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Background: PKA (protein kinase A)–mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca 2+ release and induction of cardiac Ca 2+ alternans. Methods: We performed single-cell endoplasmic reticulum Ca 2+ imaging to assess the impact of S2030 mutations on Ca 2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca 2+ imaging. Results: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca 2+ level at which Ca 2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM–wild-type increased, whereas Ca 2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca 2+ /CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM–wild-type, CaM-M, and PKA, but not CaMKII, in Ca 2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca 2+ alternans and accelerated Ca 2+ transient recovery in intact working hearts, whereas CaM–wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM–wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide–sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide–sensitive component, of isoproterenol.

PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of CaWe performed single-cell endoplasmic reticulum CaThese data demonstrate, for the first time, that the PKA phosphorylation site RyR-S2030 is an important determinant of PKA-regulated, CaM-dependent Ca

Mutations in the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. GDAP1 is an atypical glutathione S-transferase (GST) of the outer mitochondrial membrane and the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs). Here, we investigate the role of this GST in the autophagic flux and the membrane contact sites (MCSs) between mitochondria and lysosomes in the cellular pathophysiology of GDAP1 deficiency. We demonstrate that GDAP1 participates in basal autophagy and that its depletion affects LC3 and PI3P biology in autophagosome biogenesis and membrane trafficking from MAMs. GDAP1 also contributes to the maturation of lysosome by interacting with PYKfyve kinase, a pH-dependent master lysosomal regulator. GDAP1 deficiency causes giant lysosomes with hydrolytic activity, a delay in the autophagic lysosome reformation, and TFEB activation. Notably, we found that GDAP1 interacts with LAMP-1, which supports that GDAP1–LAMP-1 is a new tethering pair of mitochondria and lysosome membrane contacts. We observed mitochondria–lysosome MCSs in soma and axons of cultured mouse embryonic motor neurons and human neuroblastoma cells. GDAP1 deficiency reduces the MCSs between these organelles, causes mitochondrial network abnormalities, and decreases levels of cellular glutathione (GSH). The supply of GSH-MEE suffices to rescue the lysosome membranes and the defects of the mitochondrial network, but not the interorganelle MCSs nor early autophagic events. Overall, we showthat GDAP1 enables the proper function of mitochondrial MCSs in both degradative and nondegradative pathways, which could explain primary insults in GDAP1-related CMT pathophysiology, and highlights new redox-sensitive targets in axonopathies where mitochondria and lysosomes are involved.

Aims Atrial fibrillation (AF) has been associated with intracellular calcium disturbances in human atrial myocytes, but little is known about the potential influence of sex and we here aimed to address this issue. Methods and results Alterations in calcium regulatory mechanisms were assessed in human atrial myocytes from patients without AF or with long-standing persistent or permanent AF. Patch-clamp measurements revealed that L-type calcium current (ICa) density was significantly smaller in males with than without AF (¿1.15¿±¿0.37 vs. ¿2.06¿±¿0.29 pA/pF) but not in females with AF (¿1.88¿±¿0.40 vs. ¿2.21¿±¿0.0.30 pA/pF). In contrast, transient inward currents (ITi) were more frequent in females with than without AF (1.92¿±¿0.36 vs. 1.10¿±¿0.19 events/min) but not in males with AF. Moreover, confocal calcium imaging showed that females with AF had more calcium spark sites than those without AF (9.8¿±¿1.8 vs. 2.2¿±¿1.9 sites/µm2) and sparks were wider (3.0¿±¿0.3 vs. 2.2¿±¿0.3 µm) and lasted longer (79¿±¿6 vs. 55¿±¿8 ms), favouring their fusion into calcium waves that triggers ITIs and afterdepolarizations. This was linked to higher ryanodine receptor phosphorylation at s2808 in women with AF, and inhibition of adenosine A2A or beta-adrenergic receptors that modulate s2808 phosphorylation was able to reduce the higher incidence of ITI in women with AF. Conclusion Perturbations of the calcium homoeostasis in AF is sex-dependent, concurring with increased spontaneous SR calcium release-induced electrical activity in women but not in men, and with diminished ICa density in men only., This work was supported by grants from The Spanish Ministry of Science Innovation and Universities [SAF2017-88019-C3-1-R MICIU /AEI/ FEDER /UE] to L.H.-M. [SAF2017-88019-C3-2-R MICIU /AEI/ FEDER /UE] to R.B. and [ SAF2017-87349-R MICIU /AEI/ FEDER /UE] to F.C.; and from the Spanish Ministry of Health and Consume, ISCIII, CIBERCV [CB16/11/00276] and Fondo Europeo de Desarrollo Regional (FEDER) to J.C., Fundació Marato TV3 [20152030/31] to L.H.-M./F.C. Also supported by ISC III [PIE14/00034] and IWT [SBO-140028] to F.C., a PhD grant [FPU/01250] to S.C., a PERIS grant from Generalitat de Catalunya to A.L. and [SGR2017-1769] to L.H.-M.

Plasma-treated hydrogels have been put forward as a potential selective osteosarcoma therapy through the release of reactive species to the diseased site. To allow their translation to the clinics, it is crucial to show that the oxidative stress delivered by such hydrogels does not adversely affect healthy tissues. This is evaluated here by investigating the in vivo performance of a robocasted calcium phosphate cement infiltrated by a plasma-treated hydrogel. The plasma-treated composite implanted in a critical size bone defect of healthy rabbits revealed its safety, allowing equivalent bone ingrowth compared to the control scaffolds and to that of direct plasma treatment of the bone defect. This opens the door for using composite biomaterials containing plasma-generated reactive species in bone therapies.

Aims: It is unknown how ß-adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)-tagged RyR2 clusters. Methods: Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. Results: Spontaneous calcium release from single RyR2 clusters induced 91.4% ± 2.0% of all calcium sparks while 8.0% ± 1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two- than one-cluster sparks with a median and interquartile range for the cumulative distribution of 15.7 ± 20.1 vs 7.6 ± 5.7 a.u. (P < .01). ß2-adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two- and three-cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9 ± 23.4 a.u. vs 10.7 ± 10.9; P = .015). Conclusions: Most sparks are caused by activation of a single RyR2 cluster at baseline while ß-adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation. Keywords: calcium spark; cardiac myocyte; confocal imaging; ryanodine receptor; sarcoplasmic reticulum; ß-adrenergic., This work was supported by grants from the Spanish Ministry of Science and Innovation PID2020-116927RB-C21 and SAF2017-88019-C3-1R (to LHM) and SAF2017-88019-C3-3R (to RB); from Fundació Marató TV3, Marato-2015-2030 (to LHM); from Generalitat de Catalunya SGR2017-1769 (to LHM); from the Natural Sciences and Engineering Research Council of Canada (to SRWC); from the Canadian Institutes of Health Research (to SRWC); from the Heart and Stroke Foundation Chair in Cardiovascular Research (to SRWC), and from Spanish Ministry of Health and Consume CB16/11/00276 (to JC). SC was the recipient of a predoctoral grant (FPU18/01250) from the Spanish Ministry of Science and Innovation, and AL received a PERIS SALUT-16 grant from Generalitat de Catalunya.