Sriram Ravindran

Hydrogen sulfide post-conditioning preserves interfibrillar mitochondria of rat heart during ischemia reperfusion injury

Cardiac mitochondrial dysfunction is considered to be the main manifestation in the pathology of ischemia reperfusion injury, and by restoring its functional activity, hydrogen sulfide (H2S), a novel endogenous gaseotransmitter renders cardioprotection. Given that interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria are the two main types in the heart, the present study investigates the specific H2S-mediated action on IFM and SSM during ischemic reperfusion in the Langendorff rat heart model. Rats were randomly divided into five groups, namely normal, ischemic control, reperfusion control (I/R), ischemic post-conditioning (POC), and H2S post-conditioning (POC_H2S). In reperfusion control, cardiac contractility decreased, and lactate dehydrogenase, creatine kinase, and infracted size increased compared to both normal and ischemic group. In hearts post-conditioned with H2S and the classical method improved cardiac mechanical function and decreased cardiac markers in the perfusate and infarct size significantly. Both POC and POC_H2S exerts its cardioprotective effect of preserving the IFM, as evident by significant improvement in electron transport chain enzyme activities and mitochondrial respiration. The in vitro action of H2S on IFM and SSM from normal and I/R rat heart supports H2S and mediates cardioprotection via IFM preservation. Our study indicates that IFM play an important role in POC_H2S mediated cardioprotection from reperfusion injury.

Hydrogen sulfide preconditioning shows differential protection towards interfibrillar and subsarcolemmal mitochondria from isolated rat heart subjected to revascularization injury

BACKGROUND Hydrogen sulfide (H2S) is well known to protect the heart from ischemia reperfusion injury by specifically modulating the mitochondrial adenosine-triphosphate-linked potassium channel, thereby preserving mitochondrial function. The present study is designed to investigate the H2S preconditioning effect specifically on the mitochondrial subpopulation, namely, interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria. METHODS Isolated heart perfusion model with the method of Langendorff was used to induce reperfusion injury in rat hearts. The animals were randomly divided into five groups: normal, ischemia (ISC), reperfusion (IR), preconditioning (IPC), and H2S preconditioning (HIPC). All the groups, except normal and ischemia, were subjected to 30-min global ischemia followed by 60-min reperfusion with Krebs-Henseleit buffer. RESULTS Our study results show that H2S at a dose of 20 μM significantly (P<.05) reduced the infarct size (59%) and the creatine kinase and lactate dehydrogenase activity in cardiac tissue. DNA fragmentation as observed in ischemia reperfusion control was absent in case of H2S-preconditioned heart. On comparing the classical protection provided by IPC with H2S, a significant recovery was seen in the IFM fraction in case of HIPC, whereas the SSM could not recover as evidenced by better mitochondrial respiration rate and electron chain enzyme activities. Studies on isolated mitochondrial subpopulation from normal, IR, and IPC hearts exposed to H2S in vitro support the above observation. CONCLUSION The present study concluded that IFM shows major contribution towards H2S-mediated cardioprotection, whereas classical IPC recovered both subpopulations from IR injury.

Vascular calcification abrogates the nicorandil mediated cardio-protection in ischemia reperfusion injury of rat heart

The present study was aimed to determine the efficacy of nicorandil in treating cardiac reperfusion injury with an underlying co-morbidity of vascular calcification (VC). Adenine diet was used to induce VC in Wistar rat and the heart was isolated to induce global ischemia reperfusion (IR) by Langendorff method, with and without the nicorandil (7.5mg/kg) pre-treatment and compared with those fed on normal diet. The adenine-treated rats displayed abnormal ECG changes and altered mitochondrial integrity compared to a normal rat heart. These hearts, when subjected to IR increased the infarct size, cardiac injury (measured by lactate dehydrogenase and creatine kinase activity in the coronary perfusate) and significantly altered the hemodynamics compared to the normal perfused heart. Nicorandil pretreatment in rat fed on normal diet enhanced the hemodynamics significantly (P<0.05) along with a substantial reduction in the mitochondrial dysfunction (measured by high ADP to oxygen consumption ratio, respiratory control ratio, enzyme activities and less swelling behavior) when subjected to IR. However, this cardio-protective effect of nicorandil was absent in rat heart with underlying calcification. Our results suggest that, the protective effect of nicorandil, a known mitochondrial ATP linked K+ channel opener, against myocardial reperfusion injury was confined to normal rat heart.

Fisetin Confers Cardioprotection against Myocardial Ischemia Reperfusion Injury by Suppressing Mitochondrial Oxidative Stress and Mitochondrial Dysfunction and Inhibiting Glycogen Synthase Kinase 3β Activity

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide. Timely reperfusion is considered an optimal treatment for AMI. Paradoxically, the procedure of reperfusion can itself cause myocardial tissue injury. Therefore, a strategy to minimize the reperfusion-induced myocardial tissue injury is vital for salvaging the healthy myocardium. Herein, we investigated the cardioprotective effects of fisetin, a natural flavonoid, against ischemia/reperfusion (I/R) injury (IRI) using a Langendorff isolated heart perfusion system. I/R produced significant myocardial tissue injury, which was characterized by elevated levels of lactate dehydrogenase and creatine kinase in the perfusate and decreased indices of hemodynamic parameters. Furthermore, I/R resulted in elevated oxidative stress, uncoupling of the mitochondrial electron transport chain, increased mitochondrial swelling, a decrease of the mitochondrial membrane potential, and induction of apoptosis. Moreover, IRI was associated with a loss of the mitochondrial structure and decreased mitochondrial biogenesis. However, when the animals were pretreated with fisetin, it significantly attenuated the I/R-induced myocardial tissue injury, blunted the oxidative stress, and restored the structure and function of mitochondria. Mechanistically, the fisetin effects were found to be mediated via inhibition of glycogen synthase kinase 3β (GSK3β), which was confirmed by a biochemical assay and molecular docking studies.

The role of secretory phospholipases as therapeutic targets for the treatment of myocardial ischemia reperfusion injury

Myocardial reperfusion injury is a consequence of restoration of blood flow post ischemia. It is a complex process involving an acute inflammatory response activated by cytokines, chemokines, growth factors, and mediated by free radicals, calcium overload leading to mitochondrial dysfunction. Secretory phospholipases (sPLA2) are a group of pro-inflammatory molecules associated with diseases such as atherosclerosis, which increase the risk of reperfusion injury. This acute response leads to breakdown of phospholipids such as cardiolipin, found in the mitochondrial inner membrane, leading to disruption of energy producing enzymes of the electron transport chain. Thus the activation of secretory phospholipases has a direct link to the vascular occlusion and arrhythmia observed in myocardial reperfusion injury. Therapeutic agents targeting sPLA2 are under human trials and many are in the preclinical phase. This article reviews the pathological effects of various groups of secretory phospholipases (I, II, V and X) implicated in myocardial ischemia reperfusion injury and the phospholipase inhibitors under development. Considering the fact that human trials in this class of drugs is limited, sPLA2 as a potential target for drug development is emphasized.

Effect of Sodium Thiosulfate Postconditioning on Ischemia-Reperfusion Injury Induced Mitochondrial Dysfunction in Rat Heart

The recent research on the therapeutic applications of sodium thiosulfate (STS) has gained importance in the treatment of cardiovascular diseases. Progressively through the present work, we have demonstrated that postconditioning of isolated rat heart subjected to ischemia-reperfusion injury using STS had preserved the mitochondrial structure, function, and number. Heart comprising of two mitochondrial subpopulations interfibrillar (IFM—involved in contractile function) and subsarcolemmal (SSM—involved in metabolic function), STS postconditioning imparted a state of hypometabolism to SSM, thereby reducing the metabolic demand of the reperfused heart. The IFM, on the other hand, provided the energy required to maintain contraction. Moreover, the hypometabolic state induced in SSM can lower the free radical release in addition to STS innate ability to act as an antioxidant and radical scavenger, all of which collectively provided cardioprotection. Therefore, drugs targeting IFM specifically or those reducing the energy demand for SSM can be suitable targets for myocardial ischemia-reperfusion injury.

Role of endogenous hydrogen sulfide in cardiac mitochondrial preservation during ischemia reperfusion injury

Cardio-protective effect of hydrogen sulfide (H2S) against myocardial ischemia reperfusion injury (I/R) via preservation of mitochondria is well documented. But the distinct role of exogenous and endogenous H2S in cardio-protection and its dependency on functional cardiac mitochondria is not understood. The present study was designed to investigate the role of exogenous H2S preconditioning on cardiac mitochondrial subpopulation namely interfibrillar (IFM) and subsarcolemmal (SSM), in attenuating I/R injury in an isolated rat heart model in the absence of endogenous H2S production. The well-known inhibitor of endogenous H2S production DL-propargylglycine (cystathionine gamma lyase inhibitor) used for this purpose. Our previous studies revealed that NaSH (a H2S donor) preconditioning at a dose of 20μM significantly reduced the infarct size and preserved the functional activities of IFM. However, this protective effect significantly declined, when the heart preconditioned with NaSH in the presence of PAG. Apparently, cardiac recovery was improved hemodynamically to a minimal level by increased concentration of NaSH (40μM). The rat heart perfused with 2, 4 dinitrophenol a mitochondrial uncoupler, before NaSH preconditioning to understand the mitochondrial dependency of exogenous NaSH, we found that cardio-protective effect of NaSH was negated even with higher concentration of H2S. The result indicates that exogenous NaSH mediated cardio protection depends on functional cardiac mitochondria. However, no functional difference between the subpopulation i.e., interfibrillar and subsarcolemmal mitochondria observed with NaSH preconditioning in the presence of PAG.

Sodium thiosulfate mediated cardioprotection against myocardial ischemia-reperfusion injury is defunct in rat heart with co-morbidity of vascular calcification

Sodium thiosulfate (STS) has shown promising effects in amelioration of myocardial ischemia-reperfusion injury (IR) in a rat model and is clinically useful in the treatment of chronic kidney disease (CKD) associated calciphylaxis. As the prevalence of cardiac complications is higher in CKD, we tested the effectiveness of STS in a rat model of adenine-induced vascular calcification and subjected the heart to IR. We observed an increased infarct size (29%) by TTC staining, lactate dehydrogenase (54%) and creatine kinase (32%) release in the coronary perfusate and altered hemodynamics compared to a normal rat treated with STS and subjected to IR. As functional mitochondria are essential for preserving heart from the detrimental effects of IR, we found that calcification induced mitochondrial dysfunction (reduced RCR->80%, P/O ratio->30%, ΔΨ->10% and swelling- 27%), could not be restored efficiently by STS treatment. Therefore we used nicorandil (mitochondrial potassium channel opener) along with STS as a combination therapy to treat the diseased heart and found an improvement in cardioprotection against IR injury, compared to STS alone. Upon evaluating these hearts, we found that both the cardiac mitochondria namely interfibrillar and subsarcolemmal were functionally well preserved.

Renal mitochondria can withstand hypoxic/ischemic injury secondary to renal failure in uremic rats pretreated with sodium thiosulfate

BACKGROUND: Sodium thiosulfate (STS) is a potent drug used to treat calcific uremic arteriopathy in dialysis patients and its mode of action is envisaged by calcium chelation and antioxidant potential. STSs action on mitochondrial dysfunction, one of the major players in the pathology of vascular calcification is yet to be explored. METHODS: Adenine (0.75%, 28 days)-treated vascular calcified rat kidney was used to isolate mitochondria, where the animal was administered with or without STS for 28 days. Isolated mitochondria were subjected to physiological oxidative stress by nitrogen gas purging (hypoxia/ischemia-reperfusion injury) to assess mitochondrial recovery extent due to STS treatment in vascular calcified rat kidney. RESULTS: The results confirmed an elevated oxidative stress and deteriorated mitochondrial enzyme activities in all groups except the drug-treated group. CONCLUSION: The STS treatment, besides rendering renal protection against adenine-induced renal failure, also helped to maintain mitochondrial functional integrity in a later insult due to hypoxia/ischemia-reperfusion injury.