•Electro-mechanical models enable a thorough study of abnormalities in heart failure.•A population-based approach facilitates the identification of critical parameters.•SERCA enhancement prevents ...early afterdepolarizations in heart failure.•SERCA enhancement improves contraction in failing myocytes.
Structural and electrical remodeling in heart failure predisposes the heart to ventricular arrhythmias. Computer modeling approaches, used to complement experimental results, can provide a more mechanistic knowledge of the biophysical phenomena underlying cardiac pathologies. Indeed, previous in-silico studies have improved the understanding of the electrical correlates of heart failure involved in arrhythmogenesis; however, information on the crosstalk between electrical activity, intracellular Ca2+ and contraction is still incomplete. This study aims to investigate the electro-mechanical behavior of virtual failing human ventricular myocytes to help in the development of therapies, which should ideally target pump failure and arrhythmias at the same time.
We implemented characteristic remodeling of heart failure with reduced ejection fraction by including reported changes in ionic conductances, sarcomere function and cell structure (e.g. T-tubules disarray). Model parametrization was based on published experimental data and the outcome of simulations was validated against experimentally observed patterns. We focused on two aspects of myocardial dysfunction central in heart failure: altered force-frequency relationship and susceptibility to arrhythmogenic early afterdepolarizations. Because biological variability is a major problem in the generalization of in-silico findings based on a unique set of model parameters, we generated and evaluated a population of models.
The population-based approach is crucial in robust identification of parameters at the core of abnormalities and in generalizing the outcome of their correction. As compared to non-failing ones, failing myocytes had prolonged repolarization, a higher incidence of early afterdepolarizations, reduced contraction and a shallower force-frequency relationship, all features peculiar of heart failure. Component analysis applied to the model population identified reduced SERCA function as a relevant contributor to most of these derangements, which were largely reverted or diminished by restoration of SERCA function alone.
These simulated results encourage the development of strategies comprising SERCA stimulation and highlight the need to evaluate both electrical and mechanical outcomes.
Cardiac arrhythmias are electrical phenomena; thus, sarcolemmal ion channels have long been considered as targets of antiarrhythmic therapy. The contribution of abnormal intracellular Ca(2+) handling ...to digitalis-induced arrhythmogenesis is an old concept; however, the role of abnormal Ca(2+) handling as a common cause of arrhythmia, i.e. relevant to all arrhythmogenic mechanisms, has been fully recognized in more recent times. Stability of the intracellular Ca(2+) store (sarcoplasmic reticulum, SR) is crucial to physiological Ca(2+) handling; when it is compromised, Ca(2+) may be released independently from excitation and lead to secondary perturbation of membrane potential. Ca(2+) store stability depends on the interplay between sarcolemmal and SR "effectors" (ion channels and transports), which are mutually linked by Ca(2+)-mediated feed-back control. While instrumental to cell homeostasis, such control makes any attempt to modulate SR stability dauntingly complex. This review discusses current knowledge on the factors leading to SR instability, the mechanisms by which SR instability translates into arrhythmias and which interventions may be best suited to prevent SR instability. Although still at an initial stage of development, such interventions might represent the future of antiarrhythmic drug therapy.
The "late sodium current" (I(NaL)) is a sustained component of the fast Na+ current of cardiac myocytes and neurons. As recently appreciated, common neurological and cardiac conditions are associated ...with abnormal I(NaL) enhancement, which may contribute to the pathogenesis of both electrical and contractile dysfunction. For this reason, I(NaL) has become an appealing pharmacological target, with a potentially broad range of therapeutic indications. The recent approval by the FDA of an I(NaL) blocker (ranolazine) for clinical use justifies the increased interest in I(NaL) as a pathogenic mechanism and the rapid evolution of the information concerning it. The review focuses on cardiac aspects of I(NaL) enhancement; it deals with the origin of I(NaL), with its pathophysiological role and with the consequences of its pharmacological modulation. Both basic aspects and clinical evidence are discussed.
Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated ...with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers.
Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil.
The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to β-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.
Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca
ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy ...(DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition".
To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca
dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093).
Ca
transients (CaT) were recorded at 36 °C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN where immunolabeled in single hiPSC-CMs. Mutant preparations (MUT) were compared to isogenic wild-type ones (WT), obtained by mutation reversal.
WT and MUT differed for the following properties: (1) CaT time to peak (t
) and half-time of CaT decay were shorter in MUT; (2) several CaT profiles were identified in WT, "hyperdynamic" ones largely prevailed in MUT; (3) whereas t
rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca
rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 turned all the above properties to resemble those of MUT; when applied to MUT, PST3093 had a smaller or negligible effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs.
Functional data converge to argue for PLN p.Arg14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, possibly including dysregulation of Ca
-dependent transcription.
Abstract
Aims
In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and ...clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action.
Methods and results
We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects.
Conclusion
Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.
Abstract
Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have ...provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.
Increases in action potential duration (APD), genetic or acquired, and arrhythmias are often associated; nonetheless, the relationship between the two phenomena is inconstant, suggesting coexisting ...factors. β-adrenergic activation increases sarcoplasmic reticulum (SR) Ca
-content; angiotensin II (ATII) may increase cytosolic Ca
and ROS production, all actions stimulating RyRs opening. Here we test how APD interacts with β-adrenergic and AT-receptor stimulation in facilitating spontaneous Ca
release events (SCR).
Under "action potential (AP) clamp", guinea-pig cardiomyocytes (CMs) were driven with long (200 ms), normal (150 ms), and short (100 ms) AP waveforms at a CL of 500 ms; in a subset of CMs, all the 3 waveforms could be tested within the same cell. SCR were detected as inward current transients (I
) following repolarization; I
incidence and repetition within the same cycle were measured under increasing isoprenaline concentration (ISO) alone, or plus 100 nM ATII (30 min incubation+superfusion).
I
incidence and repetition increased with ISO; at longer APs the ISO-response curve was shifted upward and I
coupling interval was reduced. ATII increased I
incidence more at low ISO and under normal (as compared to long) APs. Efficacy of AP shortening in suppressing I
decreased in ATII-treated myocytes and at higher ISO.
AP prolongation sensitized the SR to the destabilizing actions of ISO and ATII. Summation of ISO, ATII and AP duration effects had a "saturating" effect on SCR incidence, thus suggesting convergence on a common factor (RyRs stability) "reset" by the occurrence of spontaneous Ca
release events.
The sarcoplasmic reticulum (SR) Ca
ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based ...HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na
/K
pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized.
Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects).
As expected from SERCA2a stimulation, compound 8 induced SR Ca
compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity.
The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.
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