Ablation of the cardiac ryanodine receptor phospho-site Ser2808 does not alter the adrenergic response or the progression to heart failure in mice. Elimination of the genetic background as critical variable

Abstract

Background: Phosphorylation of the cardiac ryanodine receptor (RyR2) phospho-site S2808 has been touted by the Marks group as a hallmark of heart failure (HF) and a critical mediator of the physiological fight-or-flight response of the heart. In support of this hypothesis, mice unable to undergo phosphorylation at RyR2-S2808 (S2808A) were significantly protected against HF and displayed a blunted response to adrenergic stimulation. However, the issue remains highly controversial because several groups have been unable to reproduce these findings. An important variable not considered before is the genetic background of the mice used to obtain these divergent results.

Methods and results: We backcrossed a RyR2-S2808A mouse into a congenic C57Bl/6 strain, the same strain used by the Marks group to conduct their experiments. We then performed several key experiments to confirm or discard the genetic background of the mouse as a relevant variable, including induction of HF by myocardial infarction and tests of integrity of adrenergic response. Congenic C57Bl/6 harboring the S2808A mutation showed similar echocardiographic parameters that indicated identical progression towards HF compared to wild type controls, and had a normal response to adrenergic stimulation in whole animal and cellular experiments.

Conclusions: The genetic background of the different mouse models is unlikely to be the source of the divergent results obtained by the Marks group in comparison to several other groups. Cardiac adrenergic response and progression towards HF proceed unaltered in mice harboring the RyR2-S2808A mutation. Preventing RyR2-S2808 phosphorylation does not preclude a normal sympathetic response nor mitigates the pathophysiological consequences of MI.

Keywords: Adrenergic stimulation; Heart failure; Myocardial infarction; Phosphorylation; Ryanodine receptor.

Copyright © 2017 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Experimental model and mouse survival

A. Backcrossing scheme used to produce a congenic mouse strain derived from the RyR2-S2808A mouse developed in our laboratory. B. Direct sequencing of a portion of exon 55 confirms the presence of the missense mutation in codon 2808 leading to the serine to alanine substitution. Two silent mutations are located in codons 2804 and 2805. C. General scheme used to assess cardiac function in mice undergoing myocardial infarction. In this model, each mouse acts as its own control at basal conditions. D. Kaplan-Meier plot of survival after MI induction. Survival was slightly higher in S2808A than in WT controls, but this difference is not significant (n = 11–12 per genotype, p = 0.298).

Figure 2. Cardiac function after MI

MI due to permanent LAD ligation produced a significant decrease in ejection fraction (A) and fractional shortening (B), and an increase in LV diameter in diastole (C). Septum wall thickness in diastole (E) and stroke volume (E) remained unaltered. Most parameters, measured at one and four weeks after MI, were comparable between S2808A and WT mice, except for heart rate (F) (n = 9 per genotype; * p < 0.05, ** p < 0.01 vs. same genotype basal; # p < 0.05, ## p < 0.01 vs. WT at same time-point).

Figure 3. Heart remodeling after MI

Heart weight (A), lung weight (B) and liver weight (C) as a percentage of body weight in control mice and 4 weeks after LAD ligation. Only the heart weight was increased in both groups after MI (n = 7–9 per genotype per group; * p < 0.05 vs same genotype control).

Figure 4. Excitation-contraction coupling protein expression and phosphorylation

A. Representative blots. B. Expression of RyR2 has a tendency to be decreased 4 weeks after MI. C. Phosphorylation level of RyR2-S2808 determined as a ratio between phosphorylated and total protein. Phosphorylation was absent in S2808A mice, and has a tendency to be increased in wild-type animals. D–G. Expression level of excitation-contraction coupling proteins Cav1.2, SERCA2a, NCX and PLB. Expression of NCX was nearly 2.5-fold increased after MI. H. Phosphorylation level of PLB determined as described above (n = 6–8 per genotype per group; * p < 0.05, ** p < 0.01 vs. same genotype control; ^ tendency with p < 0.1 vs. same group control).

Figure 5. Cardiac adrenergic response

A–C. IP injection of isoproterenol (2 mg/kg) in anesthetized mice produces a significant increase in cardiac function, including heart rate (A), ejection fraction (B) and fractional shortening (C) (n = 6 per genotype; * p < 0.05, ** p < 0.01 vs. same genotype basal). D–E. Langendorff-perfused hearts stimulated with 300 nM Iso show a significant increase in heart rate (C), LV-developed pressure (LVPD, D) and dP/dt (E) (n = 5 per genotype; * p < 0.05, ** p < 0.01 vs. same genotype, as indicated by the color, at t = 0).

Figure 6. Ca2+ transients and SR Ca2+ load

A. Representative traces of field stimulation-triggered Ca2+ transients. After obtaining a stable basal response, Iso 300 nM was perfused for 1 min, followed by a pulse of 10 mM caffeine to measure the SR Ca2+ load. B. Quantification of Ca2+ transient amplitude at basal conditions and with Iso stimulation. Both groups showed a significant response to Iso. C–D. Quantification of the SR Ca2+ load and fractional release (Ca2+ transient amplitude/SR Ca2+ load) under Iso stimulation. (N = 2 WT, 3 S2808A; n = 16 cells per genotype; ** p < 0.01 vs. same genotype basal).