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Abstract Background NODAL signaling plays a critical role in embryonic patterning and heart development in vertebrates. Genetic variants resulting in perturbations of the TGF-β/NODAL signaling pathway have reproducibly been shown to cause laterality defects in humans. To further explore this association and improve genetic diagnosis, the study aims to identify and characterize a broader range of NODAL variants in a large number of individuals with laterality defects. Methods We re-analyzed a cohort of 321 proband-only exomes of individuals with clinically diagnosed laterality congenital heart disease (CHD) using family-based, rare variant genomic analyses. To this cohort we added 12 affected subjects with known NODAL variants and CHD from institutional research and clinical cohorts to investigate an allelic series. For those with candidate contributory variants, variant allele confirmation and segregation analysis were studied by Sanger sequencing in available family members. Array comparative genomic hybridization and droplet digital PCR were utilized for copy number variants (CNV) validation and characterization. We performed Human Phenotype Ontology (HPO)-based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. Results Missense, nonsense, splice site, indels, and/or structural variants of NODAL were identified as potential causes of heterotaxy and other laterality defects in 33 CHD cases. We describe a recurrent complex indel variant for which the nucleic acid secondary structure predictions implicate secondary structure mutagenesis as a possible mechanism for formation. We identified two CNV deletion alleles spanning NODAL in two unrelated CHD cases. Furthermore, 17 CHD individuals were found (16/17 with known Hispanic ancestry) to have the c.778G > A:p.G260R NODAL missense variant which we propose reclassification from variant of uncertain significance (VUS) to likely pathogenic. Quantitative HPO-based analyses of the observed clinical phenotype for all cases with p.G260R variation, including heterozygous, homozygous, and compound heterozygous cases, reveal clustering of individuals with biallelic variation. This finding provides evidence for a genotypic-phenotypic correlation and an allele-specific gene dosage model. Conclusion Our data further support a role for rare deleterious variants in NODAL as a cause for sporadic human laterality defects, expand the repertoire of observed anatomical complexity of potential cardiovascular anomalies, and implicate an allele specific gene dosage model.

Abstract Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegβ deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegβ but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg β (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it’s low levels, HA-Spegβ immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegβ binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegβ display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

This article reviews progress in the field of cardiac genome editing, in particular, its potential utility in treating cardiac arrhythmias. First, we discuss genome editing methods by which DNA can be disrupted, inserted, deleted, or corrected in cardiomyocytes. Second, we provide an overview of in vivo genome editing in preclinical models of heritable and acquired arrhythmias. Third, we discuss recent advancements in cardiac gene transfer, including delivery methods, gene expression optimization, and potential adverse effects associated with therapeutic somatic genome editing. While genome editing for cardiac arrhythmias is still in its infancy, this approach holds great promise, especially for inherited arrhythmia syndromes with a defined genetic defect.

Mitochondrial permeability transition pore (MPTP)-dependent necrosis contributes to numerous pathologies in the heart, brain, and skeletal muscle. The MPTP is a non-selective pore in the inner mitochondrial membrane that is triggered by high levels of matrix Ca2+, and sustained opening leads to mitochondrial dysfunction. Although the MPTP is defined by an increase in inner mitochondrial membrane permeability, the expression of pro-apoptotic Bcl-2 family members, Bax and Bak localization to the outer mitochondrial membrane is required for MPTP-dependent mitochondrial dysfunction and subsequent necrotic cell death. Contrary to the role of Bax and Bak in apoptosis, which is dependent on their oligomerization, MPTP-dependent necrosis does not require oligomerization as monomeric/inactive forms of Bax and Bak can facilitate mitochondrial dysfunction. However, the relationship between Bax and Bak activation/oligomerization and MPTP sensitization remains to be explored. Here, we use a combination of in vitro and ex vivo approaches to determine the role of the anti-apoptotic Bcl-2 family members, which regulate Bax/Bak activity, in necrotic cell death and MPTP sensitivity. To study the role of each predominantly expressed anti-apoptotic Bcl-2 family member (i.e., Mcl-1, Bcl-2, and Bcl-xL) in MPTP regulation, we utilize various BH3 mimetics that specifically bind to and inhibit each. We determined that the inhibition of each anti-apoptotic Bcl-2 family member lowers mitochondrial calcium retention capacity and sensitizes MPTP opening. Furthermore, the inhibition of each Bcl-2 family member exacerbates both apoptotic and necrotic cell death in vitro in a Bax/Bak-dependent manner. Our findings suggests that mitochondrial Ca2+ retention capacity and MPTP sensitivity is influenced by Bax/Bak activation/oligomerization on the outer mitochondrial membrane, providing further evidence of the crosstalk between the apoptotic and necrotic cell death pathways.

Hyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrillation and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 interactome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins, with 20 being high confidence RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blots and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.

Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular diseases that often co-exist. Animal models have suggested complex AF-promoting atrial structural, electrical, and Ca2+-handling remodeling in the setting of HF, but data in human samples are scarce, particularly regarding Ca2+-handling remodeling. Here, we evaluated atrial remodeling in patients with severe left ventricular (LV) dysfunction (HFrEF), long-standing persistent (‘chronic’) AF (cAF) or both (HFrEF-cAF), and sinus rhythm controls with normal LV function (Ctl) using western blot in right-atrial tissue, sharp-electrode action potential (AP) measurements in atrial trabeculae and voltage-clamp experiments in isolated right-atrial cardiomyocytes. Compared to Ctl, expression of profibrotic markers (collagen-1a, fibronectin, periostin) was higher in HFrEF and HFrEF-cAF patients, indicative of structural remodeling. Connexin-43 expression was reduced in HFrEF patients, but not HFrEF-cAF patients. AP characteristics were unchanged in HFrEF, but showed classical indices of electrical remodeling in cAF and HFrEF-cAF (prolonged AP duration at 20% and shorter AP duration at 50% and 90% repolarization). L-type Ca2+ current (ICa,L) was significantly reduced in HFrEF, cAF and HFrEF-cAF, without changes in voltage-dependence. Potentially proarrhythmic spontaneous transient-inward currents were significantly more frequent in HFrEF and HFrEF-cAF compared to Ctl, likely resulting from increased sarcoplasmic reticulum (SR) Ca2+ load (integrated caffeine-induced current) in HFrEF and increased ryanodine-receptor (RyR2) single-channel open probability in HFrEF and HFrEF-cAF. Although expression and phosphorylation of the SR Ca2+-ATPase type-2a (SERCA2a) regulator phospholamban were unchanged in HFrEF and HFrEF-cAF patients, protein levels of SERCA2a were increased in HFrEF-cAF and sarcolipin expression was decreased in both HFrEF and HFrEF-cAF, likely increasing SR Ca2+ uptake and load. RyR2 protein levels were decreased in HFrEF and HFrEF-cAF patients, but junctin levels were higher in HFrEF and relative Ser2814-RyR2 phosphorylation levels were increased in HFrEF-cAF, both potentially contributing to the greater RyR2 open probability. These novel insights into the molecular substrate for atrial arrhythmias in HF-patients position Ca2+-handling abnormalities as a likely trigger of AF in HF patients, which subsequently produces electrical remodeling that promotes the maintenance of the arrhythmia. Our new findings may have important implications for the development of novel treatment options for AF in the context of HF.

One quarter of deaths associated with Rett syndrome (RTT), an X-linked neurodevelopmental disorder, are sudden and unexpected. RTT is associated with prolonged QTc interval (LQT), and LQT-associated cardiac arrhythmias are a potential cause of unexpected death. The standard of care for LQT in RTT is treatment with β-adrenergic antagonists; however, recent work indicates that acute treatment of mice with RTT with a β-antagonist, propranolol, does not prevent lethal arrhythmias. In contrast, acute treatment with the Na+ channel blocker phenytoin prevented arrhythmias. Chronic dosing of propranolol may be required for efficacy; therefore, we tested the efficacy of chronic treatment with either propranolol or phenytoin on RTT mice. Phenytoin completely abolished arrhythmias, whereas propranolol showed no benefit. Surprisingly, phenytoin also normalized weight and activity, but worsened breathing patterns. To explore the role of Na+ channel blockers on QT in people with RTT, we performed a retrospective analysis of QT status before and after Na+ channel blocker antiepileptic therapies. Individuals with RTT and LQT significantly improved their QT interval status after being started on Na+ channel blocker antiepileptic therapies. Thus, Na+ channel blockers should be considered for the clinical management of LQT in individuals with RTT.

IntroductionPulmonary hypertension (PH) carries significant associated morbidity and mortality and the underlying molecular mechanisms of PH are not well understood. Loss-of-function mutations in TASK-1 potassium channels are associated with PH in humans. Although TASK-1 has been considered in the development of PH for over a decade, characterization of TASK-1 knockout mice has been limited to in vitro studies or in vivo studies in room air at isolated time points. The purpose of this study was twofold. First, we sought to determine if TASK−/− male and female mice developed PH over the span of one year. Second, we sought to determine the effect of chronic hypoxia, a stimulus for PH, and its recovery on PH in TASK-1−/− mice.MethodsWe measured right ventricular systolic pressure (RVSP) and vascular remodeling in male and female C57BL/6 WT and TASK-1−/− mice at separate time points: 20–24 weeks and 1 year of age. Additionally, we measured RVSP and vascular remodeling in TASK-1−/− and wild-type mice between 13 and 16 weeks of age exposed to 10% hypoxia for 3 weeks followed by recovery to room air conditions for an additional 6 weeks.ResultsRVSP was similar between WT and TASK−/− mice. Male and female WT and TASK-1−/− mice all demonstrated age-related increases in RVSP, which correlated to age-related vascular remodeling in male mice but not in female mice. Male TASK-1−/− and WT mice exposed to chronic hypoxia demonstrated increased RVSP, which decreased following room air recovery. WT and TASK-1−/− male mice demonstrated vascular remodeling upon exposure to hypoxia that persisted in room air recovery.ConclusionFemale and male TASK-1−/− mice do not develop hemodynamic or vascular evidence for PH, but RVSP rises in an age-dependent manner independent of genotype. TASK-1−/− and WT male mice develop hypoxia-induced elevations in RVSP that decrease to baseline after recovery in room air. TASK-1−/− and WT male mice demonstrate vascular remodeling after exposure to hypoxia that persists despite recovery to room air conditions and does not correlate with RVSP normalization.

Framingham Heart Study suggests that dysfunction of steroid receptor coactivator-1 may be involved in the development of hypertension. However, there is no functional evidence linking steroid receptor coactivator-1 to the regulation of blood pressure. We used immunohistochemistry to map the expression of steroid receptor coactivator-1 protein in mouse brain, especially in regions implicated in the regulation of blood pressure. Steroid receptor coactivator-1 protein was found in central amygdala, medial amygdala, supraoptic nucleus, arcuate nucleus, ventromedial, dorsomedial, paraventricular hypothalamus, and nucleus of the solitary tract. To determine the effects of steroid receptor coactivator-1 protein on cardiovascular system we measured blood pressures, blood flow velocities, echocardiographic parameters, and aortic input impedance in female steroid receptor coactivator-1 knockout mice and their wild type littermates. Steroid receptor coactivator-1 knockout mice had higher blood pressures and increased aortic stiffness when compared to female wild type littermates. Additionally, the hearts of steroid receptor coactivator-1 knockout mice seem to consume higher energy as evidenced by increased impedance and higher heart rate pressure product when compared to female wild type littermates. Our results demonstrate that steroid receptor coactivator-1 may be functionally involved in the regulation of blood pressure and aortic stiffness through the regulation of sympathetic activation in various neuronal populations.

Atrial fibrillation (AF) is the most frequently encountered clinical arrhythmia and is associated with increased morbidity and mortality. Ectopic activity and reentry are considered major arrhythmogenic mechanisms contributing to the initiation and maintenance of AF. In addition, AF is self-reinforcing through progressive electrical and structural remodeling which stabilize the arrhythmia and make it more difficult to treat. Recent research has suggested an important role for Ca2+-dysregulation in AF. Ca2+-handling abnormalities may promote ectopic activity, conduction abnormalities facilitating reentry, and AF-related remodeling. In this review article, we summarize the Ca2+-handling derangements occurring in AF and discuss their impact on fundamental arrhythmogenic mechanisms. We focus in particular on the role of the multifunctional Ca2+/calmodulin-dependent protein kinase type-II (CaMKII), which acts as a major link between Ca2+-dysregulation and arrhythmogenesis. CaMKII expression and activity are increased in AF and promote arrhythmogenesis through phosphorylation of various targets involved in cardiac electrophysiology and excitation-contraction coupling. We discuss the implications for potential novel therapeutic strategies for AF based on CaMKII and Ca2+-handling abnormalities.

Increasing evidence suggests that microRNAs are intimately involved in the pathophysiology of heart failure. MicroRNA-22 (miR-22) is a muscle-enriched miRNA required for optimum cardiac gene transcription and adaptation to hemodynamic stress by pressure overload in mice. Recent evidence also suggests that miR-22 induces hypertrophic growth and it is oftentimes upregulated in end stage heart failure. However the scope of mRNA targets and networks of miR-22 in the heart failure remained unclear. We analyzed transgenic mice with enhanced levels of miR-22 expression in adult cardiomyocytes to identify important pathophysiologic targets of miR-22. Our data shows that forced expression of miR-22 induces a pro-hypertrophic gene expression program, and it elicits contractile dysfunction leading to cardiac dilation and heart failure. Increased expression of miR-22 impairs the Ca(2+) transient, Ca(2+) loading into the sarcoplasmic reticulum plus it interferes with transcription of estrogen related receptor (ERR) and PPAR downstream genes. Mechanistically, miR-22 postranscriptionally inhibits peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), PPARα and sirtuin 1 (SIRT1) expression via a synergistic circuit, which may account for deleterious actions of unchecked miR-22 expression on the heart.

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca(2+)-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) of β cells. This elevation in [Ca(2+)]cyt leads to activation of Ca(2+)/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca(2+)-release channel implicated in Ca(2+)-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca(2+) leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca(2+)]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca(2+) pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.

We have previously shown increased cardiac stanniocalcin-1 (STC1) in patients with idiopathic dilated cardiomyopathy. STC1 localizes to the inner mitochondrial membrane and transgenic over-expression of STC1 is associated with increased energy utilization.We examined the hypothesis that STC1 uncouples mitochondrial oxidative phosphorylation--to suppress superoxide generation and modulate neurohormonal effects on cardiomyocytes.Compared to WT mouse heart, STC1 Tg heart displays: 2-fold higher uncoupling protein 3 (UCP3) levels, but no effect on UCP2 protein; 40% lower ATP levels; but similar activities of respiratory chain complexes I-IV. In cultured adult rat and freshly-isolated mouse cardiomyocytes, rSTC1 induces UCP3, but not UCP2. Treatment of cardiomyocytes with STC1 decreases mitochondrial membrane potential and suppresses baseline and angiotensin II (Ang II)-induced superoxide generation. Furthermore, baseline superoxide generation is higher in freshly-isolated adult UCP3(-/-) mouse cardiomyocytes compared to WT, suggesting an important role for UCP3 in regulating cardiomyocyte ROS under physiologic conditions. Treatment of UCP3(-/-) cardiomyocytes with rSTC1 failed to suppress superoxide generation, suggesting that the effects of STC1 on superoxide generation in cardiomyocytes are UCP3-dependent.STC1 activates a novel anti-oxidant pathway in cardiac myocytes through induction of UCP3, and may play an important role in suppressing ROS in the heart under normal physiologic conditions and ameliorate the deleterious effects of Ang II-mediated cardiac injury. Importantly, our data point to a critical role for the mitochondria in regulating ROS generation in response to Ang II.