Abnormal Calcium Handling Research Articles

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration and counteract the dystrophic phenotype, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

Hereditary sensory neuropathy type 1-associated deoxysphingolipids cause neurotoxicity, acute calcium handling abnormalities and mitochondrial dysfunction in vitro

Hereditary sensory neuropathy type 1 (HSN-1) is a peripheral neuropathy most frequently caused by mutations in the SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first step of de novo sphingolipid synthesis. Mutations in SPT result in a change in enzyme substrate specificity, which causes the production of atypical deoxysphinganine and deoxymethylsphinganine, rather than the normal enzyme product, sphinganine. Levels of these abnormal compounds are elevated in blood of HSN-1 patients and this is thought to cause the peripheral motor and sensory nerve damage that is characteristic of the disease, by a largely unresolved mechanism. In this study, we show that exogenous application of these deoxysphingoid bases causes dose- and time-dependent neurotoxicity in primary mammalian neurons, as determined by analysis of cell survival and neurite length. Acutely, deoxysphingoid base neurotoxicity manifests in abnormal Ca2+ handling by the endoplasmic reticulum (ER) and mitochondria as well as dysregulation of cell membrane store-operated Ca2+ channels. The changes in intracellular Ca2+ handling are accompanied by an early loss of mitochondrial membrane potential in deoxysphingoid base-treated motor and sensory neurons. Thus, these results suggest that exogenous deoxysphingoid base application causes neuronal mitochondrial dysfunction and Ca2+ handling deficits, which may play a critical role in the pathogenesis of HSN-1.

Pathobiology of cardiac dyssynchrony and resynchronization therapy.

Synchronous ventricular electrical activation is a prerequisite for adequate left ventricular (LV) systolic function. Conduction abnormalities such as left bundle branch block, and ventricular pacing lead to a dyssynchronous electrical activation sequence, which may have deleterious consequences. The present review attempts to connect the various processes involved in the development of 'dyssynchronopathy', and its correction by cardiac resynchronization therapy (CRT). Abnormal electrical impulse conduction leads to abnormal contraction, characterized by regional differences in timing as well as shortening patterns and amount of external work performed. Early activated regions may show 'wasted work', which leads to inefficient action of the entire left ventricle. Moreover, both the development of heart failure (HF) in general and the regional differences in mechanical load lead to structural, electrical, and contractile remodelling processes. These have been demonstrated at the level of the myocardium (asymmetric hypertrophy, fibrosis, prolongation of activation and reduction in repolarization forces, decrease in LV ejection fraction), cell (gap junctional remodelling, derangement of the T-tubular structure), and molecule (under or overexpression of ion channels and contractile proteins subtypes and abnormal calcium handling). The myocardial adaptations to dyssynchrony are 'maladaptive'. This also explains why CRT, unlike most pharmacological treatments, continues to increase its therapeutic effect over time. Finally, better understanding of all processes involved in dyssynchrony and CRT may also lead to new pharmacological agents for treating HF and to novel pacing strategies.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

AimsSarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM.Methods and resultsCRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-β-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets.ConclusionOur holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-βMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient βMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

Xanthine Oxidase Inhibitor Allopurinol Prevents Oxidative Stress-Mediated Atrial Remodeling in Alloxan-Induced Diabetes Mellitus Rabbits.

BackgroundThere are several mechanisms, including inflammation, oxidative stress and abnormal calcium homeostasis, involved in the pathogenesis of atrial fibrillation. In diabetes mellitus (DM), increased oxidative stress may be attributable to higher xanthine oxidase activity. In this study, we examined the relationship between oxidative stress and atrial electrical and structural remodeling, and calcium handling abnormalities, and the potential beneficial effects of the xanthine oxidase inhibitor allopurinol upon these pathological changes.Methods and ResultsNinety rabbits were randomly and equally divided into 3 groups: control, DM, and allopurinol‐treated DM group. Echocardiographic and hemodynamic assessments were performed in vivo. Serum and tissue markers of oxidative stress and atrial fibrosis, including the protein expression were examined. Atrial interstitial fibrosis was evaluated by Masson trichrome staining. ICaL was measured from isolated left atrial cardiomyocytes using voltage‐clamp techniques. Confocal microscopy was used to detect intracellular calcium transients. The Ca2+ handling protein expression was analyzed by Western blotting. Mitochondrial‐related proteins were analyzed as markers of mitochondrial function. Compared with the control group, rabbits with DM showed left ventricular hypertrophy, increased atrial interstitial fibrosis, oxidative stress and fibrosis markers, ICaL and intracellular calcium transient, and atrial fibrillation inducibility. These abnormalities were alleviated by allopurinol treatment.ConclusionsAllopurinol, via its antioxidant effects, reduces atrial mechanical, structural, ion channel remodeling and mitochondrial synthesis abnormalities induced by DM‐related increases in oxidative stress.

Protein Expression Differences in Hypertrophic Cardiomyopathy Transgenic Mice

Hypertrophic Cardiomyopathy (HCM) is the most common genetic cardiac disease, occurring in 1 out of every 500 individuals. The pathologic hallmarks of HCM are myocyte hypertrophy, myocyte disarray, fibrosis and arteriolar remodeling. Majority of HCM patients have mutations in genes coding for sarcomeric proteins. However, the mechanisms whereby the mutations lead to myocyte hypertrophy are not fully elucidated. Despite successes in animal models, there is no known therapy that can prevent the cardiac phenotype in HCM patients. We hypothesized that differences in pathophysiology of the cardiac phenotype in HCM could underlie failure of clinical trials in HCM patients. Hence, in this study, we investigated 2 transgenic mouse models of HCM, namely the R403Q mutation in the alpha Myosin Heavy Chain gene (R403Q‐MHC) and the R92W mutation in the Troponin T gene (R92W‐TnT). Since energetic deficits and abnormalities in calcium handling are 2 postulated mechanisms underlying the cardiac phenotypes in HCM, we investigated expression levels of AMP‐Activated Protein Kinase (AMPK), Ca2+/Calmodulin‐Dependent Protein Kinase II (CamKII) and the mitochondrial calcium uniporter (MCU) in these 2 mouse models using western immunoblotting.MethodsTransgenic mice of age 5 weeks and 24 weeks containing the R403Q‐MHC and R92W‐TnT HCM mutations were euthanized and each heart was harvested and homogenized in 2% Triton‐Ripa buffer. Protein from whole heart homogenate was isolated by centrifugation and subjected to Western Immunoblotting.ResultsIn vitro studies have shown protein expression differences in all three transgenic mouse models, R403Q‐MHC, R92W‐TnT, and wild type, at both age 5 weeks and 24 weeks. Total target protein, AMPK, CamKII, and MCU were equally expressed in all three models; however, there was a 25% increase in phosphorylated AMPK protein expression in R403Q‐MHC transgenic mice than R92W‐TnT transgenic mice of age 5 weeks and 24 weeks. Phosphorylated CamKII (pCamKII) had 38% increase in protein expression in R92W‐TnT transgenic mice than R403Q‐MHC transgenic mice of age 5 weeks. Phosphorylated MCU had 13% increase in protein expression in R92W‐TnT transgenic mice of age 5 weeks and 26% increase in R92W‐TnT transgenic mice of age 24 weeks than R403Q‐MHC transgenic mice of age 5 weeks and 24 weeks. Although pCamKII lacked expression in transgenic mice of age 24 weeks, this result can lead us to further investigations testing other protein expression differences to gain additional insight of underlying mechanisms how mutations lead to myocyte hypertrophy.ConclusionR403Q‐MHC results in abnormal energy caused by conditions of low ATP, while R92W‐TnT results in high calcium sensitivity by an increase of calcium ions. Results confirm that there is disruption in energy and calcium handling due to the respective mutations compared to that of control. At 5 weeks, in the early development of disease, phosphorylated AMPK protein was expressed 15% lower than at 24 weeks, showing more energy stress at a later stage of disease. However, the opposite holds true for pCamKII, as it was expressed 100% higher at age of 5 weeks, as it lacked expression at age of 24 weeks. This shows that there may be abnormal calcium handling at an early developing stage of disease, but not when the disease is well developed.Support or Funding InformationThis research was supported by the JTB Foundation to Roselle Abraham.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In This Issue

HomeCirculation ResearchVol. 122, No. 6In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published16 Mar 2018https://doi.org/10.1161/RES.0000000000000203Circulation Research. 2018;122:791JNK2/CaMKII Crosstalk Underlies Atrial Arrhythmias (p 821)Inhibiting JNK2 reduces the risk of atrial fibrillation, report Yan et al.Download figureDownload PowerPointCharacterized by a rapid and irregular heartbeat, atrial fibrillation (A-Fib) is associated with hypertrophy, heart failure, ischemia, and old age. Indeed, between 10 and 15% of 70- to 80-year olds develop A-Fib. However, in addition to A-Fib, the activity of the stress–response enzyme c-Jun N-terminal kinase (JNK) also increases with age, hypertrophy, heart failure, and ischemia, which prompted Yan and colleagues to investigate whether A-Fib and JNK share a more direct link. Focusing on JNK2, which is the major JNK isoform in the heart, the team confirmed that the activity of the enzyme is positively correlated with age in both human and mouse hearts. They then showed that elevating JNK2 activity in young mice caused abnormal intracellular calcium handling in the heart, and that JNK2 phosphorylated and activated the protein kinase CamKII, a known driver of arrhythmia. Moreover, inhibition of JNK2 in older animals prevented CamKII activation, aberrant calcium handling, and, most importantly, A-Fib susceptibility. As JNK2 inhibitors are currently under clinical investigation for the treatment of cancer and arthritis, these inhibitors may be attractive candidates for preventing or treating A-Fib.CYB561 and Orthostatic Hypotension (p 846)van den Berg et al discover a new mutation underlying orthostatic hypotension.Download figureDownload PowerPointOrthostatic hypotension—a sudden drop in blood pressure upon standing after lying or sitting—can cause dizziness, fainting, and even loss of consciousness. Although mild or occasional cases are common, in some people, the condition can be severe enough to be life-threatening. Some individuals with mutations in the enzyme dopamine β-hydroxylase (DβH), for example, have impaired production of the catecholamines, norepinephrine and epinephrine, and, as a result, dangerously low blood pressure. Now, van den Berg and colleagues have uncovered a new genetic cause of orthostatic hypotension. They found that 4 patients (2 sets of sisters) with severe, life-long illness and with catecholamine levels indicative of DβH deficiency, did not in fact have DβH mutations or reduced DβH activity. Instead, genetic analysis, including whole-exome sequencing, revealed that the women had homozygous mutations in the gene encoding cytochrome b561 (CYB561)—a transmembrane electron transporter. Investigations into the functional consequences showed that these mutations perturbed electron shuttling necessary for norephinephrine synthesis. Encouragingly, treatment with the norephinerine precursor L-dihydroxyphenylserine, while producing unpleasant side effects, increased blood pressure and relieved symptoms in all 4 patients.Vascular RvDn-3 DPA Reduce Systemic Inflammation (p 855)RvDn-3 DPA tempers the daily increase in leukocyte and platelet activation, report Colas et al.Download figureDownload PowerPointSpecialized proresolving mediators (SPMs) are recently identified molecules that promote the resolution of inflammation. Armed with the knowledge that inflammation naturally waxes and wanes at different periods of night and day, Colas and colleagues investigated whether SPM levels might also exhibit diurnal variation. They found that blood obtained from healthy volunteers at regular intervals over a 24-hour period had higher level of the SPM RvDn-3 DPA at night and that these levels dropped in the morning, coincident with increased leukocyte activation. Moreover, incubation of whole blood with RvDn-3 DPA caused a decrease in neutrophil, platelet, and monocyte activation. Because such leukocyte and platelet activation are linked with cardiovascular disease (CVD)—and to increased occurrence of adverse cardiovascular events in the morning—the team measured RvDn-3 DPA in patients with CVD. They found that, compared with healthy controls, people with CVD had lower levels of RvDn-3 DPA and that the diurnal regulation of the SPM RvDn-3 DPA was impaired. Taken together, these findings suggest that RvDn-3 DPA moderates the diurnal elevations in leukocyte activation and that loss of this regulation may lead to CVD. Previous Back to top Next FiguresReferencesRelatedDetails March 16, 2018Vol 122, Issue 6 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000203 Originally publishedMarch 16, 2018 PDF download Advertisement

Next-generation sequencing of AV nodal reentrant tachycardia patients identifies broad spectrum of variants in ion channel genes.

Atrioventricular nodal reentry tachycardia (AVNRT) is the most common form of regular paroxysmal supraventricular tachycardia. This arrhythmia affects women twice as frequently as men, and is often diagnosed in patients <40 years of age. Familial clustering, early onset of symptoms and lack of structural anomaly indicate involvement of genetic factors in AVNRT pathophysiology. We hypothesized that AVNRT patients have a high prevalence of variants in genes that are highly expressed in the atrioventricular conduction axis of the heart and potentially involved in arrhythmic diseases. Next-generation sequencing of 67 genes was applied to the DNA profile of 298 AVNRT patients and 10 AVNRT family members using HaloPlex Target Enrichment System. In total, we identified 229 variants in 60 genes; 215 missenses, four frame shifts, four codon deletions, three missense and splice sites, two stop-gain variants, and one start-lost variant. Sixty-five of these were not present in the Exome Aggregation Consortium (ExAC) database. Furthermore, we report two AVNRT families with co-segregating variants. Seventy-five of 284 AVNRT patients (26.4%) and three family members to different AVNRT probands had one or more variants in genes affecting the sodium handling. Fifty-four out of 284 AVNRT patients (19.0%) had variants in genes affecting the calcium handling of the heart. We furthermore find a large proportion of variants in the HCN1-4 genes. We did not detect a significant enrichment of rare variants in the tested genes. This could be an indication that AVNRT might be an electrical arrhythmic disease with abnormal sodium and calcium handling.

P.448 - SPEG deficiency is associated with muscle weakness, triad defect, abnormal calcium handling and EC coupling

Centronuclear myopathies (CNM) are a subtype of congenital myopathies (CM) characterized by skeletal muscle weakness and an increase in the number of central myonuclei. We have identified three CNM probands, two with dilated cardiomyopathy, carrying SPEG mutations. SPEG protein localizes to the terminal cisternae of the sarcoplasmic reticulum (SR). We previously described that SPEG interacts with myotubularin, a protein also linked to a type of CNM called myotubular myopathy. Currently, the role of SPEG in muscle function is unclear. Constitutive SPEG knockout mice developed severe dilated cardiomyopathy and mostly died in utero. To overcome this, we generated a conditional SPEG-KO mouse model with Speg deletion driven by crossing them with skeletal muscle-specific cre-expressing mice that excise Speg postnatally (Mck-cre), thereby avoiding death in-utero and extending lifespan. These Speg-KO mice lived up to 3–4 months and were significantly lighter starting at one month compared to their littermate-matched controls. Muscle physiological studies in vivo demonstrated markedly reduced activity by various methods including Acti-track and inverted screen measurements. Histopathological analysis of various muscle groups revealed small fibers, abnormal structure of the triads and disrupted sarcomeric architecture. Further, SPEG-deficient muscle fibers were significantly weaker by in vitro studies, exhibiting abnormal Ca2+ release and excitation-contraction (EC) coupling, a pattern similar to myotubularin-deficient muscles. In addition, the T-tubule pattern revealed a peculiar type of alteration exhibiting local gaps in the transverse pattern at regular locations along the fibers. In summary, consistent with the human phenotype, SPEG deficiency in mice is associated with muscle weakness along with abnormal triad structure, calcium handling and EC-coupling, placing it well within the pathophysiological paradigm of other CNMs characterized by defective triadic structure and function.

CF airway smooth muscle transcriptome reveals a role for PYK2.

Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction-related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.

Functional Upregulation of STIM-1/Orai-1-Mediated Store-Operated Ca2+ Contributing to the Hypertension Development Elicited by Chronic EtOH Consumption.

Chronic ethanol (EtOH) consumption has been associated with deleterious effects on the cardiovascular system by abnormal calcium (Ca2+) handling. Store-operated Ca2+ entry (SOCE) is related to cardiovascular remodeling which leads to the hypertension development, and the coupling between STIM-1 (ER Ca2+ sensor) and Orai-1 (channel pore) is a key mechanism to control SOCE through of store-operated Ca2+ channels (SOCCs). However, the role of STIM-1/Orai-1-mediated SOCE and its cross-talk with EtOH-triggered vascular remodeling and hypertension remain poorly understood. We address this subject in the present study by evaluating how chronic EtOH consumption induces alterations in Ca2+ handling via SOCE. Male Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats were subjected to the intake of increasing EtOH concentrations (5-20%, for 30 days). Systolic blood pressure (SBP) and EtOH concentration were measured; cardiovascular remodeling was assessed by histomorphometry; and function/ expression of STIM-1/Orai-1-mediated Ca2+ influx were evaluated by isometric contraction and western blot experiments. Compared to the WKY-Control, our results show that: (1) chronic EtOH consumption caused a significant elevation of SBP in both strains; (2) cardiac hypertrophy and hypertrophic aortic wall remodeling much more pronounced in WKY-EtOH; (3) decreased capacity of ER to store and release Ca2+; (4) increased STIM-1/Orai-1-mediated SOCCs activation, which was selectively inhibited by YM-58483; and (5) increased expression of STIM-1 in WKY-EtOH and SHR-Control rats. These findings suggest that hypertrophic aortic remodeling and abnormal contraction triggered mainly by Ca2+ overload via STIM-1/Orai-1-mediated SOCE through SOCCs are involved hypertension developed by EtOH consumption.

Functional Upregulation of STIM‐1/Orai‐1‐Mediated Store‐Operated Ca2+ Entry Contributing to the Aortic Remodeling and Hypertension Development by Chronic EtOH Consumption

Chronic ethanol (EtOH) consumption has been associated with deleterious effects on the cardiovascular system by abnormal calcium (Ca2+) handling. Store‐operated Ca2+ entry (SOCE) is related to cardiovascular remodeling which leads to the hypertension development, and the coupling between STIM‐1 (Ca2+ sensor) and Orai‐1 (channel pore) is a key mechanism to control SOCE through of store‐operated Ca2+ channels (SOCCs). However, the role of STIM‐1/Orai‐1‐mediated SOCE and its cross‐talk with EtOH‐triggered vascular remodeling and hypertension remain poorly understood. We address this subject in the present study by evaluating how chronic EtOH consumption induces alterations in Ca2+ handling via SOCE. Male Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats were subjected to the intake of increasing EtOH concentrations (5–20%, for 30 days). Systolic blood pressure (SBP) and EtOH concentration were measured; cardiovascular remodeling was assessed by histomorphometry; and function/expression of STIM‐1/Orai‐1‐mediated Ca2+ influx were evaluated by isometric contraction and western blot experiments. Compared to the WKY‐Control, our results show that: (1) chronic EtOH consumption caused a significant elevation of SBP in both strains; (2) cardiac hypertrophy and hypertrophic aortic wall remodeling much more pronounced in WKY‐EtOH; (3) decreased capacity of reticulum to store and release Ca2+; (4) increased STIM‐1/Orai‐1‐mediated SOCCs activation, which was selectively inhibited by YM‐58483; and (5) increased expression of STIM‐1 in WKY‐EtOH and SHR‐Control rats. These findings suggest that hypertension developed by EtOH consumption has as one of its origins the hypertrophic aortic remodeling and abnormal contraction triggered mainly by Ca2+ overload via STIM‐1/Orai‐1‐mediated SOCE through SOCCs.Support or Funding InformationThis work was supported by the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior‐CAPES (BEX 8477/13‐2); the Fundação de Amparo a Pesquisa do Estado de São Paulo‐FAPESP (2014/01569‐0); the Ministerio de Economía y Competitividad, España (SAF: 2013‐44108‐P and FPI BES‐2014‐069005).

PN06 - Mitochondrial dysfunction and abnormal calcium handling in cell models of Hereditary Sensory Neuropathy type I

PN06 - Mitochondrial dysfunction and abnormal calcium handling in cell models of Hereditary Sensory Neuropathy type I

Boosting the reserves: additive regulation of cardiac repolarisation.

Since the first description of the mechanism of digitalis toxicity in this journal in 1978 (Kass et al. 1978), abnormal calcium handling has been established as a key player across a spectrum of ventricular arrhythmias. This article is protected by copyright. All rights reserved

Abstract 11235: Improvement of Defective Channel Function on RyR2 Mediated by CaMKII Activation Suppresses Arrhythmogenesis in TnT-related Hypertrophic Cardiomyopathy

Backgrounds: In Familial Hypertrophic Cardiomyopathy (FHC) caused by cardiac troponin T (cTnT) mutations, Ca 2+ sensitivity in myofilaments is increased, results in leading to sudden cardiac death. The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) is known to be one of the key elements for the alteration of calcium handling in heart failure, presumably mediated through defective channel function of cardiac ryanodine receptor (RyR2). However, how cTnT mutations lead to lethal arrhythmias and abnormal calcium handling through defective RyR2 remains elusive. Here, we investigated the pathogenic role of CaMKII on aberrant Ca 2+ release through RyR2 for arrhythmogenesis in FHC hearts and therapeutic effects of dantrolene, which was found to correct inter-domain interactions between N-terminal and central domain of RyR2 in FHC-related cTnT-mutated transgenic mouse (TG; cTnT-delta160E). TG have been reported myocardial disarray in histology and increased Ca 2+ sensitivity in myofilaments. Methods and results: On confocal Ca 2+ imaging by fluo4-AM exposed to isoproterenol (ISO; 10 nmol/L), the Ca 2+ spark frequency (SpF:s-1·100μm-1) was much higher in TG cardiomyocytes (ISO-TG: n=11: 7.8±0.6; p&lt;0.05) than in non-TG (n=8: 3.4±0.4). It was largely reversed by CaMKII inhibitor {KN-93(1μM), n=7: 5.2±0.5; p&lt;0.05}, but not by PKA inhibitor (PKI){H-89(1μM), n=7: 7.3±0.4}. The significant increase of spontaneous Ca 2+ transient (sCaT), which was measured after sequential pacing at 5 Hz was appeared in ISO-TG (42.1%; p&lt;0.05 vs ISO-treated non-TG 14.0%), whereas it was again attenuated by CaMKII inhibitor (15.8%), but not by PKI (33.0%). These abnormal events in ISO-TG were reproduced by adding EGTA-AM into ISO-treated non-TG, suggesting that increased Ca 2+ buffering capacity, causing an increase in diastolic [Ca 2+ ], predisposes to aberrant Ca 2+ release events. Moreover, after the treatment of dantrolene (1μM), ISO-induced SpF (n=6: 5.2±0.3; p&lt;0.05 vs ISO-TG) and sCaT (25.9%) were decreased in TG. Conclusions: CaMKII activation may play a role in the development of abnormal calcium handling in FHC-linked cTnT mutated hearts, possibly through defective inter-domain interaction between N-terminal and central domain of RyR2.

Hepatic Inositol 1,4,5 Trisphosphate Receptor Type 1 Mediates Fatty Liver.

Fatty liver is the most common type of liver disease, affecting nearly one third of the U.S. population and more than half a billion people worldwide. Abnormalities in endoplasmic reticulum (ER) calcium handling and mitochondrial function each have been implicated in abnormal lipid droplet formation. Here we show that the type 1 isoform of the inositol 1,4,5‐trisphosphate receptor (InsP3R1) specifically links ER calcium release to mitochondrial calcium signaling and lipid droplet formation in hepatocytes. Moreover, liver‐specific InsP3R1 knockout mice have impaired mitochondrial calcium signaling, decreased hepatic triglycerides, and reduced lipid droplet formation and are resistant to the development of fatty liver. Patients with nonalcoholic steatohepatitis, the most malignant form of fatty liver, have increased hepatic expression of InsP3R1, and the extent of ER–mitochondrial colocalization correlates with the degree of steatosis in human liver biopsies. Conclusion: InsP3R1 plays a central role in lipid droplet formation in hepatocytes, and the data suggest that it is involved in the development of human fatty liver disease. (Hepatology Communications 2017;1:23–35)

Dexamethasone-induced cardiac deterioration is associated with both calcium handling abnormalities and calcineurin signaling pathway activation.

Dexamethasone is a potent and widely used anti-inflammatory and immunosuppressive drug. However, recent evidences suggest that dexamethasone cause pathologic cardiac remodeling, which later impairs cardiac function. The mechanism behind the cardiotoxic effect of dexamethasone is elusive. The present study aimed to verify if dexamethasone-induced cardiotoxicity would be associated with changes in the cardiac net balance of calcium handling protein and calcineurin signaling pathway activation. Wistar rats (~400g) were treated with dexamethasone (35µg/g) in drinking water for 15days. After dexamethasone treatment, we analyzed cardiac function, cardiomyocyte diameter, cardiac fibrosis, and the expression of proteins involved in calcium handling and calcineurin signaling pathway. Dexamethasone-treated rats showed several cardiovascular abnormalities, including elevated blood pressure, diastolic dysfunction, cardiac fibrosis, and cardiomyocyte apoptosis. Regarding the expression of proteins involved in calcium handling, dexamethasone increased phosphorylation of phospholamban at threonine 17, reduced protein levels of Na+/Ca2+ exchanger, and had no effect on protein expression of Serca2a. Protein levels of NFAT and GATA-4 were increased in both cytoplasmic and nuclear faction. In addition, dexamethasone increased nuclear protein levels of calcineurin. Altogether our findings suggest that dexamethasone causes pathologic cardiac remodeling and diastolic dysfunction, which is associated with impaired calcium handling and calcineurin signaling pathway activation.

A Review of the Potential Pathogenicity and Management of Frequent Premature Ventricular Contractions.

Very frequent premature ventricular complexes (PVCs) may be a reversible cause of dilated cardiomyopathy. Literature on this largely unrecognized entity has increased in the last 15 years. This paper reviews the literature on the consequences of frequent PVCs on myocardial function and management of PVC-associated cardiomyopathy. The authors reviewed articles published in English before June 2015 describing pathophysiology, risk factors, symptoms, time course, treatment, and outcome of cardiomyopathy associated with PVCs. The search was conducted using Medline and Embase. Keywords included: cardiomyopathy, catheter ablation, antiarrhythmic drug (AAD), pathophysiology, and ventricular premature contractions or synonyms. PVC-associated cardiomyopathy is associated with a high burden of PVC (over 20% of heartbeats). Other risk factors include electrophysiological characteristics, such as PVC QRS width, presence of ventricular tachycardia, retrograde P waves, interpolation, polymorphic PVCs, and longer coupling intervals. Symptoms include palpitations, light-headedness, dyspnea, cough, and dysphagia. The systolic dysfunction and chamber dilatation progress over a few years. Once the PVCs are suppressed by radiofrequency ablation or AADs, the cardiomyopathy usually resolves within 6 months. The pathophysiology remains unknown, but hypotheses mainly include ventricular dyssynchrony resulting in hemodynamic disturbances and abnormalities in calcium handling and oxygen consumption. PVC-associated cardiomyopathy remains a largely unrecognized entity. It is a reversible cause of dilated cardiomyopathy that results from abnormal calcium and oxygen handling within the myocyte, dyssynchrony, and hemodynamic compromise from inefficient heartbeats. Suppression of the PVCs improves myocardial function, cardiac chamber sizes, and patient's symptoms.

Cyclic nucleotide regulation of cardiac sympatho-vagal responsiveness.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are now recognized as important intracellular signalling molecules that modulate cardiac sympatho-vagal balance in the progression of heart disease. Recent studies have identified that a significant component of autonomic dysfunction associated with several cardiovascular pathologies resides at the end organ, and is coupled to impairment of cyclic nucleotide targeted pathways linked to abnormal intracellular calcium handling and cardiac neurotransmission. Emerging evidence also suggests that cyclic nucleotide coupled phosphodiesterases (PDEs) play a key role limiting the hydrolysis of cAMP and cGMP in disease, and as a consequence this influences the action of the nucleotide on its downstream biological target. In this review, we illustrate the action of nitric oxide-CAPON signalling and brain natriuretic peptide on cGMP and cAMP regulation of cardiac sympatho-vagal transmission in hypertension and ischaemic heart disease. Moreover, we address how PDE2A is now emerging as a major target that affects the efficacy of soluble/particulate guanylate cyclase coupling to cGMP in cardiac dysautonomia.

Mitochondrial oxidative stress during cardiac lipid overload causes intracellular calcium leak and arrhythmia

Diabetes and obesity are associated with an increased risk of arrhythmia and sudden cardiac death. Abnormal lipid accumulation is observed in cardiomyocytes of obese and diabetic patients, which may contribute to arrhythmia, but the mechanisms are poorly understood. A transgenic mouse model of cardiac lipid overload, the peroxisome proliferator-activated receptor-γ (PPARg) cardiac overexpression mouse, has long QT and increased ventricular ectopy. The purpose of this study was to evaluate the hypothesis that the increase in ventricular ectopy during cardiac lipid overload is caused by abnormalities in calcium handling due to increased mitochondrial oxidative stress. Ventricular myocytes were isolated from adult mouse hearts to record sparks and calcium transients. Mice were implanted with heart rhythm monitors for in vivo recordings. PPARg cardiomyocytes have more frequent triggered activity and increased sparks compared to control. Sparks and triggered activity are reduced by mitotempo, a mitochondrial-targeted antioxidant. This is explained by a significant increase in oxidation of RyR2. Calcium transients are increased in amplitude, and sarcoplasmic reticulum (SR) calcium stores are increased in PPARg cardiomyocytes. Computer modeling of the cardiac action potential demonstrates that long QT contributes to increased SR calcium. Mitotempo decreased ventricular ectopy in vivo. During cardiac lipid overload, mitochondrial oxidative stress causes increased SR calcium leak by oxidizing RyR2 channels. This promotes ventricular ectopy, which is significantly reduced in vivo by a mitochondrial-targeted antioxidant. These results suggest a potential role for mitochondrial-targeted antioxidants in preventing arrhythmia and sudden cardiac death in obese and diabetic patients.