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S100A1, a small homodimeric EF-hand Ca2+-binding protein (~21 kDa), plays an important regulatory role in Ca2+ signaling pathways involved in various biological functions including Ca2+ cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca2+ release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca2+. Ca2+-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca2+-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca2+-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca2+ release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) are critical mediators of blood pressure (BP) regulation, primarily via the generation and release of vasorelaxants, including nitric oxide (NO). NO is produced in ECs by endothelial NO synthase (eNOS),which is activated by both calcium(Ca2+)-dependent and independent pathways. Here, we report that intracellular Ca2+ release from the endoplasmic reticulum(ER) via inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca2+-dependent eNOS activation. EC-specific type 1 1,4,5-trisphosphate receptor knockout (IP3R1-/-) mice are hypertensive and display blunted vasodilation in response to acetylcholine (ACh). Moreover, eNOS activity is reduced in both isolated IP3R1-deficient murine ECs and human ECs following IP3R1 knockdown. IP3R1 is upstream of calcineurin, a Ca2+/calmodulinactivated serine/threonine protein phosphatase. We show here that the calcineurin/nuclear factor of activated T cells (NFAT) pathway is less active and eNOS levels are decreased in IP3R1-deficient ECs. Furthermore, the calcineurin inhibitor cyclosporin A, whose use has been associated with the development of hypertension, reduces eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 plays a crucial role in the ECmediated vasorelaxation and the maintenance of normal BP. [ABSTRACT FROM AUTHOR]

The ryanodine receptor (RyR)/calcium-release channel on the sarcoplasmic reticulum mediates intracellular calcium release required for striated muscle contraction. RyR2, the predominant isoform in cardiac myocytes, comprises a macromolecular complex that includes calstabin2 (FKBP12.6). Calstabin2, an 11.8-kDa cis-trans peptidyl-prolyl isomerase (apparent molecular mass 12.6 kDa), stabilizes the closed state of the RyR2 channel, but the mechanism by which it achieves this regulation is not fully understood. Protein kinase A (PKA) phosphorylation of RyR2 decreases the affinity of calstabin2 for the RyR2 channel complex. In the present study we identified key aspartic acid residues on calstabin2 that are involved in binding to RyR2 and likely play a role in PKA phosphorylation-induced dissociation of calstabin2 from RyR2. We show that a mutant calstabin2 in which a key negatively charged residue (Asp-37) has been neutralized binds to a mutant RyR2 channel that mimics constitutively PKA-phosphorylated RyR2 (RyR2-S2808D). Furthermore, using wild-type and genetically altered murine models of heart failure induced by myocardial infarction, we show that manipulating the stoichiometry between calstabin2 and RyR2 can restore normal cardiac function in vivo. [ABSTRACT FROM AUTHOR]

Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have Previously shown that cardiac ryanodine receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-2-/- mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 ± 5.1%) compared with placebo (31.1 ± 3.1%; p < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WI mice. However, JTV519 did not show any of these beneficial effects in calstabin- 2-/- mice with MI. Additionally, JTVS19 improved skeletal muscle fatigue in WT and calstabin-2-/- mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WI but not calstabin- 2-/- mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR. [ABSTRACT FROM AUTHOR]