Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

Abstract

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.

Fig. 1

Site-specific phosphorylation of RyR2. Representative immunoblots and overall results of SR membrane vesicles dephosphorylated by treatment with alkaline phosphatase (AP) and phosphorylated in vitro by the catalytic subunit of PKA in the presence of EGTA (PKA) or by the endogenous SR-associated CaMKII in the presence of the PKA inhibitor PKI (CaMKII) as described under Materials and methods. For the control samples, Ca²⁺ (1 mM EGTA present), calmodulin and the catalytic subunit of PKA were omitted (Ctrl). SR proteins were separated on SDS-PAGE and transferred to PVDF membranes. Blots were probed with anti-PS2809-RyR2 from Badrilla (A), anti-PS2809-RyR2 (B) and anti-PS2815-RyR2 (C) from X. Wehrens and A. Marks laboratories and anti-RyR2 from Affinity Bioreagents (D). Results are expressed as percentage of the phosphorylation obtained in Ctrl. *p<0.05 with respect to Ctrl.

Fig. 2

β-adrenergic-dependent effects on left ventricular function and phosphorylation of RyR2 in the perfused rat heart in the absence and the presence of low [Ca]_o and KN-93. Maximal rate of rise of pressure (+dP/dt, A), phosphorylation of Ser2809 (B) and of Ser2815 (C) of RyR2 in response to isoproterenol in the absence (closed circles) and presence of low [Ca]_o and nifedipine (open circles), to avoid Ca²⁺ influx to the cells. Isoproterenol increased contractility and the phosphorylation of both Ser2809 and Ser2815 sites of RyR2. Low [Ca]_o plus nifedipine (Low [Ca]_o) abolished contractility in the absence and presence of isoproterenol (+dP/dt, A) and significantly diminished basal Ser2809 phosphorylation and that produced by the lowest isoproterenol concentrations (0.3 and 3 nM) without significantly affecting the phosphorylation evoked by the highest concentrations of the β-agonist (B). Low [Ca]_o plus nifedipine did not decrease basal phosphorylation of Ser2815 but significantly diminished to basal values the phosphorylation of Ser2815 at all isoproterenol concentrations tested (C). 5 μM of the CaMKII inhibitor KN-93 (crosses) did not affect the increase in contractility and phosphorylation of Ser2809 induced by 300 nM isoproterenol, but mimicked the effects of low [Ca]_o plus nifedipine in the phosphorylation of Ser2815 induced by 300 nM isoproterenol. Mean±SEM, n=3–10 experiments. ^#p<0.05 with respect to hearts perfused with normal [Ca]_o in the absence of drugs. *p<0.05 with respect to the corresponding situation in the presence of normal [Ca]_o.

Fig. 3

Activation of PKA is required for the CaMKII-dependent phosphorylation of Ser2815 of RyR2 and Thr17 of PLN. Representative immunoblots and overall results of SR vesicles isolated from hearts perfused without (Ctrl) and with 30 nM isoproterenol in the absence (Iso) and the presence of 30 μM of the PKA inhibitor H-89 (Iso-H-89) and with 1 μM of forskolin (FK). Phosphorylation of Ser2815 of RyR2 and Thr17 of PLN is shown as ratio between the signals of the site-specific phospho-antibody and the antibody used to recognized the non-phosphorylated form. *p<0.05 with respect to Ctrl. ^#p<0.05 with respect to Iso-treated hearts.

Fig. 4

[³H]-ryanodine binding in SR vesicles isolated from treated hearts. Ca²⁺ dependence of [³H]-ryanodine binding in cardiac SR vesicles isolated from hearts perfused in the absence (Control) and the presence of 300 nM isoproterenol (Iso) and in the presence of Iso, either with low [Ca]_o plus nifedipine (Iso-low Ca) or with 5 μM KN-93 (Iso-KN). Isoproterenol increased [₃H]-ryanodine binding. This increase did not occur in the presence of KN-93 or low [Ca]_o plus nifedipine. Of note, at 300 nM isoproterenol, the decrease in Ca²⁺ influx to the cell or the inhibition of CaMKII did not affect the phosphorylation of Ser2809 site but significantly decreased the phosphorylation of Ser2815 site (see Fig. 2). Neither KN-93 nor low [Ca]_o plus nifedipine modified [³H]-ryanodine binding in the absence of isoproterenol. ^#p<0.05 with respect to control hearts.

Fig. 5

Effect of isoproterenol on Ca²⁺ release kinetics in isolated hearts. Release kinetic was measured following the change in fluorescence of Calcium Green 5 and was fitted to the double exponential function: A₁ [1−exp (−k₁t)]+A₂ [1−exp (−k₂t)]. Representative Ca²⁺ release records in SR vesicles isolated from hearts submitted to the different experimental protocols. The increase in Ca²⁺ release kinetics produced by isoproterenol (Iso, A) was significantly decreased bylow [Ca]_o plus nifedipine (Iso-low Ca, B) or by CaMKII-inhibition (Iso-KN, C). Records shown are the average of 5 to 7 determinations.

Fig. 6

Ca²⁺ release rate constants (k₁ and k₂) and phosphorylation of RyR2 sites. The rate constant of the fast-exponential release (k₁, A) increased 5.8-fold in isoproterenol-treated hearts (Iso) and this increase was prevented in hearts perfused with low [Ca]_o plus nifedipine (Iso-low Ca) or the CaMKII inhibitor KN-93 (Iso-KN). k₂ increased with Iso and it did no decrease by inhibition of CaMKII (B). Changes in the phosphorylation of PSer2815 (A) and PSer2809 sites (B) in the same experimental conditions are shown for comparison. ^#p<0.05 with respect to control hearts; *p<0.05 with respect to Iso.