Abstract-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 Ca 2ϩ release by augmenting the sensitivity of the RyR2 channel to diastolic Ca 2ϩ . 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 ␤-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 ␤-adrenergic stimulation and PKA phosphorylation of Ser2808 in heart failure progression and cellular Ca 2ϩ handling. We found that the RyR2-S2808A mutation does not alter the ␤-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 [Ca 2ϩ ]. 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.

The intracellular Ca 2+ ([Ca 2+ ] i ) level of skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process 1 . However, the signaling components involved in such rapid Ca 2+ movement are not fully understood. Here, we report that mice deficient in the novel phosphatidylinositol phosphate (PIP) phosphatase MIP displayed muscle weakness and fatigue. Muscles isolated from MIP −/− mice produced less contractile force, markedly prolonged relaxation, and exhibited exacerbated fatigue. Further analyses revealed that MIP deficiency resulted in spontaneous Ca 2+ leak from the internal store -the sarcoplasmic reticulum (SR). This was attributed to the decreased metabolism/dephosphorylation and the subsequent accumulation of MIP substrates, especially PI(3,5)P 2 and PI(3,4)P 2 . Furthermore, we found that PI(3,5)P 2 and PI(3,4)P 2 bound to and directly activated the Ca 2+ release channel/ryanodine receptor (RyR1) of the SR. These studies provide the first evidence that finely controlled PIP levels in muscle cells are essential for maintaining Ca 2+ homeostasis and muscle performance.During our systematic genome-wide survey for tyrosine/dual specificity phosphatases (unpublished work), we discovered a novel phosphatase by hidden Markov database mining using the conserved catalytic motif ([V/I][V/I]HCXXGXXR[T/S]) as the bait sequence. Both human (BC035690) and mouse (BC018294) homologies were identified. They share 90% identity in amino acid sequences ( Supplementary Information, Fig. S1). Northern blotting analyses illustrated that this phosphatase was predominantly expressed in skeletal muscle and heart (Fig. 1a). Immunostaining indicates that it is primarily localized in the cytoplasm (data not shown). To verify its phosphatase property, we generated a GST fusion protein and tested its catalytic activity using pNPP (p-Nitrophenyl Phosphate), a widely used non-specific 7Correspondence should be addressed to: C.K.Q. (e-mail: E-mail: cxq6@case.edu). 6 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSJ.S., W.M. Y., M.B., J.A.S., and C.S. conducted the research and summarized the data. C.K.Q., M.B., H.H.V., T.M.N., and C.G. designed the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. (Fig. 1b). Instead, it dephosphorylated a variety of PIPs, especially PI(3,5) P 2 (Fig. 1c), similar to PTEN and myotubularin and myopathy related (MTMR) phosphatases that also favor PIPs as substrates despite containing tyrosine phosphatase domains 2 . As this new phosphatase is mainly expressed in skeletal muscle and heart, we named it MIP (musclespecific inositol phosphatase). While our gene knockout work on MIP was ongoing, the Mustalin group also identified this phosphatase (FLJ20133) in their comprehensive collection of tyrosine phosphatases from the human genome and listed it as the 14 th member of the MTMR family (MTMR14) based on the homology of its catalytic motif to myotubularin 3 . More recently, ina...

Abstract-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 2ϩ 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 2ϩ current (I CaL ), sarcoplasmic reticulum Ca 2ϩ 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 2ϩ 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. (Circ Res. 2008;102:e65-e72.)

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