Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression

Abstract

Defective regulation of the cardiac ryanodine receptor (RyR2)/calcium release channel, required for excitation-contraction coupling in the heart, has been linked to cardiac arrhythmias and heart failure. For example, diastolic calcium "leak" via RyR2 channels in the sarcoplasmic reticulum has been identified as an important factor contributing to impaired contractility in heart failure and ventricular arrhythmias that cause sudden cardiac death. In patients with heart failure, chronic activation of the "fight or flight" stress response leads to protein kinase A (PKA) hyperphosphorylation of RyR2 at Ser-2808. PKA phosphorylation of RyR2 Ser-2808 reduces the binding affinity of the channel-stabilizing subunit calstabin2, resulting in leaky RyR2 channels. We developed RyR2-S2808A mice to determine whether Ser-2808 is the functional PKA phosphorylation site on RyR2. Furthermore, mice in which the RyR2 channel cannot be PKA phosphorylated were relatively protected against the development of heart failure after myocardial infarction. Taken together, these data show that PKA phosphorylation of Ser-2808 on the RyR2 channel appears to be a critical mediator of progressive cardiac dysfunction after myocardial infarction.

Fig. 1.

Lack of PKA phosphorylation of RyR2 in RyR2-S2808A mice. (A) The wild-type locus of the murine RyR2 gene containing exons 51–55. (B) The targeting construct containing 2.4- and 5.4-kb homologous regions (horizontal gray lines). The S2808A mutation (*) is engineered in exon 55. (C) The homologous recombinant mutant allele containing the RyR2-S2808A mutation and the ACN selection marker cassette. (D) Final RyR2-S2808A allele after excision of the ACN selection marker. (E) Representative Western blot analysis by using a phospho-specific antibody recognizing PKA-phosphorylated Ser-2808 on RyR2 and PKA kinasing reaction both demonstrate that RyR2-S2808A channels from mice cannot be PKA-phosphorylated. RyR2 immunoprecipitated from cardiac lysates from WT, heterozygous WT/RyR2-S2808A, and RyR2-S2808A mice were treated with alkaline phosphatase (AP) or protein kinase A (PKA), respectively (Left). In addition, WT and RyR2-S2808A (SA) mice were injected with isoproterenol (Right). (F) Representative confocal images of isolated cardiomyocytes immunolabeled with antibodies detecting the RyR2 protein (Top) or the PKA-phosphorylated form of RyR2 at Ser²⁸⁰⁸ (Center and Bottom). Center and Bottom represent isolated cardiomyocytes untreated or treated with isoproterenol, respectively.

Fig. 2.

PKA phosphorylation of Ser-2808 but not Ser-2031 modulates RyR2 single-channel activity. (A) Representative WT and mutant recombinant human RyR2 channels, coexpressed with calstabin2 (FKBP12.6), were PKA-phosphorylated in the presence or absence of the PKA-inhibitor PKI_5–24. Alanine substitution of Ser-2808, but not Ser-2031, prevents PKA phosphorylation of RyR2. Moreover, PKA phosphorylation of WT and RyR2-S2031A channel reduced the binding affinity of the RyR2 subunit calstabin2, whereas calstabin2 did not dissociate from RyR2-S2808A channels treated with PKA. (B) Single-channel recordings of RyR2-WT and mutant RyR2. PKA phosphorylation of RyR2-WT and RyR2-S2031A channels increased Po at low-cytosolic Ca²⁺ concentrations (150 nM), whereas the mutant RyR2-S2808A channels did not exhibit PKA phosphorylation-induced increase in Po. The Asp substitution of Ser-2031 functionally mimicked PKA phosphorylation of this residue but did not cause an increase in Po of the RyR2-S2031D channels. In contrast, RyR2-S2808D channels mimicked constitutively PKA-phosphorylated RyR2-WT channels, confirming that Ser-2808 is the only functional PKA site on RyR2. (C) Summary values of Po of single-channel recordings described in B (n = 4–9 channels in each group).

Fig. 3.

Increased cardiac contractility in RyR2-S2808A mice 4 weeks after MI. (A) Quantification of M-mode echocardiograms showing increased ejection fraction (EF) in RyR2-S2808A mice compared with WT. *, P < 0.05. Number of mice as indicated. (B) Pressure-volume loops showing increased cardiac contractility in RyR2-S2808A mice compared with WT. dP/dt, maximum slope of the derivative of change in systolic pressure over time. (C) Echocardiographic quantification of the end-systolic diameter (ESD) showing reduced cardiac remodeling in RyR2-S2808A mice compared with WT.

Fig. 4.

MI does not cause PKA hyperphosphorylation of RyR2 and calstabin2 dissociation in RyR2-S2808A mice. Equivalent amounts of RyR2 were immunoprecipitated from cardiac lysates by using an anti-RyR2 antibody. Representative immunoblots (A) and bar graphs (B) show the amount of PKA and CaMKII phosphorylation of RyR2 as well as the amount of calstabin2 associated with RyR2 (Left). In contrast to WT mice, RyR2-S2808A mice did not develop PKA hyperphosphorylation of RyR2 after MI. A slight reduction in SERCA2a and PLB expression was observed in both WT and S2808A mice, whereas PLB was hypophosphorylated at Ser-16 in both infarcted WT and S2808A mice (n = 9, P = N.S.).

Fig. 5.

Normalized RyR2 channel function in RyR2-S2808A mice after MI. (A) RyR2 channels isolated from hearts 28 days after MI showed reduced Po in RyR2-S2808A mice compared with WT. Representative single-channel tracings are shown at 150 nM Ca²⁺. Fo, frequency of channel opening (s^–1); To, average open time (ms); and Tc, average closed time (ms); values correspond to the representative tracing shown. Duration upper tracings, 5 s; bottom tracings, 0.5 s. Current amplitude of full openings is 4 pA; c, closed state of channel. (B) Bar graphs show mean values for WT (n = 7 channels) and RyR2-S2808A (n = 5 channels). *, P < 0.05.

Fig. 6.

Proposed model of mechanism by which inhibition of PKA phosphorylation of RyR2 prevents intracellular Ca²⁺ leak in heart failure. (Left) The cardiac ryanodine receptor exists in clusters of tetrameric calcium-release channels located on the SR membrane. Each RyR2 monomer contains one PKA phosphorylation site Ser-2808 (S) and binds one PKA enzyme complex (in the cartoon, only one PKA complex is shown per tetrameric channel). (Middle) During heart failure, persistent activation of the fight-or-flight stress response causes chronic activation of the β-adrenergic signaling pathway and PKA hyperphosphorylation of RyR2 (stars depict posttranslational modification by PO₄ molecules). Chronic PKA hyperphosphorylation of RyR2 is associated with calstabin2 depletion of the channel complex (symbolized by dissociation of octagons from RyR2). (Right) In RyR2-S2808A mice, substitution of Ser-2808 by Ala prevents RyR2 PKA hyperphosphorylation and SR Ca²⁺ leak that has beneficial effects, including reduced maladaptive remodeling after MI.