Local calcium release activation by DHPR calcium channel openings in rat cardiac myocytes

The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current (ICa) controls the intensity of calcium release is not clear because of the stochastic nature of voltage-dependent gating of calcium channels (DHPRs) and of calcium-dependent gating of ryanodine receptors (RyRs). To disclose the relation between DHPR openings and the probability of calcium release, local calcium release activation by ICa was investigated in rat ventricular myocytes using patch-clamp and confocal microscopy. Calcium spikes were activated by temporally synchronized DHPR calcium current triggers, generated by instantaneous ‘tail’ICa and modulated by prepulse duration, by tail potential, and by the DHPR agonist BayK 8644. The DHPR–RyR coupling fidelity was determined from the temporal distribution of calcium spike latencies using a model based on exponentially distributed DHPR open times. The analysis provided a DHPR mean open time of ∼0.5 ms, RyR activation time constant of ∼0.6 ms, and RyR activation kinetics of the 4th order. The coupling fidelity was low due to the inherent prevalence of very short DHPR openings but was increased when DHPR openings were prolonged by BayK 8644. The probability of calcium release activation was high, despite low coupling fidelity, due to the activation of many DHPRs at individual release sites. We conclude that the control of calcium release intensity by physiological stimuli can be achieved by modulating the number and duration of DHPR openings at low coupling fidelity, thus avoiding the danger of inadvertently triggering calcium release events

Journal of Leukocyte Biology / The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6phosphate/insulinlike growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is upregulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogeninduced efferocytosis. The level of uptake of plasminogencoated apoptotic cells inversely correlates with the TNF production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an uptonow undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.(VLID)341171

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation

RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca2+ release channel/type 2 ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2R420Q. OBJECTIVE: To determine the arrhythmogenic mechanisms of this mutation. METHODS AND RESULTS: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca2+ waves during confocal Ca2+ recording in both mouse and human RyR2R420Q cardiomyocytes. In addition, Ca2+-induced Ca2+-release, as well as a rough indicator of fractional Ca2+ release, were higher and Ca2+ sparks longer in the RyR2R420Q-expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electronic microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2R420Q binding to junctophilin-2, as there were less junctophilin-2 coimmunoprecipitated with RyR2R420Q. At the single current level, the RyR2R420Q channel dwells longer in the open state at low intracellular Ca2+ ([Ca2+]i), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. CONCLUSIONS: The RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction. GRAPHIC ABSTRACT: An online graphic abstract is available for this article.This work was funded by Inserm and University Paris-Sud, and grants from ANR (ANR-19-CE14-0031-01 to A.M. Gómez), LabEx LERMIT (ANR-10-LABX-33), “Instituto de Salud Carlos III”; FEDER “Union Europea, Una forma de hacer Europa” (PI18/01582), La Fe Biobank (PT17/0015/0043) and Memorial Nacho Barberá to E. Zorio; Swiss National Science Foundation (SNSF grants no. 31003A 179325 and 310030 156375 to E. Niggli), British Heart Foundation (FS/15/30/31494) to S. Zissimopoulos; Italian Telethon ONLUS Foundation (GGP19231) and Italian MIUR (PRIN no. 2015ZZR4W3) to F. Protasi; National Institutes of Health (NIH) grants to J. Ramos-Franco (R01GM111397) and H.H. Valdivia–A.M. Gómez (2R01HL055438-22); and European Union H2020 (MSCA-RISE AMD-734931- 6) to A.M. Gómez. H.H. Valdivia was recipient of a Fullbright-Tockeville chair to work on A.M. Gómez laboratory. J.L. Álvarez and C.R. Valdivia were recipient of a visiting program Alambert from University Paris-Sud to work in A.M. Gómez laboratory. A. Zahradnikova was recipient of a postdoctoral position from University Paris-Sud. R. Rizzetto was recipient of a postdoctoral fellowship from CORDDIM (Région Ile de France). L. Yin was recipient of the CSC (Chinese Scholarship council)