Calcium Mishandling Research Articles

IRS2 Signaling Protects Against Stress-Induced Arrhythmia by Maintaining Ca2+ Homeostasis.

The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of IRS2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease. A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice. The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²+ transient amplitudes, increased spontaneous Ca²+ sparks, and reduced sarcoplasmic reticulum Ca²+ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca2+/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice. Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

Modeling the atrioventricular conduction axis using human pluripotent stem cell-derived cardiac assembloids.

The atrioventricular (AV) conduction axis provides electrical continuity between the atrial and ventricular chambers. The "nodal" cardiomyocytes populating this region (AV canal in the embryo, AV node from fetal stages onward) propagate impulses slowly, ensuring sequential contraction of the chambers. Dysfunction of AV nodal tissue causes severe disturbances in rhythm and contraction, and human models that capture its salient features are limited. Here, we report an approach for the reproducible generation of AV canal cardiomyocytes (AVCMs) with invivo-like gene expression and electrophysiological profiles. We created the so-called "assembloids" composed of atrial, AVCM, and ventricular spheroids, which effectively recapitulated unidirectional conduction and the "fast-slow-fast" activation pattern typical for the vertebrate heart. We utilized these systems to reveal intracellular calcium mishandling as the basis of LMNA-associated AV conduction block. In sum, our study introduces novel cell differentiation and tissue construction strategies to facilitate the study of complex disorders affecting heart rhythm.

Abstract Mo011: Selective silencing of p38δ is cardioprotective in female mice during doxorubicin chemotherapy

Introduction: Dose-dependent cardiotoxicity limits the efficacy of chemotherapeutic agent doxorubicin (DOX). p38δ is one of four p38 mitogen-activated protein kinase isoforms that regulate DOX-induced cardiotoxicity (DIC). p38δ genetic deletion protects female mice from DIC. In the absence of selective pharmacologic inhibitors of p38δ, docosanoic acid (DCA) conjugate-mediated delivery of chemically engineered small interfering RNA (siRNA) provides a novel avenue to achieve selective, efficient, durable, and nontoxic in vivo p38δ silencing in the heart. Hypothesis: The selective silencing of p38δ is cardioprotective from DIC in female mice. Methods: Following the screening of a panel of p38δ-selective siRNA sequences, the lead siRNA si-p38δ-1, with the highest silencing efficacy and potency in vitro, was identified. 15-week-old female mice were subcutaneously injected with 20 mg/kg of DCA-conjugated chemically stabilized si-p38δ-1 (n = 3) or the non-targeting control (NTC) siRNA (n = 3) twice 10 hours apart. p38δ silencing in mouse hearts was measured 14 days later by assessing mRNA and protein expression. To evaluate the effect of p38δ silencing after acute DOX treatment, the mice were administered si-p38δ-1 (n = 5) or NTC (n = 5), followed by DOX treatment (30 mg/kg) six days later. The study concluded 11 days after DOX treatment. Morbidity was assessed by assigning health scores daily. Electrocardiography, echocardiography, and optical mapping were performed to evaluate cardiac function. Results: si-p38δ-1 led to 80% silencing of p38δ in mouse hearts 14 days after administration, without any cardiotoxic effects. si-p38δ-1-treated female mice exhibited diminished DOX-induced morbidity (p = 0.003) and were protected from developing calcium alternans (CAs) 11 days post-DOX treatment, compared to NTC-treated counterparts. Thus, DOX-induced CAs, indicative of calcium mishandling, were observed in 75% (3/4) NTC-treated mice and 0% (0/5) si-p38δ-1-treated mice. Conclusion: Efficient and well-tolerated in vivo p38δ silencing by si-p38δ-1 in mouse hearts mitigated DOX-induced morbidity and calcium mishandling in female mice, highlighting the therapeutic potential of p38δ silencing for alleviating DIC in female cancer patients.

Abstract Mo042: Poison Peptides May Contribute to Dysregulation of SERCA in Heart Failure

Proteolysis increases in heart disease, resulting in an accumulation of degraded membrane protein fragments in heart cells. Previous studies have shown that diverse hydrophobic alpha helices can bind and non-specifically inhibit the cardiac calcium pump SERCA. Thus, we hypothesized that partially degraded fragments of transmembrane proteins may directly inhibit SERCA or compete with native peptide regulators of the pump, like such as phospholamban. The resulting dysregulation of SERCA may contribute to calcium mishandling in heart failure. To test these hypotheses, purified cell membranes from non-failing or dilated cardiomyopathy human hearts were subjected to SDS-PAGE and mass spectrometry to identify protein fragments smaller than 26 kD. 1800 proteins were detected, of which 92 were upregulated >3 fold in the failing heart samples, consistent with increased production of small fragments from these membrane proteins. 9 of the upregulated fragments containing transmembrane segments were selected for further analysis based on sequence hydrophobicity and structural similarity to endogenous micropeptide regulators of SERCA. Of these 9 fragments, 6 showed ER localization, suggesting potential for a toxic interaction with SERCA. The relative binding of these candidates to SERCA was quantified with FRET microscopy. Fragments of three membrane proteins (SEC61B, VAMP3, VAMP8) showed an avid interaction with SERCA. To measure SERCA inhibition, we utilized a calcium activity assay was performed usingin which HEK293T cells were transfected with SERCA, ryanodine receptor, candidate peptides and rCepia as an ER calcium indicator. ER-calcium level was normalized to minimum fluorescence after caffeine addition and an ionomycin induced maximum fluorescence after addition of ionomycin and calcium and caffeine induced minimum. Unregulated Candidate poison peptides were compared to SERCA alone and SERCA-PLB phospholamban controls were also performed. . SEC61B demonstrated the a strongest inhibition, most (similar to phospholamban, ) while VAMP3 and VAMP8’s effect was more modest. This type of non-nativeThus, we propose accumulation of toxic transmembrane peptide fragments may inhibition could be contributinginhibit SERCA and contribute to the calcium mishandling seen in heart failure. Future experiments will be performed to quantify the relative abundance of the fragments compared to endogenous regulatory micropeptides.

Cardioprotective effect of the anti-ageing protein Klotho on ischemic heart disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII, Ministerio de Universidades, Education and Research Council of Madrid, Biomedicine Network Comunidad de Madrid Background Cardiovascular diseases are the leading cause of death and disability worldwide. Among them, ischemic heart disease, due to myocardial infarction (MI) remains the first cause of cardiovascular death. The anti-ageing protein Klotho is mainly expressed in membrane at renal level. However, Klotho also has a soluble form with different pleiotropic functions that are still undetermined, including a possible cardioprotective role that is currently the target of several studies. Purpose Firstly, we evaluated circulating Klotho levels related to cardiac damage in a cohort of 146 patients after ST-elevation myocardial infarction (STEMI) and in an experimental post-myocardial infarction (PMI) model. Secondly, we investigated soluble Klotho supplementation as a potential novel therapeutic strategy for the prevention of cardiac dysfunction, deleterious remodeling, cardiomyocyte calcium (Ca2+) mishandling and arrhythmias in PMI mice. Methods Study with human samples: we analysed Klotho and NT-proBNP plasma levels in STEMIpatients with preserved renal function. Experimental study: C57BL6 mice subjected to PMI by ligation of the left anterior descending coronary artery were treated with vehicle solution or recombinant Klotho (10 mg/Kg/day) for 15 days after MI. Cardiac function was studied through non-invasive methods including electrocardiogram (ECG) and cardiac magnetic resonance imaging (CMRI). Intracellular Ca2+ handling was analysed using confocal microscopy in isolated ventricular cardiomyocytes. Cardiac remodeling was studied through histological and biomolecular analysis of several markers of cardiac damage and target proteins involved in Ca2+ handling. Results Plasma Klotho levels decrease rapidly after MI in both STEMI patients and PMI mice. Additionally, MI patients in the lowest Klotho tertile showed significantly elevated NT-proBNP levels. Klotho treatment decreased the infarct area in PMI mice accompanied by a decrease in both cardiac hypertrophy and fibrosis. Moreover, reduction in ejection fraction and MI-related ECG alterations were reduced in PMI mice treated with Klotho, including increased QRS, JT, QTc intervals and occurrence of premature ventricular contractions. Klotho also prevented systolic Ca2+ release and cell shortening disturbances in cardiomyocytes from PMI mice without Klotho. Increased diastolic Ca2+ leak was prevented in mice treated with Klotho via CaMKII-dependent pathway by avoiding its phosphorylation and the RyR2 phosphorylation at CaMKII site. Conclusions Klotho supplementation protect against MI by avoiding deleterious cardiac remodeling and ameliorating one of the main complications of MI such as arrhythmias. Thus, recombinant Klotho prevents the intracardiomyocyte Ca2+ mishandling after MI. Our translational findings support the importance of Klotho as a novel biomarker of ventricular damage just after MI and as a novel therapeutic strategy for prevention cardiac events associated to ischemic heart disease.

Role of NOD1 in atrial myopathy associated with heart failure

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funding: Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ISCIII [PI17/01344 and PI20/01482]), Ministry of Education (FPU19/01973), SEC, Proyecto Traslacional 2019, FEDER, FSE, CIBER-CV, a network funded by ISCIII Introduction Heart failure (HF) is a complex syndrome that constitutes one of the leading causes of cardiovascular death worldwide. Although multifactorial mechanisms appear to be responsible of atrial and ventricular HF-myopathy, the innate immune system and the derived inflammatory response have gained much attention in the last years. Previous studies from our group have demonstrated that the nucleotide-binding oligomerization domain 1 (NOD1), a receptor of the innate immune system, plays a role in HF left ventricular (LV) dysfunction. However, its role in HF-atrial myopathy remains completely unknown. The main purpose of our study is to determine the role of NOD1 in human and experimental HF-atrial myopathy. Methods and Results The study was performed in two cohorts of individuals who underwent heart valve repair surgery, 51 non-failing individuals (NF) with LV ejection fraction (LVEF)>50% and NT-ProBNP levels<200pg/mL and 41 subjects with medium to severe reduced EF (HF)<50% and NT-ProBNP levels>200pg/mL. An informed consent was signed by all patients. Atrial myocardial tissue was excised from the atrial appendage and prepared for biochemical and histological studies. Echocardiography analysis showed that left and right atrial areas (LA and RA) were significantly increased in the HF group compared to NF, while LA and RA reservoir functions were significantly decreased in HF. Immunostaining demonstrated NOD1 location in atrial cardiomyocytes, being the protein levels of NOD1 and its specific adaptor RIP2 significantly increased in atrial tissue obtained from HF vs. NF. Although the prevalence of atrial fibrillation (AF) was significantly higher in the HF group compared to NF, the upregulation of NOD1 was not conditioned by the presence of AF. Similarly, pigs that underwent transverse aortic constriction (TAC) or proximal left circumflex coronary artery occlusion involving the LA branch (LAI), showed an upregulation of NOD1 pathway in atrial tissue, compared with the sham operated animals. Interestingly, an atrial upregulation of NOD1 axis was also observed in wild-type TAC mice, whereas Nod1-/- knockout mice showed a prevention in both structural and functional atrial remodelling induced by TAC, mainly by a prevention of intracellular calcium mishandling in isolated atrial cardiomyocytes. Finally, wild-type mice injected i.p. for 3 days with 3.3 mg/kg with a selective activator of NOD1, showed increased structural atrial remodelling and intracellular calcium mishandling compared to the vehicle-treated group. Conclusions Our results demonstrate that NOD1 axis is upregulated in human, swine and murine atrial failing myocardium. Genetic deletion of NOD1 prevented HF-atrial structural and functional remodelling, mainly by avoiding intracellular calcium mishandling, whereas its pharmacological activation induced atrial dilation and intracellular Ca2+ mishandling. All these data uncover NOD1 as a new player in atrial HF-remodelling.

Integrated multi-omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex.

Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions. Skeletal muscles from 2-month-old SPEG-deficient (Speg-CKO) and wild-type (WT) mice were used for RNA sequencing (n=4 per genotype) to profile transcriptomics and mass spectrometry (n=4 for WT; n=3 for Speg-CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg-CKO mice. Based on the multi-omics results, we performed quantitative real-time PCR, co-immunoprecipitation and immunoblot to verify the findings. We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22±3% for CMYA5 [P<0.05] and 18±3% for FSD2 [P<0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55±15% at S2902 in Speg-CKO mice; P<0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90±0.24-fold], S162 [1.61±0.37-fold] and S165 [1.66±0.13-fold]; decreased phosphorylation at S228 and S231 [39±6%], S234 [50±12%], S593 [48±3%] and S613 [66±10%]; P<0.05 for S162 and P<0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P<1e-15) and peroxisome proliferator-activated receptor signalling (P<9e-14). We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG-interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi-omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.

Cardiac dysfunction in sucrose-fed rats is associated with alterations of phospholamban phosphorylation and TNF-α levels

IntroductionHigh sucrose intake is linked to cardiovascular disease, a major global cause of mortality worldwide. Calcium mishandling and inflammation play crucial roles in cardiac disease pathophysiology. ObjectiveEvaluate if sucrose-induced obesity is related to deterioration of myocardial function due to alterations in the calcium-handling proteins in association with proinflammatory cytokines. MethodsWistar rats were divided into control and sucrose groups. Over eight weeks, Sucrose group received 30% sucrose water. Cardiac function was determined in vivo using echocardiography and in vitro using papillary muscle assay. Western blotting was used to detect calcium handling protein; ELISA assay was used to assess TNF-α and IL-6 levels. ResultsSucrose led to cardiac dysfunction. RYR2, SERCA2, NCX, pPBL Ser16 and L-type calcium channels were unchanged. However, pPBL-Thr17, and TNF-α levels were elevated in the S group. ConclusionSucrose induced cardiac dysfunction and decreased myocardial contractility in association with altered pPBL-Thr17 and elevated cardiac pro-inflammatory TNF-α.

Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function.

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it "bigheart." Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

The Role of Pro-Inflammatory Cytokines in the Pathogenesis of Cardiovascular Disease.

With cardiovascular disease (CVD) being a primary source of global morbidity and mortality, it is crucial that we understand the molecular pathophysiological mechanisms at play. Recently, numerous pro-inflammatory cytokines have been linked to several different CVDs, which are now often considered an adversely pro-inflammatory state. These cytokines most notably include interleukin-6 (IL-6),tumor necrosis factor (TNF)α, and the interleukin-1 (IL-1) family, amongst others. Not only does inflammation have intricate and complex interactions with pathophysiological processes such as oxidative stress and calcium mishandling, but it also plays a role in the balance between tissue repair and destruction. In this regard, pre-clinical and clinical evidence has clearly demonstrated the involvement and dynamic nature of pro-inflammatory cytokines in many heart conditions; however, the clinical utility of the findings so far remains unclear. Whether these cytokines can serve as markers or risk predictors of disease states or act as potential therapeutic targets, further extensive research is needed to fully understand the complex network of interactions that these molecules encompass in the context of heart disease. This review will highlight the significant advances in our understanding of the contributions of pro-inflammatory cytokines in CVDs, including ischemic heart disease (atherosclerosis, thrombosis, acute myocardial infarction, and ischemia-reperfusion injury), cardiac remodeling (hypertension, cardiac hypertrophy, cardiac fibrosis, cardiac apoptosis, and heart failure), different cardiomyopathies as well as ventricular arrhythmias and atrial fibrillation. In addition, this article is focused on discussing the shortcomings in both pathological and therapeutic aspects of pro-inflammatory cytokines in CVD that still need to be addressed by future studies.

JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes.

Prolonged stimulation of β-adrenergic receptor (β-AR) can lead to sympathetic overactivitythat causes pathologic cardiac hypertrophy and fibrosis, ultimately resulting in heart failure. Recent studies suggest that abnormal protein ubiquitylation may contribute to the pathogenesis of cardiac hypertrophy and remodeling. In this study, we demonstrated that deficiency of a deubiquitinase, Josephin domain-containing protein 2 (JOSD2), ameliorated isoprenaline (ISO)- and myocardial infarction (MI)-induced cardiac hypertrophy, fibrosis, and dysfunction both in vitro and in vivo. Conversely, JOSD2 overexpression aggravated ISO-induced cardiac pathology. Through comprehensive mass spectrometry analysis, we identified that JOSD2 interacts with Calcium-calmodulin-dependent protein kinase II (CaMKIIδ). JOSD2 directly hydrolyzes the K63-linked polyubiquitin chains on CaMKIIδ, thereby increasing the phosphorylation of CaMKIIδ and resulting in calcium mishandling, hypertrophy, and fibrosis in cardiomyocytes. In vivo experiments showed that the cardiac remodeling induced by JOSD2 overexpression could be reversed by the CaMKIIδ inhibitor KN-93. In conclusion, our study highlights the role of JOSD2 in mediating ISO-induced cardiac remodeling through the regulation of CaMKIIδ ubiquitination, and suggests its potential as a therapeutic target for combating the disease. Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary. All have been checked.

Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

Diabetic cardiomyopathy, an increasingly global epidemic and a major cause of heart failure with preserved ejection fraction (HFpEF), is associated with hyperglycemia, insulin resistance, and intracardiomyocyte calcium mishandling. Here we identify that, in db/db mice with type 2 diabetes-induced HFpEF, abnormal remodeling of cardiomyocyte transverse-tubule microdomains occurs with downregulation of the membrane scaffolding protein cardiac bridging integrator 1 (cBIN1). Transduction of cBIN1 by AAV9 gene therapy can restore transverse-tubule microdomains to normalize intracellular distribution of calcium-handling proteins and, surprisingly, glucose transporter 4 (GLUT4). Cardiac proteomics revealed that AAV9-cBIN1 normalized components of calcium handling and GLUT4 translocation machineries. Functional studies further identified that AAV9-cBIN1 normalized insulin-dependent glucose uptake in diabetic cardiomyocytes. Phenotypically, AAV9-cBIN1 rescued cardiac lusitropy, improved exercise intolerance, and ameliorated hyperglycemia in diabetic mice. Restoration of transverse-tubule microdomains can improve cardiac function in the setting of diabetic cardiomyopathy and can also improve systemic glycemic control.

Remodeling of lipid landscape in high fat fed very-long chain acyl-CoA dehydrogenase null mice favors pro-arrhythmic polyunsaturated fatty acids and their downstream metabolites

Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid β-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD−/− mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD−/− mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD−/− mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.

Abstract P2077: FoxO3 Transcription Factor Regulates Myocyte Contractility And Calcium Homeostasis In Diabetic Cardiomyopathy

Diabetes is a risk factor for heart failure with preserved ejection fractions (HFpEF). Calcium (Ca 2+ ) mishandling and diastolic abnormalities are the characteristics of diabetic cardiomyopathy (DCM), but the molecular mechanisms of these are elusive. FoxO transcription factors (FoxOs) are suppressed by insulin and this study evaluated the role of FoxO3 in myocardial contractility and Ca 2+ handling in the heart of a mouse model of type 1 diabetes. We show that cardiomyocyte-restricted deletion of FoxO3 (H-FoxO3 KO) prevented reduction in the peak rate of left ventricular (LV) pressure (dP/dt max) and prevented the increase in the minimum rate of fall in LV pressure (dP/dt min) that was evident after 4-7 weeks of Streptozotocin (STZ) diabetes. Time for pressure decay of LV while relaxation (Tau) was partially increased in STZ hearts and tended to decrease in STZ H-FoxO3 KO. Echocardiography showed LVEF was unchanged in control and H-FoxO3 KO animals treated with/without STZ. Ca 2+ imaging in perfused hearts found that Ca 2+ transient amplitude was decreased and decay kinetics prolonged (indicates decreased SERCA activity) in STZ hearts, but these changes were prevented in STZ H-FoxO3 KO. Phosphorylation of SERCA at Thr484 by SPEG (Striated muscle preferentially expressed protein kinase) controls Ca 2+ re-uptake and cardiac contractility. We show that STZ diabetes decreases SPEG protein and pSERCA2a Thr484 , but levels of each were normalized or increased in STZ H-FoxO3 KO. Mechanistically, overexpression of FoxO3 in neonatal cardiomyocytes increases high-molecular weight SPEG adducts when proteasome degradation was inhibited with MG-132, suggesting that ubiquitin-mediated degradation of SPEG is a mechanism by which FoxO3 controls Ca 2+ handling via SERCA phosphorylation. SERCA function requires ATP, thus, we evaluated mitochondrial energetics in these animals. Respiratory capacity in saponin-permeabilized cardiac fibers was unchanged in hearts from controls and H-FoxO3 KO treated with/without STZ. ATP generation was decreased in STZ hearts but was not normalized in STZ-H FoxO3 KO. These data indicate FoxO3 regulates myocyte contractility via SPEG degradation to control intracellular Ca 2+ via pSERCA2a Thr484 in the heart in a type 1 diabetes model.

Sustained high-fat diet increases ventricular susceptibility to arrhythmias and impairs myocardial calcium cycling

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Medizinische Fakultät, Heinrich Heine Universität Düsseldorf Background – Obesity is one of the most relevant factors for cardiovascular disease in western societies. Independent of coronary heart disease, obesity promotes a multimodal arrhythmic substrate and increases the susceptibility to ventricular arrhythmias (VA) eventually leading to sudden cardiac death. Purpose – Aim of the present study was to develop an ex vivo mouse model to characterize obesity-mediated VAs and left ventricular (LV) myocardial disruption of calcium homeostasis. Methods – All experiments were performed ex vivo in retrogradely perfused hearts from 32 – 36 week-old C57Bl/6J mice (n=14). Five mice received a normal chow diet (NCD), 9 mice were fed a sustained high-fat diet (HFD) over a 6-month period to generate diet-induced-obesity (DIO). We combined an endocardial catheter-based technique to assess VA susceptibility with an epicardial dual fluorescence imaging approach employing a potentiometric (Rh237) and a calcium sensitive (Rhod2) dye to assess action potential and calcium cycling characteristics. Optical mapping data was analysed using a MATLAB-based algorithm. VA susceptibility was quantified by an established scoring system. Results – Mice receiving a sustained HFD were significantly heavier than those receiving NCD (NCD: 34.8±2.1g, HFD: 53.3±3.2g, P&amp;lt;0.0001). The number of induced VAs and VA susceptibility were increased in HFD compared to NCD (number of induced VAs: NCD: 1.4±0.7, HFD: 6.3±0.8, P=0.005, Figure 1A; VA susceptibility: NCD: 1.4±0.7, HFD: 18.1±2.3, P=0.001, Figure 1B). Ventricular tachycardias (VT) were only inducible in HFD, additionally, those hearts presented mostly polymorphic VTs (VT inducibility: NCD: 0.0 %, HFD: 88.9 %, P=0.003, Figure 1C; VT morphology: monomorphic VT: 25 %, polymorphic VT: 75 %). Epicardial optical mapping of the anterior LV wall showed a prolonged action potential duration (APD) and prolonged calcium transient duration (CaTD) in HFD (APD90 at CL 100 ms: NCD: 45.1±1.5 ms, HFD: 50.8±1.8 ms, P=0.03; CaTD90 at 100 ms pacing cycle length (PCL): NCD: 61.0±2.3 ms, HFD: 66.6±0.6 ms, P=0.02, Figure 2A). Furthermore, CaT alternans (ALT) threshold defined as amplitude alternans &amp;gt; 10 % of two consecutive beats was decreased (PCL ALT threshold: NCD: 60.0±1.6 ms, DIO: 67.8±1.2 ms, P=0.006, Figure 2B), with mean ALT being increased in HFD (mean ATL at 60 ms PCL: NCD: 15.4±2.4 %, HFD: 25.7±3.0 %, P=0.04). Conclusions – Our findings show that sustained HFD in mice increases ventricular susceptibility to VTs, especially polymorphic VTs indicating heterogeneity in wave propagation. DIO leads to disruption of LV myocardial calcium homeostasis as demonstrated by a decreased ALT threshold and increased mean ALT. Our data suggest that LV calcium mishandling may be a driver for arrhythmogenesis in DIO and modulation may present a perspective therapeutical approach. Further experiments will address molecular mechanisms and identify potential targets of modulation.

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling.

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Iron and atrial fibrillation: A review.

Atrial fibrillation (AF), one of the most common arrhythmias in clinical practice, is classified into paroxysmal, persistent, and permanent AF according to its duration. The development of AF is associated with increased cardiovascular morbidity and mortality. However, the exact etiology of this disease remains poorly understood. Recent studies found disorders of iron metabolism might be involved in the progression of AF. Abnormal iron metabolism in cardiomyocytes provides arrhythmogenic substrates through a variety of mechanisms, including calcium mishandling, ion channel remodeling, and oxidative stress overaction. Interestingly, in AF patients with iron overload, interventions on iron metabolism, such as iron chelators and ferroptosis inhibitors, has been shown to prevent AF via reducing ferroptosis. Herein, we review the possible mechanisms, consequences, and therapeutic implications of altered atrial iron handling for AF pathophysiology.

Calcium Handling in Inherited Cardiac Diseases: A Focus on Catecholaminergic Polymorphic Ventricular Tachycardia and Hypertrophic Cardiomyopathy.

Calcium (Ca2+) is the major mediator of cardiac contractile function. It plays a key role in regulating excitation-contraction coupling and modulating the systolic and diastolic phases. Defective handling of intracellular Ca2+ can cause different types of cardiac dysfunction. Thus, the remodeling of Ca2+ handling has been proposed to be a part of the pathological mechanism leading to electrical and structural heart diseases. Indeed, to ensure appropriate electrical cardiac conduction and contraction, Ca2+ levels are regulated by several Ca2+-related proteins. This review focuses on the genetic etiology of cardiac diseases related to calcium mishandling. We will approach the subject by focalizing on two clinical entities: catecholaminergic polymorphic ventricular tachycardia (CPVT) as a cardiac channelopathy and hypertrophic cardiomyopathy (HCM) as a primary cardiomyopathy. Further, this review will illustrate the fact that despite the genetic and allelic heterogeneity of cardiac defects, calcium-handling perturbations are the common pathophysiological mechanism. The newly identified calcium-related genes and the genetic overlap between the associated heart diseases are also discussed in this review.

Poison peptides interactions with the sarcoplasmic reticulum Ca-ATPase (SERCA).

Maintaining cellular integrity requires maintenance of protein quality, so when damaged/misfolded proteins fail to be repaired, they are degraded via proteolysis. Proteolysis increases in heart disease, overwhelming the proteosome, resulting in an accumulation of protein fragments in the cell. We propose that undegraded transmembrane protein fragments found in heart failure may persist in the membrane and interact with SERCA, competing with and displacing the physiological regulatory micropeptide phospholamban. These interactions with “poison peptides” may contribute to calcium mishandling in heart failure. To test this hypothesis, we performed mass spectrometry of membrane fractions isolated from failing human myocardium. We detected approximately 1000 protein fragments of interest in the failing heart, then narrowed our selection to high-abundance transmembrane protein fragments ranging from 20-40 amino acids. We used fluorescence resonance energy transfer (FRET) to test whether seven candidate poison proteins could interact with SERCA. Some peptides did not interact with SERCA at all, some bound weakly, and some bound with a high affinity. The highest affinity candidates bound SERCA as avidly as some native regulatory micropeptides. The candidate peptides tested here include transmembrane fragments of phospholamban (PLB), phospholemman (PLM), calcium voltage-gated channel subunit alpha1 E (CACNA1E) and sarcolemma associated protein (SLMAP). We also tested whether the putative poison peptides altered the structure of SERCA, using time-correlated single photon counting to quantify intramolecular FRET in a “2-color SERCA” construct labeled with mCyRFP-1 and mMaroon1. Overall, the results suggest that transmembrane peptide fragments generated in heart failure can interact with SERCA and may disrupt calcium handling in the failing heart.

Partial Genetic Deletion of Klotho Aggravates Cardiac Calcium Mishandling in Acute Kidney Injury.

Acute kidney injury (AKI) is associated with an elevated risk of cardiovascular major events and mortality. The pathophysiological mechanisms underlying the complex cardiorenal network interaction remain unresolved. It is known that the presence of AKI and its evolution are significantly associated with an alteration in the anti-aging factor klotho expression. However, it is unknown whether a klotho deficiency might aggravate cardiac damage after AKI. We examined intracellular calcium (Ca2+) handling in native ventricular isolated cardiomyocytes from wild-type (+/+) and heterozygous hypomorphic mice for the klotho gene (+/kl) in which an overdose of folic acid was administered to induce AKI. Twenty-four hours after AKI induction, cardiomyocyte contraction was decreased in mice with the partial deletion of klotho expression (heterozygous hypomorphic klotho named +/kl). This was accompanied by alterations in Ca2+ transients during systole and an impairment of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) function in +/kl mice after AKI induction. Moreover, Ca2+ spark frequency and the incidence of Ca2+ pro-arrhythmic events were greater in cardiomyocytes from heterozygous hypomorphic klotho compared to wild-type mice after AKI. A decrease in klotho expression plays a role in cardiorenal damage aggravating cardiac Ca2+ mishandling after an AKI, providing the basis for future targeted approaches directed to control klotho expression as novel therapeutic strategies to reduce the cardiac burden that affects AKI patients.