Hyperphosphorylation of the cardiac ryanodine receptor at serine 2808 is not involved in cardiac dysfunction after myocardial infarction

Abstract

Rationale: Abnormal behavior of the cardiac ryanodine receptor (RyR2) has been linked to cardiac arrhythmias and heart failure (HF) after myocardial infarction (MI). It has been proposed that protein kinase A (PKA) hyperphosphorylation of the RyR2 at a single residue, Ser-2808, is a critical mediator of RyR dysfunction, depressed cardiac performance, and HF after MI.

Objective: We used a mouse model (RyRS2808A) in which PKA hyperphosphorylation of the RyR2 at Ser-2808 is prevented to determine whether loss of PKA phosphorylation at this site averts post MI cardiac pump dysfunction.

Methods and results: MI was induced in wild-type (WT) and S2808A mice. Myocyte and cardiac function were compared in WT and S2808A animals before and after MI. The effects of the PKA activator Isoproterenol (Iso) on L-type Ca(2+) current (I(CaL)), contractions, and [Ca(2+)](I) transients were also measured. Both WT and S2808A mice had depressed pump function after MI, and there were no differences between groups. MI size was also identical in both groups. L type Ca(2+) current, contractions, Ca(2+) transients, and SR Ca(2+) load were also not significantly different in WT versus S2808A myocytes either before or after MI. Iso effects on Ca(2+) current, contraction, Ca(2+) transients, and SR Ca(2+) load were identical in WT and S2808A myocytes before and after MI at both low and high concentrations.

Conclusions: These results strongly support the idea that PKA phosphorylation of RyR-S2808 is irrelevant to the development of cardiac dysfunction after MI, at least in the mice used in this study.

Figure 1. Cardiac structure and function are not different in WT and S2808A animals before or after MI

Ejection fraction (A), fractional shortening (B) LV internal diameter (C) and septum wall thickness (D) were measured with Echocardiography before and 1,4 weeks after MI. * p< 0.05 and ** p<0.01 between Pre-MI and Post-MI within groups.

Figure 2. MI Causes cardiac remodeling and fibrosis in WT and S2808A Hearts

A–C. Heart weight (HW), lung weight (LW) and Liver weight (LiverW) normalized to body weight (BW) (HW/BW, LW/BW, LiverW/BW) in sham- and MI-operated mice after 1 and 4 weeks surgery. D. Infarct length 4 weeks after MI. E. Fibrotic area (blue) in remote zones of trichrome-stained cardiac histological sections was measured 4 weeks after MI. $ P <0.05 between WT and S2808A mice. **p<0.01 between sham and MI

Figure 3. Western analysis of Ca²⁺ regulatory proteins in WT and S2808A mice 4 weeks after MI

Total RyR (RyRt), S2808 phosphorylated RyR (RyRp), LTCC alpha1C, SERCa2, and phosphorylated Phospholamban (PT17) are shown. The phosphorylated forms of RyR2 and PT17 increased in MI hearts. *p<0.05 and **p<0.01 vs sham.

Figure 4. L-type Ca²⁺ current (I_Ca,L) in Sham and Post-MI myocytes

A. Representative I_Ca,L in sham or post-MI myocytes from WT and S2808A hearts. B. Representative I_Ca,L after 1umol/L isoproterenol in sham or post-MI myocytes from WT and S2808A hearts. C–D. Current-voltage relationships in sham and MI myocytes +/− Iso. E–F. Peak I_Ca,L and Voltage dependence of I_Ca,L activation in sham or post-MI myocytes +/− Iso from WT and S2808A hearts. # p<0.05 between +/− Iso.

Figure 5. Low and high [Iso] had identical effects on contractions of WT and S2808A myocytes from sham and MI hearts

A–B. Myocyte contractions −/+ Iso in sham and post-MI myocytes. C–D. Average Half Width (ms) of fractional shortening −/+ Iso in WT and S2808A myocytes from sham or post-MI hearts. E–F. Average Time to 50% Relaxation (ms) of fractional shortening −/+ Iso in WT and S2808A myocytes from sham or post-MI hearts. * P<0.05 sham versus MI, # P<0.05 between −/+ Iso. . 10 and 100 nMol/L Iso were used in this study.

Figure 6. Low and high [Iso] had identical effects on Ca²⁺ transients of WT and S2808A myocytes from sham and MI hearts

A–B. Myocyte peak Ca²⁺ transients −/+ Iso in sham and post-MI myocytes. C–D. Half-width of Ca²⁺ transients −/+ Iso in myocytes from sham or post-MI hearts. E–F. Average Tau of Ca²⁺ transients −/+ Iso in myocytes from sham or post-MI hearts. *P<0.05 between sham and MI, # P<0.05 between −/+ Iso.

Figure 7. Sarcoplasmic Reticulum (SR) Ca²⁺ content is identical in WT and S2808A myocytes from Sham and MI hearts

Caffeine-induced SR Ca²⁺ release was measured in WT and S2808A myocytes +/− Iso (10 nM) from sham or MI mice. A–B. Examples of Ca²⁺ transients elicited by 0.5Hz of electrical field stimulation (the first four traces) followed by Ca²⁺ transients induced by caffeine (the fifth large trace; the peak indicates SR Ca²⁺ content). C. Average amplitudes of caffeine induced Ca²⁺ transients −/+ Iso in WT and S2808A myocytes. *p<0.05 between sham and MI and # p<0.05 between −/+ Iso.