Intact beta-adrenergic response and unmodified progression toward heart failure in mice with genetic ablation of a major protein kinase A phosphorylation site in the cardiac ryanodine receptor.

Increased phosphorylation of the cardiac ryanodine receptor (RyR)2 by protein kinase A (PKA) at the phosphoepitope encompassing Ser2808 has been advanced as a central mechanism in the pathogenesis of cardiac arrhythmias and heart failure. In this scheme, persistent activation of the sympathetic system during chronic stress leads to PKA "hyperphosphorylation" of RyR2-S2808, which increases Ca2+ release by augmenting the sensitivity of the RyR2 channel to diastolic Ca2+. This gain-of-function is postulated to occur with the unique participation of RyR2-S2808, and other potential PKA phosphorylation sites have been discarded. Although it is clear that RyR2 is among the first proteins in the heart to be phosphorylated by beta-adrenergic stimulation, the functional impact of phosphorylation in excitation-contraction coupling and cardiac performance remains unclear. We used gene targeting to produce a mouse model with complete ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Whole-heart and isolated cardiomyocyte experiments were performed to test the role of beta-adrenergic stimulation and PKA phosphorylation of Ser2808 in heart failure progression and cellular Ca2+ handling. We found that the RyR2-S2808A mutation does not alter the beta-adrenergic response, leaves cellular function almost unchanged, and offers no significant protection in the maladaptive cardiac remodeling induced by chronic stress. Moreover, the RyR2-S2808A mutation appears to modify single-channel activity, although modestly and only at activating [Ca2+]. Taken together, these results reveal some of the most important effects of PKA phosphorylation of RyR2 but do not support a major role for RyR2-S2808 phosphorylation in the pathogenesis of cardiac dysfunction and failure.

Ryanodine receptor channelopathies.

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.

Regulation of cardiac excitation-contraction coupling by sorcin, a novel modulator of ryanodine receptors.

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (I(Ca)) gives rise to Ca(2+)-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. Sorcin, a 21.6 kDa Ca(2+)-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the I(Ca)-triggered activity of RyRs that gives rise to [Ca2+]i transients. Since sorcin decreases the amplitude of the [Ca2+]i transient without affecting the amplitude of I(Ca), the overall effect of sorcin is to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = approximately 200 microM) appears to be reached only within the dyadic space. Thus, sorcin is a potent inhibitor of both spontaneous and I(Ca)-triggered RyR activity and may play a role in helping terminate the positive feedback loop of CICR.

Regulation of cardiac excitation-contraction coupling by sorcin, a novel modulator of ryanodine receptors

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (ICa) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. Sorcin, a 21.6 kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the ICa-triggered activity of RyRs that gives rise to [Ca2+]i transients. Since sorcin decreases the amplitude of the [Ca2+]i transient without affecting the amplitude of ICa, the overall effect of sorcin is to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = ~200 mM) appears to be reached only within the dyadic space. Thus, sorcin is a potent inhibitor of both spontaneous and ICa-triggered RyR activity and may play a role in helping terminate the positive feedback loop of CICR.

Sorcin inhibits calcium release and modulates excitation-contraction coupling in the heart.

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (ICa) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. We reported previously (Lokuta, A. J., Meyers, M. B., Sander, P. R., Fishman, G. I., and Valdivia, H. H. (1997) J. Biol. Chem. 272, 25333-25338) that sorcin, a 22-kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Here we show that sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the ICa-triggered activity of RyRs that gives rise to [Ca2+]i transients. Because sorcin decreased the amplitude of the [Ca2+]i transient without affecting the amplitude or kinetics of ICa, the overall effect of sorcin was to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = approximately 200 microM) appears to be reached only within the dyadic space. Rapid injection of 5 microM sorcin onto the cytosolic face of RyRs reconstituted in lipid bilayers resulted in complete inhibition of channel activity in < or = 20 ms. Thus, sorcin is a potent inhibitor of both spontaneous and ICa-triggered RyR activity and is kinetically capable of playing a role in terminating the positive feedback loop of CICR.

Abnormal Ca2+ release, but normal ryanodine receptors, in canine and human heart failure.

Sarcoplasmic reticulum (SR) Ca2+ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca2+ transport. Intracellular Ca2+ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca2+]i transient were reduced in HF. Ca2+ uptake assays showed 44+/-8% reduction of Vmax in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30+/-5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P(o) of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor beta-adrenergic stimulation of Ca2+ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca2+ uptake, more than Ca2+ release, contributing to the depressed and slow Ca2+ transient characteristic of HF.