Diastolic Calcium Levels Research Articles

Studying Pathogenetic Contribution of a Variant of Unknown Significance, p.M659I (c.1977G > A) in MYH7, to the Development of Hypertrophic Cardiomyopathy Using CRISPR/Cas9-Engineered Isogenic Induced Pluripotent Stem Cells.

Hypertrophic cardiomyopathy (HCM) is a cardiovascular pathology that is caused by variants in genes encoding sarcomere-associated proteins. However, the clinical significance of numerous variants in HCM-associated genes is still unknown. CRISPR/Cas9 is a tool of nucleotide sequence editing that allows for the unraveling of different biological tasks. In this study, introducing a mutation with CRISPR/Cas9 into induced pluripotent stem cells (iPSCs) of a healthy donor and the directed differentiation of the isogenic iPSC lines into cardiomyocytes were used to assess the pathogenicity of a variant of unknown significance, p.M659I (c.1977G > A) in MYH7, which was found previously in an HCM patient. Using two single-stranded donor oligonucleotides with and without the p.M659I (c.1977G > A) mutation, together with CRISPR/Cas9, an iPSC line heterozygous at the p.M659I (c.1977G > A) variant in MYH7 was generated. No CRISPR/Cas9 off-target activity was observed. The iPSC line with the introduced p.M659I (c.1977G > A) mutation in MYH7 retained its pluripotent state and normal karyotype. Compared to the isogenic control, cardiomyocytes derived from the iPSCs with the introduced p.M659I (c.1977G > A) mutation in MYH7 recapitulated known HCM features: enlarged size, elevated diastolic calcium level, changes in the expression of HCM-related genes, and disrupted energy metabolism. These findings indicate the pathogenicity of the variant.

Dock10 Regulates Cardiac Function under Neurohormonal Stress.

Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the α-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte.

Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae.

In vivo models of cardiac function maintain the complex relationship of cardiomyocytes with other heart cells, as well as the paracrine and mechanoelectrical feedback mechanisms. We aimed at imaging calcium transients simultaneously with heart contraction in zebrafish larvae.Methods: To image calcium in beating hearts, we generated a zebrafish transgenic line expressing the FRET-based ratiometric biosensor Twitch-4. Since emission ratioing canceled out the motion artifacts, we did not use myosin inhibitors or tnnt2a morpholinos to uncouple contraction from changes in calcium levels. We wrote an analysis program to automatically calculate kinetic parameters of the calcium transients. In addition, the ventricular diameter was determined in the fluorescence images providing a real-time measurement of contraction correlated with calcium.Results: Expression of Twitch-4 did not affect the force of contraction, the size of the heart nor the heart rate in 3- and 5-days post-fertilization (dpf) larvae. Comparison of 3 and 5 dpf larvae showed that calcium levels and transient amplitude were larger at 5 dpf, but the fractional shortening did not change. To validate the model, we evaluated the effect of drugs with known effects on cardiomyocytes. Calcium levels and the force of contraction decreased by the L-type calcium channel blocker nifedipine, whereas they increased with the activator Bay-K 8644. Caffeine induced bradycardia, markedly decreased ventricular diastolic calcium levels, increased the size of the calcium transients, and caused an escape rhythm in some larvae.Conclusions: The Tg(myl7:Twitch-4) line provides a physiological approach to image systolic and diastolic calcium levels in the heart of zebrafish larvae. Since the heart is beating, calcium levels and contraction can be correlated. This line will be a useful tool to address pathophysiological mechanisms in diseases like heart failure and arrhythmia, in cardiotoxicity studies and for drug screening.

The α2-isoform of the Na+/K+-ATPase protects against pathological remodeling and β-adrenergic desensitization after myocardial infarction.

The role of the Na+/K+-ATPase (NKA) in heart failure associated with myocardial infarction (MI) is poorly understood. The elucidation of its precise function is hampered by the existence of two catalytic NKA isoforms (NKA-α1 and NKA-α2). Our aim was to analyze the effects of an increased NKA-α2 expression on functional deterioration and remodeling during long-term MI treatment in mice and its impact on Ca2+ handling and inotropy of the failing heart. Wild-type (WT) and NKA-α2 transgenic (TG) mice (TG-α2) with a cardiac-specific overexpression of NKA-α2 were subjected to MI injury for 8 wk. As examined by echocardiography, gravimetry, and histology, TG-α2 mice were protected from functional deterioration and adverse cardiac remodeling. Contractility and Ca2+ transients (Fura 2-AM) in cardiomyocytes from MI-treated TG-α2 animals showed reduced Ca2+ amplitudes during pacing or after caffeine application. Ca2+ efflux in cardiomyocytes from TG-α2 mice was accelerated and diastolic Ca2+ levels were decreased. Based on these alterations, sarcomeres exhibited an enhanced sensitization and thus increased contractility. After the acute stimulation with the β-adrenergic agonist isoproterenol (ISO), cardiomyocytes from MI-treated TG-α2 mice responded with increased sarcomere shortenings and Ca2+ peak amplitudes. This positive inotropic response was absent in cardiomyocytes from WT-MI animals. Cardiomyocytes with NKA-α2 as predominant isoform minimize Ca2+ cycling but respond to β-adrenergic stimulation more efficiently during chronic cardiac stress. These mechanisms might improve the β-adrenergic reserve and contribute to functional preservation in heart failure.NEW & NOTEWORTHY Reduced systolic and diastolic calcium levels in cardiomyocytes from NKA-α2 transgenic mice minimize the desensitization of the β-adrenergic signaling system. These effects result in an improved β-adrenergic reserve and prevent functional deterioration and cardiac remodeling.

Molecular- and Cellular-Level Effects of a Troponin Mutation Linked to Dilated Cardiomyopathy

Familial cardiomyopathies are the leading cause of sudden cardiac death among young people, and pediatric-onset disease is particularly devastating. Dilated cardiomyopathy (DCM) is characterized by dilation of the left ventricular chamber and impaired cardiac contractility. DCM can be caused by mutations in proteins that regulate cardiac muscle contraction, including troponin T; however, it is not well-understood how mutation-induced changes in protein biophysics affect cardiac contraction and contribute to the disease phenotype. By understanding the biophysical underpinnings of specific mutations, it might be possible to develop targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. Here, we examined the molecular- and cellular-level impacts of a troponin T mutation implicated in pediatric-onset DCM, R134G. In vitro motility assays revealed decreased calcium sensitivity with the R134G mutant, which would be expected to reduce force production in cardiomyocytes. To probe the molecular mechanism underlying this change in sensitivity, we performed stopped-flow and steady-state fluorescence measurements to determine the equilibrium constants that define thin filament activation. Unexpectedly, these biochemical experiments revealed increased activation of thin filaments containing R134G troponin T at diastolic calcium levels, in contrast to the frequently cited hypothesis that DCM is caused by molecular hypocontractility. We also demonstrate that R134G decreases the cooperativity of myosin binding to regulated thin filaments, which could be responsible for the shift in calcium sensitivity. Consistent with the molecular studies, cardiomyocytes carrying the R134G mutation were hypocontractile; however, they also showed hallmarks of DCM that lie downstream of the initial molecular insult, including disorganized sarcomeres, cellular hypertrophy, and expression of DCM-associated genes. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and suggest the value of precision medicine approaches for the treatment of DCM.

Phosphorylation of cardiac myosin–binding protein-C contributes to calcium homeostasis

Cardiac myosin-binding protein-C (cMyBP-C) is highly phosphorylated under basal conditions. However, its phosphorylation level is decreased in individuals with heart failure. The necessity of cMyBP-C phosphorylation for proper contractile function is well-established, but the physiological and pathological consequences of decreased cMyBP-C phosphorylation in the heart are not clear. Herein, using intact adult cardiomyocytes from mouse models expressing phospho-ablated (AAA) and phosphomimetic (DDD) cMyBP-C as well as controls, we found that cMyBP-C dephosphorylation is sufficient to reduce contractile parameters and calcium kinetics associated with prolonged decay time of the calcium transient and increased diastolic calcium levels. Isoproterenol stimulation reversed the depressive contractile and Ca2+-kinetic parameters. Moreover, caffeine-induced calcium release yielded no difference between AAA/DDD and controls in calcium content of the sarcoplasmic reticulum. On the other hand, sodium-calcium exchanger function and phosphorylation levels of calcium-handling proteins were significantly decreased in AAA hearts compared with controls. Stress conditions caused increases in both spontaneous aftercontractions in AAA cardiomyocytes and the incidence of arrhythmias in vivo compared with the controls. Treatment with omecamtiv mecarbil, a positive cardiac inotropic drug, rescued the contractile deficit in AAA cardiomyocytes, but not the calcium-handling abnormalities. These findings indicate a cascade effect whereby cMyBP-C dephosphorylation causes contractile defects, which then lead to calcium-cycling abnormalities, resulting in aftercontractions and increased incidence of cardiac arrhythmias under stress conditions. We conclude that improvement of contractile deficits alone without improving calcium handling may be insufficient for effective management of heart failure.

Examination of the Changes in Calcium Homeostasis in the Delayed Antiarrhythmic Effect of Sodium Nitrite.

We have evidence that the intravenous infusion of sodium nitrite (NaNO2) results in an antiarrhythmic effect when given 24 h prior to an ischemia and reperfusion (I/R) insult in anaesthetized dogs. This protection was associated with the reduction of reactive oxygen species resulting from I/R through the attenuation of mitochondrial respiration. Here, we examined whether the changes in calcium, which also contributes to arrhythmia generation, play a role in the NaNO2-induced effect. On the first day, 30 anaesthetized dogs were treated either with saline or NaNO2 (0.2 µmol/kg/min) for 20 min. Some animals were subjected to a 25 min LAD (anterior descending branch of the left coronary artery) occlusion and 2 min reperfusion (I/R = 4; NaNO2-I/R = 6), or the heart was removed 24 h later. We have shown that nitrite prevented the I/R-induced increase in cellular and mitochondrial calcium deposits. During simulated I/R, the amplitude of the calcium transient and the diastolic calcium level were significantly lower in the nitrite-treated hearts and the ERP (effective refractory period) fraction of the action potential was significantly increased. Furthermore, nitrite also enhanced the mitochondrial respiratory response and prevented the MPTPT opening during calcium overload. These results suggest that nitrite can reduce the harmful consequences of calcium overload, perhaps directly by modulating ion channels or indirectly by reducing the mitochondrial ROS (reactive oxygen species) production.

Abstract 334: Cardiac Myosin Binding Protein C Phosphorylation Regulates Calcium Homeostasis

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is heavily phosphorylated to regulate normal cardiac function under basal conditions. However, its phosphorylation level is significantly decreased in patients with heart failure and atrial fibrillation. Furthermore, decreased cMyBP-C phosphorylation causes reduced myofilament contractility and decreased calcium sensitivity. The impact of such decreases in cMyBP-C phosphorylation and myofilament calcium sensitivity on the overall calcium handling and potential induction of arrhythmogenesis is not known. Objective: To determine the necessity and sufficiency of cMyBP-C phosphorylation to regulate calcium cycling and contractility at the isolated cardiomyocyte level. Methods and Results: Contractile properties and calcium kinetics were measured in intact cardiomyocytes isolated from 3-month-old, mixed sex, mice expressing phospho-ablated (S273A/S282A/S302A) cMyBP-C (AAA) or phospho-mimetic (S273D/S282D/S302D) cMyBP-C (DDD) and nontransgenic (NTG) control mice. AAA cells displayed a significant decrease in fractional shortening compared to NTG and DDD cells (9.4% vs 12.8% in NTG, and 12.3% in DDD, p<0.0001). Similarly, AAA myocytes demonstrated abnormal calcium kinetics with increased diastolic calcium levels (20%, p<0.001 vs . NTG and DDD) and prolonged decay time of the calcium transient (26%, p<0.01 vs NTG and DDD) when compared with NTG and DDD myocytes. Caffeine-induced calcium release in AAA myocytes indicated no change in SR calcium content, while sodium-calcium exchanger function, assessed as the time constant (τau) of calcium decline, was increased (89%, p<0.01 vs NTG). However, these depressive effects in AAA myocytes were relieved by isoproterenol (100 nmol/L) stimulation. Furthermore, stress conditions (2 Hz + ISO) increased after-contractions in AAA cardiomyocytes (60% in AAA vs 13% in NTG, p<0.001 and 15% in DDD, p<0.001). In addition, preliminary data indicate increased susceptibility of the AAA mice to arrhythmias under stress conditions in vivo. Conclusion: Dephosphorylation of cMyBP-C is sufficient to reduce sarcomere contractility and impair calcium cycling resulting in spontaneous after-contractions and arrhythmias under stress conditions.

A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin-binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Isogenic iPSC lines were generated from HCM patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing. Comprehensive phenotypic and transcriptome analyses were performed in the iPSC-CMs. We observed aberrant calcium handling properties with prolonged decay kinetics and elevated diastolic calcium levels in the absence of structural abnormalities or contracile dysfunction in HCM iPSC-CMs as compared to isogenic controls. The mRNA expression levels of MYBPC3 were significantly reduced in mutant iPSC-CMs, but the protein levels were comparable among isogenic iPSC-CMs, suggesting that haploinsufficiency of MYBPC3 does not contribute to the pathogenesis of HCM in vitro. Furthermore, truncated MYBPC3 peptides were not detected. At the molecular level, the nonsense-mediated decay pathway was activated, and a set of genes involved in major cardiac signaling pathways was dysregulated in HCM iPSC-CMs, indicating an HCM gene signature in vitro. Specific inhibition of the nonsense-mediated decay pathway in mutant iPSC-CMs resulted in reversal of the molecular phenotype and normalization of calcium-handling abnormalities. iPSC-CMs carrying MYBPC3 PTC mutations displayed aberrant calcium signaling and molecular dysregulations in the absence of significant haploinsufficiency of MYBPC3 protein. Here we provided the first evidence of the direct connection between the chronically activated nonsense-mediated decay pathway and HCM disease development.

Empagliflozin directly improves diastolic function in human heart failure.

Empagliflozin, a clinically used oral antidiabetic drug that inhibits the sodium-dependent glucose co-transporter 2, has recently been evaluated for its cardiovascular safety. Surprisingly, empagliflozin reduced mortality and hospitalization for heart failure (HF) compared to placebo. However, the underlying mechanisms remain unclear. Therefore, our study aims to investigate whether empagliflozin may cause direct pleiotropic effects on the myocardium. In order to assess possible direct myocardial effects of empagliflozin, we performed contractility experiments with in toto-isolated human systolic end-stage HF ventricular trabeculae. Empagliflozin significantly reduced diastolic tension, whereas systolic force was not changed. These results were confirmed in murine myocardium from diabetic and non-diabetic mice, suggesting independent effects from diabetic conditions. In human HF cardiomyocytes, empagliflozin did not influence calcium transient amplitude or diastolic calcium level. The mechanisms underlying the improved diastolic function were further elucidated by studying myocardial fibres from patients and rats with diastolic HF (HF with preserved ejection fraction, HFpEF). Empagliflozin beneficially reduced myofilament passive stiffness by enhancing phosphorylation levels of myofilament regulatory proteins. Intravenous injection of empagliflozin in anaesthetized HFpEF rats significantly improved diastolic function measured by echocardiography, while systolic contractility was unaffected. Empagliflozin causes direct pleiotropic effects on the myocardium by improving diastolic stiffness and hence diastolic function. These effects were independent of diabetic conditions. Since pharmacological therapy of diastolic dysfunction and HF is an unmet need, our results provide a rationale for new translational studies and might also contribute to the understanding of the EMPA-REG OUTCOME trial.

Abstract 383: Modulating Calcium Dysregulation in Troponin T Linked Hypertrophic Cardiomyopathy

Mutations in cardiac troponin T (cTnT) account for approximately 5-10% of all Hypertrophic Cardiomyopathy (HCM). Residue Arg92 of cTnT represents a HCM-mutational hotspot, including Arg92Leu (R92L) and Arg92Trp (R92W). These mutations, although differing by only a single amino acid, present with varying degrees of cardiac remodeling and disease severity in the human population. Studying the differential effects of the R92 mutations on Ca 2+ homeostasis revealed an age-dependent disruption in down-stream calcium handling in R92W mice, including differential diastolic calcium levels and SERCA2a uptake activity. To determine the effects of treating this differential calcium handling disruption, R92L and R92W mice were treated with diltiazem hydrochloride, an L-type Ca 2+ channel blocker. While R92L mice showed a progressive worsening in diastolic function and remodeling throughout treatment, R92W mice showed improvement in diastolic function but no change in cardiac remodeling, suggesting an improvement but not amelioration of disease with diltiazem treatment. Of note, previous studies also suggested an increase in phospholamban (PLB) phosphorylation at Thr17 (CaMK-II-mediated) over time in R92W mice only. Thus, we hypothesized a mutation-specific role of CaMK-II in cTnT-linked HCM progression. We tested this via genetic partial inhibition of CaMK-II (AC3-I peptide) in both R92W and R92L transgenic mice. Both R92W and AC3-IxRW hearts exhibited a normalization of SERCA2a activity to non-transgenic levels over time. This improved Ca 2+ re-uptake was coupled with a decrease in atrial mass, improved ventricular relaxation, and normalized diastolic function only in AC3-IxRW animals, consistent with blunting of disease progression in vivo . In contrast, AC3-IxRL mice showed depressed SERCA2a activity, increased atrial mass and ventricular thickening, and diastolic dysfunction as compared to R92L alone mice, indicating a CaMK-II independent progression to HCM. These results suggest that closely related primary mutations can result in specific ventricular remodeling and targeting these discrete molecular secondary effects represent a novel targeted treatment approach in HCM.

Abstract 308: An in vitro Model of Cardiac Stem Cell Therapy to Study the Coupling of Primary and Stem Cell-derived Cardiomyocytes

For cardiac cell therapy to be effective, newly formed immature cardiomyocytes need to structurally and functionally integrate with the existing myocardium. Unfortunately, testing the electro-chemo-mechanical coupling of mature and immature cardiomyocytes in vivo is difficult. Here we engineered two cell μtissues containing combinations of mouse neonate, ES-derived, and iPS-derived cardiac myocytes on flexible substrates and utilized ratiometric calcium detection and traction force microscopy to measure excitation-contraction coupling in individual cells and in the pairs. We found that SC-derived cardiac myocytes can structurally couple with neonate cardiomyocytes to functionally support synchronous contraction, yet diastolic calcium levels were reduced in SC-derived cardiomyocytes. Consistently, neonate cardiomyocytes exerted peak systolic forces that were ~1.5-fold higher than that generated by SC-derived myocytes, yielding an imbalance in tension within the pair that was dissipated by focal adhesion-like structures at the cell-cell boundary. Finally we developed a finite element model of two-cell tissue contraction to demonstrate that an imbalance in isometric tension is sufficient to limit force transmission across cell-cell boundaries. Taken together, these results suggest that reduced force transmission between poorly coupled immature and native cardiomyocytes may explain the incomplete repair of ejection fraction observed in several clinical studies of cardiac cell therapy.

Abnormal calcium homeostasis in heart failure with preserved ejection fraction is related to both reduced contractile function and incomplete relaxation: an electromechanically detailed biophysical modeling study.

Heart failure with preserved ejection fraction (HFpEF) accounts for about 50% of heart failure cases. It has features of incomplete relaxation and increased stiffness of the left ventricle. Studies from clinical electrophysiology and animal experiments have found that HFpEF is associated with impaired calcium homeostasis, ion channel remodeling and concentric left ventricle hypertrophy (LVH). However, it is still unclear how the abnormal calcium homeostasis, ion channel and structural remodeling affect the electro-mechanical dynamics of the ventricles. In this study we have developed multiscale models of the human left ventricle from single cells to the 3D organ, which take into consideration HFpEF-induced changes in calcium handling, ion channel remodeling and concentric LVH. Our simulation results suggest that at the cellular level, HFpEF reduces the systolic calcium level resulting in a reduced systolic contractile force, but elevates the diastolic calcium level resulting in an abnormal residual diastolic force. In our simulations, these abnormal electro-mechanical features of the ventricular cells became more pronounced with the increase of the heart rate. However, at the 3D organ level, the ejection fraction of the left ventricle was maintained due to the concentric LVH. The simulation results of this study mirror clinically observed features of HFpEF and provide new insights toward the understanding of the cellular bases of impaired cardiac electromechanical functions in heart failure.

Delayed AV-conduction in pressure-overloaded Scn5a-1798insD+/- mice rescued by genetic deletion of the calcium-dependent transcription factor Nfatc2

Introduction: Transgenic Scn5a-1798insD+/- mice display atrio-ventricular conduction disturbances and develop susceptibility to arrhythmias with age. Isolated cardiomyocytes from Scn5a-1798insD+/- mouse hearts exhibit increased sustained inward sodium current which is associated with elevated intracellular sodium (Na+), intracellular diastolic calcium ([Ca2+]i) levels and Ca2+ transient amplitudes. We investigated whether the Scn5a-1798insD+/- mutation leads to activation of Ca2+-dependent calcineurin/Nfat signaling pathway, and assessed its impact on atrio-ventricular conduction. Results: Despite increased [Ca2+]i, activation of the calcineurin/Nfat pathway was not observed in Scn5a-1798insD+/- hearts at baseline (as evidenced by unaltered nuclear levels of Nfat protein as compared to wild type hearts). Wild-type and Scn5a-1798insD+/- mice displayed a similar hypertrophic response to 2 weeks of TAC (as evidenced by similar increases in heart weight and Anf mRNA levels), indicating equal activation of the calcineurin/Nfat pathway. However, Scn5a-1798insD+/–TAC mice displayed increased incidence of sudden death preceded by progressive bradycardia and AV-block. Furthermore, isolated Scn5a-1798insD+/–TAC hearts showed AV-block and significantly prolonged AV-delay as compared to wild-type-TAC hearts. Since Nfatc2 is a well-described downstream effector of the calcineurin pathway, we next investigated whether the observed electrical abnormalities after TAC could be rescued by crossing Scn5a-1798insD+/- with Nfatc2-KO mice. Scn5a-1798insD+/-/Nfatc2-KO mice showed a lower response to TAC as Scn5a-1798insD+/–TAC mice (as evidenced by lower increase in heart weight and Anf mRNA levels). The ex vivo AV-delay after TAC was attenuated in Scn5a-1798insD+/-/Nfatc2-KO TAC hearts, indicating rescue of the AV-conduction disturbances in Scn5a-1798insD+/–TAC mice by Nfatc2 deficiency. Moreover, His-ventricular conduction was unaffected by TAC, indicating that the increase in AV-delay in Scn5a-1798insD+/–TAC mice occurs proximal to the His bundle. Conclusions: Scn5a-1798insD+/- mice show intracellular Na+/Ca2+-dysregulation at baseline and severe AV-conduction abnormalities and sudden death upon increased activation of the calcineurin/Nfat-pathway by TAC challenge. Nfatc2 deficiency prevents these electrical disturbances secondary to TAC in Scn5a-1798insD+/-/Nfatc2-KO mice, indicating a role of the calcineurin/Nfat pathway on atrio-ventricular conduction.

Abstract 279: Loss Of Rad GTPase Produces A Sympathomimetic Cardiac Phenotype Leading To Calcium Overload And Arrhythmia.

Background: Rad is a small GTPase that regulates inward calcium current in excitable cells. In human heart failure, calcium dysregulation is accompanied by lowered levels of Rad expression, and dampened responsiveness to beta-adrenergic stimulation. However, the effects of Rad ablation on calcium cycling in the heart have yet to be examined. Hypothesis: Loss of Rad emulates beta-adrenergic stimulation in cardiac myocytes, elevating calcium levels, and promoting after depolarizations. Methods: The role of Rad was evaluated using knockout mice (RadKO)at several levels of biological organization. Inward calcium current, action potentials, fractional shortening, and calcium transients were measured in isolated ventricular myocytes. Functional parameters were recorded in the isolated working heart, and telemetry was used to monitor ECGs in intact freely-roaming mice. Results: Isolated ventricular myoctyes from Rad null mice display increased ICaL density, greater inward current at low voltages, and after depolarizations at low frequencies. Isolated cells exhibit increased diastolic calcium levels and increased dynamic calcium changes in transient amplitude during pacing. Further, RadKO cardiomyocytes develop a significantly greater number of spontaneous calcium transients than wildtype cohorts. Ventricular myocytes and intact working hearts from RadKO mice fail to respond to beta-adrenergic stimulation at the level of channel activation, calcium transient kinetics, and exhibit a significantly dampened change in +dP/dt in the isolated heart. Consistent with these effects, PKA substrates are phosphorylated at baseline in RadKO cardiomyocytes, suggesting tonic PKA activation. Conclusion: These data suggest the new provocative hypothesis that beta-adrenergic-mediated changes in excitation, calcium handling, and heart contraction involve relief of Rad-dependent negative regulation of channel function.

Abstract 19085: Cross-bridge Activation at Diastolic Calcium Levels in Human Myocardium

Tropomyosin is positioned on actin in dynamic equilibrium as a function of free calcium concentration. However, it is estimated that ∼5% of the myosin binding sites on actin are exposed at low calcium levels (e.g. pCa 8) raising the possibility of myosin cross-bridge formation at diastolic calcium levels. We tested this hypothesis in detergent-treated (skinned) strips dissected from epicardial biopsies obtained from male patients during coronary bypass grafting. Patients (n=15) had normal left ventricular (LV) ejection fraction and wall motion. Eight control (C) patients had neither hypertension (HTN) nor diabetes mellitus. Seven patients had HTN and concentric remodeling with increased LV wall thickness/radius ratio. Western blot analysis of skinning supernatant revealed no loss of tropomyosin or troponin-I that would implicate loss of normal thin filament regulation during the skinning process. Myofilament calcium sensitivity at sarcomere length 2.2 μm was similar between C and HTN (pCa 50 6.07±0.07 vs 6.06±0.04). Tension at pCa 8 was similar between C and HTN (4.26±0.52 vs 4.30±0.51 mN.mm -2 ), but was significantly reduced by application the cross-bridge inhibitor 2,3-butanedione monoximine (BDM) (2.15±0.24 vs 3.01±0.40 mN.mm -2 , p<0.05). The higher tension in HTN with BDM reflects a greater inherent stiffness in HTN independent of cross-bridges. We then used sinusoidal length perturbations (0.125-250 Hz) to detect the detachment rate of cycling cross-bridges. In both C and HTN strips, frequency-dependent viscoelastic characteristics of cycling cross-bridges were apparent at pCa 8 and abolished with BDM. At pCa 8 cross-bridge detachment rate was significantly greater in C than HTN (106.1±12.5 vs 74.6±9.3 s -1 , p<0.05). These results indicate that there are cycling cross-bridges in normal and concentrically remodeled human myocardium at diastolic calcium levels that contribute to diastolic tension. Furthermore, the rate of cross-bridge detachment under relaxed conditions is slower in concentrically remodeled myocardium from HTN compared with C patients. The presence and lifetime of cross-bridges formed in addition to cross-bridge independent stiffness must be considered as significant contributors to diastolic dysfunction in HTN.

Dissociation Between Force and Calcium after a Step Change in Frequency

The force frequency response (FFR) is one of the major physiological regulators which govern alterations in contractile function in the body in which an increase in frequency of pacing leads to an increase in force production and a decrease in rate of contractile kinetics. Typically studies on FFR have been conducted at steady state where the changes in contractile function have been elicited by changes in myofilament phosphorylation. Here we aim to determine the role of alterations in calcium homeostasis and myofilament sensitization during the transition from one steady state of contractile function to another.Trabeculae, excised from the right ventricular free wall of New Zealand white rabbits were loaded with Rhod-AM calcium indicator dye and stimulated to contract from 1 to 4 Hz. Data was analyzed through customized Labview software.We hypothesize that the transition from one steady state to another occurs in two phases; the rapid/early phase is due to increases in calcium transient amplitude and diastolic calcium levels, and the gradual/latent phase is due to alterations in phosphorylation state of myofilament proteins such as troponin I (TnI) and myosin light chain 2 (MLC2). During our experiments we are able to measure calcium and developed force simultaneously. Despite previous studies which show alterations in myofilament calcium sensitivity occurring at steady state, we are able to show that during the immediate phase calcium and force change with each other, while at the latent phase there is an increase in diastolic calcium level that is not paralleled by the diastolic force. This suggests a myofilament desensitization during this latent phase that is not seen in the early transitory period as originally thought.

Response to the Letter of Hester Den Ruijter and Ruben Coronel Regarding the Article “The Role of n-3 PUFAs in Preventing the Arrhythmic Risk in Patients with Idiopathic Dilated Cardiomyopathy”

We thank Drs Hester M. Den Ruijter and Ruben Coronel for their valuable comments [1]. Consumption of fish or fish oil is associated with lower risk of arrhythmic outcomes including sudden death and atrial fibrillation [2]. However, the mechanisms underlying these relationships are not well established and may include direct or indirect effects on myocardial electrophysiology, which may apply to numerous potential arrhythmogenic substrates (heart failure, acute ischemia, infarct scar and hibernated/ stunned myocardium). Of note, n-3 PUFAs may worsen arrhytmic events based on re-entry circuits activation as in the presence of large infarct scar or hibernated/stunned myocardium, but may also reduce risk of ventricular arrhythmias in implantable cardioverter defibrillator patients without coronary artery disease [3]. Trials evaluating fish oil have shown controversial results, possibly due to different sample size and duration of intake or variable pharmacological doses of fish oil, dietary intake of n-6 fatty acids and concomitant medication [4]. Different arrhythmogenic substrates may be a cause of divergent results, as well. In our patients, with idiopathic dilated cardiomyopathy, the predominant arrhythmogenic substrate is a nonreentrant mechanism that likely involves triggered activity from delayed after depolarizations in response to catecholamines. Recent work by Drs Den Ruijter and Coronel has shown that triggered arrhythmias are inhibited by fish oil in isolated myocytes of patients with end-stage heart failure as a result of cardiac action potential shortening, decreased diastolic and systolic intracellular calcium levels and a reduced response to β-adrenergic stimulation [5]. Probably in heart failure patients also other favorable biological effects of n-3 PUFAs, as we observed in our study (reduction of heart rate, inflammatory pathways and catecholamine plasma levels, increase of heart rate variability, etc), may indirectly contribute to antiarrhythmic result of the fish oil treatment. Certainly, as Dr Raitt has emphasized in his editorial, an improvement in markers of risk may not translate to improvement in outcome and large prospective trials assessing the n-3 PUFA effects on mortality or severe ventricular arrhythmias need to be performed [6]. We agree with Drs Den Ruijter and Coronel and hypothesize that habitual consumption of fish and longchain n-3 fatty acid intake would be associated with more favorable outcomes just in selected populations. According to the available evidence, trial designs in heart failure patients have to take into account not only the different Cardiovasc Drugs Ther (2009) 23:335–336 DOI 10.1007/s10557-009-6174-7

The Response to Fish Oil in Patients with Heart Disease Depends on the Predominant Arrhythmia Mechanism

Dear sir, We have read with interest the paper by Nodari et al [1] and the editorial by Dr. Raitt [2] on fish oil fatty acids and arrhythmic risk in heart failure patients caused by idiopathic dilated cardiomyopathy. In both publications, the mechanism by which fish oils may be both pro- and antiarrhythmic are mentioned but remain un-clarified. The heterogeneous response to fish oil in patients with heart disease may be explained by the arrhythmia mechanism. The GISSI Trial showed that long-term supplementation with fish oil reduced cardiovascular mortality following a myocardial infarction [3]. This could be attributed to a 45% reduction in sudden death, suggesting a direct antiarrhythmic effect of fish oil in these patients [4]. Subsequently, the effect of fish oil on arrhythmias was studied in patients with implanted cardiac defibrillators that suffered from a variety of cardiac pathologies [5–7]. The results were controversial; proarrhythmic and antiarrhythmic effects as well as absence of effects were documented. The recent meta-analysis by Jenkins and co-workers [8] confirmed heterogeneous outcomes in fish oil trials and concludes that fish oils need to be prescribed with caution. The question remains: Why is the response to fish oil heterogeneous? One possibility is that the diverse patient populations have different arrhythmogenic substrates (heart failure, acute ischemia, infarcted myocardium). Circulating and membrane-bound fish oils shorten the cardiac action potential duration by modulating several ion channels (for review see [9]). Shortening of the action potential, as seen in fish oil fed pigs [10], is proarrhythmic during acute myocardial ischemia [11]. Blockade of the cardiac sodium channel by fish oil may slow conduction, and favor reentrant activation. In contrast, the cardiac action potential is prolonged during heart failure, leading to triggered arrhythmias [12]. Action potential or repolarization time shortening in this setting is antiarrhythmic. In the patients with idiopathic dilated cardiomyopathy that were studied by Nodari and coworkers [1], the predominant arrhythmia mechanism likely is triggered activity. These triggered arrhythmias are inhibited by fish oil in isolated myocytes of patients with end-stage heart failure as a result of cardiac action potential shortening in combination with lower diastolic calcium levels and a reduced response to β-adrenergic stimulation [13]. Fish oils also affect heart rate, lipoprotein levels, inflammatory pathways and hypertrophy and thus also indirectly may determine whether the heart develops arrhythmia. Altogether this leads to a complex situation; fish oil is antiarrhythmic under conditions of heart failure, and proarrhythmic under condition of acute myocardial ischemia. Patients, especially those with heart disease, may have both types of arrhythmia mechanisms. This makes the outcome of dietary supplementation with fish oil difficult to predict. The paper of Nodari [1] nicely demonstrates that the effect of fish oil can be best studied in a well defined patient population. We are of the opinion that fish oil supplementation can be safely administered in these patients with idiopathic dilated cardiomyopathy. Whether or not this is the case for other heart disease patients depends on the main arrhythmia mechanism.

The L-type Ca 2+ Channel as a Potential Mediator of Pathology During Alterations in Cellular Redox State

The L-type Ca(2+) channel is the main route for calcium influx into cardiac myocytes and an important determinant of calcium homeostasis. There is now considerable evidence that the function of the L-type Ca(2+) channel is influenced by the cell's redox state. Reactive oxygen species such as hydrogen peroxide and superoxide can regulate biological function by directly altering the thiol redox state of proteins. Under conditions where cellular redox state varies, L-type Ca(2+) channel function and diastolic calcium levels can be significantly altered. This article will present the evidence for alterations in L-type Ca(2+) channel function by reactive oxygen species and the potential role for the channel in development of acute electrophysiological instability or chronic pathological remodelling under conditions of persistent oxidative stress.