Nieves Gomez-Hurtado

Essential Role of Calmodulin in RyR Inhibition by Dantrolene

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca2+ release. Although dantrolene inhibits Ca2+ release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca2+ release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca2+] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca2+ release in the absence of CaM, but reduced Ca2+ wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin Variants in Long QT Syndrome and Functional Characterization of a Novel Long QT Syndrome-Associated Calmodulin Missense Variant, E141G.

Background—Calmodulin (CaM) is encoded by 3 genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca2+ and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results—Thirty-eight genetically elusive LQTS cases underwent whole-exome sequencing to identify CaM variants. Nonsynonymous CaM variants were over-represented significantly in this heretofore LQTS cohort (13.2%) compared with exome aggregation consortium (0.04%; P<0.0001). When the clinical sequelae of these 5 CaM-positive cases were compared with the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 10 months, an average corrected QT interval of 676 ms, and a high prevalence of cardiac arrest. Functional characterization of 1 novel variant, E141G-CaM, revealed an 11-fold reduction in Ca2+-binding affinity and a functionally dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions—Overall, 13% of our genetically elusive LQTS cohort harbored nonsynonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history.

Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin (CaM) Variants in Long QT Syndrome (LQTS) and Functional Characterization of a Novel LQTS-Associated CaM Missense Variant, E141G

Background—Calmodulin (CaM) is encoded by 3 genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca2+ and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results—Thirty-eight genetically elusive LQTS cases underwent whole-exome sequencing to identify CaM variants. Nonsynonymous CaM variants were over-represented significantly in this heretofore LQTS cohort (13.2%) compared with exome aggregation consortium (0.04%; P<0.0001). When the clinical sequelae of these 5 CaM-positive cases were compared with the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 10 months, an average corrected QT interval of 676 ms, and a high prevalence of cardiac arrest. Functional characterization of 1 novel variant, E141G-CaM, revealed an 11-fold reduction in Ca2+-binding affinity and a functionally dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions—Overall, 13% of our genetically elusive LQTS cohort harbored nonsynonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history.

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Rationale: Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation–contraction coupling, and inefficient Ca-induced Ca release remain major limitations. Objective: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation–contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). Methods and Results: hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation–contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. Conclusions: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation–contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

DIOL Triterpenes Block Profibrotic Effects of Angiotensin II and Protect from Cardiac Hypertrophy

Background The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied. Methodology/Principal Findings The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals. Conclusions/Significance Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ.

Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks

Background— Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). CaM mutations are found in 13% of genotype-negative long QT syndrome patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically diagnosed CPVT. Methods and Results— We performed mutational analysis of CALM1, CALM2, and CALM3 gene-coding regions, in vitro measurement of CaM-Ca2+ (Ca)-binding affinity, ryanodine receptor 2–CaM binding, Ca handling, L-type Ca current, and action potential duration. We identified a novel CaM mutation—A103V—in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction versus WT-CaM), but did not alter CaM binding to ryanodine receptor 2. In permeabilized cardiomyocytes, A103V-CaM (100 nmol/L) promoted spontaneous Ca wave and spark activity, a cellular phenotype of ryanodine receptor 2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared with long QT syndrome D96V-CaM, A103V-CaM had significantly less effects on L-type Ca current inactivation, did not alter action potential duration, and caused delayed afterdepolarizations and triggered beats in intact cardiomyocytes. Conclusions— We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via ryanodine receptor 2 dysregulation, which explains the autosomal dominant inheritance.

Prolonged leptin treatment increases transient outward K + current via upregulation of Kv4.2 and Kv4.3 channel subunits in adult rat ventricular myocytes

Circulating leptin levels are elevated in obesity and hyperleptinaemia has been postulated to be an independent risk factor for the development of cardiovascular diseases. Although many studies have been published on the mechanisms involved in the effects of leptin on cardiac function and pathological remodeling, scarce information is currently available analyzing the influence of prolonged leptin treatment on ionic cardiac channels remodeling in adult ventricular myocytes. Enzymatically isolated adult rat ventricular myocytes were treated with leptin or vehicle for 48h. Real-Time RT-PCR were used to analyze mRNA expression of Kir2.1, Cav1.2, Cav 3.1, Kv4.2 and Kv4.3 α-subunits and KChIP2 auxiliary subunit. The fast transient outward potassium channels (Itof) α-subunits Kv4.2, Kv4.3 and KChIP2 were analyzed by Western-blot. The fast transient outward potassium current and the action potentials were recorded in isolated myocytes by the whole-cell patch-clamp technique. Leptin treatment induced an up-regulation of Kv4.2, Kv4.3 and KChIP2 subunits mRNA expression. However, transcriptional levels of Kir2.1, Cav1.2, or Cav3.1 α-subunit channels were unmodified by leptin. Protein expression levels of Kv4.2, Kv4.3 and KChIP2 subunits were also increased by leptin. The electrophysiological study showed that leptin increases the fast transient outward potassium current amplitudes and densities shortening action potential duration. In addition, leptin activated Akt signaling in cardiomyocytes and this mechanism was involved in the effect of leptin on Itof channels. In conclusión, leptin increases both the expression and function of Itof channels in adult ventricular myocytes and this mechanism involves Akt signaling. Altogether these data suggest that leptin could exert beneficial or detrimental effects depending on the initial ventricular myocyte repolarizing reserve.

Role of NOD1 in Heart Failure Progression via Regulation of Ca2+ Handling

BACKGROUND Heart failure (HF) is a complex syndrome associated with a maladaptive innate immune system response that leads to deleterious cardiac remodeling. However, the underlying mechanisms of this syndrome are poorly understood. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a newly recognized innate immune sensor involved in cardiovascular diseases. OBJECTIVES This study evaluated the role of NOD1 in HF progression. METHODS NOD1 was examined in human failing myocardium and in a post-myocardial infarction (PMI) HF model evaluated in wild-type (wt-PMI) and Nod1-/- mice (Nod1-/--PMI). RESULTS The NOD1 pathway was up-regulated in human and murine failing myocardia. Compared with wt-PMI, hearts from Nod1-/--PMI mice had better cardiac function and attenuated structural remodeling. Ameliorated cardiac function in Nod1-/--PMI mice was associated with prevention of Ca2+ dynamic impairment linked to HF, including smaller and longer intracellular Ca2+ concentration transients and a lesser sarcoplasmic reticulum Ca2+ load due to a down-regulation of the sarcoplasmic reticulum Ca2+-adenosine triphosphatase pump and by augmented levels of the Na+/Ca2+ exchanger. Increased diastolic Ca2+ release in wt-PMI cardiomyocytes was related to hyperphosphorylation of ryanodine receptors, which was blunted in Nod1-/--PMI cardiomyocytes. Pharmacological blockade of NOD1 also prevented Ca2+ mishandling in wt-PMI mice. Nod1-/--PMI mice showed significantly fewer ventricular arrhythmias and lower mortality after isoproterenol administration. These effects were associated with lower aberrant systolic Ca2+ release and with a prevention of the hyperphosphorylation of ryanodine receptors under isoproterenol administration in Nod1-/--PMI mice. CONCLUSIONS NOD1 modulated intracellular Ca2+ mishandling in HF, emerging as a new target for HF therapy.

Beneficial effects of leptin treatment in a setting of cardiac dysfunction induced by transverse aortic constriction in mouse

Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart. Controversy exists regarding the adverse and beneficial effects of leptin on the heart We analysed the effect of a non‐hypertensive dose of leptin on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction. We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype. These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca2+ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca2+ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca2+ handling parameters.

Remodeling of energy metabolism and absence of electrophysiological changes in the heart of obese hyperleptinemic mice. New insights into the pleiotropic role of leptin.

Dietary treatment with high-fat diets (HFD) triggers diabetes and hyperleptinemia, concomitantly with a partial state of leptin resistance that affects hepatic and adipose tissue but not the heart. In this context, characterized by widespread steatosis, cardiac lipid content remains unchanged. As previously reported, HFD-evoked hyperleptinemia could be a pivotal element contributing to increase fatty-acid (FA) metabolism in the heart and to prevent cardiac steatosis. This metabolic adaptation might theoretically reduce energy efficiency in cardiomyocytes and lead to cardiac electrophysiological remodeling. Therefore the aim of the current study has been to investigate the impact of long-term HFD on cardiac metabolism and electrophysiological properties of the principal ionic currents responsible of the action potential duration in mouse cardiomyocytes. Male C57BL/6J mice were fed a control (10 kcal% from fat) or HFD (45 kcal% from fat) during 32 weeks. Quantification of enzymatic activities regulating mitochondrial uptake of pyruvate and FA showed an increase of both carnitine-palmitoyltransferase and citrate synthase activities together with a decrease of lactate dehydrogenase and pyruvate dehydrogenase activities. Increased expression of uncoupling protein-3, Mn-, and Cu/Zn-superoxide dismutases and catalase were also detected. Total glutathione/oxidized glutathione ratios were unaffected by HFD. These data suggest that HFD triggers adaptive mechanisms aimed at (i) facilitating FA catabolism, and (ii) preventing oxidative stress. All these changes did not affect the duration of action potentials in cardiomyocytes and only slightly modified electrocardiographic parameters.