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Background: Transverse (t)-tubules drive the rapid and synchronous Ca 2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca 2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca 2+ ., Methods: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca 2+ , and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure., Results: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca 2+ release in the cell interior. Systolic Ca 2+ , I Ca-L , sarcoplasmic reticulum Ca 2+ content, and sarcoendoplasmic reticulum Ca 2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca 2+ transient, the rate of Ca 2+ removal, and the peak L-type Ca 2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria., Conclusions: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of I Ca-L , sarcoplasmic reticulum Ca 2+ content, and systolic Ca 2+ . We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy., Competing Interests: None.

Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which limits pharmacological therapy development. Prior work has demonstrated the importance of reactive oxygen species (ROS) and mitochondrial dysfunction in the development of AF. Mitochondrial structure, interactions with other organelles such as sarcoplasmic reticulum (SR) and T-tubules (TT), and degradation of dysfunctional mitochondria via mitophagy are important processes to understand ultrastructural changes due to AF. However, most analysis of mitochondrial structure and interactome in AF has been limited to two-dimensional (2D) modalities such as transmission electron microscopy (EM), which does not fully visualize the morphological evolution of the mitochondria during mitophagy. Herein, we utilize focused ion beam-scanning electron microscopy (FIB-SEM) and perform reconstruction of three-dimensional (3D) EM from murine left atrial samples and measure the interactions of mitochondria with SR and TT. We developed a novel 3D quantitative analysis of FIB-SEM in a murine model of AF to quantify mitophagy stage, mitophagosome size in cardiomyocytes, and mitochondrial structural remodeling when compared with control mice. We show that in our murine model of spontaneous and continuous AF due to persistent late sodium current, left atrial cardiomyocytes have heterogenous mitochondria, with a significant number which are enlarged with increased elongation and structural complexity. Mitophagosomes in AF cardiomyocytes are located at Z-lines where they neighbor large, elongated mitochondria. Mitochondria in AF cardiomyocytes show increased organelle interaction, with 5X greater contact area with SR and are 4X as likely to interact with TT when compared to control. We show that mitophagy in AF cardiomyocytes involves 2.5X larger mitophagosomes that carry increased organelle contents. In conclusion, when oxidative stress overcomes compensatory mechanisms, mitophagy in AF faces a challenge of degrading bulky complex mitochondria, which may result in increased SR and TT contacts, perhaps allowing for mitochondrial Ca 2+ maintenance and antioxidant production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy., Methods: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging., Results: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca 2+ -induced Ca 2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca 2+ channels and ryanodine receptor Ca 2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components., Conclusions: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload., Competing Interests: None.

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca 2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 ( Mfn2 ), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Atrial Ca 2+ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca 2+ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca 2+ signals of atria were collected in isolated perfused hearts by optical mapping. Ca 2+ transient duration (CaD), alternan, and the recovery properties of Ca 2+ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca 2+ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca 2+ -RyR pathway, and thus exerts therapeutic effect on POAF.

Background: Duchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age., Objectives: The authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs., Methods: Two-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old., Results: Conventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca 2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca 2+ -sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation., Conclusions: GRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036., Competing Interests: Funding Support and Author Disclosures This study was supported by the Association Française contre les Myopathies (AFM) (OC N°11590, AL N°15083, SB N° 14389, 15208, 15632, 16396, and 17124) and by Institut de Recherches Servier (Suresnes, France). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

We compared the expression of Са 2+ -ATPase (SERCA2a), calsequestrin (CASQ2), ryanodine receptors (RyR2) proteins and their genes (ATP2A2, CASQ2, and RYR2) in coronary heart disease (CHD) patients with and without comorbid type 2 diabetes mellitus. All studies were performed on the right atrial appendages resected during coronary bypass surgeries. Expression of SERCA2a and RyR2 proteins and their ATP2A2 (p=0.046) and RYR2 genes in comorbid pathology was significantly (p=0.042) higher (by 1.2 and 2 times; p=0.025). The expression of CASQ2 protein and its gene did not differ significantly between the groups (p=0.82 and p=0.066, respectively). It was concluded that the expression of SERCA2a and RyR2 proteins and their genes (but not CASQ2 and its gene) is elevated in CHD associated with type 2 diabetes mellitus. Expression of the studied proteins correlated with the expression of their genes. Increased expression of CASQ2 protein and its gene can probably prevent imbalance of the Ca 2+ -transporting systems in cardiomyocytes and contractile dysfunction of the myocardium, even in CHD associated with type 2 diabetes mellitus., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Metabolic syndrome (MetS) is associated with additional cardiovascular risk in mammalians while there are relationships between hyperglycemia-associated cardiovascular dysfunction and increased platelet P2Y12 receptor activation. Although P2Y12 receptor antagonist ticagrelor (Tica) plays roles in reduction of cardiovascular events, its beneficial mechanism remains poorly understood. Therefore, we aimed to clarify whether Tica can exert a direct protective effect in ventricular cardiomyocytes from high-carbohydrate diet-induced MetS rats, at least, through affecting sarcoplasmic reticulum (SR)-mitochondria (Mit) miscommunication. Tica treatment of MetS rats (150 mg/kg/day for 15 days) significantly reversed the altered parameters of action potentials by reversing sarcolemmal ionic currents carried by voltage-dependent Na + and K + channels, and Na + /Ca 2+ -exchanger in the cells, expressed P2Y12 receptors. The increased basal-cytosolic Ca 2+ level and depressed SR Ca 2+ load were also reversed in Tica-treated cells, at most, though recoveries in the phosphorylation levels of ryanodine receptors and phospholamban. Moreover, there were marked recoveries in Mit structure and function (including increases in both autophagosomes and fragmentations) together with recoveries in Mit proteins and the factors associated with Ca 2+ transfer between SR-Mit. There were further significant recoveries in markers of both ER stress and oxidative stress. Taken into consideration the Tica-induced prevention of ER stress and mitochondrial dysfunction, our data provided an important document on the pleiotropic effects of Tica in the electrical activity of the cardiomyocytes from MetS rats. This protective effect seems through recoveries in SR-Mit miscommunication besides modulation of different sarcolemmal ion-channel activities, independent of P2Y12 receptor antagonism., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscular weakness because of the loss of dystrophin. Extracellular Ca2+ flows into the cytoplasm through membrane tears in dystrophin-deficient myofibers, which leads to muscle contracture and necrosis. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) takes up cytosolic Ca2+ into the sarcoplasmic reticulum, but its activity is decreased in dystrophic muscle. Here, we show that an allosteric SERCA activator, CDN1163, ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. The administration of CDN1163 prevented exercise-induced muscular damage and restored mitochondrial function. In addition, treatment with CDN1163 for 7 weeks enhanced muscular strength and reduced muscular degeneration and fibrosis in mdx mice. Our findings provide preclinical proof-of-concept evidence that pharmacological activation of SERCA could be a promising therapeutic strategy for DMD. Moreover, CDN1163 improved muscular strength surprisingly in wild-type mice, which may pave the new way for the treatment of muscular dysfunction., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca 2+ release. Muscles from older DG mice display ER stress, ER expansion, increased mTOR signaling, inadequate clearance of aggregated proteins by the proteasomes, and elevation of protein aggregates and lysosomes. This study suggests that the myopathy associated with the D244G mutation in CASQ1 is driven by CASQ1 mislocalization, reduced SR Ca 2+ release, CASQ1 misfolding/aggregation and ER stress. The subsequent maladaptive increase in protein synthesis and decreased protein aggregate clearance are likely to contribute to disease progression., (© 2021 Federation of American Societies for Experimental Biology.)

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca 2+ -handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to β-adrenergic stimulation. To test the hypothesis, [Ca 2+ ] i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca 2+ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca 2+ signal amplitude and recovery time. In support of the depleted SR Ca 2+ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the β1-adrenergic receptor (ADRβ1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed β-adrenergic responsiveness., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Since its first identification as a cardiac transverse tubule (t-tubule) protein, followed by the cloning of the cardiac isoform responsible for t-tubule membrane microdomain formation, cardiac bridging integrator 1 (cBIN1) and its organized microdomains have emerged as a key mechanism in maintaining normal beat-to-beat heart contraction and relaxation. The abnormal remodeling of cBIN1-microdomains occurs in stressed and diseased cardiomyocytes, contributing to the pathophysiology of heart failure. Due to the homeostatic turnover of t-tubule cBIN1-microdomains via microvesicle release into the peripheral circulation, plasma cBIN1 can be assayed as a liquid biopsy of cardiomyocyte health. A new blood test cBIN1 score (CS) has been developed as a dimensionless inverse index derived from plasma cBIN1 concentration with a diagnostic and prognostic power for clinical outcomes in stable ambulatory patients with heart failure with reduced or preserved ejection fraction (HFrEF or HFpEF). Recent evidence further indicates that exogenous cBIN1 introduced by adeno-associated virus 9-based gene therapy can rescue cardiac contraction and relaxation in failing hearts. The therapeutic potential of cBIN1 gene therapy is enormous given its ability to rescue cardiac inotropy and provide lusitropic protection in the meantime. These unprecedented capabilities of cBIN1 gene therapy are shifting the current paradigm of therapy development for heart failure, particularly HFpEF.

We investigated the immunohistochemical localization of several proteins related to excitation-contraction coupling and ultrastructural alterations of the sarcotubular system in biopsied muscles from a father and a daughter in a family with permanent myopathy with hypokalemic periodic paralysis (PMPP) due to a mutation in calcium channel CACNA1S; p. R1239H hetero. Immunostaining for L-type calcium channels (LCaC) showed linear hyper-stained regions indicating proliferation of longitudinal t-tubules. The margin of vacuoles was positive for ryanodine receptor, LCaC, calsequestrin (CASQ) 1, CASQ 2, SR/ER Ca2+-ATPase (SERCA) 1, SERCA2, dysferlin, dystrophin, α-actinin, LC3, and LAMP 1. Electron microscopy indicated that the vacuoles mainly originated from the sarcoplasmic reticulum (SR). These findings indicate impairment of the muscle contraction system related to Ca2+ dynamics, remodeling of t-tubules and muscle fiber repair. We speculate that PMPP in patients with a CACNA1S mutation might start with abnormal SR function due to impaired LCaC. Subsequent induction of muscular contractile abnormalities and the vacuoles formed by fused SR in the repair process including autophagy might result in permanent myopathy. Our findings may facilitate prediction of the pathomechanisms of PMPP seen on morphological observation., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Risk of cardiovascular disease (CVD) increases considerably as renal function declines in chronic kidney disease (CKD). Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) has emerged as a novel innate immune receptor involved in both CVD and CKD. Following activation, NOD1 undergoes a conformational change that allows the activation of the receptor-interacting serine/threonine protein kinase 2 (RIP2), promoting an inflammatory response. We evaluated whether the genetic deficiency of Nod1 or Rip2 in mice could prevent cardiac Ca 2+ mishandling induced by sixth nephrectomy (Nx), a model of CKD. We examined intracellular Ca 2+ dynamics in cardiomyocytes from Wild-type ( Wt ), Nod1 -/- and Rip2 -/- sham-operated or nephrectomized mice. Compared with Wt cardiomyocytes, Wt -Nx cells showed an impairment in the properties and kinetics of the intracellular Ca 2+ transients, a reduction in both cell shortening and sarcoplasmic reticulum Ca 2+ load, together with an increase in diastolic Ca 2+ leak. Cardiomyocytes from Nod1 -/- -Nx and Rip2 -/- -Nx mice showed a significant amelioration in Ca 2+ mishandling without modifying the kidney impairment induced by Nx. In conclusion, Nod1 and Rip2 deficiency prevents the intracellular Ca 2+ mishandling induced by experimental CKD, unveiling new innate immune targets for the development of innovative therapeutic strategies to reduce cardiac complications in patients with CKD.

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that patients with HD also exhibit cardiac and respiratory dysfunction, including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a potentially novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via posttranslationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated, and leaky in brain, heart, and diaphragm both in patients with HD and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor, and respiratory functions were ameliorated by treatment with a novel Rycal small-molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac, and diaphragmatic pathophysiology in HD, and RyRs are a potential novel therapeutic target.

Aims: Nakajo-Nishimura syndrome (NNS) is an autosomal recessive disease caused by biallelic mutations in the PSMB8 gene that encodes the immunoproteasome subunit β5i. There have been only a limited number of reports on the clinicopathological features of the disease in genetically confirmed cases., Methods: We studied clinical and pathological features of three NNS patients who all carry the homozygous p.G201V mutations in PSMB8. Patients' muscle specimens were analysed with histology and immunohistochemistry., Results: All patients had episodes of typical periodic fever and skin rash, and later developed progressive muscle weakness and atrophy, similar to previous reports. Oral corticosteroid was used for treatment but showed no obvious efficacy. On muscle pathology, lymphocytes were present in the endomysium surrounding non-necrotic fibres, as well as in the perimysium perivascular area. Nearly all fibres strongly expressed MHC-I in the sarcolemma. In the eldest patient, there were abnormal protein aggregates in the sarcoplasm, immunoreactive to p62, TDP-43 and ubiquitin antibodies., Conclusions: These results suggest that inflammation, inclusion pathology and aggregation of abnormal proteins underlie the progressive clinical course of the NNS pathomechanism., (© 2020 British Neuropathological Society.)

Objectives: Investigation of the effect of SGLT2 inhibition by empagliflozin on left ventricular function in a model of diabetic cardiomyopathy., Background: SGLT2 inhibition is a new strategy to treat diabetes. In the EMPA-REG Outcome trial empagliflozin treatment reduced cardiovascular and overall mortality in patients with diabetes presumably due to beneficial cardiac effects, leading to reduced heart failure hospitalization. The relevant mechanisms remain currently elusive but might be mediated by a shift in cardiac substrate utilization leading to improved energetic supply to the heart., Methods: We used db/db mice on high-fat western diet with or without empagliflozin treatment as a model of severe diabetes. Left ventricular function was assessed by pressure catheter with or without dobutamine stress., Results: Treatment with empagliflozin significantly increased glycosuria, improved glucose metabolism, ameliorated left ventricular diastolic function and reduced mortality of mice. This was associated with reduced cardiac glucose concentrations and decreased calcium/calmodulin-dependent protein kinase (CaMKII) activation with subsequent less phosphorylation of the ryanodine receptor (RyR). No change of cardiac ketone bodies or branched-chain amino acid (BCAA) metabolites in serum was detected nor was cardiac expression of relevant catabolic enzymes for these substrates affected., Conclusions: In a murine model of severe diabetes empagliflozin-dependent SGLT2 inhibition improved diastolic function and reduced mortality. Improvement of diastolic function was likely mediated by reduced spontaneous diastolic sarcoplasmic reticulum (SR) calcium release but independent of changes in cardiac ketone and BCAA metabolism., Competing Interests: Declaration of competing interest N.M. has received support for clinical trial leadership from Boehringer Ingelheim, N.M. has served as a consultant to Amgen, Bayer, Boehringer Ingelheim, Sanofi-Aventis, MSD, BMS, AstraZeneca, NovoNordisk and has received grant support from Boehringer Ingelheim and MSD. In addition, N.M. has served as speaker for Amgen, Bayer, Boehringer Ingelheim, Sanofi-Aventis, MSD, BMS, AstraZeneca, Lilly, NovoNordisk. N.M. declines all personal compensation from pharma or device companies. M.L. has received grant support for experimental and clinical studies from Boehringer Ingelheim and MSD; has served as a consultant to Boehringer Ingelheim, Sanofi-Aventis, MSD, AstraZeneca, Lilly, NovoNordisk, Amgen and Bayer. Has served as a speaker for Boehringer Ingelheim Sanofi-Aventis, MSD, AstraZeneca, Lilly, NovoNordisk and Bayer. S.V. has received speaker honoraria from Abbott, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Eli Lilly, Janssen, Merck, NovoNordisk, and Sanofi; and received research grant support from Amgen, AstraZeneca, Boehringer Ingelheim, and Eli Lilly. G.D.L.: shareholder in Metabolic Modulators Research Ltd., received grant support from Servier, Boehringer Ingelheim, Sanofi, REMED Biopharmaceuticals. K.S. has received grant support for experimental studies from Boehringer Ingelheim and has served as speaker for Boehringer Ingelheim, Amgen, MSD, Omniamed and NovoNordisk. All other authors report no relevant disclosures., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease which contributes to cardiac contractile dysfunction when activated during myocardial ischaemia-reperfusion (IR) injury. MMP-2 is localized to several subcellular sites inside cardiac myocytes; however, its role in the sarcoplasmic reticulum (SR) is unknown. The Ca2+ ATPase SERCA2a, which pumps cytosolic Ca2+ into the SR to facilitate muscle relaxation, is degraded in cardiac IR injury; however, the protease responsible for this is unclear. We hypothesized that MMP-2 contributes to cardiac contractile dysfunction by proteolyzing SERCA2a, thereby impairing its activity in IR injury., Methods and Results: Isolated rat hearts were subjected to IR injury in the presence or absence of the selective MMP inhibitor ARP-100, or perfused aerobically as a control. Inhibition of MMP activity with ARP-100 significantly improved the recovery of cardiac mechanical function and prevented the increase of a 70 kDa SERCA2a degradation fragment following IR injury, although 110 kDa SERCA2a and phospholamban levels appeared unchanged. Electrophoresis of IR heart samples followed by LC-MS/MS confirmed the presence of a SERCA2a fragment of ∼70 kDa. MMP-2 activity co-purified with SR-enriched microsomes prepared from the isolated rat hearts. Endogenous SERCA2a in SR-enriched microsomes was proteolyzed to ∼70 kDa products when incubated in vitro with exogenous MMP-2. MMP-2 also cleaved purified porcine SERCA2a in vitro. SERCA activity in SR-enriched microsomes was decreased by IR injury; however, this was not prevented with ARP-100., Conclusion: This study shows that MMP-2 activity is found in SR-enriched microsomes from heart muscle and that SERCA2a is proteolyzed by MMP-2. The cardioprotective actions of MMP inhibition in myocardial IR injury may include the prevention of SERCA2a degradation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

This work discusses active and passive electrical properties of transverse (T-)tubules in ventricular cardiomyocytes to understand the physiological roles of T-tubules. T-tubules are invaginations of the lateral membrane that provide a large surface for calcium-handling proteins to facilitate sarcomere shortening. Higher heart rates correlate with higher T-tubular densities in mammalian ventricular cardiomyocytes. We assess ion dynamics in T-tubules and the effects of sodium current in T-tubules on the extracellular potential, which leads to a partial reduction of the sodium current in deep segments of a T-tubule. We moreover reflect on the impact of T-tubules on macroscopic conduction velocity, integrating fundamental principles of action potential propagation and conduction. We also theoretically assess how the conduction velocity is affected by different T-tubular sodium current densities. Lastly, we critically assess literature on ion channel expression to determine whether action potentials can be initiated in T-tubules., (Copyright © 2019. Published by Elsevier B.V.)

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca 2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.

Key Points: Changes in intramuscular Ca 2+ handling contribute to development of fatigue and disease-related loss of muscle mass and function. To date, no data on human intact living muscle fibres have been described. We manually dissected intact single fibres from human intercostal muscle and simultaneously measured force and myoplasmic free [Ca 2+ ] at physiological temperature. Based on their fatigue resistance, two distinct groups of fibres were distinguished: fatigue sensitive and fatigue resistant. Force depression in fatigue and during recovery was due to impaired sarcoplasmic reticulum Ca 2+ release in both groups of fibres. Acidification did not affect force production in unfatigued fibres and did not affect fatigue development in fatigue-resistant fibres. The current study provides novel insight into the mechanisms of fatigue in human intercostal muscle., Abstract: Changes in intracellular Ca 2+ handling of individual skeletal muscle fibres cause a force depression following physical activity and are also implicated in disease-related loss of function. The relation of intracellular Ca 2+ handling with muscle force production and fatigue tolerance is best studied in intact living single fibres that allow continuous measurements of force and myoplasmic free [Ca 2+ ] during repeated contractions. To this end, manual dissections of human intercostal muscle biopsies were performed to isolate intact single fibres. Based on the ability to maintain tetanic force at >40% of the initial value during 500 fatiguing contractions, fibres were classified as either fatigue sensitive or fatigue resistant. Following fatigue all fibres demonstrated a marked reduction in sarcoplasmic reticulum Ca 2+ release, while myofibrillar Ca 2+ sensitivity was either unaltered or increased. In unfatigued fibres, acidosis caused a reduction in myofibrillar Ca 2+ sensitivity that was offset by increased tetanic myoplasmic free [Ca 2+ ] so that force remained unaffected. Acidification did not affect the fatigue tolerance of fatigue-resistant fibres, whereas uncertainties remain whether or not fatigue-sensitive fibres were affected. Following fatigue, a prolonged force depression at preferentially low-frequency stimulation was evident in fatigue-sensitive fibres and this was caused exclusively by an impaired sarcoplasmic reticulum Ca 2+ release. We conclude that impaired sarcoplasmic reticulum Ca 2+ release is the predominant mechanism of force depression both in the development of, and recovery from, fatigue in human intercostal muscle., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Atrial fibrillation (AF), defined by disorganized atrial cardiac rhythm, is the most prevalent cardiac arrhythmia worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with AF risk, including the cardiogenic transcription factor genes TBX5, GATA4, and NKX2-5. We report that Tbx5 and Gata4 interact with opposite signs for atrial rhythm controls compared with cardiac development. Using mouse genetics, we found that AF pathophysiology caused by Tbx5 haploinsufficiency, including atrial arrhythmia susceptibility, prolonged action potential duration, and ectopic cardiomyocyte depolarizations, were all rescued by Gata4 haploinsufficiency. In contrast, Nkx2-5 haploinsufficiency showed no combinatorial effect. The molecular basis of the TBX5/GATA4 interaction included normalization of intra-cardiomyocyte calcium flux and expression of calcium channel genes Atp2a2 and Ryr2. Furthermore, GATA4 and TBX5 showed antagonistic interactions on an Ryr2 enhancer. Atrial rhythm instability caused by Tbx5 haploinsufficiency was rescued by a decreased dose of phospholamban, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, consistent with a role for decreased sarcoplasmic reticulum calcium flux in Tbx5-dependent AF susceptibility. This work defines a link between Tbx5 dose, sarcoplasmic reticulum calcium flux, and AF propensity. The unexpected interactions between Tbx5 and Gata4 in atrial rhythm control suggest that evaluating specific interactions between genetic risk loci will be necessary for ascertaining personalized risk from genetic association data.

Atrial fibrillation (AF) is associated with oxidative stress and Ca 2+ -handling abnormalities in atrial myocytes. Our prior study has demonstrated the involvement of CD44, a membrane receptor for hyaluronan (HA), in the pathogenesis of AF. This study further evaluated whether CD44 and its related signaling mediate atrial tachycardia-induced oxidative stress and Ca 2+ -handling abnormalities. Tachypacing in atrium-derived myocytes (HL-1 cell line) induced the activation of CD44-related signaling, including HA and HA synthase (HAS) expression. Blocking HAS/HA/CD44 signaling attenuated tachypacing-induced oxidative stress (NADPH oxidase [NOX] 2/4 expression) and Ca 2+ -handling abnormalities (oxidized Ca 2+ /calmodulin-dependent protein kinase II [ox-CaMKII] and phospho-ryanodine receptor type 2 [p-RyR2] expression) in HL-1 myocytes. Furthermore, a direct association between CD44 and NOX4 was documented in tachy-paced HL-1 myocytes and atrial tissues from AF patients. In vitro, Ca 2+ spark frequencies in atrial myocytes isolated from CD44 -/- mice were lower than those from wild-type mice. Furthermore, administration of an anti-CD44 blocking antibody in atrial myocytes isolated from wild-type mice diminished the frequency of Ca 2+ spark. Ex vivo tachypacing models of CD44 -/- mice exhibited a lower degree of oxidative stress and expression of ox-CaMKII/p-RyR2 in their atria than those of wild-type mice. In vivo, burst atrial pacing stimulated a less inducibility of AF in CD44 -/- mice than in wild-type mice. In conclusion, atrial tachypacing-induced Ca 2+ -handling abnormalities are mediated via CD44/NOX4 signaling, which provides a possible explanation for the development of AF., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Heart failure (HF) is characterized, among other factors, by a progressive loss of contractile function and by the formation of an arrhythmogenic substrate, both aspects partially related to intracellular Ca2+ cycling disorders. In failing hearts both electrophysiological and structural remodeling, including fibroblast proliferation, contribute to changes in Ca2+ handling which promote the appearance of Ca2+ alternans (Ca-alt). Ca-alt in turn give rise to repolarization alternans, which promote dispersion of repolarization and contribute to reentrant activity. The computational analysis of the incidence of Ca2+ and/or repolarization alternans under HF conditions in the presence of fibroblasts could provide a better understanding of the mechanisms leading to HF arrhythmias and contractile function disorders., Methods and Findings: The goal of the present study was to investigate in silico the mechanisms leading to the formation of Ca-alt in failing human ventricular myocytes and tissues with disperse fibroblast distributions. The contribution of ionic currents variability to alternans formation at the cellular level was analyzed and the results show that in normal ventricular tissue, altered Ca2+ dynamics lead to Ca-alt, which precede APD alternans and can be aggravated by the presence of fibroblasts. Electrophysiological remodeling of failing tissue alone is sufficient to develop alternans. The incidence of alternans is reduced when fibroblasts are present in failing tissue due to significantly depressed Ca2+ transients. The analysis of the underlying ionic mechanisms suggests that Ca-alt are driven by Ca2+-handling protein and Ca2+ cycling dysfunctions in the junctional sarcoplasmic reticulum and that their contribution to alternans occurrence depends on the cardiac remodeling conditions and on myocyte-fibroblast interactions., Conclusion: It can thus be concluded that fibroblasts modulate the formation of Ca-alt in human ventricular tissue subjected to heart failure-related electrophysiological remodeling. Pharmacological therapies should thus consider the extent of both the electrophysiological and structural remodeling present in the failing heart., Competing Interests: The authors have declared that no competing interests exist.

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca 2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca 2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca 2+ -ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.

Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca2+ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca2+ load, trigger L-type Ca2+ current, or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca2+ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, 2 Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation.

Rationale: SPEG (Striated muscle preferentially expressed protein kinase) has 2 kinase-domains and is critical for cardiac development and function. However, it is not clear how these 2 kinase-domains function to maintain cardiac performance., Objective: To determine the molecular functions of the 2 kinase-domains of SPEG., Methods and Results: A proteomics approach identified SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr 484 on SERCA2a, promote its oligomerization and increase calcium reuptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium-transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a-Thr 484 phosphorylation and sarcoplasmic reticulum calcium reuptake. Moreover, overexpression of SERCA2a Thr484Ala mutant protein also slowed sarcoplasmic reticulum calcium reuptake in neonatal rat cardiomyocytes. In contrast, domain mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently identified substrate JPH2 (junctophilin-2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a-Thr 484 phosphorylation and its oligomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium-transporting activity and impaired calcium reuptake into the sarcoplasmic reticulum in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice., Conclusions: Our data demonstrate that the 2 kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.

Background: Senescent cardiomyocytes exhibit a mismatch between energy demand and supply that facilitates their transition toward failing cells. Altered calcium transfer from sarcoplasmic reticulum (SR) to mitochondria has been causally linked to the pathophysiology of aging and heart failure., Methods: Because advanced glycation-end products accumulate throughout life, we investigated whether intracellular glycation occurs in aged cardiomyocytes and its impact on SR and mitochondria., Results: Quantitative proteomics, Western blot and immunofluorescence demonstrated a significant increase in advanced glycation-end product-modified proteins in the myocardium of old mice (≥20months) compared with young ones (4-6months). Glyoxalase-1 activity (responsible for detoxification of dicarbonyl intermediates) and its cofactor glutathione were decreased in aged hearts. Immunolabeling and proximity ligation assay identified the ryanodine receptor (RyR2) in the SR as prominent target of glycation in aged mice, and the sites of glycation were characterized by quantitative mass spectrometry. RyR2 glycation was associated with more pronounced calcium leak, determined by confocal microscopy in cardiomyocytes and SR vesicles. Interfibrillar mitochondria-directly exposed to SR calcium release-from aged mice had increased calcium content compared with those from young ones. Higher levels of advanced glycation-end products and reduced glyoxalase-1 activity and glutathione were also present in atrial appendages from surgical patients ≥75 years as compared with the younger ones. Elderly patients also exhibited RyR2 hyperglycation and increased mitochondrial calcium content that was associated with reduced myocardial aerobic capacity (mitochondrial O 2 consumption/g) attributable to less respiring mitochondria. In contracting HL-1 cardiomyocytes, pharmacological glyoxalase-1 inhibition recapitulated RyR2 glycation and defective SR-mitochondria calcium exchange of aging., Conclusions: Mitochondria from aging hearts develop calcium overload secondary to SR calcium leak. Glycative damage of RyR2, favored by deficient dicarbonyl detoxification capacity, contributes to calcium leak and mitochondrial damage in the senescent myocardium.

As a consequence of impaired glucose or fatty acid metabolism, bioenergetic stress in skeletal muscles may trigger myopathy and rhabdomyolysis. Genetic mutations causing loss of function of the LPIN1 gene frequently lead to severe rhabdomyolysis bouts in children, though the metabolic alterations and possible therapeutic interventions remain elusive. Here, we show that lipin1 deficiency in mouse skeletal muscles is sufficient to trigger myopathy. Strikingly, muscle fibers display strong accumulation of both neutral and phospholipids. The metabolic lipid imbalance can be traced to an altered fatty acid synthesis and fatty acid oxidation, accompanied by a defect in acyl chain elongation and desaturation. As an underlying cause, we reveal a severe sarcoplasmic reticulum (SR) stress, leading to the activation of the lipogenic SREBP1c/SREBP2 factors, the accumulation of the Fgf21 cytokine, and alterations of SR-mitochondria morphology. Importantly, pharmacological treatments with the chaperone TUDCA and the fatty acid oxidation activator bezafibrate improve muscle histology and strength of lipin1 mutants. Our data reveal that SR stress and alterations in SR-mitochondria contacts are contributing factors and potential intervention targets of the myopathy associated with lipin1 deficiency., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Cardiomyocytes both cause and experience continual cyclic deformation. The exact effects of this deformation on the properties of intracellular organelles are not well characterized, although they are likely to be relevant for cardiomyocyte responses to active and passive changes in their mechanical environment. In the present study we provide three-dimensional ultrastructural evidence for mechanically induced mitochondrial deformation in rabbit ventricular cardiomyocytes over a range of sarcomere lengths representing myocardial tissue stretch, an unloaded "slack" state, and contracture. We also show structural indications for interaction of mitochondria with one another, as well as with other intracellular elements such as microtubules, sarcoplasmic reticulum and T-tubules. The data presented here help to contextualize recent reports on the mechanosensitivity and cell-wide connectivity of the mitochondrial network and provide a structural framework that may aide interpretation of mechanically-regulated molecular signaling in cardiac cells. Anat Rec, 302:146-152, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists., (© 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

The tumor-suppressive role of p53, a transcription factor that regulates the expression of many genes, has been linked to cell cycle arrest, apoptosis, and senescence. The noncanonical function or the pathogenic role of p53 has more recently been implicated in pulmonary vascular disease. We previously reported that rapid nuclear accumulation of hypoxia-inducible factor (HIF)-1α in pulmonary arterial smooth muscle cells (PASMCs) upregulates transient receptor potential channels and enhances Ca 2+ entry to increase cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). Also, we observed differences in HIF-1α/2α expression in PASMCs and pulmonary arterial endothelial cells (PAECs). Here we report that p53 is increased in PAECs, but decreased in PASMCs, isolated from mice with hypoxia-induced pulmonary hypertension (PH) and rats with monocrotaline (MCT)-induced PH (MCT-PH). The increased p53 in PAECs from rats with MCT-PH is associated with an increased ratio of Bax/Bcl-2, while the decreased p53 in PASMCs is associated with an increased HIF-1α. Furthermore, p53 is downregulated in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension compared with PASMCs from normal subjects. Overexpression of p53 in normal PASMCs inhibits store-operated Ca 2+ entry (SOCE) induced by passive depletion of intracellularly stored Ca 2+ in the sarcoplasmic reticulum, while downregulation of p53 enhances SOCE. These data indicate that differentially regulated expression of p53 and HIF-1α/2α in PASMCs and PAECs and the cross talk between p53 and HIF-1α/2α in PASMCs and PAECs may play an important role in the development of PH via, at least in part, induction of PAEC apoptosis and PASMC proliferation.

Aims: Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia-reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress., Methods and Results: TRPV4 protein expression increased in cardiomyocytes of Aged (24-27 months) mice compared with Young (3-6 months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1 μmol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4-/-) mice. In Aged, pharmacological inhibition of TRPV4 (1 μmol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage., Conclusions: Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.

Background: Disruption of Ca 2+ homeostasis is a key pathomechanism in heart failure. CaMKII-dependent hyperphosphorylation of ryanodine receptors in the sarcoplasmic reticulum (SR) increases the arrhythmogenic SR Ca 2+ leak and depletes SR Ca 2+ stores. The contribution of conversely acting serine/threonine phosphatases [protein phosphatase 1 (PP1) and 2A (PP2A)] is largely unknown., Methods and Results: Human myocardium from three groups of patients was investigated: (i) healthy controls (non-failing, NF, n = 8), (ii) compensated hypertrophy (Hy, n = 16), and (iii) end-stage heart failure (HF, n = 52). Expression of PP1 was unchanged in Hy but greater in HF compared to NF while its endogenous inhibitor-1 (I-1) was markedly lower expressed in both compared to NF, suggesting increased total PP1 activity. In contrast, PP2A expression was lower in Hy and HF compared to NF. Ca 2+ homeostasis was severely disturbed in HF compared to Hy signified by a higher SR Ca 2+ leak, lower systolic Ca 2+ transients as well as a decreased SR Ca 2+ load. Inhibition of PP1/PP2A by okadaic acid increased SR Ca 2+ load and systolic Ca 2+ transients but severely aggravated diastolic SR Ca 2+ leak and cellular arrhythmias in Hy. Conversely, selective activation of PP1 by a PP1-disrupting peptide (PDP3) in HF potently reduced SR Ca 2+ leak as well as cellular arrhythmias and, importantly, did not compromise systolic Ca 2+ release and SR Ca 2+ load., Conclusion: This study is the first to functionally investigate the role of PP1/PP2A for Ca 2+ homeostasis in diseased human myocardium. Our data indicate that a modulation of phosphatase activity potently impacts Ca 2+ cycling properties. An activation of PP1 counteracts increased kinase activity in heart failure and successfully seals the arrhythmogenic SR Ca 2+ leak. It may thus represent a promising future antiarrhythmic therapeutic approach., (© 2018 The Authors. European Journal of Heart Failure © 2018 European Society of Cardiology.)

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca 2+ -handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca 2+ -sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca 2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca 2+ sensor of Ca 2+ dynamics in the junctional space. The sensor was used to assess the basal Ca 2+ -handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca 2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca 2+ handling under defined ionic conditions., Competing Interests: The authors declare no conflict of interest.

Key Points: Cigarette smoke components directly alter muscle fatigue resistance and intracellular muscle fibre Ca 2+ handling independent of a change in lung structure. Changes in muscle vascular structure are associated with a depletion of satellite cells. Sarcoplasmic reticulum Ca 2+ uptake is substantially impaired in myofibres during fatiguing contractions in mice treated with cigarette smoke extract., Abstract: Cigarette smokers exhibit exercise intolerance before a decline in respiratory function. In the present study, the direct effects of cigarette smoke on limb muscle function were tested by comparing cigarette smoke delivered to mice by weekly injections of cigarette smoke extract (CSE), or nose-only exposure (CS) 5 days each week, for 8 weeks. Cigarette smoke delivered by either route did not alter pulmonary airspace size. Muscle fatigue measured in situ was 50% lower in the CSE and CS groups than in control. This was accompanied by 34% and 22% decreases in soleus capillary-to-fibre ratio of the CSE and CS groups, respectively, and a trend for fewer skeletal muscle actin-positive arterioles (P = 0.07). In addition, fewer quiescent satellite cells (Nes+Pax7+) were associated with soleus fibres in mice with skeletal myofibre VEGF gene deletion (decreased 47%) and CS exposed (decreased 73%) than with control fibres. Contractile properties of isolated extensor digitorum longus and soleus muscles were impaired. In flexor digitorum brevis myofibres isolated from CSE mice, fatigue resistance was diminished by 43% compared to control and CS myofibres, and this was accompanied by a pronounced slowing in relaxation, an increase in intracellular Ca 2+ accumulation, and a slowing in sarcoplasmic reticulum Ca 2+ uptake. These data suggest that cigarette smoke components may impair hindlimb muscle vascular structure, fatigue resistance and myofibre calcium handling, and these changes ultimately affect contractile efficiency of locomotor muscles independent of a change in lung function., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

The cellular control of glucose uptake and glycogen metabolism in mammalian tissues is in part mediated through the regulation of protein-serine/threonine kinases including CK2. Although it participates to several cellular signaling processes, however, its subcellular localization is not well-defined while some documents mentioned its localization change under pathological conditions. The activation/phosphorylation of some proteins including Zn 2+ -transporter ZIP7 in cardiomyocytes is controlled with CK2α, thereby, inducing changes in the level of intracellular free Zn 2+ ([Zn 2+ ] i ). In this regard, we aimed to examine cellular localization of CK2α in cardiomyocytes and its possible subcellular migration under hyperglycemia. Our confocal imaging together with biochemical analysis in isolated sarco(endo)plasmic reticulum [S(E)R] and nuclear fractions from hearts have shown that CK2α localized highly to S(E)R and Golgi and weakly to nuclear fractions in physiological condition. However, it can migrate from nuclear fractions to S(E)R under hyperglycemia. This migration can further underlie phosphorylation of a target protein ZIP7 as well as some endogenous kinases and phosphatases including PKA, CaMKII, and PP2A. We also have shown that CK2α activation is responsible for hyperglycemia-associated [Zn 2+ ] i increase in diabetic heart. Therefore, our present data demonstrated, for the first time, the physiological relevance of CK2α in cellular control of Zn 2+ -distribution via inducing ZIP7 phosphorylation and activation of these above endogenous actors in hyperglycemia/diabetes-associated cardiac dysfunction. Moreover, our present data also emphasized the multi-subcellular compartmental localizations of CK2α and a tightly regulation of these localizations in cardiomyocytes. Therefore, taken into consideration of all data, one can emphasize the important role of the subcellular localization of CK2α as a novel target-pathway for understanding of diabetic cardiomyopathy.

Myocardial stunning is a temporary post-ischemic cardiac mechanical dysfunction. As such, it is a heterogeneous entity and different conditions can promote its occurrence. Transient coronary occlusion, increased production of catecholamines and endothelin, and myocardial inflammation are all possible causes of myocardial stunning. Possible underlying mechanisms include an oxyradical hypothesis, calcium overload, decreased responsiveness of myofilaments to calcium, and excitation-contraction uncoupling due to sarcoplasmic reticulum dysfunction. The aim of this review is to summarize the clinical conditions that may be responsible for stunned myocardium., (Copyright © 2018 Elsevier Inc. All rights reserved.)

For arrhythmia triggers that are secondary to dysfunctional intracellular Ca 2+ cycling, there are few, if any, agents that specifically target the Ca 2+ handling machinery. However, several candidates have been proposed in the literature. Here we review the idea that these agents or their derivatives will prove invaluable in clinical applications in the future., (Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Aims: The mechanisms responsible for cardiac damage in the early stages of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with cellular myofibrils, with the cytoskeleton functioning as a linkage coordinator, and closely associated to the calcium release sites of the sarcoplasmic reticulum (SR). We hypothesized that early MetS is characterized by mitochondria-related myocardial damage, associated with altered cytoskeletal-mitochondria-SR interaction., Methods and Results: Domestic pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondrial-targeted peptide elamipretide (ELAM; 0.1 mg/kg SC q.d), or Lean controls (n = 6/group). Cardiac remodeling and function were assessed by fast comuted tomography. Myocardial mitochondrial structure, SR-mitochondria interaction, calcium handling, cytoskeletal proteins, oxidative stress, and apoptosis were studied ex-vivo. MetS pigs developed hyperlipidemia, hypertension, and insulin resistance, yet cardiac function was preserved. MetS-induced mitochondrial disorganization, decreased (C18:2)4 cardiolipin, disrupted ATP/ADP balance, and decreased cytochrome-c oxidase (COX)-IV activity. MetS also increased mitochondrial hydrogen peroxide (H2O2) production, decreased nicotinamide adenine dinucleotide phosphate (NADPH)/NADP and GSH/GSSG, and decreased myocardial desmin and β2 tubulin immunoreactivity, and impaired SR-mitochondrial interaction and mitochondrial calcium handling, eliciting myocardial oxidative stress and apoptosis. ELAM improved mitochondrial organization and cardiolipin species profile, restored ATP/ADP ratio and COX-IV activity, decreased H202 production, and improved generation of NADPH and GSH. ELAM also improved cytoskeletal-mitochondria-SR interaction and mitochondrial calcium handling, attenuating oxidative stress, and apoptosis., Conclusions: Disorganization of cardiomyocyte cytoskeletal-mitochondria-SR network is associated with cardiac reversible changes in early MetS, preceding overt cardiac dysfunction. These findings may introduce novel therapeutic targets for blunting cardiac damage in early MetS., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.)

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic β cyto - or γ cyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific β cyto - and γ cyto -actin KO mice. We found β cyto - and γ cyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking β cyto - and/or γ cyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific β cyto - and γ cyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old β cyto - and γ cyto -actin muscle-specific KO mice. Our data suggest that impaired Ca 2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy., (© 2017 Federation of European Biochemical Societies.)

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca 2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

Background: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Preservation of contractile function and protection against adverse changes in ventricular architecture (cardiac remodeling) are key factors to limiting progression of this condition to heart failure. Consequently, new therapeutic targets are urgently required to achieve this aim. Expression of the Runx1 transcription factor is increased in adult cardiomyocytes after MI; however, the functional role of Runx1 in the heart is unknown., Methods: To address this question, we have generated a novel tamoxifen-inducible cardiomyocyte-specific Runx1 -deficient mouse. Mice were subjected to MI by means of coronary artery ligation. Cardiac remodeling and contractile function were assessed extensively at the whole-heart, cardiomyocyte, and molecular levels., Results: Runx1 -deficient mice were protected against adverse cardiac remodeling after MI, maintaining ventricular wall thickness and contractile function. Furthermore, these mice lacked eccentric hypertrophy, and their cardiomyocytes exhibited markedly improved calcium handling. At the mechanistic level, these effects were achieved through increased phosphorylation of phospholamban by protein kinase A and relief of sarco/endoplasmic reticulum Ca 2+ -ATPase inhibition. Enhanced sarco/endoplasmic reticulum Ca 2+ -ATPase activity in Runx1-deficient mice increased sarcoplasmic reticulum calcium content and sarcoplasmic reticulum-mediated calcium release, preserving cardiomyocyte contraction after MI., Conclusions: Our data identified Runx1 as a novel therapeutic target with translational potential to counteract the effects of adverse cardiac remodeling, thereby improving survival and quality of life among patients with MI., (© 2017 The Authors.)

Ectopic heartbeats can trigger reentrant arrhythmias, leading to ventricular fibrillation and sudden cardiac death. Such events have been attributed to perturbed Ca2+ handling in cardiac myocytes leading to spontaneous Ca2+ release and delayed afterdepolarizations (DADs). However, the ways in which perturbation of specific molecular mechanisms alters the probability of ectopic beats is not understood. We present a multiscale model of cardiac tissue incorporating a biophysically detailed three-dimensional model of the ventricular myocyte. This model reproduces realistic Ca2+ waves and DADs driven by stochastic Ca2+ release channel (RyR) gating and is used to study mechanisms of DAD variability. In agreement with previous experimental and modeling studies, key factors influencing the distribution of DAD amplitude and timing include cytosolic and sarcoplasmic reticulum Ca2+ concentrations, inwardly rectifying potassium current (IK1) density, and gap junction conductance. The cardiac tissue model is used to investigate how random RyR gating gives rise to probabilistic triggered activity in a one-dimensional myocyte tissue model. A novel spatial-average filtering method for estimating the probability of extreme (i.e. rare, high-amplitude) stochastic events from a limited set of spontaneous Ca2+ release profiles is presented. These events occur when randomly organized clusters of cells exhibit synchronized, high amplitude Ca2+ release flux. It is shown how reduced IK1 density and gap junction coupling, as observed in heart failure, increase the probability of extreme DADs by multiple orders of magnitude. This method enables prediction of arrhythmia likelihood and its modulation by alterations of other cellular mechanisms.

Abnormal intracellular Ca2+ handling is the major contributor to the depressed cardiac contractility observed in heart failure. The electrophysiological remodeling associated with this pathology alters both the action potential and the Ca2+ dynamics, leading to a defective excitation-contraction coupling that ends in mechanical dysfunction. The importance of maintaining a correct intracellular Ca2+ concentration requires a better understanding of its regulation by ionic mechanisms. To study the electrical activity and ionic homeostasis of failing myocytes, a modified version of the O'Hara et al. human action potential model was used, including electrophysiological remodeling. The impact of the main ionic transport mechanisms was analyzed using single-parameter sensitivity analyses, the first of which explored the modulation of electrophysiological characteristics related to Ca2+ exerted by the remodeled parameters. The second sensitivity analysis compared the potential consequences of modulating individual channel conductivities, as one of the main effects of potential drugs, on Ca2+ dynamic properties under both normal conditions and in heart failure. The first analysis revealed the important contribution of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) dysfunction to the altered Ca2+ homeostasis, with the Na+/Ca2+ exchanger (NCX) and other Ca2+ cycling proteins also playing a significant role. Our results highlight the importance of improving the SR uptake function to increase Ca2+ content and restore Ca2+ homeostasis and contractility. The second sensitivity analysis highlights the different response of the failing myocyte versus the healthy myocyte to potential pharmacological actions on single channels. The result of modifying the conductances of the remodeled proteins such as SERCA and NCX in heart failure has less impact on Ca2+ modulation. These differences should be taken into account when designing drug therapies.

Besides its role in calcium (Ca 2+ ) homeostasis, the sarco-endoplamic reticulum (SR/ER) controls protein folding and is tethered to mitochondria. Under pathophysiological conditions the unfolded protein response (UPR) is associated with disturbance in SR/ER-mitochondria crosstalk. Here, we investigated whether ER stress altered SR/ER-mitochondria links, Ca 2+ handling and muscle damage in WT (Wild Type) and mdx mice, the murine model of Duchenne Muscular Dystrophy (DMD). In WT mice, the SR/ER-mitochondria links were decreased in isolated FDB muscle fibers after injection of ER stress activator tunicamycin (TM). Ca 2+ imaging revealed an increase of cytosolic Ca 2+ transient and a decrease of mitochondrial Ca 2+ uptake. The force generating capacity of muscle dropped after TM. This impaired contractile function was accompanied by an increase in autophagy markers and calpain-1 activation. Conversely, ER stress inhibitors restored SR/ER-mitochondria links, mitochondrial Ca 2+ uptake and improved diaphragm contractility in mdx mice. Our findings demonstrated that ER stress-altered SR/ER-mitochondria links, disturbed Ca 2+ handling and muscle function in WT and mdx mice. Thus, ER stress may open up a prospect of new therapeutic targets in DMD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sarcoplasmic reticulum (SR) Ca 2+ leak induced by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is centrally involved in atrial and ventricular arrhythmogenesis as well as heart failure remodeling. Consequently, treating SR Ca 2+ leak has been proposed as a novel therapeutic paradigm, but compounds for use in humans are lacking. SMP-114 ("Rimacalib") is a novel, orally available CaMKII inhibitor developed for human use that has already entered clinical phase II trials to treat rheumatoid arthritis. We speculated that SMP-114 might also be useful to treat cardiac SR Ca 2+ leak. SMP-114 significantly reduces SR Ca 2+ leak (as assessed by Ca 2+ sparks) in human atrial (0.72 ± 0.33 sparks/100 µm/s vs. control 3.02 ± 0.91 sparks/100 µm/s) and failing left ventricular (0.78 ± 0.23 vs. 1.69 ± 0.27 sparks/100 µm/s) as well as in murine ventricular cardiomyocytes (0.30 ± 0.07 vs. 1.50 ± 0.28 sparks/100 µm/s). Associated with lower SR Ca 2+ leak, we found that SMP-114 suppressed the occurrence of spontaneous arrhythmogenic spontaneous Ca 2+ release (0.356 ± 0.109 vs. 0.927 ± 0.216 events per 30 s stimulation cessation). In consequence, post-rest potentiation of Ca 2+ -transient amplitude (measured using Fura-2) during the 30 s pause was improved by SMP-114 (52 ± 5 vs. 37 ± 4%). Noteworthy, SMP-114 has these beneficial effects without negatively impairing global excitation-contraction coupling: neither systolic Ca 2+ release nor single cell contractility was compromised, and also SR Ca 2+ reuptake, in line with resulting cardiomyocyte relaxation, was not impaired by SMP-114 in our assays. SMP-114 demonstrated potential to treat SR Ca 2+ leak and consequently proarrhythmogenic events in rodent as well as in human atrial cardiomyocytes and cardiomyocytes from patients with heart failure. Further research is necessary towards clinical use in cardiac disease.

Atherosclerosis is a disease caused by a build-up of fatty plaques and cholesterol in the arteries. The lumen of the vessels is obliterated resulting in restricted blood supply to tissues. In ischemic conditions, the cytosolic Ca 2+ level of skeletal muscle may increase, indicating the alteration of Ca 2+ removal mechanisms. Ca 2+ is transported from cytosol into the sarcoplasmic reticulum by Ca 2+ ATPase (SERCA), with its 1a isoform expressed in adult, while its 1b isoform in neonatal and regenerating fast-twitch skeletal muscle. To investigate the role of these isoforms in ischemic skeletal muscle, biopsies from musculus biceps femoris of patients who underwent amputation due to atherosclerosis were examined. Samples were removed from the visibly healthy and hypoxia-affected tissue. Significantly increased SERCA1a expression was detected under the ischemic conditions (246 ± 69%; p < 0.05) compared with the healthy tissue. Furthermore, the ratio of SERCA1a-positive fibers was slightly increased (46 ± 4% in healthy tissue and 60 ± 5% in ischemic tissue; p > 0.05), whereas SERCA2a did not change. In addition, in primary cultures derived from hypoxia-affected tissue, the diameter and fusion index of myotubes were significantly increased (30 ± 1.6 µm vs. 41 ± 2.4 µm and 31 ± 4% vs. 45 ± 3%; p < 0.05). We propose that the increased SERCA1a expression indicates the existence and location of compensating mechanisms in ischemic muscle.

Objective: Triadin is a component of the calcium release complex of cardiac and skeletal muscle. Our objective was to analyze the skeletal muscle phenotype of the triadin knockout syndrome., Methods: We performed clinical evaluation, analyzed morphologic features by light and electron microscopy, and immunolocalized triadin in skeletal muscle., Results: A 6-year-old boy with lifelong muscle weakness had a triadin knockout syndrome caused by compound heterozygous null mutations in triadin. Light microscopy of a deltoid muscle specimen shows multiple small abnormal spaces in all muscle fibers. Triadin immunoreactivity is absent from type 1 fibers and barely detectable in type 2 fibers. Electron microscopy reveals focally distributed dilation and degeneration of the lateral cisterns of the sarcoplasmic reticulum and loss of the triadin anchors from the preserved lateral cisterns., Conclusions: Absence of triadin in humans can result in a congenital myopathy associated with profound pathologic alterations in components of the sarcoplasmic reticulum. Why only some triadin-deficient patients develop a skeletal muscle phenotype remains an unsolved question., (© 2017 American Academy of Neurology.)

Intracellular calcium [Ca 2+ ] i dysregulation plays an important role in the pathophysiology of iron overload cardiomyopathy. Although either iron chelators or antioxidants provide cardioprotection, a comparison of the efficacy of deferoxamine (DFO), deferiprone (DFP), deferasirox (DFX), N-acetyl cysteine (NAC) or a combination of DFP plus NAC on cardiac [Ca 2+ ] i homeostasis in chronic iron overload has never been investigated. Male Wistar rats were fed with either a normal diet or a high iron (HFe) diet for 4 months. At 2 months, HFe rats were divided into 6 groups and treated with either a vehicle, DFO (25 mg/kg/day), DFP (75 mg/kg/day), DFX (20 mg/kg/day), NAC (100 mg/kg/day), or combined DFP plus NAC. At 4 months, the number of cardiac T-type calcium channels was increased, whereas cardiac sarcoplasmic-endoplasmic reticulum Ca 2+ ATPase (SERCA) was decreased, leading to cardiac iron overload and impaired cardiac [Ca 2+ ]i homeostasis. All pharmacological interventions restored SERCA levels. Although DFO, DFP, DFX or NAC alone shared similar efficacy in improving cardiac [Ca 2+ ]i homeostasis, only DFP + NAC restored cardiac [Ca 2+ ]i homeostasis, leading to restoring left ventricular function in the HFe-fed rats. Thus, the combined DFP + NAC was more effective than any monotherapy in restoring cardiac [Ca 2+ ] i homeostasis, leading to restored myocardial contractility in iron-overloaded rats.