Hyperphosphorylation Of RyR2 Research Articles

Cardiac Ryanodine Receptor Function and Regulation in Heart Disease

The cardiac ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR) controls intracellular Ca(2+) release and muscle contraction in the heart. Ca(2+) release via RyR2 is regulated by several physiological mediators. Protein kinase (PKA) phosphorylation dissociates the stabilizing FKBP12.6 subunit (calstabin2) from the RyR2 complex, resulting in increased contractility and cardiac output. Congestive heart failure is associated with elevated plasma catecholamine levels, and chronic stimulation of beta-adrenergic receptors leads to PKA hyperphosphorylation of RyR2 in failing hearts. PKA hyperphosphorylation results in calstabin2-depleted RyR2 that displays altered channel gating and may cause aberrant SR Ca(2+) release, depletion of SR Ca(2+) stores, and reduced myocardial contractility in heart failure. Calstabin2-depleted RyR2 may also trigger cardiac arrhythmias that cause sudden cardiac death. In patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), RyR2 missense mutations cause reduced calstabin2 binding to RyR2. Increased RyR2 phosphorylation and pathologically increased calstabin2 dissociation during exercise results in aberrant diastolic calcium release, which may trigger ventricular arrhythmias and sudden cardiac death. In conclusion, heart failure and exercise-induced sudden cardiac death have been linked to defects in RyR2-calstabin2 regulation, and this may represent a novel target for the prevention and treatment of these forms of heart disease.

Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6).

Dissociation of FKBP12.6 from the cardiac Ca2+-release channel (RyR2) as a consequence of protein kinase A (PKA) hyperphosphorylation of RyR2 at a single amino acid residue, serine-2808, has been proposed as an important mechanism underlying cardiac dysfunction in heart failure. However, the issue of whether PKA phosphorylation of RyR2 can dissociate FKBP12.6 from RyR2 is controversial. To additionally address this issue, we investigated the effect of PKA phosphorylation and mutations at serine-2808 of RyR2 on recombinant or native FKBP12.6-RyR2 interaction. Site-specific antibodies, which recognize the serine-2808 phosphorylated or nonphosphorylated form of RyR2, were used to unambiguously correlate the phosphorylation state of RyR2 at serine-2808 with its ability to bind FKBP12.6. We found that FKBP12.6 can bind to both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2. The S2808D mutant thought to mimic constitutive phosphorylation also retained the ability to bind FKBP12.6. Complete phosphorylation at serine-2808 by exogenous PKA disrupted neither the recombinant nor native FKBP12.6-RyR2 complex. Furthermore, binding of site-specific antibodies to the serine-2808 phosphorylation site did not dissociate FKBP12.6 from or prevent FKBP12.6 from binding to RyR2. Taken together, our results do not support the notion that PKA phosphorylation at serine-2808 dissociates FKBP12.6 from RyR2.

Molecular determinants of altered contractility in heart failure

Heart failure remains a leading cause of mortality in the Western world. An important hallmark of heart failure is reduced myocardial contractility. Alterations in intracellular Ca2+handling play a major role in the pathophysiology of these contractile abnormalities. Several defects in the excitation‐contraction (EC) coupling system have been identified in patients with heart failure. Alterations in the density and function of proteins relevant for EC coupling have been reported. Chronic stimulation of the beta‐adrenergic signaling pathway leads to protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2), which dissociates FKBP12.6 from RyR2, thereby altering channel gating and promoting diastolic sarcoplasmic reticulum (SR) Ca2+release. This may deplete the SR Ca2+stores, which may reduce myocardial contractility. Clinical studies have demonstrated that beta‐adrenergic receptor blockers reduce morbidity and mortality in all grades of congestive heart failure. Our experimental data indicate that beta‐blockers reverse RyR2 hyperphosphorylation and normalize channel gating, which is associated with increased contractility in heart failure. In conclusion, chronic hyperactivity of the beta‐adrenergic signaling pathway impairs intracellular Ca2+handling, which leads to reduced contractility in patients with heart failure.

Immunophilins and coupled gating of ryanodine receptors.

The ryanodine receptor (RyR) is the major calcium (Ca(2+)) release channel in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle and is required for excitation-contraction (EC) coupling. The 565 kDa RyR protein forms a tetrameric channel that is part of a macromolecular signaling complex that also includes four FK506 binding proteins (FKBPs). The RyR channel complex is localized on specialized regions of the SR, such that the large RyR cytoplasmic domain is closely opposed to the transverse tubule (T-tubule) of the plasma membrane. RyR channel complexes are organized in regular arrays such that neighboring RyRs are in physical contact with each other. We have shown that physical and functional association between RyR1 or RyR2 channels results in coordinated gating behavior termed coupled gating. Coupled gating requires FKBP12 or FKBP12.6 in the RyR1 or RyR2 macromolecular complexes, respectively. FKBPs are known to stabilize single RyR channel function. Coupled gating describes an additional role for FKBPs in the functional coordination of RyR channel complexes that allows clusters of channels to function as "Ca2+ release units" (CRU). In addition, the FKBP-RyR interaction is regulated by PKA phosphorylation. In failing hearts PKA hyperphosphorylation of RyR2 causes depletion of FKBP12.6 from the channel macromolecular complex and may contribute to contractile dysfunction by impairing EC coupling. As FKBPs are potent modulators of RyR channel function, the FKBP-RyR interaction is a focus for determining molecular mechanisms of coupled gating and presents an exciting pharmacologic target for restoration of RyR complex function in diseased states.

Progression of Heart Failure

The cardiac ryanodine receptor (RyR2)/Ca2+ release channel on the sarcoplasmic reticulum (SR) is regulated by evolutionarily highly conserved signaling pathways that control excitation-contraction (EC) coupling in the heart. Phosphorylation of RyR2 by cAMP-dependent protein kinase (PKA) plays a key role in regulating the channel in response to stress via activation of the sympathetic nervous system (the “fight-or-flight response”).1 Maladaptive PKA hyperphosphorylation of RyR2 in failing hearts alters channel function, which may cause depletion of SR Ca2+ and diastolic release of SR Ca2+. This can initiate delayed afterdepolarizations that trigger ventricular arrhythmias.1 Mutations in RyR2 recently have been identified in patients with catecholaminergic induced sudden cardiac death (SCD).2–4⇓⇓ There may be a direct link between the PKA hyperphosphorylation of RyR2 that occurs during the progression of heart failure and fatal cardiac arrhythmias. Regulation of cardiac EC coupling by the release of Ca2+ from the SR via RyR2 in cardiomyocytes, known as Ca2+-induced Ca2+ release (CICR), has been appreciated for more than a decade.5,6⇓ Furthermore, it is well known that the amplitude of the Ca2+ transient generated by SR Ca2+ release determines contractile force in cardiomyocytes. The systems that regulate SR Ca2+ release include: (1) the triggers (predominantly Ca2+ influx through the voltage-gated Ca2+ channel on the plasma membrane); (2) the SR Ca2+ release channel or type 2 RyR2; and (3) the SR Ca2+ reuptake pump (SERCA2a) and its regulator phospholamban. These systems (trigger, release, and reuptake) are modulated by signaling pathways, including the β-adrenergic receptor (β-AR) signaling pathway (ie, phosphorylation by PKA). Activation of the sympathetic nervous system in response to stress results in elevation of cAMP levels and activation of PKA. Phosphorylation of RyR2 may not correlate directly …

Ryanodine receptors, FKBP12, and heart failure

RyR2 function is regulated by highly conserved signaling pathways that modulate excitation-contraction (EC) coupling. cAMP dependent protein kinase (PKA) phosphorylation of RyR2 plays an important role in regulating channel function in response to stress signaled by the sympathetic nervous system (the classic "fight or flight response") (1). PKA phosphorylation of RyR2 induces dissociation of the regulatory protein FKBP12.6 resulting in channels with increased sensitivity to Ca2+-induced Ca2+ release. Under normal physiological conditions (no cardiac damage) PKA phosphorylation of RyR2 is part of an integrated physiological response that leads to increased EC coupling gain and increased cardiac output. PKA-hyperphosphorylation of RyR2 in failing hearts is a maladaptive response that results in depletion of FKBP12.6 from the RyR2 macromolecular complex and defective channel function (pathologically increased sensitivity to Ca2+-induced Ca2+ release) that may cause depletion of SR Ca2+ and diastolic release of SR Ca2+ that can initiate delayed after depolarizations (DADs) that trigger ventricular arrhythmias (1). RyR2 mutations in patients with catecholaminergic induced sudden cardiac death provide further evidence linking the sympathetic nervous system, RyR2 and ventricular arrhythmias (2-4). The chronic hyperadrenergic state of heart failure is associated with defective Ca2+ signaling in part due to PKA hyperphosphorylation of RyR2.

Ryanodine receptors, FKBP12, and heart failure.

RyR2 function is regulated by highly conserved signaling pathways that modulate excitation-contraction (EC) coupling. cAMP dependent protein kinase (PKA) phosphorylation of RyR2 plays an important role in regulating channel function in response to stress signaled by the sympathetic nervous system (the classic "fight or flight response") (1). PKA phosphorylation of RyR2 induces dissociation of the regulatory protein FKBP12.6 resulting in channels with increased sensitivity to Ca2+-induced Ca2+ release. Under normal physiological conditions (no cardiac damage) PKA phosphorylation of RyR2 is part of an integrated physiological response that leads to increased EC coupling gain and increased cardiac output. PKA-hyperphosphorylation of RyR2 in failing hearts is a maladaptive response that results in depletion of FKBP12.6 from the RyR2 macromolecular complex and defective channel function (pathologically increased sensitivity to Ca2+-induced Ca2+ release) that may cause depletion of SR Ca2+ and diastolic release of SR Ca2+ that can initiate delayed after depolarizations (DADs) that trigger ventricular arrhythmias (1). RyR2 mutations in patients with catecholaminergic induced sudden cardiac death provide further evidence linking the sympathetic nervous system, RyR2 and ventricular arrhythmias (2-4). The chronic hyperadrenergic state of heart failure is associated with defective Ca2+ signaling in part due to PKA hyperphosphorylation of RyR2.

Beta-adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure.

beta-Adrenergic receptor blockade is one of the most effective treatments for heart failure, a leading cause of mortality worldwide. The use of beta-adrenergic receptor blockers in patients with heart failure is counterintuitive, however, because they are known to decrease contractility in normal hearts. The ryanodine receptor (RyR2) on cardiac sarcoplasmic reticulum is the key calcium release channel required for excitation-contraction coupling. In failing hearts, the stoichiometry and function of the RyR2 macromolecular complex is altered. Decreased levels of phosphatases (PP1 and PP2A) and hyperphosphorylation by protein kinase A result in dissociation of the regulatory protein FKBP12.6 and channels with increased open probability. Here, we show that systemic oral administration of a beta-adrenergic receptor blocker reverses protein kinase A hyperphosphorylation of RyR2, restores the stoichiometry of the RyR2 macromolecular complex, and normalizes single-channel function in a canine model of heart failure. These results may, in part, explain the improved cardiac function observed in heart failure patients treated with beta-adrenergic receptor blockers.