Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Extracellular ATP is known to augment cardiac contractility by increasing intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes; however, the status of ATP-mediated Ca2+ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subjected to 10–30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca2+]i was measured in purified cardiomyocytes by fura-2 microfluorometry. Reperfusion for 30 min of 20-min ischemic hearts, unlike 10-min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca2+]i; no changes in basal [Ca2+]i were evident in 10- or 20-min I/R preparations. On the other hand, reperfusion of 30-min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca2+]i and a dramatic increase in basal [Ca2+]i. The positive inotropic effect of extracellular ATP was attenuated, and the maximal binding characteristics of 35S-labeled adenosine 5′-[γ-thio]triphosphate with crude membranes from hearts undergoing I/R was decreased. ATP-induced increase in [Ca2+]i in cardiomyocytes was depressed by verapamil and Cibacron Blue in both control and I/R hearts; however, this response in I/R hearts, unlike control hearts, was not affected by ryanodine. I/R-induced alterations in cardiac function and ATP-induced increase in [Ca2+]i were attenuated by treatment with an antioxidant mixture and by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2. The results suggest an impairment of extracellular ATP-induced Ca2+ mobilization in I/R hearts, and this defect appears to be mediated through oxidative stress.

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Ischemia–reperfusion-induced changes in sarcolemmal Na+/K+-ATPase are due to the activation of calpain in the heartThis article is one of a selection of papers published in a Special Issue on Oxidative Stress in Health and Disease.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y10-012 ◽

2010 ◽

Vol 88 (3) ◽

pp. 388-397 ◽

Cited By ~ 16

Author(s):

Raja B. Singh ◽

Naranjan S. Dhalla

Keyword(s):

Oxidative Stress ◽

Cardiac Function ◽

Atpase Activity ◽

Protein Content ◽

Ischemia Reperfusion ◽

Specific Inhibitor ◽

Calpain Activity ◽

Rat Hearts ◽

Intracellular Ca ◽

Calpain Inhibitors

Depression in cardiac performance due to ischemia–reperfusion (I/R) injury is associated with the development of oxidative stress and decreased sarcolemmal (SL) Na+/K+-ATPase activity. Since both I/R and oxidative stress have been reported to promote the occurrence of intracellular Ca2+ overload and activate proteases such as calpain, this study was undertaken to investigate whether the activation of calpain in I/R hearts is associated with alterations in the SL Na+/K+-ATPase activity and its isoform content. For this purpose, isolated rat hearts treated with and without 2 different calpain inhibitors (leupeptin and MDL28170) were subjected to 30 min ischemia followed by 60 min of reperfusion, and the cardiac function, SL Na+/K+-ATPase activity, Na+/K+-ATPase isoform protein content, and calpain activity were measured. The I/R-induced depressions in cardiac function, Na+/K+-ATPase activity, and protein content of Na+/K+-ATPase isoforms were associated with an increase in calpain activity , but were prevented by treatment of hearts with leupeptin. Incubation of SL membranes with calpain decreased the Na+/K+-ATPase activity and protein content of its isoforms; these changes were also attenuated by leupeptin. The I/R-induced alterations in cardiac function and the activity of SL Na+/K+-ATPase and calpain were Ca2+-dependent and were prevented by MDL28170, a specific inhibitor of calpain. The I/R-induced translocation of calpain isoforms (I and II) from the cytosol to SL and the changes in distribution of calpastatin were also attenuated by treatment with calpain inhibitors. These results suggest that the depression in cardiac function and SL Na+/K+-ATPase activity in I/R hearts may be due to changes in the activity and translocation of calpain.

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Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia–reperfused heart may be mediated through oxidative stress

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y11-128 ◽

2012 ◽

Vol 90 (2) ◽

pp. 249-260 ◽

Cited By ~ 24

Author(s):

Raja B. Singh ◽

Larry Hryshko ◽

Darren Freed ◽

Naranjan S. Dhalla

Keyword(s):

Oxidative Stress ◽

Cardiac Function ◽

Atpase Activity ◽

Proteolytic Enzymes ◽

Ischemia Reperfusion ◽

The Other ◽

Calpain Activity ◽

Rat Hearts ◽

Induced Changes ◽

Isolated Rat

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia–reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na+/K+-ATPase, Mg2+-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na+/K+-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H2O2 and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na+/K+-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na+/K+-ATPase activity. These results suggest that the I/R-induced depression in Na+/K+-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.

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Dexmedetomidine Protects against Myocardial Ischemia/Reperfusion Injury by Ameliorating Oxidative Stress and Cell Apoptosis through the Trx1-Dependent Akt Pathway

BioMed Research International ◽

10.1155/2020/8979270 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Zhi-lin Wu ◽

Jacques Robert Jeppe Davis ◽

Yi Zhu

Keyword(s):

Oxidative Stress ◽

Myocardial Ischemia ◽

Cardiac Function ◽

Cell Apoptosis ◽

Ischemia Reperfusion ◽

Cardioprotective Effect ◽

Akt Pathway ◽

Rat Hearts ◽

Myocardial Ischemia Reperfusion ◽

Isolated Rat

Dexmedetomidine (Dex) was reported to reduce oxidative stress and protect against myocardial Ischemia/Reperfusion (I/R) injury. However, the molecular mechanism involved in its antioxidant property is not fully elucidated. The present study was aimed at investigating whether the Trx1/Akt pathway participated in the cardioprotective effect of Dex. In the present study, I/R-induced myocardial injury in isolated rat hearts and OGD/R-induced injury in H9c2 cardiomyocytes were established. Our findings suggested that Dex ameliorated myocardial I/R injury by improving cardiac function, reducing myocardial apoptosis and oxidative stress, which was manifested by increased GSH and SOD contents, decreased ROS level, and MDA generation in both the isolated rat hearts and OGD/R-treated H9C2 cells. More importantly, it was found that the level of Trx1 was preserved, and Akt phosphorylation was significantly upregulated by Dex treatment. However, these effects of Dex were abolished by PX-12 (a specific Trx1 inhibitor) administration. Taken together, this study suggests that Dex plays a protective role in myocardial I/R injury, improves cardiac function, and relieves oxidative stress and cell apoptosis. Furthermore, our results present a novel signaling mechanism that the cardioprotective effect of Dex is at least partly achieved through the Trx1-dependent Akt pathway.

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Endoxin-mediated myocardial ischemia reperfusion injury in rats in vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-041 ◽

2004 ◽

Vol 82 (6) ◽

pp. 402-408 ◽

Cited By ~ 2

Author(s):

Yong-Sheng Ke ◽

He-Gui Wang ◽

De-Guo Wang ◽

Gen-Bao Zhang

Keyword(s):

Myocardial Ischemia ◽

Cardiac Function ◽

Atpase Activity ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Rat Hearts ◽

Myocardial Ischemia Reperfusion Injury ◽

Myocardial Ischemia Reperfusion ◽

Isolated Rat

Myocardial ischemia reperfusion results in an increase in intracellular sodium concentration, which secondarily increases intracellular calcium via Na+-Ca2+ exchange, resulting in cellular injury. Endoxin is an endogenous medium of digitalis receptor and can remarkably inhibit Na+/K+-ATPase activity. Although the level of plasma endoxin is significantly higher during myocardial ischemia, its practical significance is unclear. This research is to investigate whether endoxin is one of important factors involved in myocardial ischemia reperfusion injury. Ischemia reperfusion injury was induced by 30 min of global ischemia and 30 min of reperfusion in isolated rat hearts. Heart rate (HR), left ventricular developed pressure (LVDP), and its first derivative (±dp/dtmax) were recorded. The endoxin contents, intramitochondrial Ca2+ contents, and the Na+/K+-ATPase activity in myocardial tissues were measured. Myocardial damages were evaluated by electron microscopy. The endoxin and intramitochondrial Ca2+ contents in myocardial tissues were remarkably higher, myocardial membrane ATPase activity was remarkably lower, the cardiac function was significantly deteriorated, and myocardial morphological damages were severe in myocardial ischemia reperfusion group vs. control. Anti-digoxin antiserum (10, 30 mg/kg) caused a significant improvement in cardiac function (LVDP and ±dp/dtmax), Na+/K+-ATPase activity, and myocardial morphology, and caused a reduction of endoxin and intramitochondrial Ca2+ contents in myocardial tissues. In the present study, the endoxin antagonist, anti-digoxin antiserum, protected the myocardium against the damages induced by ischemia reperfusion in isolated rat hearts. The results suggest that endoxin might be one of main factors mediating myocardial ischemia reperfusion injury.Key words: endoxin, anti-digoxin antiserum, myocardial reperfusion injury, morphological evaluation, Na+/K+-exchanging ATPase.

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Effects of intermedin1–53 on cardiac function and ischemia/reperfusion injury in isolated rat hearts

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.12.071 ◽

2005 ◽

Vol 327 (3) ◽

pp. 713-719 ◽

Cited By ~ 57

Author(s):

Jing-Hui Yang ◽

Yue-Xia Jia ◽

Chun-Shui Pan ◽

Jing Zhao ◽

Ming Ouyang ◽

...

Keyword(s):

Cardiac Function ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Rat Hearts ◽

Isolated Rat

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Mechanisms of cardiodepression by an Na+–H+ exchange inhibitor methyl-N-isobutyl amiloride (MIA) on the heart: lack of beneficial effects in ischemia–reperfusion injuryThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigators' Forum.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y06-099 ◽

2007 ◽

Vol 85 (1) ◽

pp. 67-78 ◽

Cited By ~ 4

Author(s):

Harjot K. Saini ◽

Vijayan Elimban ◽

A. Tanju Ozcelikay ◽

Naranjan S. Dhalla

Keyword(s):

Sarcoplasmic Reticulum ◽

Cardiac Function ◽

Atpase Activity ◽

Direct Effect ◽

Positive Inotropic Effect ◽

Ischemia Reperfusion ◽

Negative Inotropic Effect ◽

Inotropic Effect ◽

Beneficial Effects ◽

Intracellular Ca

Although Na+–H+ exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular Ca2+ mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HCO3–-free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HCO3–-containing buffer. MIA increased the basal concentration of intracellular Ca2+ ([Ca2+]i) and augmented the KCl-mediated increase in [Ca2+]i. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils Ca2+-stimulated ATPase activity. MIA also increased Ca2+ uptake by mitochondria without having any direct effect on sarcoplasmic reticulum Ca2+ uptake. In addition, MIA did not protect the hearts subjected to mild Ca2+ paradox as well as ischemia–reperfusion-mediated injury. These results suggest that the increase in [Ca2+]i in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial Ca2+ overloading as well as indirect depression of myofibrillar Ca2+ ATPase activity. Thus the accumulation of [H+]i as well as occurrence of intracellular and mitochondrial Ca2+ overload may explain the lack of beneficial effects of MIA in preventing the ischemia–reperfusion-induced myocardial injury.

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Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01153.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. H2260-H2270 ◽

Cited By ~ 48

Author(s):

Harjot K. Saini ◽

Naranjan S. Dhalla

Keyword(s):

Oxidative Stress ◽

Cardiac Function ◽

Ischemia Reperfusion ◽

Cardiac Performance ◽

The Other ◽

Calcium Handling ◽

Subcellular Organelles ◽

Rat Hearts ◽

Intracellular Ca

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10–30 min of ischemia followed by 5–30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(−)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon β-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.

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