Effects of timolol on phosphorylation and protein levels of both SR Ca²⁺ release channel (RyR2) and some Ca²⁺ handling regulators.

Data presented in (A) and (B) are obtained from homogenates from control (CON), diabetic (DM), and timolol treated diabetic (DM+TIM) rat hearts. (A) Left: representative Western blotting for 565 kDa phospho-RyR2-Ser²⁸⁰⁸ (pRyR2) and total RyR2, 42 kDa phospho-PKA-Thr198 (pPKA) and PKA, 50 kDa phospho-CaMKII-Thr286 (pCaMKII) and CaMKII, 12 kDA FKBP12.6, and 45 kDa β-actin, respectively. Right: quantification for the ratio of pRyR2 to RyR2, FKBP12.6 to β-actin pPKA to PKA, and pCaMKII to CaMKII, respectively. (B) Left: Western blotting for 120 kDa sarcolemmal NCX, 27 kDa phopho-PLN (Thr-17) and 6 kDa total PLN (L-15), 100 kDa SERCA2 (N-19), and 45 kDa β-actin, respectively. Right: quantification for the ratio of pPLN/actin, PLN/SERCA, and NCX/actin, respectively. The parameters given in section (C) are presented for ratio of 565 kDa pRyR2 to total RyR2, pPKA/PKA, and pCaMKII/CaMKII, respectively (right) in the isolated cardiomyocytes from the diabetic (DM) rats incubated with either 10-μM TIM (+TIM) or 4-mM glutathione, GSH (+GSH) (1 hour). Left: representative Western blotting for 565 kDa pRyR2 and total RyR2, 42 kDa pPKA (Thr198) and PKA, and 50 kDa pCaMKII-Thr286 and CaMKII, respectively. Bars represent mean ± SEM, n = 5–6 for hearts/group/protocol (double assays in each sample from each group for each type of measurement). Significant at ^*p<0.05 vs. CON and ^†p<0.05 vs. DM.