Dwarf open reading frame (DWORF) peptide is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

ABSTRACTThe cardiac sarcoplasmic reticulum calcium pump, SERCA, sequesters calcium in the sarco-endoplasmic reticulum (SR/ER) and plays a critical role in the contraction-relaxation cycle of the heart. A well-known regulator of SERCA in cardiac muscle is phospholamban (PLN), which interacts with the pump and reduces its apparent calcium affinity. A newly discovered SERCA regulatory subunit in cardiac muscle, dwarf open reading frame (DWORF), has added a new level of SERCA regulation. In this report, we modeled the structure of DWORF and evaluated it using molecular dynamics simulations. DWORF structure was modeled as a discontinuous helix with an unwound region at Pro15. This model orients an N-terminal amphipathic helix along the membrane surface and leaves a relatively short C-terminal transmembrane helix. We determined the functional regulation of SERCA by DWORF using a membrane reconstitution system. Surprisingly, we observed that DWORF directly activated SERCA by increasing its turnover rate. Furthermore, in-cell imaging of calcium dynamics demonstrated that DWORF increased SERCA-dependent ER calcium load, calcium reuptake rate, and spontaneous calcium release. Together, these functional assays suggest opposing effects of DWORF and PLN on SERCA function. The results agree with fluorescence resonance energy transfer experiments, which revealed changes in the affinity of DWORF for SERCA at low versus high cytosolic calcium concentrations. We found that DWORF has a higher affinity for SERCA in the presence of calcium, while PLN had the opposite behavior, a higher affinity for SERCA in low calcium. We propose a new mechanism for DWORF regulation of cardiac calcium handling in which DWORF directly enhances SERCA turnover by stabilizing the conformations of SERCA that predominate during elevated cytosolic calcium.

Abstract MP135: Cardiac Sarcoplasmic Reticulum Calcium Cycling is Regulated by Kinase Independent Function of Pi3kgamma

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular contractility. We investigated whether the lack of PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Isolated cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine induced calcium release indicating that PI3Kγ locally regulates the function of SR. The intracellular calcium remained at elevated levels in the cardiomyocytes of PI3Kγ -/- for a prolonged period after caffeine treatment. This could be due to changes in phosphorylation of SERCA2, Ryanodine receptor (RyR 2 ) or phospholamban (PLN). In fact, when we looked at phosphorylation of PLN in cardiac lysates, a major regulator of cardiac contractility and relaxation, PI3Kγ -/- mice showed significantly reduced PLN phosphorylation compared to littermate controls. Previous studies from our laboratory suggested that absence of PI3Kγ leads to increase in protein phosphatase (PP) activity which could be possible reason for rapid dephosphorylation of PLN, resulting in inhibition of SERCA2 pump. We observed increased SR associated PP activity and PLN associated PP activity in PI3Kγ -/- mice. We also observed increased association of PP-1 and PP2A with PLN in the absence of PI3Kγ. The altered calcium handling in the cardiomyocytes of PI3Kγ -/- mice could be restored to the level of WT controls by okadaic acid mediated inhibition of PP, suggesting that PI3Kγ plays a role in regulating PP activity associated with SR. To test whether PI3Kγ activity is required for PLN dephosphorylation and SR calcium cycling, we used mice with cardiac specific overexpression of kinase dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ). PI3Kγ inact /PI3Kγ -/- mice showed restored PLN phosphorylation, improved caffeine induced calcium reuptake, decreased SR and PLN associated PP activity. These studies show a novel regulation of PP and SR calcium regulation by kinase independent function of PI3Kγ. The underlying mechanism of PP regulation by PI3Kγ will be presented.

Exercise-induced increases in the expression and activity of cardiac sarcoplasmic reticulum calcium ATPase 2 is attenuated in AMPKα2kinase-dead mice

Exercise enhances cardiac sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) function through unknown mechanisms. The present study tested the hypothesis that the positive effects of exercise on SERCA2a expression and function in the left ventricle is dependent on adenosine-monophosphate-activated protein kinase (AMPK) α2 function. AMPKα2kinase-dead (KD) transgenic mice, which overexpress inactivated AMPKα2subunit, and wild-type C57Bl/6 (WT) mice were randomized into sedentary groups or groups with access to running wheels. After 5 months, exercised KD mice exhibited shortened deceleration time compared with sedentary KD mice. In left ventricular tissue, the ratio of phosphorylated AMPKαThr172:total AMPKα was 65% lower (P < 0.05) in KD mice compared with WT mice. The left ventricle of KD mice had 37% lower levels of SERCA2a compared with WT mice. Although exercise increased SERCA2a protein levels in WT mice by 53%, this response of exercise was abolished in exercised KD mice. Exercise training reduced total phospholamban protein content by 23% in both the WT and KD mice but remained 20% higher overall in KD mice. Collectively, these data suggest that AMPKα influences SERCA2a and phospholamban protein content in the sedentary and exercised heart, and that exercise-induced changes in SERCA2a protein are dependent on AMPKα function.

Perspectives on “Control of Ca release from within the cardiac sarcoplasmic reticulum”

Five groups of experts unravel the complex modulation of a function crucial for the beating heart.

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca2+ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca2+ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca2+]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca2+]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca2+ transient was normal, despite the reduced SR Ca2+ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca2+ transients because they are incapable of properly increasing the amount of Ca2+ stored in the SR because of a larger SR Ca2+ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca2+ channel current (ICa-L) and SR Ca2+ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca2+ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca2+ content, but cannot compensate for the inability to increase SR Ca2+ content during inotropic challenge.

Abstract 16191: Phospholamban Regulates Perinuclear/nuclear Calcium Dynamics in Cardiomyocytes

Introduction: Phospholamban (PLB) regulates cardiac sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), thus modulating SR Ca 2+ dynamics. Recent studies demonstrated that SERCA is involved in Ca 2+ uptake into the lumen of nuclear envelope (NE) of cardiomyocytes (CMs). However, the regulatory role of PLB on Ca 2+ uptake into NE remains unknown. Hypothesis: We hypothesize that PLB is also responsible for modulating nuclear Ca 2+ dynamics. Methods: Confocal immunofluorescence microscopy was used to determine subcellular expression of PLB. By using fluo-4 based confocal line-scan Ca 2+ imaging, we measured spontaneous Ca 2+ waves (SCWs) across both cytoplasmic and nuclear regions in isolated permeabilized mouse CMs. Results: Several anti-PLB antibodies strongly stained PLB at both SR and the perinuclear membranes in CMs. A PLB peptide (residues 1-31) eliminated all these anti-PLB antibody stains. To identify the functional role of PLB expressed in the perinuclear membranes, we took advantage of our recently established method that a Fab fragment of anti-PLB monoclonal antibody (Fab) reversed PLB inhibition specifically and increased SR Ca 2+ uptake and release. SCWs through the nuclear regions had typically relative low fluorescent amplitude (F/F 0 ) and slow decay time (t 1/2 ) compared to that in the cytoplasmic region. At the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) of 400 nM, Fab (100 μg/mL) significantly enhanced F/F 0 and decreased t 1/2 of SCWs in both cytoplasmic and nuclear regions. After addition of Fab, F/F 0 of SCWs through the nuclear regions increased from 0.91±0.16 to 1.27±0.19 (n=9, p<.05) while t 1/2 decreased from 137.6±18.5 ms to 105.0±11.3 ms, (p<.05). Similar effects were also observed after phosphorylation of PLB by addition of 20 μM cAMP (F/F 0 =1.43±0.11 vs. 1.04±0.14 in control, p<.05; t 1/2 =107.82±10.9 ms vs. 139.21±20.1 ms in control, n=6, p<.05). At high [Ca 2+ ] i of 1000 nM where PLB does not inhibit SERCA2a, addition of cAMP or Fab had no significant effect on SCWs in both cytoplasmic and nuclear regions. Conclusions: We demonstrated that PLB is expressed in and around NE. Acute removal of PLB inhibition increased perinuclear/nuclear Ca 2+ uptake and release. PLB is critically involved in nuclear Ca 2+ signaling modulation.