Calcium‐calmodulin‐dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance

Abstract

The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca /calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca spontaneous events in the absence of SR Ca load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [ H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favour Ca transit between the two organelles.

Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2

“…[ 3 H]Ryanodine binding assays. Binding assays were carried out following a modified version of a protocol previously described (53). Binding mixtures were prepared containing 50-100 μg of cell lysate, 0.2 M KCl, 20 mM Na-HEPES (pH 7.4), 6.5 nM [ 3 H]ryanodine (NET950, PerkinElmer), and enough CaCl 2 to set free [Ca 2+ ] between 100 nM-100 μM.…”

Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage

“…(D) Histological samples (upper right) show a closer distance between mitochondria and SR in FRD vs. CD treated mice, which might be due to an alteration in structural proteins involved in SR-mitochondria communication. The cartoon in the left side of (D) represents this SR-mito interaction (reproduced with permission from Federico et al, 2017). *p < 0.05. mitochondrial cytochrome c release and consequent activation of the caspase-3 pathway (Cai et al, 2002).…”

“…Intriguingly, db/db female mouse hearts showed no changes in RyR2 expression associated with a decrease in PKA and CaMKII RyR2 phosphorylation sites (Pereira et al, 2014). In a model of fructoserich diet (FRD) applied to male rats and mice, our laboratory has described that this hypercaloric diet, which also induces insulin resistance, increases CaMKII activation and RyR2 dysfunction due to Ser 2814 phosphorylation (Sommese et al, 2016;Federico et al, 2017). On the other hand, experiments in vitro revealed that during acute hyperglycemia, RyR2 activity can also be altered.…”

Physiological and unappreciated roles of CaMKII in the heart

“…In the diseased myocardium, CaMKII plays central roles in processes such as maladaptive remodeling [1, 3, 44, 45, 48, 50, 72, 115, 117], arrhythmogenesis [63], interstitial fibrosis [3, 45] and apoptosis [21, 22, 103, 112]. As such, CaMKII is a promising target for pharmacological inhibition and the development of inhibitory compounds is racing ahead [73].…”