Kinematics but not kinetics alterations to single-leg drop jump movements following a subject-tailored fatiguing protocol suggest an increased risk of ACL injury.

Introduction: Neuromuscular fatigue causes a transient reduction of muscle force, and alters the mechanisms of motor control. Whether these alterations increase the risk of anterior cruciate ligament (ACL) injury is still debated. Here we compare the biomechanics of single-leg drop jumps before and after the execution of a fatiguing exercise, evaluating whether this exercise causes biomechanical alterations typically associated with an increased risk of ACL lesion. The intensity of the fatiguing protocol was tailored to the aerobic capacity of each participant, minimizing potential differential effects due to inter-individual variability in fitness. Methods: Twenty-four healthy male volunteers performed single leg drop jumps, before and after a single-set fatiguing session on a cycle ergometer until exhaustion (cadence: 65-70 revolutions per minute). For each participant, the intensity of the fatiguing exercise was set to 110% of the power achieved at their anaerobic threshold, previously identified by means of a cardiopulmonary exercise test. Joint angles and moments, as well as ground reaction forces (GRF) before and after the fatiguing exercise were compared for both the dominant and the non-dominant leg. Results: Following the fatiguing exercise, the hip joint was more extended (landing: Δ=-2.17°, p = 0.005; propulsion: Δ=-1.83°, p = 0.032) and more abducted (landing: Δ=-0.72°, p = 0.01; propulsion: Δ=-1.12°, p = 0.009). Similarly, the knee joint was more extended at landing (non-dominant leg: Δ=-2.67°, p < 0.001; dominant: Δ=-1.4°, p = 0.023), and more abducted at propulsion (both legs: Δ=-0.99°, p < 0.001) and stabilization (both legs: Δ=-1.71°, p < 0.001) hence increasing knee valgus. Fatigue also caused a significant reduction of vertical GRF upon landing (Δ=-0.21 N/kg, p = 0.003), but not during propulsion. Fatigue did not affect joint moments significantly. Conclusion: The increased hip and knee extension, as well as the increased knee abduction we observed after the execution of the fatiguing exercise have been previously identified as risk factors for ACL injury. These results therefore suggest an increased risk of ACL injury after the execution of the participant-tailored fatiguing protocol proposed here. However, the reduced vertical GRF upon landing and the preservation of joint moments are intriguing, as they may suggest the adoption of protective strategies in the fatigued condition to be evaluated in future studied.

Early consequences of the phospholamban mutation PLN-R14del+/- in a transgenic mouse model.

AIMS: The heterozygous phospholamban (PLN) mutation R14del (PLN R14del+/- ) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected. METHODS: Ca2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy, and redox homeostasis were measured in ventricular myocytes of 8-12 weeks-old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analyzed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated. RESULTS: The mutation was characterized by hyperdynamic Ca2+ handling, compatible with a loss of SERCA2a inhibition by PLN. All components of energy metabolism were depressed; myocyte energy charge was preserved under quiescence but reduced during stimulation. Cytosolic Ca2+ buffering or SERCA2a blockade reduced O2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca2+ dynamics and response to stress. CONCLUSIONS: (1) PLN R14del+/- loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a pathogenetic mechanism; (2) depressed energy metabolism, its enhanced dependency on Ca2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model.

Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ 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. METHODS: 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). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ 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. CONCLUSIONS: 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.

25 years of basic and translational science in EP Europace: novel insights into arrhythmia mechanisms and therapeutic strategies.

In the last 25 years, EP Europace has published more than 300 basic and translational science articles covering different arrhythmia types (ranging from atrial fibrillation to ventricular tachyarrhythmias), different diseases predisposing to arrhythmia formation (such as genetic arrhythmia disorders and heart failure), and different interventional and pharmacological anti-arrhythmic treatment strategies (ranging from pacing and defibrillation to different ablation approaches and novel drug-therapies). These studies have been conducted in cellular models, small and large animal models, and in the last couple of years increasingly in silico using computational approaches. In sum, these articles have contributed substantially to our pathophysiological understanding of arrhythmia mechanisms and treatment options; many of which have made their way into clinical applications. This review discusses a representative selection of EP Europace manuscripts covering the topics of pacing and ablation, atrial fibrillation, heart failure and pro-arrhythmic ventricular remodelling, ion channel (dys)function and pharmacology, inherited arrhythmia syndromes, and arrhythmogenic cardiomyopathies, highlighting some of the advances of the past 25 years. Given the increasingly recognized complexity and multidisciplinary nature of arrhythmogenesis and continued technological developments, basic and translational electrophysiological research is key advancing the field. EP Europace aims to further increase its contribution to the discovery of arrhythmia mechanisms and the implementation of mechanism-based precision therapy approaches in arrhythmia management.

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.

Insights from an electro-mechanical heart failure cell model: Role of SERCA enhancement on arrhythmogenesis and myocyte contraction.

BACKGROUND AND OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These simulated results encourage the development of strategies comprising SERCA stimulation and highlight the need to evaluate both electrical and mechanical outcomes.

Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.

Highly Selective SERCA2a Activators: Preclinical Development of a Congeneric Group of First-in-Class Drug Leads against Heart Failure.

The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na+/K+ ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na+/K+ ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na+/K+ ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.

Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition". AIM: To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093). METHODS: Ca2+ 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. RESULTS: WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) 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 tpeak rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca2+ 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. CONCLUSIONS: 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 Ca2+-dependent transcription.

ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research.

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.

NOS1AP polymorphisms reduce NOS1 activity and interact with prolonged repolarization in arrhythmogenesis.

AIMS: NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. METHODS AND RESULTS: In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by IKs blockade (JNJ303) and ß-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1-A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced ICaL and INaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed 'transient inward current' events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and ICaL larger; NOS1AP and NOS1 expression and co-localization were decreased. CONCLUSION: The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes.

MTMR4 SNVs modulate ion channel degradation and clinical severity in congenital long QT syndrome: insights in the mechanism of action of protective modifier genes.

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.

Repolarization instability and arrhythmia by IKr block in single human-induced pluripotent stem cell-derived cardiomyocytes and 2D monolayers.

AIMS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. METHODS AND RESULTS: Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation-contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. CONCLUSION: Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.