Further study on the role of HSP70 on C[a.sup.2+] homeostasis in rat ventricular myocytes subjected to simulated ischemia

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of C[a.sup.2+] at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RYR), sarco(endo)plasmic reticulum C[a.sup.2+]-ATPase (SERCA), and N[a.sup.+]/C[a.sup.2+] exchanger (NCX) handled C[a.sup.2+] in rat ventricular myocytes preconditioned with a [kappa]-opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the C[a.sup.2+] transients induced by electrical stimulation and caffeine, which provide the overall picture of C[a.sup.2+] homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) C[a.sup.2+] fluxes via NCX by the use of [45.sup.C[a.sup.2+] in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of C[a.sup.2+] from the sarcoplasmic reticulum via RyR and in the removal of C[a.sup.2+] from the cytoplasm by reuptake of C[a.sup.2+] to the SR via SERCA and by extrusion of C[a.sup.2+] out of the cell via NCX. UP also reduced mitochondrial C[a.sup.2+] accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired C[a.sup.2+] homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated C[a.sup.2+] overload, improved recovery in contractile function, and cardioprotection. intracellular C[a.sup.2+], [kappa]-opioid receptor; N[a.sup.+]/C[a.sup.2+] exchanger; ryanodine receptor; sarco(endo)plasmic reticulum C[a.sup.2+]-ATPase