Insulin protects cardiomyocytes against reoxygenation-induced hypercontracture by a survival pathway targeting SR Ca2+ storage.
OBJECTIVE
Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the underlying mechanisms are still unknown. In this study, we investigated in isolated rat cardiomyocytes subjected to hypoxia and reoxygenation whether administration of insulin during reoxygenation reduces reoxygenation-induced hypercontracture, a hallmark of acute reperfusion injury. The effects of insulin on potential pro-survival kinases, i.e., PI 3-kinase, NO synthase (eNOS), and cGMP-dependent protein kinase (PKG), and on cytosolic Ca2+ control in reoxygenated cardiomyocytes were investigated.


RESULTS
Administration of insulin (10 mU/L) during reoxygenation protected cardiomyocytes against hypercontracture development (cell length as % of end-hypoxic length: control 61.6+/-3.2; insulin 76.3+/-2.9; n=26; p<0.05 vs. control). Cytosolic [Ca2+] recovery during the first 2 min of reoxygenation was accelerated (fura-2 ratio after 2 min of reoxygenation; control 1.01+/-0.05; insulin 0.79+/-0.04; n=26; p<0.05 vs. control). The beneficial effects of insulin on cytosolic [Ca2+] recovery and hypercontracture were suppressed in the presence of inhibitors of PI 3-kinase (LY294002, 1 microM), eNOS (L-NMMA, 100 microM), PKG (KT 5823, 1 microM), or sarcoplasmic reticulum Ca2+ pump (SERCA) (thapsigargin, 150 nM). Insulin increased phosphorylation and activity of eNOS and augmented phospholamban phosphorylation in reoxygenated cardiomyocytes. Correlated with phospholamban phosphorylation, insulin also augmented SR Ca2+ load.


CONCLUSIONS
Insulin protects cardiomyocytes against reoxygenation-induced hypercontracture. This is due to acceleration of cytosolic [Ca2+] recovery by enhanced Ca2+ sequestration into the sarcoplasmic reticulum via SERCA activation. This protective mechanism is activated through the survival pathway consisting of PI 3-kinase, eNOS, and PKG.
Institute of Physiology, Justus-Liebig-University, D-35392 Giessen, Germany. yaser.abdallah@physiologie.med.uni-giessen.de
Yaser
Y
Abdallah
Anna
A
Gkatzoflia
Dragan
D
Gligorievski
Sascha
S
Kasseckert
Gerhild
G
Euler
Klaus-Dieter
KD
Schlüter
Matthias
M
Schäfer
Hans-Michael
HM
Piper
Claudia
C
Schäfer
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D007150:Immunohistochemistry / Q000379:methods; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D007328:Insulin; D052250:Nitric Oxide Synthase Type III; D000099211:Phospholamban