CaMKII and osmolarity are involved in the arrhythmogenic effect of mouse cardiomyocytes exposed to extracellular high glucose

CELY ORTIZ, ALEJANDRA; MATTIAZZI, RAMONA ALICIA; CIOCCI PARDO, ALEJANDRO; MOSCA, SUSANA

Mar del Plata

Congreso; Reunión Anual Conjunta de AIC SAI SAFIS 2018; 2018

SAIC SAI SAFIS

Although the factors contributing to I/R injury are complex, experimental evidence reveals that loss of Ca2+ homeostasis is one of the major contributing mechanisms. Previous experiments indicate that there are two main factors related to Ca2+ handling involved in I/R cardiac injury: SR Ca2+ leak due to phosphorylation of the ryanodine receptors (RyR2) by the Ca-calmodulin dependent protein kinase (CaMKII) at the onset of reperfusion; and the increased Ca2+ sequestration/load that involves CaMKII-dependent phosphorylation of phospholamban (PLN), which, when phosphorylated, increases SR Ca-ATPase activity and Ca2+ sequestration. In an attempt to dissect the relative roles of these factors on mitochondrial dysfunction during I/R, experiments were performed in wild type (WT) mice, double mutant mice (SDKO), with increased SR Ca2+ leak, due to constitutive pseudophosphorylation of RyR2 (aspartic acid replaces serine at RyR2?2814, the CaMKII site) and increased SR Ca2+ reuptake by PLN ablation, and double mutant mice (SAKO) with non-phosphorylatable CaMKII site at the RyR2, by replacement of Ser2814 site by Ala and PLN ablation. Mitochondria were isolated from hearts non-submitted and submitted to I/R (15/10 min). After isolation, mitochondrial membrane potential (ΔΨm) expressed in mV and measured by rhodamine-123 fluorescence quenching) and mitochondrial swelling (light scattering decrease (LSD, in au)), were evaluated. In mitochondria not submitted to I/R there was no differences in ΔΨm among the groups. The short protocol of I/R significantly increased mitochondrial depolarization in SDKO (-114.6±3.1) vs. WT (-137.4±1.4). ΔΨm returned to WT values in SAKO (-143.2±2.28). LSD produced by addition of Ca2+ was also significantly lower in SDKO (0.21±0.04) vs. WT (1.04±0.12) and SAKO (0.95±0.03) after I/R. The results indicated that the enhanced Ca2+ leak due to CaMKII-dependent phosphorylation of RyR2 is more important in determining mitochondrial dysfunction in I/R than the increase in SR Ca2+ reuptake by PLN ablation.