Ca2+/calmodulin-dependent protein kinase II regulation by inhibitor 1 of protein phosphatase 1 alleviates necroptosis in high glucose-induced cardiomyocytes injury

Abstract

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) plays an important role in the cardiovascular system. However, the potential protective role of inhibitor 1 of protein phosphatase 1 (I1PP1), which is able to regulate CaMKII, in high glucose-induced cardiomyocytes injury remains unknown. In the present study, cardiomyocytes were transfected with I1PP1 adenovirus to inhibit protein phosphatase 1 (PP1) expression. After the cardiomyocytes were subjected to high glucose stimulation for 48 h, quantitative real-time PCR was used to detect CaMKIIδ alternative splicing. Lactate dehydrogenase (LDH) release and adenosine triphosphate (ATP) level were measured to assess cell damage and energy metabolism respectively. CaMKII activity was represented as phospholamban (PLB) phosphorylation, CaMKII phosphorylation (p-CaMKII) and oxidation (ox-CaMKII). Dihydroethidium (DHE), MitoSOX and JC-1 staining were used to assess oxidative stress and mitochondrial membrane potential. Necroptosis was evaluated by receptor interacting protein kinase 3 (RIPK3) expression, TUNEL and cleaved-caspase 3 levels. RIPK3, mixed lineage kinase domain like protein (MLKL) and dynamin-related protein 1 (DRP1) expressions were also detected. We found that high glucose disordered CaMKIIδ alternative splicing. I1PP1 over-expression suppressed PLB phosphorylation, ox-CaMKII, DRP1, RIPK3 and cleaved-caspase 3 proteins expression, decreased LDH release, attenuated necroptosis, increased ATP level, inhibited oxidative stress, and elevated mitochondrial membrane potential in high glucose-stimulated cardiomyocytes. However, there was no effect on phosphorylation of MLKL (p-MLKL), p-CaMKII, and receptor interacting protein kinase 1 (RIPK1) expression. Altogether, I1PP1 over-expression alleviated CaMKIIδ alternative splicing disorder, suppressed CaMKII oxidation, reduced reactive oxygen species (ROS) accumulation and inhibited necroptosis to attenuate high glucose-induced cardiomyocytes injury.

Keywords: Ca(2+)/calmodulin-dependent protein kinase II; High glucose; Inhibitor 1 of protein phosphatase 1; Necroptosis; Oxidative stress.