JTV519 (K201) reduces sarcoplasmic reticulum Ca²⁺ leak and improves diastolic function in vitro in murine and human non‐failing myocardium

Abstract

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca 2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca 2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. EXPERIMENTAL APPROACH SR Ca2+ leak was induced by ouabain in murine cardiomyocytes. [Ca 2+ ]-transients, SR Ca 2+ load and RyR2-mediated Ca 2+ leak (sparks/waves) were quantified, with or without JTV519 (1 mmol·L-1 ). Contribution of Ca 2+-/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser 2814 phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. ], Ca 2+ leak was significantly reduced by JTV519, but it had no effect on fractional Ca 2+ release or Ca 2+ wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. 2+ removal varies among species between 7% (rats), 28% (rabbits) and up to about 50% (failing human hearts) (Dipla et al., 1999;Bers, 2001). KEY RESULTS Ouabain CONCLUSIONS AND IMPLICATIONSLeak of Ca 2+ from the SR through RyR2s during diastole has been implicated in arrhythmias and contractile dysfunction in heart failure (Marx et al., 2000;Lindner et al., 2002), myocardial ischaemia (Hirose et al., 2008), atrial fibrillation (Vest et al., 2005) and congenital arrhythmias (Mohamed et al., 2007). Several mechanisms have been proposed to explain RyR2 dysfunction, including altered RyR2 gating due to point mutations in the RyR2 molecule (Cerrone et al., 2005), alterations in RyR2 phosphorylation (Witcher et al., 1991;Wehrens et al., 2004b;Xiao et al., 2005) MethodsThe use of donor hearts and research protocols for human samples were approved by the local ethics committee (ref. nr. 20-277 ex 08/09). Investigations on human tissue conform with the principles outlined in the Declaration of Helsinki. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (McGrath et al., 2010). Animals were treated according to the Guidelines for the Care and Use of Laboratory Animals (National Institute of Health, USA). Cell isolationAdult FVB/N mice (11-16 weeks old; total = 43) provided by the Abt. f. Labortierkunde, Medical University of Vienna, Austria, were used for all experiments. Ventricular cardiomyocytes were isolated as previously described (Heinzel et al., 2002;Sedej et al., 2010). Mice were anesthetized with isoflu- . Only quiescent, rod-shaped ventricular cardiomyocytes were used for experiments. Confocal [Ca 2+ ] i measurementsCells were studied in a perfusion chamber mounted on the stage of an ...

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle

“…Some aspects of K201-mediated RyR2 stabilization and prevention of SR leak are controversial, including the ideas that K201 promotes FKBP12.6 binding to the channels and/or modulates phosphorylation status (Yano et al, 2003;Wehrens et al, 2005;Xiao et al, 2007;Blayney et al, 2010;Sacherer et al, 2012). In our hands, the effects of K201 on RyR2 open probability were not directly affected by adding FKBP12.6, which is in line with our previous reports of FKBP12.6 not being a direct modulator of RyR2 Xiao et al, 2007).…”

“…Other groups have further confirmed K201's cardioprotective properties in various heart pathologies, including ischemia and arrhythmogenesis in various animal models (Kaneko, 1994;Ito et al, 2000;Kawabata et al, 2002;Wehrens et al, 2004;Lisy and Burnett, 2006;Loughrey et al, 2007;Otani et al, 2013), in addition to human cardiac cells (Toischer et al, 2010;Sacherer et al, 2012). The mechanism of action of K201 is complex and includes various effects, including block of annexin V-mediated Ca 21 transport ) and plasmalemma currents mediated by K 1 , Na 1 , or Ca 21 channels (Kimura et al, 1999;Loughrey et al, 2007).…”

Intracellular Dyssynchrony of Diastolic Cytosolic [Ca 2+ ] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

“…15 Confocal line scan images of cytosolic [Ca 2+ ] transients in cardiomyocytes from a porcine model of chronic myocardial ischemia reported earlier 8 were analyzed for subcellular dyssynchrony in cytosolic [Ca 2+ ] decay. 2 1, HEPES 10, and glucose 10; pH was adjusted to 7.35 with NaOH. Cells were transferred to laminin-coated culture dishes on the stage of a confocal microscope (Zeiss, LSM 510 Meta, Jena, Germany), and superfused with Tyrode solution (37°C), containing blebbistatin (10 µmol/L, Tocris Bioscience, Bristol, United Kingdom) or butanedione monoxime (10 mmol/L) to avoid cell movement.…”

“…1 Dyssynchronous activation of ventricular tissue, as it occurs in bundle-branch block, leads to less efficient contraction and worsening of heart failure, whereas resynchronization therapy improves cardiac contractility, morbidity, and mortality in these patients. 2 Dyssynchronous contraction is also observed in hearts with diastolic or systolic heart failure in the absence of overt electric conduction defects, 3 suggesting dysfunction at the level of the cardiomyocyte.…”

Atrial Structure and Function and its Implications for Current and Emerging Treatments for Atrial Fibrillation

“…30 This drug is currently the focus of phase II investigative clinical trial and may have a broader role in remodelling processes given the strong overlap of calcium handling pathways in electrical and early structural remodelling. 31 Neural mediated electrical remodelling Autonomic cardiac inputs are well described and originate both centrally (extrinsic) and locally (intrinsic), contain both cholinergic and adrenergic inputs and have been recently expertly reviewed by Chen et al 23 Extrinsic inputs arise from the medulla (vagal nerve) or the paravertebral ganglia (superior cervical, middle cervical and cervicothoracic or stellate ganglion).…”