019; 14: 107-115 tion CCD camera (Nikon DSFi2) and NIS-elements documentation tool. Image J analysis tool (USA) was used to estimate the percentage of infarct tissue (Mensah et al., 2005). Anti-oxidant enzymes The activities of glutathione peroxidase and glutathione reductase were measured in the heart mitochondrial fraction as per the pre-described protocols (Blanken- berg et al., 2003; Goldberg and Spooner, 1983). The level of reduced glutathione in the heart mitochondrial fraction was estimated to determine the oxidative stress by the method described elsewhere (Beutler and Kelly, 1963). Catalase activity was measured by following the rate of hydrogen peroxide consumption according to the method described elsewhere (Baudhuin et al., 1964). Total superoxide dismutase activity was measured by the pre-established procedure (Nandi and Chatterjee, 1988). Mitochondrial electron transport chain and citric acid cycle enzyme activities Electron transport chain enzyme activities were mea- sured spectrophotometrically in both the mitochondrial subpopulations by using specific donor acceptors to evaluate the mitochondria’s integrity. Complex I and II activities were assessed by rotenone–sensitive NADH- oxidoreductase (NQR) and succinate decylubiquinone DCPIP reductase (SQR) respectively. Ubiquinol-cyto- chrome-C reductase (QCR) was used to assess complex III and cytochrome c oxidase (Complex IV) activity was measured as per the protocol described (Frazier and Thorburn, 2012). The citric acid cycle enzymes malate dehydrogenase and succinate dehydrogenase, and NADH dehydrogenase activity, were measured spec- trophotometrically. Statistical analysis Data were presented using ± SD. For statistical analysis, GraphPad Prism 5.0 was used. One-way analysis of variance (ANOVA) followed by Dunnet’s test was carried out to know the difference between groups. p<0.05 was considered as statistically significant. Results Effect of mito-KATP channel opener or closer on cardiac hemodynamics and injury The cardiac physiological performance in ischemia reperfusion-treated rat heart was significantly different from the normal rat heart (Table I). The use of mito- chondrial KATP channel closer (glibenclamide) and opener (diazoxide) in ischemia-reperfusion showed improved hemodynamics but prominent recovery was observed by diazoxide as measured by end diastolic pressure, developed pressure and rate pressure pro- ducts respectively. Cardiac injury was assessed via measuring triphenyl- tetrazolium chloride staining, where glibenclamide or diazoxide pretreatment exhibited 36 and 86% decline in cell death from ischemia reperfusion control respec- tively. This result was supported by significant decline of lactate dehydrogenase level (2.9 ± 0.4 and 1.8 ± 0.2 respectively) and creatine kinase level (0.4 ± 0.1 and 0.4 ± 0.2 respectively) activity in the myocardium (Figure 1). Influence of NaSH on cardiac hemodynamics and injury in presence of mito-KATP channel opener or closer After confirming the cardioprotective effect of hydro- gen sulfide via hemodynamics (rate pressure products for hydrogen sulfide pretreated group, 83 ± 3 vs nor- mal 95 ± 2), the protection in presence of mitochondrial KATP channel closer and opener were evaluated (Table I). The protective effect of hydrogen sulfide was not influenced by the presence of mitochondrial KATP channel closer or opener. There was no significant difference in cardiac infarct size between the glibenclamide plus hydrogen sulfide (5.9 ± 0.5) and diazoxide plus hydrogen sulfide (4.9 ± Table I Hemodynamic parameters Pretreatment End diastolic pressure Developed pressure Rate pressure product (mmHg beats/min x 103) Procedure Buffer - - 4 ± 2 98 ± 4 95 ± 2 Buffer Ischemia Reperfusion 43 ± 5a 42 ± 3a 32 ± 3a Hydrogen sulfide Ischemia Reperfusion 23 ± 3 93 ± 3 83 ± 3 Glibenclamide Ischemia Reperfusion 50 ± 5a 59 ± 3a 57 ± 2a Glibenclamide plus hydrogen sulfide Ischemia Reperfusion 37 ± 4 93 ± 2 90 ± 3 Diazoxide Ischemia Reperfusion 25 ± 2 89 ± 2 87 ± 2 Diazoxide plus hydrogen sulfide Ischemia Reperfusion 39 ± 3a 86 ± 1 84 ± 4 Ischemia for 30 min followed by reperfusion for 60 min; Values are expressed as mean ± SD of n = 6 rats/group. aStatistically significant from normal perfusion (p<0.05) Bangladesh J Pharmacol 2019; 14: 107-115 109 0.3) (Table II; Figure 2). This observations were further validated by level of cardiac markers in the myocar- dium and coronary perfusate and are in agreement with the above results. Impact of mito-KATP channel modulators on the mito- chondrial electron transport chain enzyme activities Mitochondrial enzymes (NADH, SDH and MDH) and the electron transport chain enzyme activities (NQR, SQR, COX, and QCR) were measured in both the mitochondrial subpopulation namely interfibrillar mitochondria and subsarcolemmal mitochondria from rat hearts subjected to ischemia-reperfusion (Figure 3). The efficiency of mitochondrial electron flux through respiratory enzymes were measured via NQR, SQR, COX and QCR activities in both sub-populations (Figure 3A, B, C and D) respectively. NQR activity representing the transfer of electrons through complex-I declined significantly in ischemia-reperfusion by 90% in subsarcolemmal mitochondria and 60% in interfibrillar mitochondria, in glibenclamide plus hydrogen sulfide- treated groups by 92% in subsarcolemmal mitochondria and 62% in interfibrillar mitochondria and diazoxide plus hydrogen sulfide-treated groups by 1.3% in subsarcolemmal mitochondria and 7.3% in interfibrillar mitochondria respectively when compared with the normal (Figure 3A). The electron transfer through complex-II represented by SQR activity decreased in Gli IR by 62 and 55% with no difference in their activity with respect to interfibrillar mitochondria and subsarcolemmal mitochondria (Figure 3B). But SQR activity was preserved in Dia IR group indicate the protective effect. The final electron acceptor in the electron transport chain (ETC), cytochrome c oxidase (COX) activity was found significantly low in both IR (63 and 60% in subsarcolemmal mitochondria and interfibrillar mitochondria respectively) and Gli IR groups (79 and 69% in subsarcolemmal mitochondria µ M o f N AD H o xi di ze d/ m in /m g pr ot ei n 5 4 3 2 1 0 Lactate dehydrogenaseA a a µ M o f i no rg an ic p ho sp ho ro us pr od uc ed /m in /m g pr ot ei n 0.8 0.6 0.4 0.2 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC Creatinine kinase B a Figure 1: Effect of hydrogen sulfide on ischemia reperfusion- induced myocardial injury: hydrogen sulfide preconditioning effect on cardiac injury markers across the groups was repre- sented by the activities of (A) lactate dehydrogenase and (B) creatine kinase. Values were expressed as mean ± SD of n=6 rats/group. astatistically significant from normal perfusion (p<0.05) Figure 2: Infarct size measurement in heart using triphenylte- trazolium chloride (TTC) staining in different conditions Dia HIPC Gli IR Gli HIPC Normal IR Dia IR Table II Infarct size measurement Pretreatment Procedure Infarct size (% of total heart) Buffer - - 3.5 ± 0.5 Buffer Ischemia Reperfusion 32.2 ± 0.7a Glibenclamide Ischemia Reperfusion 20.5 ± 0.3a Glibenclamide plus hydrogen sulfide Ischemia Reperfusion 5.9 ± 0.5 Diazoxide Ischemia Reperfusion 4.5 ± 0.5 Diazoxide plus hydrogen sulfide Ischemia Reperfusion 4.9 ± 0.3 Data represent the heart infarct size calculated as percentage area affected, from TTC staining images. Values are expressed as mean ± SD of n = 4 rats/group. aStatistically significant from normal perfusion (p<0.05) 110 Bangladesh J Pharmacol 2019; 14: 107-115 and interfibrillar mitochondria respectively). Similar to SQR activity, COX activity was found to be preserved in Dia IR groups. Effect of hydrogen sulfide in the activities of electron transport chain enzymes in mitochondrial sub-popula- tion in presence of mito-KATP channel modulators Previous publications from our lab confirmed the ability of hydrogen sulfide to preserve the ETC enzy- mes from interfibrillar mitochondria and subsarco- lemmal mitochondria with a higher preference on interfibrillar mitochondria fraction. In the present study, we demarcated the effect of HIPC in presence of mito-KATP channel modulators to find the rationale for the HIPC preference on interfibrillar mitochondria. The improved NQR activity by HIPC protocol on ischemia reperfusion challenged rat was maintained in both glibenclamide plus hydrogen sulfide and Dia HIPC, but only in subsarcolemmal mitochondria fraction. Even though glibenclamide plus hydrogen sulfide-induced heart showed improvement in interfibrillar mitochondria, the recovery was not similar as that of HIPC and Dia HIPC. However, an opposite effect was observed in SQR activity, where HIPC protocol was not effective to regain SQR activity from IR challenge, but in presence of mito-KATP channel modulators, the µ M o f N AD H o xi di ze d/ m in /m g pr ot ei n 0.4 0.3 0.2 0.1 0 NQRA a a a a µ M o f D C P IP re du ce d/ m in /m g pr ot ei n 0.8 0.6 0.4 0.2 0 SQRB a a a a SSM IFM µ M o f c yt oc hr om e C o xi di ze d/ m in /m g pr ot ei n 2.0 1.5 1.0 0.5 0 COX C a aa a a a a a µ M o f c yt oc hr om e C re du ce d/ m in /m g pr ot ei n 2.5 2.0 1.5 1.0 0.5 0 QCR D a a a a a a µ M o f N AD H o xi di ze d/ m in /m g pr ot ei n 50 40 30 20 10 0 NADH E a a a a a µ M K 3 F c(C N ) 6 re du ce d/ m in /m g pr ot ei n 60 40 20 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC SDHF a aa a a a µ M o f N AD H o xi di ze d/ m in /m g pr ot ei n 20 15 10 5 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC MDH G a Figure 3: Effect of hydrogen sulfide and KATP modulators on cardiac mitochondrial enzyme activities: Effects of hydrogen sulfide on mitochondrial functional activities in the presence of KATP modulators were evaluated from the mitochondrial enzyme and electron transport chain enzyme activities: (A) NQR, (B) SQR, (C) COX and (D) QCR (E) NADH, (F) succinate dehydrogenase (SDH), (G) malate dehydrogenase (MDH). Results are expressed as mean ± SD of n=6 rats/group. aStatistically significant from normal perfusion (p<0.05) from the normal control Bangladesh J Pharmacol 2019; 14: 107-115 111 activity was significantly improved, without any prominent difference between the subpopulations. A similar pattern of changes was observed in COX activity as well. Effect of hydrogen sulfide in the activities of NADH dehydrogeanse, malate dehydrogenase and succinate dehydrogenase Reperfusion injury induced a significant decline in the mitochondrial enzyme activities like NADH dehydro- genase (33.7% in subsarcolemmal mitochondria and 30% in interfibrillar mitochondria), SDH (9% in subsarcolemmal mitochondria and 62% in interfibrillar mitochondria) and MDH (45% in subsarcolemmal mitochondria and 32% in interfibrillar mitochondria) when compared with the normal. A similar pattern of mitochondrial enzyme activities were found with GliIR groups as well (Figure 3E, F and G). However, these enzymes were significantly improved in both subpopu- lations in the heart from Gli HIPC. Oxidative stress experienced by mitochondrial sub- population: Role of mito-KATP channel modulators The oxidative stress experienced by the mitochondrial subpopulation was evaluated through the activities of antioxidant enzymes (catalase, SOD, GPX and GR) and level of reduced glutathione, represented in Figure 4. Isolated mitochondria from ischemia reperfusion, glebenclamide or diazoxide ischemia-reperfusion groups experienced significant oxidative stress, evident from the activities of anti-oxidant enzymes. Reduced activity in catalase (17 and 24%, 12 and 23%, 5 and 6% µ M o f H 2O 2 o xi di ze d/ m in / m g pr ot ei n 10 8 6 4 2 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC CatalaseA a aa S O D ( u n it s/ m g pr ot ei n) 2.0 1.5 1.0 0.5 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC SODB aa a a SSM IFM µ M o f g lu ta th io ne u til iz ed /m in / m g pr ot ei n 0.3 0.2 0.1 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC GPX C aa a a a a µ M o f N AD PH o xi di ze d/ m in / m g pr ot ei n 6 4 2 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC GR D a a a G S H ( µ M /m g of p ro te in 0.15 0.10 0.05 0 Normal IR HIPC Gli IR Gli HIPC Dia IR Dia HIPC GSH E a a a a a Figure 4: Effect of H2S and KATP modulators on IR induced oxidative stress in cardiac mitochondrial subpopulation: Effect of H2S and KATP modulators on IR induced oxidative stress in cardiac mitochondrial subpopulation were evaluated from the activities of (A) Catalase, (B) Superoxide Dismutase (SOD), (C) Glutathione Peroxidase (GPx) and (D) Glutathione Reductase (GR), (E) Gluta- thione level (GSH). Results are expressed as mean ± SD of n = 6 rats/group. aStatistically significant from normal perfusion (p<0.05) a a aa a a a a 112 Bangladesh J Pharmacol 2019; 14: 107-115 in subsarcolemmal mitochondria and interfibrillar mitochondria), SOD (24 and 13%, 26 and 18%, 20 and 18% in subsarcolemmal mitochondria and interfibrillar mitochondria), GPx (43 and 39%, 11 and 42%, 49 and 42% in subsarcolemmal mitochondria and interfibrillar mitochondria) and GR (1 and 47%, 16 and 47%, 70 and 74% in subsarcolemmal mitochondria and interfibrillar mitochondria) were found in ischemia reperfusion, glebenclamide or diazoxide ischemia-reperfusion groups respectively, when compared with the control group. Similarly anti-oxidant potential measured by reduced glutathione level showed significantly low concentration by 11 and 36%, 26 and 43%, 49 and 65% in subsarcolemmal mitochondria and interfibrillar mito- chondria of ischemia reperfusion, glebenclamide or diazoxide ischemia-reperfusion groups respectively than normal control group. In fact, hydrogen sulfide preconditioning, glebenclamide or diazoxide ischemia- reperfusion reversed the negative effect of reperfuison injury on mitochondrial oxidative stress. Glibenclamide plus hydrogen sulfide preconditioning recovered the antioxidant enzymes level like catalase by 58 and 76% in subsarcolemmal mitochondria and interfibrillar mitochondria respectively, SOD by 61% in subsarcolemmal mitochondria and 73% in interfibrillar mitochondria respectively when compared with Gli IR. Discussion The present study had shown that hydrogen sulfide- mediated cardioprotection against ischemia-reperfu- sion injury was centered mainly on interfibrillar fraction than the subsarcolemmal mitochondria frac- tion of mitochondria (Banu et al., 2016). In the present study, it was aimed to evaluate whether hydrogen sulfide’s preference for interfibrillar over subsarco- lemmal mitochondria depends on the ATP-sensitive potassium channel, where the latter act as the thera- peutic site for the hydrogen sulfide-mediated cardio- protection. The findings from the current study indi- cate that: 1) the action of hydrogen sulfide on interfibri- llar and subsarcolemmal mitochondria fraction of mitochondria are similar in the presence of mitochon- drial potassium ATP channel inhibitor. Hydrogen sulfide has protected the ischemic heart from the reperfusion injury by modulating the mitochondrial KATP channel present in both the interfibrillar and subsarcolemmal mitochondria. Thus, the effective pre- servation of interfibrillar fraction (the key player for the myocardial contractility) of mitochondria by hydrogen sulfide may be linked to its spatial location and associated effector molecule responsible for the physiological contractile recovery. 2) Persistent open- ing of KATP channel via diazoxide had an adverse effect on the hydrogen sulfide-linked cardioprotection. Hydrogen sulfide preconditioning rendered the cardio- protection against myocardial ischemia-reperfusion injury by reducing apoptosis, preserving mitochondria via stimulating different cardioprotective signalling like reperfusion injury salvage kinase pathway and survival activated factor enhancement pathway (Heng-Fei et al., 2012). From the literature, it is evident that mitochon- drial preservation is one of the prime requirements for the intact myocardial contractile function (Kurian et al., 2012), which is governed by the electrogenic proton ejection via membrane potential that depends on K+ entry through KATP channel into the organelle. The potassium entry improves the functional coupling bet- ween creatine kinase and adenine nucleotide translo- case in mitochondria, thereby contribute to the protec- tion (Oldenburg et al., 2002). Mitochondrial KATP channel opening mediates the protection via other mediators also like the low concentration of reactive oxygen species, the resistance of calcium overload and by stimulation of cardioprotective signalling molecules, thereby improving the ATP production as well. How- ever, the mitochondria’s capacity of oxidative phospho- rylation, protein synthesis, protein and lipid composi- tion and sensitivity to metabolic challenge differs in the two different cardiac mitochondrial fractions name- ly interfibrillar and subsarcolemmal mitochondria (Boengler et al., 2017). A recent study demonstrated an enhanced salvaging effect of hydrogen sulfide on interfibrillar over subsarcolemmal mitochondria in IR challenged rat heart (Ansari and Kurian, 2016). Simi- larly, another study suggests that the response of inter- fibrillar and subsarcolemmal mitochondria towards diazoxide, KATP channel opener are different and the effect was more prominent in the subsarcolemmal mitochondria (Holmuhamedov et al., 2012). But accor- ding to the present study, mitochondrial KATP channel modulators (inhibitor/opener) did not show distinct differences between the interfibrillar and subsarco- lemmal mitochondria fraction in ischemia-reperfusion heart. These results underline the similarity of mito- chondrial KATP channel in both the interfibrillar and subsarcolemmal mitochondria. The interaction of hydrogen sulfide with KATP channel is already well established by using the patch-clamp technique and mutagenesis approach (Jiang et al., 2010). Both interfibrillar and subsarcolemmal mitochondria possess KATP channel and hydrogen sulfide action was found to be similar in interfibrillar and subsarco- lemmal mitochondria fraction of mitochondria in the presence of mitochondrial potassium ATP channel inhibitor based on the present data. The insignificant difference observed between the interfibrillar and subsarcolemmal mitochondria’s response towards mito -chondrial KATP channel modulators in the heart makes it unable to explain the distinct impact of hydrogen sulfide on interfibrillar over subsarcolemmal mito- chondria in the present study. It is evident from the literature that the cardiac KATP channel’s pharmaco- Bangladesh J Pharmacol 2019; 14: 107-115 113 logy, molecular composition and properties varies bet- ween the different cardiovascular components like ventricle, atrium, conducting system, endothelium and cell organelles like mitochondria and sarcoplasmic reti- culum (Foster and Coetzee, 2016). However, the proper- ties and pharmacological regulation of the KATP chan- nel in different cardiac cell types are not well established. The similar response of interfibrillar and subsarcolemmal mitochondria in the p interfibrillar presence of potassium ATP channel inhibitors from this study may be attributed to the non-specific modulation of potassium ATP channel by the chemical (gliben- clamide, diazoxide) in different cardiac cell types (Foster and Coetzee, 2016), that may influence the distinct preservation of interfibrillar by hydrogen sulfide during ischemia-reperfusion. By using diazoxide (KATP channel openers), the present study demonstrated the reversal of hydrogen sulfide- mediated cardioprotection and the observed adverse effect was associated with the persistence opening of the channel. This was well in agreement with previous work by Pomerantz et al, who showed tran- sient opening of mitochondrial KATP channel with diazoxide provided protection to human atrial trabe- culae against ischemia-reperfusion injury and a loss of cardioprotection was observed when diazoxide treat- ment was clubbed with ischemic preconditioning, due to the continuous opening of the potassium channel (Pomerantz et al., 2000). KATP channel modulates the mitochondrial function by changing the matrix volume, membrane potential and oxygen consumption that resulted in the release of varying concentration of reactive oxygen species and calcium ions. The concentration of diazoxide/glibenclamide plays a key role in determining the transient or permanent opening of the KATP channel. For instinct, for the pharmacological action, glibenclamide of concentration 1.2 nM is required in atrial KATP channel, whereas 1-2 µM is required to induce the effect on KATP channel in mitochondria (Foster and Coetzee, 2016). Thus, the sensitivity of the chemicals will change depending on the metabolic activity of the cardiac cell type that may be varied in different diseased conditions. The hydro- gen sulfide being a potassium channel open- er works similar to diazoxide, it is highly recommended to determine a threshold dose for the transient opening of the mKATP channel, which is essential for its clinical success. Based on the above observations, we emphasized the importance of mitochondrial subpopulations in under- standing the underlying mechanism of cardioprotective drug or procedure that works via stimulating cardiac mitochondria. Previously, investigators were more con- cerned about the healthy mitochondrial population for the drug action. But now, scientists are forced to think the number, composition and the unique contribution of the subpopulations in the healthy mitochondrial population in ischemia-reperfusion challenged the heart. The biggest challenge ahead in this area of research is to develop unique reliable marker to distin- guish the mitochondrial subpopulations and its utiliza- tion in bedside via identifying its signature in blood samples. Conclusion Targeting KATP channel may not be good option to target interfibrillar mitochondria/subsarcolemmal mitochondria against ischemia-reperfusion injury. Ethical Issue All the animal experimental protocols were approved by Institutional Animal Ethical Committee of SASTRA University, Thanjavur, Tamilnadu. Experiments were done in accordance with the guidelines of Control and Supervision of Experiments on Animals, Chennai, India (CPCSEA Approval No.: 347/ SASTRA/IAEC/RPP). Conflict of Interest The authors declare no conflicts of interest. Acknowledgement This study was funded by Department of Science and Technology, New Delhi, Government of India (No. DST-SR/ SO/HS-0255/2012) and Indian Council for Medical Research, New Delhi, Government of India (No. ICMR-5/4/1-14/12- NCD-II). References Ansari SB, Kurian GA. Hydrogen sulfide modulates sub- cellular susceptibility to oxidative stress induced by myocardial ischemic reperfusion injury. Chem Biol Interact. 2016; 252: 28-35. Banu SA, Ravindran S, Kurian GA. Hydrogen sulfide post- conditioning preserves interfibrillar mitochondria of rat heart during ischemia-reperfusion injury. Cell Stress Chaperones. 2016; 21: 571-82. Baudhuin P, Beaufay H, Rahman-Li Y, Sellinger OZ, Wattiaux R, Jacques P, De Duve C. Tissue fractionation studies: Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, d-amino acid oxidase and catalase in rat-liver tissue. Biochem J. 1964; 92: 179-84. Beutler E, Kelly BM. The effect of sodium nitrite on red cell GSH. Experientia 1963; 19: 96-97. 114 Bangladesh J Pharmacol 2019; 14: 107-115 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ansari%20SB%5BAuthor%5D&cauthor=true&cauthor_uid=27041072 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kurian%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=27041072 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ansari%20SB%5BAuthor%5D&cauthor=true&cauthor_uid=27041072 https://www.ncbi.nlm.nih.gov/pubmed/?term=Banu%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=26951457 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ravindran%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26951457 https://www.ncbi.nlm.nih.gov/pubmed/?term=Banu%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=26951457 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ravindran%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26951457 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kurian%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=26951457 https://www.ncbi.nlm.nih.gov/pubmed/?term=Banu%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=26951457 https://www.ncbi.nlm.nih.gov/pubmed/clipboard https://www.ncbi.nlm.nih.gov/pubmed/?term=Sellinger%20OZ%5BAuthor%5D&cauthor=true&cauthor_uid=4378796 https://www.ncbi.nlm.nih.gov/pubmed/?term=Wattiaux%20R%5BAuthor%5D&cauthor=true&cauthor_uid=4378796 https://www.ncbi.nlm.nih.gov/pubmed/?term=Jacques%20P%5BAuthor%5D&cauthor=true&cauthor_uid=4378796 Author Info Gino A Kurian (Principal contact) e-mail: kurian@scbt.sastra.edu Blankenberg S, Rupprecht HJ, Bickel C, Torzewski M, Hafner G, Tiret L, Smieja M, Cambien F, Meyer J, Lackner KJ. Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease. N Engl J Med. 2003; 349: 1605-13. Boengler K, Kosiol M, Mayr M, Schulz R, Rohrbach S. Mitochondria and ageing: Role in heart, skeletal muscle and adipose tissue. J Cachexia Sarcopenia Muscle. 2017; 8: 349-69. Chevion M, Jiang Y, Har-E1 R, Berenshtein E, Uretzky G, Kitrossky N. Copper and iron are mobilized following myocardial ischemia: Possible predictive criteria for tissue injury. Proc Natl Acad Sci USA. 1993; 90: 1102-06. Foster MN, Coetzee WA. KATP Channels in the cardiovascular system. Physiol Rev. 2016; 96: 177-252. Frazier AE, Thorburn DR. Biochemical analyses of the electron transport chain complexes by spectrophotometry. Methods Mol Biol. 2012; 837: 49–62. Goldberg DM, Spooner RJ. Assay of glutathione reductase. Methods Enzyme Anal. 1983; 3: 258-365. Heng-Fei L, Zhi-Bin Z, Qi-Hong Z, Pin Z, Ming-Yu Xiu, Yong Ji. Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway. Brazilian J Med Biol Res. 2012; 45: 898-905. Holmuhamedov EL, Oberlin A, Short K, Terzic A, Jahangir A. Cardiac subsarcolemmal and interfibrillar mitochondria dis- play distinct responsiveness to protection by diazo- xide. PLoS One. 2012; 7: e44667. Jiang B, Tang G, Cao K, Wu L, Wang R. Molecular mechanism for H2S-induced activation of K(ATP) channels. Antioxid Redox Signal. 2010; 12: 1167-78. Judith AF, Perviz A, Darrel PF. Mortality from ischemic heart disease by country, region, and age: Statistics from World Health Organisation and United Nations. Int J Cardiol. 2013; 168: 934-45. Kaasik A, Safiulina D, Zharkovsky A, Veksler V. Regulation of mitochondrial matrix volume. Am J Physiol Cell Physiol. 2007; 292: 157-63. Kurian GA, Berenshtein E, Saada A, Chevion M. Rat cardiac mitochondrial sub-populations show distinct features of oxidative phosphorylation during ischemia, reperfusion and ischemic preconditioning. Cell Physiol Biochem. 2012; 30: 83- 94. Kurian GA, Philip S, Varghese T. Effect of aqueous extract of desmodium gangeticum root in the severity of isoproterenol- induced myocardial infarcted rats. J Ethnopharmacol. 2005; 97: 457-61. Kuznetsov AV, Margreiter R. Heterogeneity of mitochondria and mitochondrial function within cells as another level of mitochondrial complexity. Int J Mol Sci. 2009; 10: 1911-29. Luan HF, Zhao ZB, Zhao QH, Zhu P, Xiu MY, Ji Y. Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/ STAT3 survival pathway. Braz J Med Biol Res. 2012; 45: 898- 905. Mensah K, Mocanu MM, Yellon DM. Failure to protect the myocardium against ischemia/reperfusion injury after chro- nic atorvastatin treatment is recaptured by acute atorvastatin treatment: A potential role for phosphatase and tensin homolog deleted on chromosome ten. J Am Coll Cardiol. 2005; 45: 1287–91. Murphy MP, Hartley RC. Mitochondria as a therapeutic target for common pathologies. Nat Rev Drug Discov. 2018; 17: 865 -86. Mustafa AK, Sikka G, Gazi SK, Steppan J, Jung SM, Bhunia AK, Barodka VM, Gazi FK, Barrow RK, Wang R, Amzel LM, Berkowitz DE, Snyder SH. Hydrogen sulfide as endothelium -derived hyperpolarizing factor sulfhydrates potassium channels. Circ Res. 2011; 109: 1259-68. Nandi A, Chatterjee IB. Assay of superoxide dismutase activity in animal tissues. J Biosci. 1988; 13: 305-15. O’Rourke B. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circ Res. 2004; 94: 420-32. Oldenburg O, Cohen MV, Yellon DM, Downey JM. Mitochon- drial KATP channels: Role in cardioprotection. Cardiovasc Res. 2002; 55: 429-37. Palmer JW, Tandler B, Hoppel CL. Biochemical properties of ubsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem. 1977; 252: 8731–39. Pollesello P, Mebazaa A. ATP-dependent potassium channels as a key target for the treatment of myocardial and vascular dysfunction. Curr Opin Crit Care. 2004; 10: 436-41. Pomerantz BJ, Robinson TN, Heimbach JK, Calkins CM, Miller SA, Banerjee A, Harken AH. Selective mitochondrial KATP channel opening controls human myocardial precondi- tioning (too much of a good thing?). Surgery 2000; 128: 368- 73. Ravindran S, Banu SA, Kurian GA. Hydrogen sulfide precon- ditioning shows differential protection towards interfibrillar and subsarcolemmal mitochondria from isolated rat heart subjected to revascularization injury. Cardiovasc Pathol. 2016; 25: 306-15. Walewska A, Szewczyk A, Koprowski P. Gas signaling mole- cules and mitochondrial potassium channels. Int J Mol Sci. 2018; 19: 3227. Wang R. The gasotransmitter role of hydrogen sulfide. Antioxid Redox Signal. 2004; 5: 493-501. Bangladesh J Pharmacol 2019; 14: 107-115 115 mailto:kurian@scbt.sastra.edu https://www.ncbi.nlm.nih.gov/pubmed/?term=Blankenberg%20S%5BAuthor%5D&cauthor=true&cauthor_uid=14573732 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hafner%20G%5BAuthor%5D&cauthor=true&cauthor_uid=14573732 https://www.ncbi.nlm.nih.gov/pubmed/?term=Tiret%20L%5BAuthor%5D&cauthor=true&cauthor_uid=14573732 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kaasik%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16870828 https://www.ncbi.nlm.nih.gov/pubmed/?term=Safiulina%20D%5BAuthor%5D&cauthor=true&cauthor_uid=16870828 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kaasik%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16870828 https://www.ncbi.nlm.nih.gov/pubmed/?term=Safiulina%20D%5BAuthor%5D&cauthor=true&cauthor_uid=16870828 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kaasik%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16870828 https://www.ncbi.nlm.nih.gov/pubmed/?term=Safiulina%20D%5BAuthor%5D&cauthor=true&cauthor_uid=16870828 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kurian%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=22759958 https://www.ncbi.nlm.nih.gov/pubmed/?term=Berenshtein%20E%5BAuthor%5D&cauthor=true&cauthor_uid=22759958 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kurian%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=22759958 https://www.ncbi.nlm.nih.gov/pubmed/?term=Berenshtein%20E%5BAuthor%5D&cauthor=true&cauthor_uid=22759958 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kurian%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=15740881 https://www.ncbi.nlm.nih.gov/pubmed/?term=Philip%20S%5BAuthor%5D&cauthor=true&cauthor_uid=15740881 https://www.ncbi.nlm.nih.gov/pubmed/?term=Varghese%20T%5BAuthor%5D&cauthor=true&cauthor_uid=15740881 https://www.ncbi.nlm.nih.gov/pubmed/?term=Luan%20HF%5BAuthor%5D&cauthor=true&cauthor_uid=22948409 https://www.ncbi.nlm.nih.gov/pubmed/?term=Zhao%20ZB%5BAuthor%5D&cauthor=true&cauthor_uid=22948409 https://www.ncbi.nlm.nih.gov/pubmed/?term=Murphy%20MP%5BAuthor%5D&cauthor=true&cauthor_uid=30393373 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hartley%20RC%5BAuthor%5D&cauthor=true&cauthor_uid=30393373 https://www.ncbi.nlm.nih.gov/pubmed/30393373 http://www.jbc.org/ https://www.ncbi.nlm.nih.gov/pubmed/?term=Pollesello%20P%5BAuthor%5D&cauthor=true&cauthor_uid=15616383 https://www.ncbi.nlm.nih.gov/pubmed/?term=Mebazaa%20A%5BAuthor%5D&cauthor=true&cauthor_uid=15616383 https://www.ncbi.nlm.nih.gov/pubmed/clipboard https://www.ncbi.nlm.nih.gov/pubmed/?term=Calkins%20CM%5BAuthor%5D&cauthor=true&cauthor_uid=10923018 https://www.ncbi.nlm.nih.gov/pubmed/?term=Miller%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=10923018 https://www.ncbi.nlm.nih.gov/pubmed/?term=Banerjee%20A%5BAuthor%5D&cauthor=true&cauthor_uid=10923018 https://www.ncbi.nlm.nih.gov/pubmed/?term=Miller%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=10923018 https://www.ncbi.nlm.nih.gov/pubmed/?term=Banerjee%20A%5BAuthor%5D&cauthor=true&cauthor_uid=10923018 https://www.ncbi.nlm.nih.gov/pubmed/?term=Harken%20AH%5BAuthor%5D&cauthor=true&cauthor_uid=10923018 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ravindran%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27167777 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ansari%20Banu%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27167777 https://www.ncbi.nlm.nih.gov/pubmed/?term=Ravindran%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27167777