Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats

Abstract

Autonomous Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca²⁺ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca²⁺ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca²⁺ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations. MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca²⁺ leak (in the form of spontaneous Ca²⁺ waves) under basal conditions and that Ca²⁺ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [³H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca²⁺ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca²⁺ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.

Keywords: Ca(2+)/calmodulin-dependent protein kinase type II; Cardiac ryanodine receptor phosphorylation; Diastolic Ca(2+)leak; High sucrose diet; Metabolic syndrome; β-adrenergic stimulation.

Figure 1.. Exacerbated diastolic Ca²⁺ leak induced by the β-adrenergic stimulus in isolated MetS cardiomyocytes.

Representative pseudo-colored confocal images of Ca²⁺ spark (A) and Ca²⁺ waves (B) recorded in quiescent Fluo-3 loaded cardiomyocytes from CTL and MetS rats in the presence of isoproterenol (ISO, 100 nM) or in its absence. C. Bar graph of average Ca²⁺ spark frequency. D. Ca²⁺ wave frequency (events/s). E. Caffeine-evoked Ca²⁺ transient amplitude (ΔF/F₀) of Fluo-3 loaded cardiomyocytes from CTL and MetS rats, perfused with recording solution in the presence of isoproterenol (ISO, 100 nM) or in its absence. F. Bar graph of diastolic [Ca²⁺]_i determined in cardiomyocytes loaded with Fura-2 of CTL and MetS rats before and after ISO administration. All values are presented as mean ± SE. Ca²⁺ sparks and Ca²⁺ waves data are obtained from n=30 cells from N= 6 rats for each experimental condition. Caffeine-evoked Ca²⁺ transient data are obtained from n_CTL= 16 cells, n_CTL+ISO= 11 cells, n_MetS= 13 cells and n_MetS+ISO= 9 cells, from N = 4 rats for each experimental condition. [Ca²⁺]_i data are obtained from n_CTL= 9 cells, n_CTL+ISO= 8 cells, n_MetS= 10 cells and n_MetS+ISO= 8 cells, from N = 3 rats for each experimental condition. *P ≤ 0.05 with respect to the CTL group, † P ≤ 0.05 with respect to the CTL+ISO group and # P ≤ 0.05 with respect to the MetS group.

Figure 2.. Increased activity of Ryanodine Receptors in MetS associated with its phosphorylation levels.

A. Normalized [³H]-Ryanodine binding Bmax determined at pCa 5, pH 7.2 in CTL and MetS heart homogenates in the presence (1 μM) or absence of isoproterenol (ISO). B. Representative Western Blot images of total and phosphorylated RyR2 at S2808, S2814 and GAPDH, obtained from heart homogenates from CTL and MetS rats in the presence (1 μM) or absence of ISO. C. Total ryanodine receptor (RyR) expression normalized against corresponding GAPDH from N = 5 rats for each experimental condition. D. Phosphorylation levels of RyR at S2808 normalized against corresponding total RyR from N = 6 rats for each experimental condition. E. Phosphorylation levels of RyR at S2814 normalized against corresponding total RyR from N= 5 rats for each experimental condition. Data are expressed as mean ± SE. *P ≤ 0.05 with respect to CTL group and † P ≤ 0.05 with respect to the CTL + ISO group.

Figure 3.. SERCA pump ATPase activity is increased in MetS hearts under resting conditions.

A. SERCA pump ATPase hydrolytic activity evaluated by enzymatic assay in heart homogenates of CTL and MetS rats in the presence (1 μM) or absence of isoproterenol (ISO) B. Bar graph of SERCA2 pump reaction rate obtained from enzymatic assay. C. SERCA2 protein expression normalized against corresponding GAPDH and representative Western blot. Data are expressed as mean ± SE, N= 5 rats for each experimental condition. *P ≤ 0.05 and **P ≤ 0.001 respect to the CTL group.

Figure 4.. Augmented T17-PLN phosphorylation in MetS.

A. Representative Western Blot images of total SERCA2, PLN, and phosphorylated PLN at S16 and T17, obtained from heart homogenates from CTL and MetS rats in the presence (1 μM) or absence of isoproterenol (ISO). B. Phosphorylation levels of PLN at S16 normalized against corresponding total PLN. C. Phosphorylation levels of PLN at T17 normalized against corresponding total PLN. Data are expressed as mean ± SE, N= 5 animals for each experimental condition. *P ≤ 0.05 with respect to the CTL group † P ≤ 0.05 with respect to the CTL + ISO group, and # P ≤ 0.05 with respect to the MetS group.

Figure 5.. Autonomous activation of CaMKII is increased in MetS hearts and is unchanged by ISO.

A. Representative Western Blot images of total and phosphorylated CaMKII at T287 and GAPDH, obtained from heart homogenates from CTL and MetS rats in the presence (1 μM) or absence of isoproterenol (ISO). B. CaMKII protein expression normalized against corresponding GAPDH C. Phosphorylation levels of CaMKII at T287 normalized against corresponding total CaMKII. Data are expressed as mean ± SE, N= 5 animals for each experimental condition. *P ≤ 0.05 with respect to the CTL group.

Figure 6.. Incubation with AIP prevents exacerbated diastolic Ca²⁺ leak induced by the β-adrenergic stimulus in isolated MetS cardiomyocytes.

Representative pseudo-colored confocal images of Ca²⁺ sparks (A) and Ca²⁺ waves (B) recorded in quiescent Fluo-3 loaded cardiomyocytes from CTL and MetS rats pre-incubated with AIP 10 μM, in the presence or absence of isoproterenol (ISO, 100 nM). Bar graphs of Ca²⁺ spark frequencies (C, in events/s * 100 μm) and Ca²⁺ wave frequencies (D, events/s) for the different experimental conditions. E. Caffeine-evoked Ca²⁺ transient amplitude (ΔF/F₀) of Fluo 3-loaded cardiomyocytes from CTL and MetS rats pretreated with AIP (10 μM), and perfused with recording solution in the presence of isoproterenol (ISO, 100 nM) or in its absence. F. Bar graph of basal fluorescence (F₀) determined in Fluo 3-loaded, AIP-treated cardiomyocytes of CTL and MetS rats before and after ISO administration. All values are presented as mean ± SE. Ca²⁺ sparks, Ca²⁺ waves and F₀ data are obtained from n=25 cells from N= 6 rats for each experimental condition. Caffeine-evoked Ca²⁺ transient data are obtained from n_CTL= 9 cells, n_CTL+ISO= 6 cells, n_MetS= 9 cells and n_MetS+ISO= 9 cells, from N = 3 rats for each experimental condition. *P ≤ 0.05 respect to the CTL group, † P ≤ 0.05 respect to the CTL + ISO group and # P ≤ 0.05 respect to the MetS group.