Increase of free intracellular calcium [Ca 2+]i plays a crucial role in cardiomyocyte ischemic injury. Here we demonstrate that overexpression of the sarcoplasmic reticulum stress-protein GRP94 reduces myocyte necrosis due to calcium overload or simulated ischemia. Selective 3 to 8 fold GRP94 increase, with no change in Grp78 or calreticulin, was achieved by stable transfection in skeletal C2C12 and cardiac H9c2 muscle cell. After exposure to the calcium ionophore A23187, LDH release from five different GRP94-overexpressing clones of either C2C12 and H9c2 origin was significantly lower than that of controls and [Ca 2+]i increase was significantly delayed. The number of necrotic cells, evaluated by propidium iodide uptake, was reduced when cells from the GRP94-overexpressing H9c2 clone were exposed to conditions simulating ischemia. Experiments performed in neonatal rat cardiomyocytes, co-transfected with GRP94 and the green fluorescent protein GFP, validated the protective effect of GRP94 overexpression. A lower percentage of propidium-iodide positive/GFP-fluorescent myocytes co-expressing exogenous GRP94, with respect to myocytes expressing GFP alone, was observed after exposure either to A23187 (6.6% vs. 14.0%, respectively), or to simulated ischemia (8.5% vs 17.7%, respectively). In conclusion, the selective increase in Grp94 protects cardiomyocytes from both ischemia and calcium overload counteracting [Ca2+]I elevations.
Overexpression of the stress-protein Grp94 reduces cardiomyocyte necrosis due to calcium overload and simulated ischemia.
Vitadello M;Petronilli V;
2003
Abstract
Increase of free intracellular calcium [Ca 2+]i plays a crucial role in cardiomyocyte ischemic injury. Here we demonstrate that overexpression of the sarcoplasmic reticulum stress-protein GRP94 reduces myocyte necrosis due to calcium overload or simulated ischemia. Selective 3 to 8 fold GRP94 increase, with no change in Grp78 or calreticulin, was achieved by stable transfection in skeletal C2C12 and cardiac H9c2 muscle cell. After exposure to the calcium ionophore A23187, LDH release from five different GRP94-overexpressing clones of either C2C12 and H9c2 origin was significantly lower than that of controls and [Ca 2+]i increase was significantly delayed. The number of necrotic cells, evaluated by propidium iodide uptake, was reduced when cells from the GRP94-overexpressing H9c2 clone were exposed to conditions simulating ischemia. Experiments performed in neonatal rat cardiomyocytes, co-transfected with GRP94 and the green fluorescent protein GFP, validated the protective effect of GRP94 overexpression. A lower percentage of propidium-iodide positive/GFP-fluorescent myocytes co-expressing exogenous GRP94, with respect to myocytes expressing GFP alone, was observed after exposure either to A23187 (6.6% vs. 14.0%, respectively), or to simulated ischemia (8.5% vs 17.7%, respectively). In conclusion, the selective increase in Grp94 protects cardiomyocytes from both ischemia and calcium overload counteracting [Ca2+]I elevations.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.