Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca(2+) homeostasis.

The intracellular Ca(2+) concentration ([Ca(2+)](i)) in skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process. However, the signalling components involved in such rapid Ca(2+) movement are not fully understood. Here we report that mice deficient in the newly identified PtdInsP (phosphatidylinositol phosphate) phosphatase MIP/MTMR14 (muscle-specific inositol phosphatase) show muscle weakness and fatigue. Muscles isolated from MIP/MTMR14(-/-) mice produced less contractile force, had markedly prolonged relaxation and showed exacerbated fatigue relative to normal muscles. Further analyses revealed that MIP/MTMR14 deficiency resulted in spontaneous Ca(2+) leakage from the internal store - the sarcoplasmic reticulum. This was attributed to decreased metabolism (dephosphorylation) and the subsequent accumulation of MIP/MTMR14 substrates, especially PtdIns(3,5)P(2) and PtdIns (3,4)P(2). Furthermore, we found that PtdIns(3,5)P(2) and PtdIns(3,4)P(2) bound to, and directly activated, the Ca(2+) release channel (ryanodine receptor 1, RyR1) of the sarcoplasmic reticulum. These studies provide the first evidence that finely controlled PtdInsP levels in muscle cells are essential for maintaining Ca(2+) homeostasis and muscle performance.

Adrenergic regulation of cardiac contractility does not involve phosphorylation of the cardiac ryanodine receptor at serine 2808.

The sympathetic nervous system is a critical regulator of cardiac function (heart rate and contractility) in health and disease. Sympathetic nervous system agonists bind to adrenergic receptors that are known to activate protein kinase A, which phosphorylates target proteins and enhances cardiac performance. Recently, it has been proposed that protein kinase A-mediated phosphorylation of the cardiac ryanodine receptor (the Ca(2+) release channel of the sarcoplasmic reticulum at a single residue, Ser2808) is a critical component of sympathetic nervous system regulation of cardiac function. This is a highly controversial hypothesis that has not been confirmed by several independent laboratories. The present study used a genetically modified mouse in which Ser2808 was replaced by alanine (S2808A) to prevent phosphorylation at this site. The effects of isoproterenol (a sympathetic agonist) on ventricular performance were compared in wild-type and S2808A hearts, both in vivo and in isolated hearts. Isoproterenol effects on L-type Ca(2+) current (I(CaL)), sarcoplasmic reticulum Ca(2+) release, and excitation-contraction coupling gain were also measured. Our results showed that isoproterenol caused significant increases in cardiac function, both in vivo and in isolated hearts, and there were no differences in these contractile effects in wild-type and S2808A hearts. Isoproterenol increased I(CaL), the amplitude of the Ca(2+) transient and excitation-contraction coupling gain, but, again, there were no significant differences between wild-type and S2808A myocytes. These results show that protein kinase A phosphorylation of ryanodine receptor Ser2808 does not have a major role in sympathetic nervous system regulation of normal cardiac function.

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.