Abstract
Heat shock protein 70 (HSP70) mediates delayed cardioprotection of preconditioning. Cytosolic calcium ([Ca2+])i overload precipitates injury, whereas attenuation of [Ca2+]i overload is believed to be responsible for cardioprotection. There is evidence suggesting a link between HSP70 and [Ca2+]i homeostasis. We hypothesize that activation of HSP70 by preconditioning may restore [Ca2+]i homeostasis altered by ischemic insults. To test the hypothesis, we determined the effects of preconditioning with metabolic inhibition or pretreating with U50,488H [trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (a κ-opioid receptor agonist)] on viability and injury, HSP70 expression, and [Ca2+]i in ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), with blockade of HSP70 synthesis. In myocytes with vehicle pretreatment, the percentage of dead cells determined by trypan blue exclusion, the injury reflected by release of lactate dehydrogenase, and the resting [Ca2+]i measured by spectrofluorometry significantly increased, whereas the amplitude of electrically induced [Ca2+]i transient decreased, after 10 min with 10 mM 2-deoxy-d-glucose and 10 mM sodium dithionite, known to cause MI/A. However, when myocytes were subjected for 30 min to either 20 mM lactate and 10 mM 2-deoxy-d-glucose (MIP) or 30 μM U50,488H (UP) 20 h before MI/A, the changes in viability and injury, and [Ca2+]i responses were significantly attenuated. These were accompanied by a significantly increased HSP70 expression. Furthermore, blockade of HSP70 synthesis with selective antisense oligonucleotides abolished the beneficial effects of MIP or UP. This study provides first evidence that activation of HSP70 induced by preconditioning, which conferred delayed cardioprotection, restored partially the [Ca2+]i homeostasis altered by ischemic insults.
Footnotes
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This study was supported by the Research Grant Councils, Hong Kong Grant HKU 7488103M (to T.M.W.). J.L. was supported by postgraduate studentship of the University of Hong Kong.
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DOI: 10.1124/jpet.104.067926.
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ABBREVIATIONS: MIP, preconditioned with metabolic inhibition; κ-OR, κ-opioid receptor; UP, preconditioned with U50,488H; HSP70, heat shock protein 70; AS, antisense; [Ca2+]i, cytosolic calcium; IR, ischemia and reperfusion; BAPTA-2AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-2 acetoxymethyl ester; AM, acetoxymethyl ester; MI/A, metabolic inhibition and anoxia; 2-DOG, 2-deoxy-d-glucose; nor-BNI, nor-binaltorphimine; VP, vehicle pretreatment; S, sense; RE, reperfusion; LDH, lactate dehydrogenase; TBS, Tris-buffered saline; IP, ischemic preconditioning; U50,488H, trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide.
- Received March 3, 2004.
- Accepted March 26, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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