Activation of proteases and changes in Na+-K+-ATPase subunits in hearts subjected to ischemia-reperfusion

2013 ◽  
Vol 114 (3) ◽  
pp. 351-360 ◽  
Author(s):  
Alison L. Müller ◽  
Darren Freed ◽  
Naranjan S. Dhalla
Keyword(s):  
Atpase Activity ◽  
Protein Content ◽  
Cardiac Dysfunction ◽  
Ischemia Reperfusion ◽  
Global Ischemia ◽  
Calpain Inhibitor ◽  
Mmp Inhibitor ◽  
Rat Hearts ◽  
Atpase Subunits

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and changes in sarcolemmal Na+-K+-ATPase subunits and activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, as well as reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 30–60 min of global ischemia were accompanied by augmented activities of both calpain and matrix metalloproteinases (MMPs) and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. Compared with control values, the activities of both calpain and MMP-2 were increased, whereas the activity and protein content for all subunits of Na+-K+-ATPase were decreased upon reperfusion for 5–40 min, except that α1- and α2-subunit content was not depressed in 5 min I/R hearts. MDL28170, a calpain inhibitor, was more effective in attenuating the I/R-induced alterations in cardiac contracture, Na+-K+-ATPase activity, and α2-subunit than doxycycline, an MMP inhibitor. Incubation of control sarcolemma preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1-, α2-, and β2-subunits, without changes in the β1-subunit. These results support the view that activation of both calpain and MMP-2 are involved in depressing Na+-K+-ATPase activity and degradation of its subunits directly or indirectly in hearts subjected to I/R injury.

Role of oxidative stress in ischemia–reperfusion-induced alterations in myofibrillar ATPase activities and gene expression in the heartThis article is one of a selection of papers from the NATO Advanced Research Workshop on Translational Knowledge for Heart Health (published in part 1 of a 2-part Special Issue).

2009 ◽  
Vol 87 (2) ◽  
pp. 120-129 ◽  
Author(s):  
Srilekha Maddika ◽  
Vijayan Elimban ◽  
Donald Chapman ◽  
Naranjan S. Dhalla
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Atpase Activity ◽  
Cardiac Dysfunction ◽  
Ischemia Reperfusion ◽  
Myosin Heavy Chain Isoforms ◽  
Minute Period ◽  
Research Workshop ◽  
Rat Hearts

Ischemia–reperfusion (IR) in the heart has been shown to produce myofibrillar remodeling and depress Ca2+ sensitivity of myofilaments; however, the mechanisms for these alterations are not clearly understood. In view of the role of oxidative stress in cardiac dysfunction due to IR, isolated rat hearts were subjected to global ischemia for 30 min followed by a 30-minute period of reperfusion. IR was found to induce cardiac dysfunction, as reflected by depressed LVDP, +dP/dt, and –dP/dt, and elevated LVEDP, and to reduce myofibrillar Ca2+-stimulated ATPase activity. These changes were simulated by perfusing the hearts with a mixture of xanthine plus xanthine oxidase, which is known to generate oxyradicals. The alterations in cardiac function and myofibrillar Ca2+-stimulated ATPase in IR hearts were attenuated by pretreatment with antioxidants (superoxide dismutase plus catalase, and N-acetylcysteine) and leupeptin, an inhibitor of Ca2+-dependent protease. The levels of mRNA for myosin heavy chain isoforms (α-MHC and β-MHC) and myosin light chain (MLC1) were depressed in IR hearts. These changes in gene expression due to IR were prevented upon perfusing the hearts with superoxide plus catalase, with N-acetylcysteine, or with leupeptin. The results suggest that oxidative stress due to IR injury and associated proteolysis play an important role in inducing changes in myofibrillar Ca2+-stimulated ATPase activity and gene expression in the heart.

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.

2010 ◽  
Vol 88 (3) ◽  
pp. 388-397 ◽  
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.

scholarly journals Neonatal Rat Hearts Cannot Be Protected by Ischemic Postconditioning

2015 ◽  
pp. 789-794 ◽  
Author(s):  
J. DOUL ◽  
Z. CHARVÁTOVÁ ◽  
I. OŠŤÁDALOVÁ ◽  
M. KOHUTIAR ◽  
H. MAXOVÁ ◽  
...  
Keyword(s):  
Isometric Force ◽  
Ischemia Reperfusion ◽  
Force Transducer ◽  
Global Ischemia ◽  
Ischemic Postconditioning ◽  
Neonatal Rat ◽  
Postnatal Life ◽  
Protective Effects ◽  
Rat Hearts ◽  
Ldh Release

Although there are abundant data on ischemic postconditioning (IPoC) in the adult myocardium, this phenomenon has not yet been investigated in neonatal hearts. To examine possible protective effects of IPoC, rat hearts isolated on days 1, 4, 7 and 10 of postnatal life were perfused according to Langendorff. Developed force (DF) of contraction was measured by an isometric force transducer. Hearts were exposed to 40 or 60 min of global ischemia followed by reperfusion up to the maximum recovery of DF. IPoC was induced by three cycles of 10, 30 or 60 s periods of global ischemia/reperfusion. To further determine the extent of ischemic injury, lactate dehydrogenase (LDH) release was measured in the coronary effluent. Tolerance to ischemia did not change from day 1 to day 4 but decreased to days 7 and 10. None of the postconditioning protocols tested led to significant protection on the day 10. Prolonging the period of sustained ischemia to 60 min on day 10 did not lead to better protection. The 3x30 s protocol was then evaluated on days 1, 4 and 7 without any significant effects. There were no significant differences in LDH release between postconditioned and control groups. It can be concluded that neonatal hearts cannot be protected by ischemic postconditioning during first 10 days of postnatal life.

scholarly journals Hydrogen sulfide-mediated cardioprotection against ischemia reperfusion is linked to KATP channel for mitochondrial preservation but not for its distinct preference on interfibrillar mitochondria

2019 ◽  
Vol 14 (2) ◽  
pp. 107-115 ◽  
Author(s):  
Priyadharshini Chandrasekaran ◽  
Sriram Ravindran ◽  
Sri Rahavi Boovarahan ◽  
Gino A. Kurian
Keyword(s):  
Hydrogen Sulfide ◽  
Reperfusion Injury ◽  
Katp Channel ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Rat Hearts ◽  
Significant Difference ◽  
Interfibrillar Mitochondria ◽  
Subsarcolemmal Mitochondria

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 subsarcolemmal mitochondria.  In conclusion, targeting KATP channel may not be good option to target interfibrillar mitochondria/subsarcolemmal mitochondria against ischemia-reperfusion injury.

scholarly journals Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

2010 ◽  
Vol 298 (2) ◽  
pp. H570-H579 ◽  
Author(s):  
Chengqun Huang ◽  
Wayne Liu ◽  
Cynthia N. Perry ◽  
Smadar Yitzhaki ◽  
Youngil Lee ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Enhanced Recovery ◽  
Ischemia Reperfusion ◽  
Dominant Negative ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Kinase C ◽  
Rat Hearts

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15 ). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor of autophagy, Tat-Atg5K130R. Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5K130R. Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia.

scholarly journals Critical Role of AT1Receptor Expression After Ischemia/Reperfusion in Isolated Rat Hearts

1998 ◽  
Vol 83 (5) ◽  
pp. 552-559 ◽  
Author(s):  
B. C. Yang ◽  
M. I. Phillips ◽  
Y. C. Zhang ◽  
B. Kimura ◽  
L. P. Shen ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Critical Role ◽  
Rat Hearts ◽  
Isolated Rat

Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion

2009 ◽  
Vol 106 (6) ◽  
pp. 1909-1916 ◽  
Author(s):  
Ebru Gürel ◽  
Kirsten M. Smeele ◽  
Otto Eerbeek ◽  
Anneke Koeman ◽  
Cihan Demirci ◽  
...  
Keyword(s):  
Protein Content ◽  
Ischemic Preconditioning ◽  
Ischemia Reperfusion ◽  
Glycolytic Enzyme ◽  
Cardiac Ischemia ◽  
Biphasic Response ◽  
Control Groups ◽  
Rat Hearts ◽  
Before And After ◽  
Perfused Rat Hearts

The glycolytic enzyme hexokinase (HK) is suggested to play a role in ischemic preconditioning (IPC). In the present study we determined how ischemic preconditioning affects HK activity and HKI and HKII protein content at five different time points and three different subcellular fractions throughout cardiac ischemia-reperfusion. Isolated Langendorff-perfused rat hearts (10 groups of 7 hearts each) were subjected to 35 min ischemia and 30 min reperfusion (control groups); the IPC groups were pretreated with 3 times 5-min ischemia. IPC was without effect on microsomal HK activity, and only decreased cytosolic HK activity at 35 min ischemia, which was mimicked by decreased cytosolic HKII, but not HKI, protein content. In contrast, mitochondrial HK activity at baseline and during reperfusion was elevated by IPC, without changes during ischemia. No effect of IPC on mitochondrial HK I protein content was observed. However, mitochondrial HK II protein content during reperfusion was augmented by IPC, albeit not following the IPC stimulus. It is concluded that IPC results in decreased cytosolic HK activity during ischemia that could be explained by decreased HKII protein content. IPC increased mitochondrial HK activity before ischemia and during reperfusion that was only mimicked by increased HK II protein content during reperfusion. IPC was without effect on the phosphorylation status of HK before ischemia. We conclude that IPC is associated with 1) a biphasic response of increased mitochondrial HK activity before and after ischemia, 2) decreased cytosolic HK activity during ischemia, and 3) cellular redistribution of HKII but not HKI.

Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants

2003 ◽  
Vol 285 (3) ◽  
pp. H1198-H1205 ◽  
Author(s):  
M. Said ◽  
L. Vittone ◽  
C. Mundiña-Weilenmann ◽  
P. Ferrero ◽  
E. G. Kranias ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Phosphorylation Sites ◽  
Adrenergic Stimulation ◽  
Rat Hearts ◽  
Adrenergic Blocker ◽  
Perfused Rat Hearts ◽  
A Site ◽  
Contractile Recovery ◽  
Mechanical Recovery

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on β-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 μM) or the β-adrenergic blocker dl-propranolol (1 μM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.

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