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Preconditioning cultured human pediatric myocytes requires adenosine and protein kinase C
J S Ikonomidis1, T Shirai, R D Weisel
1Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
Insights
Pediatric myocytes preconditioned by brief ischemia release adenosine, activating protein kinase C (PKC) for protection. This adenosine-mediated pathway, involving PKC activation, confers resistance to subsequent prolonged ischemia-reperfusion injury.
Area of Science:
- Cardiology
- Cellular Biology
- Biochemistry
Background:
- Pediatric myocyte cultures can be preconditioned against ischemic damage.
- The molecular mechanisms underlying this protective effect require further elucidation.
Purpose of the Study:
- To investigate the roles of adenosine and protein kinase C (PKC) in ischemic preconditioning of pediatric myocytes.
- To determine if adenosine release and PKC activation mediate the protective effects.
Main Methods:
- Assessing mediator release in the supernatant of preconditioned myocytes.
- Evaluating the effects of adenosine receptor agonists and antagonists.
- Investigating the impact of PKC agonists and antagonists.
- Measuring PKC translocation and phosphorylation rates.
Main Results:
- Preconditioned myocytes release protective factors, including adenosine, into the supernatant.
- Adenosine receptor antagonism inhibits preconditioning; exogenous adenosine confers protection.
- PKC activation mimics preconditioning effects and is inhibited by antagonists.
- Ischemic and adenosine preconditioning induce PKC translocation and increase phosphorylation.
Conclusions:
- Adenosine released from ischemic pediatric myocytes mediates preconditioning.
- The protective effect is achieved through the activation of protein kinase C (PKC).
- This pathway offers a potential target for therapeutic interventions against myocardial injury.
Abstract:
We showed previously that 20 min of low-volume anoxia ("ischemia") and 20 min of "reperfusion" preconditions quiescent pediatric myocyte cultures against damage resulting from 90 min of subsequent prolonged ischemia and 30 min of reperfusion. The purpose of this study was to assess the roles of adenosine and protein kinase C (PKC) in this preconditioning model. Our results suggest that 1) preconditioned myocytes secrete a protective mediator(s) into the "ischemic" supernatant that is transferable to other cells, and adenosine is released into the supernatant in quantities sufficient for adenosine-receptor activation (2) preconditioning is inhibited by adenosine-receptor antagonism, and myocyte protection similar to preconditioning can be achieved with exogenously administered adenosine or adenosine-receptor stimulation; (3) brief ischemic and adenosine-induced myocyte preconditioning is mimicked by the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PKC agonist) and inhibited by PKC antagonists; and (4) brief ischemic and adenosine-induced myocyte preconditioning both induce PKC translocation to myocyte membranes and increase the PKC phosphorylation rate. These data suggest that adenosine released from ischemic human pediatric myocytes mediates preconditioning through activation of PKC.
