Adrenergic regulation of cardiac contractility does not involve phosphorylation of the cardiac ryanodine receptor at serine 2808

Abstract

The sympathetic nervous system is a critical regulator of cardiac function (heart rate and contractility) in health and disease. Sympathetic nervous system agonists bind to adrenergic receptors that are known to activate protein kinase A, which phosphorylates target proteins and enhances cardiac performance. Recently, it has been proposed that protein kinase A-mediated phosphorylation of the cardiac ryanodine receptor (the Ca(2+) release channel of the sarcoplasmic reticulum at a single residue, Ser2808) is a critical component of sympathetic nervous system regulation of cardiac function. This is a highly controversial hypothesis that has not been confirmed by several independent laboratories. The present study used a genetically modified mouse in which Ser2808 was replaced by alanine (S2808A) to prevent phosphorylation at this site. The effects of isoproterenol (a sympathetic agonist) on ventricular performance were compared in wild-type and S2808A hearts, both in vivo and in isolated hearts. Isoproterenol effects on L-type Ca(2+) current (I(CaL)), sarcoplasmic reticulum Ca(2+) release, and excitation-contraction coupling gain were also measured. Our results showed that isoproterenol caused significant increases in cardiac function, both in vivo and in isolated hearts, and there were no differences in these contractile effects in wild-type and S2808A hearts. Isoproterenol increased I(CaL), the amplitude of the Ca(2+) transient and excitation-contraction coupling gain, but, again, there were no significant differences between wild-type and S2808A myocytes. These results show that protein kinase A phosphorylation of ryanodine receptor Ser2808 does not have a major role in sympathetic nervous system regulation of normal cardiac function.

Figure 1

Effects of ISO on cardiac function in vivo. A, Representative echocardiographic measurements in WT and S2808A animals before and after ISO. B, ISO caused significant increases in heart rate in both WT and S2808A mice. There were no differences in the response between the 2 groups. C, ISO caused significant increases in ejection fraction (EF) in WT and S2808A mice. There were no differences in ISO effects between groups. D, ISO caused significant increases in fractional shortening in WT and S2808A mice, with no differences between the response in the 2 groups.

Figure 2

Effects of ISO on cardiac function in isovolumic Langendorff perfused hearts. A, Representative LV pressure (mm Hg) and ±dP/dt (mm Hg/sec) tracings acquired during both steady-state baseline (end-diastolic pressure, 10 mm Hg; 8.0Hz) and ISO (10 nmol/L) infusions. Boxed areas are shown at an expanded time scale. B, Group averages. No differences in baseline performance or peak ISO responses were observed between WT (n=5) and RyR-S2808A (n=6) mice. Data are presented as means±SEM. *P<0.01 vs respective group baseline.

Figure 3

Western blot analysis of Ca²⁺ regulatory proteins. Two representative Western blots are shown. S2808A animals were identified by the failure of binding of an antibody (RyRpS2808) that detects phosphorylation at S2808. The following were measured: Na⁺/Ca²⁺ exchanger (NCX); the α subunit of the L-type Ca²⁺ channel (α1C); SERCa; and RyR (total) (RyRt). There were no significant differences in Na⁺/Ca²⁺ exchanger, α1C, and SERCa abundance (normalized to either GAPDH or cardiac actin) between samples from WT and S2808A (labeled WT and S/A, respectively) hearts.

Figure 4

Effects of ISO on Ca²⁺ current, contraction, Ca²⁺ transients, and ECC gain. A, Peak systolic [Ca²⁺]_i (F/F₀) plotted against test voltage in WT (n=9) and knock-in (S2808A) (n=11) myocytes. The amplitude of the Ca²⁺ transient before and after ISO treatment are shown. B, I_CaL density plotted against voltage in WT (n=9) and S2808A (n=11) myocytes before and after ISO treatment. C, Voltage dependence of cell shortening in WT (n=7) and S2808A (n=6) and the ISO effect on cell shortening. D, Time constant of [Ca²⁺]_i decay obtained by fitting of the decay fluorescence trace to a single exponential function plotted vs test voltage before and after ISO treatment. E, ECC gain calculated for each voltage dividing the F/F₀ by the integral of I_CaL (pC/pF). Black bars represent WT cells; red bars, RyR-S2808A cells before and after ISO treatment. Similar results were obtained when peak I_CaL was used in the ECC gain calculation. *P≤0.05 before ISO addition within group.