Background: Cardiac myosin inhibitors (CMIs) demonstrate advantages over other guideline-directed therapy for patients with obstructive hypertrophic cardiomyopathy (oHCM). By reducing hypercontractility, CMIs abrogate excessive systolic function and improve diastolic function; diminish hypertrophy of the left ventricle (LV); and improve exercise capacity, functional class, and symptoms. Whether CMIs are therapeutic in heart failure with preserved ejection fraction (HFpEF) is of interest because a significant subset of these patients demonstrate supranormal ejection fractions and abnormal LV structure, characteristics in common with HCM, where CMIs have proved effective. Objectives: Our goal was to characterize the mechanism of myosin inhibition for ulacamten and determine its efficacy in a rodent model of HFpEF. Methods: Ulacamten was characterized using biophysical and biochemical approaches, cardiomyocytes from humans and the ZSF1 obese rat model of HFpEF, hypercontractile human-engineered heart tissues, and echocardiography in the ZSF1 rat model. Results: Unlike the other CMIs, aficamten and mavacamten, ulacamten binds outside the S1 domain of myosin and requires the regulatory light chain domain to bind and inhibit the activity of 2-headed myosin. Ulacamten only partially inhibits the myosin ATPase activity in both myofibrillar and protein systems, but inhibition of contractility was nearly complete in cardiomyocytes. Improvement in relaxation was demonstrated in hypercontractile-engineered heart tissues, and chronic treatment of ZSF1 obese rats showed benefits in both cardiac structure and function. Conclusions: Ulacamten inhibits myosin in a manner distinct from aficamten and mavacamten, potentially broadening the mechanistic properties of CMIs available for treatment of hypercontractile cardiac dysfunction. ### Competing Interest Statement Funding support was provided by Cytokinetics, Inc. S.S.S., M.A.R., J.J.H., D.T.H., A.B.-E., L.K., C.C., A.D., S.E., Y.W., L.Y., A.N.M., B.P.M., and F.I.M. are, or have been, employees of and potential stockholders of Cytokinetics, Inc. C.R. is a paid employee of Propria LLC, which was contracted by Cytokinetics, Incorporated to work on this study. N.A.-G., J.R., and D.M. are paid employees of AnaBios Corporation, which was contracted by Cytokinetics, Incorporated to work on this study. Cytokinetics, Inc

Cyclin A2 (CCNA2), a master cell cycle regulator, is silenced in postnatal mammalian cardiomyocytes. We have previously demonstrated its ability to promote cardiac repair in small and large animals when delivered to the heart via a viral vector. However, the effect of CCNA2 gene delivery on cytokinesis in isolated cardiomyocytes from adult human hearts has not been explored. We designed a human gene therapy vector featuring a replication-deficient adenovirus encoding human CCNA2 driven by the cardiac Troponin T promoter to enable the expression of CCNA2 in freshly isolated human cardiomyocytes. Time-lapse live imaging of adult human primary cardiomyocytes from a 21-year-old male, a 41-year-old female, and a 55-year-old male demonstrated the induction of complete cytokinesis in human adult cardiomyocytes with preservation of sarcomere integrity in the resulting daughter cells with active calcium mobilization in redifferentiated cardiomyocytes. To elucidate the transcriptional mechanisms underlying this response, we conducted single-nucleus transcriptomics analysis of hearts isolated from adult transgenic mice that constitutively express CCNA2 in cardiomyocytes (CCNA2-Tg) and non-transgenic mice (nTg). This revealed a cardiomyocyte subpopulation enriched with cytokinesis, proliferative, and reprogramming genes in hearts obtained from CCNA2-Tg mice as compared to nTg mice. Ultra-deep bulk RNA sequencing of human adult and fetal hearts identified key reprogramming genes relevant to understanding the mechanisms of CCNA2-induced effects observed in our experimental models. These findings provide a promising path for the clinical development of CCNA2-based cardiac regenerative therapy.

Cyclin A2 (CCNA2), a master cell cycle regulator silenced in postnatal cardiomyocytes, promotes cardiac repair in animal models. However, its effect on cytokinesis in adult human cardiomyocytes was previously unknown. We engineered a replication-deficient adenoviral vector encoding human CCNA2 under the cardiac Troponin T promoter and delivered it to freshly isolated cardiomyocytes from adult human hearts. Time-lapse live imaging revealed the induction of complete cytokinesis with preservation of sarcomeres and calcium mobilization in redifferentiated daughter cardiomyocytes. Single-nucleus transcriptomic profiling of CCNA2-transgenic and non-transgenic mouse hearts uncovered a cardiomyocyte subpopulation characterized by enrichment of cytokinesis, proliferation, and reprogramming gene signatures. Ultra-deep bulk RNA sequencing of adult and fetal human hearts further highlighted reprogramming pathways relevant to CCNA2-induced effects. Together, these findings demonstrate that CCNA2 can reinitiate cytokinesis in adult human cardiomyocytes, illuminating conserved molecular programs that support its promise as a regenerative gene therapy for the heart.

Cardiotoxicity, including QTc prolongation and contractility alterations, is one of the leading causes of drug attrition. Hence, implementing human-based pre-clinical assays with improved prediction of cardiotoxicity early in drug discovery could accelerate the development of safe new chemical entities (NCEs). Monolayers of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) proved to be efficient in defining pro-arrhythmic risk of NCEs but showed limitations in predicting contractility liabilities. Here, hiPSC-CMs and adult human primary ventricular myocytes (hCMs) were used to measure Ca2+ dynamics (Hamamatsu FDSS platform) and contractility (CardioPRIME®), respectively. Changes in parameters of hiPSC-CM Ca2+ dynamics (transient duration at 90 % decay -CTD90) and incidence of early afterdepolarization (EADs) were used to infer drug-induced pro-arrhythmia, whilst changes in contractility parameters for drug-induced inotropic effect (sarcomere shortening) and pro-arrhythmia (aftercontraction) in hCMs. The effects of the following 11 reference drugs (including positive/negative inotropes) were evaluated blindly (four concentrations per compound, covering/exceeding free Cmax): dofetilide, sparfloxacin, quinidine, flecainide, mexiletine, cetirizine, ranolazine, levocromakalin, PA-6, milrinone, digoxin. hiPSC-CMs and hCMs treated with vehicle solutions (DMSO 0.1 %) or cetirizine (negative control) did not show significant changes in the parameters assessed. Pro-arrhythmic drugs, sparfloxacin and dofetilide prolonged CTD90, induced EADs (hiPSC-CMs) and after-contractions (hCMs), without altering contractility in hCMs. Ranolazine increased CTD90 and caused EADs in hiPSC-CMs, while it decreased contractility in hCMs, without causing aftercontractions. In hiPSC-CMs, levcromakalim induced CTD90 shortening and quiescence, while quinidine and flecainide induced CTD90 prolongation and EADs. In hCMs, flecainide and levcromakalim decreased amplitude of contraction (without aftercontractions), while quinidine had no effect at tested concentrations. Milrinone and digoxin induced an increase in contractility amplitude in hCMs. In hiPSC-CMs, milrinone had no effect. Digoxin increased Ca2+-transient amplitude, induced CTD90 shortening and quiescence. Mexiletine induced quiescence in hiPSC-CMs and a decrease in contractility amplitude in hCMs. PA-6 induced after-contractions in hCMs and prolonged CTD90 and caused quiescence in hiPSC-CMs. In conclusion, while hiPSC-CMs are consistently predictive of early proarrhythmic risk, the addition of a hCMs-based assay could be useful in identifying inotropic and proarrhythmic properties of some compounds.

It has been hypothesized that concomitant inhibition of the late sodium current (INaL) or L-type calcium current (ICaL) could mitigate hERG block-mediated QTC prolongation. Using available patch clamp data at the time, dofetilide and moxifloxacin were chosen as selective hERG blockers, mexiletine and lidocaine as INaL inhibitors, and diltiazem as ICaL inhibitor to test this hypothesis. The results showed that hERG block-mediated QTC prolongation was shortened by co-administration of mexiletine or lidocaine but not diltiazem, challenging this hypothesis. As patch clamp results are impacted by experimental conduct, this study reexamined the effects of the aforementioned drugs plus nifedipine on hERG current, ICaL, and INaL and on action potential (AP) of ventricular trabeculae from organ donors to provide insight into the previously observed discrepancy. Whole-cell patch-clamp recordings were performed on cell lines that stably expressed hERG, CaV1.2, and NaV1.5 proteins to re-evaluate individual drugs' effects using more physiologically relevant protocols consistent with ICH S7B Q&A 2.1 best practices. Sharp-electrode ventricular AP recordings were measured to test drug combinations. Patch-clamp results show that mexiletine inhibited INaL (IC50:15 μM) and ICaL (22.6 μM) with similar potencies, in contrast with prior results showing selective INaL inhibition. Mexiletine and lidocaine reduced dofetilide-induced increase in AP duration (APD), consistent with the clinical QTC data. Patch-clamp experiments are ongoing to determine whether lidocaine profile is like mexiletine. For diltiazem, ICaL potency and separation between ICaL and hERG potencies (ICaL:1.3 μM; hERG:8.9 μM) are reduced compared to prior studies (ICaL:0.1 μM; hERG:6.6 μM). These new findings are consistent with AP shortening and triangulation – a signature of multi-channel block. A greater separation of ICaL (0.01 μM) and hERG (34.8 μM) was observed for nifedipine along with AP shortening without triangulation. The new patch-clamp and AP recordings suggest that the previous discrepancy between clinical-nonclinical data is due to insufficient inhibition of ICaL or concomitant inhibition of hERG by diltiazem at clinical concentrations (0.05 uM). Integrating data from different nonclinical models and following best practices can improve nonclinical to clinical translation – especially important when evaluating drug or drug combinations that impact multiple cardiac ionic currents.

Clinical trials of cardiac myosin inhibitors, including mavacamten and aficamten , have been shown to induce cardiac remodeling and improve exercise capacity of patients with hypertrophic cardiomyopathy (HCM) with left ventricular outflow tract obstruction. A subset of patients with heart failure with preserved ejection fraction (HFpEF) have overlapping features with HCM including hypercontractility (EF >65%), thickened heart walls and impaired diastolic function. This similarity suggests myosin inhibitors could be therapeutic in treating HFpEF. Here, we describe the preclinical characterization of a novel cardiac myosin inhibitor, CK-4021586 (CK-586), which has a distinct mechanism of action from both mavacamten and aficamten . CK-586 is a partial inhibitor of cardiac myofibrillar ATPase activity (EC50: 2.9 µM, maximal biochemical inhibition ~50%) which requires the presence of two-headed heavy meromyosin (HMM) and the regulatory light chain. CK-586 potently inhibited sarcomere shortening in both rat and human adult ventricular cardiomyocytes, with IC50 values of 3.2 µM and 2.8 µM respectively, achieving over 80% inhibition at 10 µM. Importantly, even at high concentrations up to 20 µM, CK-586 did not affect calcium transients in rat myocytes. Similarly, CK-586 effectively normalized contractile function in engineered heart tissues derived from human iPSC-cardiomyocytes carrying the HCM-associated R403Q myosin mutation. At 2 µM, the compound restored contractile force and relaxation time (RT90) to near-normal levels, while showing >80% force inhibition at 10 µM. In the ZSF1 obese HFpEF rat model, 16 weeks of CK-586 treatment reduced fractional shortening (vehicle: 52.7 ± 2.9% vs. CK-586: 48.1 ± 2.6%) and as a measure of diastolic function, improved isovolumic relaxation time (vehicle: 27.0 ± 2.2 ms vs. CK-586: 23.3 ± 2.3 ms). ZSF1 obese rats treated with CK-586 also demonstrated approximately 50% lower interstitial fibrosis compared to untreated controls. In conclusion, CK-586 is a novel, small molecule, cardiac myosin inhibitor that reduces contractility in vitro and in vivo . These findings suggest that targeting cardiac myosin to reduce contractility could help reverse some of the structural changes associated with HFpEF and improve cardiac relaxation.

QT interval prolongation is incontrovertibly associated with increased risk of arrhythmias and action potential (AP) recordings from human cardiomyocytes remain one of the most integrative readouts to predict drug-induced prolongation of the QT interval. Rapid differentiation of primary cardiomyocytes in culture or immaturity of human induced pluripotent stem cell-derived cardiomyocytes hamper prediction of drug-induced QT prolongation. Thus, we investigated AP recordings from adult human primary cardiomyocytes, which were shipped post-isolation from California to France using an air freight service, as a model to assess cardiotoxic potential of novel drugs. APs were recorded at 1 Hz pacing frequency between days 5 and 14 post-isolation with the whole-cell current-clamp mode of the patch-clamp technique using appropriate intra- and extra-cellular solutions under control conditions and in the presence of dofetilide, a hERG channel blocker known to prolong QT and induce pro-arrhythmia. Results are expressed as mean ± s.e.m. Like non-shipped cardiomyocytes, shipped cells were found to be Ca2+-tolerant, retain rod-shaped morphology and exhibit cross striations with 65 % viability at day 5 post-isolation and approximately 60 % success rate in recording AP signals. AP characteristics were in agreement with the human AP literature: resting membrane potential (RMP) −72.3 ± 1.1 mV, amplitude (AMP) 123.1 ± 3.0 mV, AP durations at 20 % (APD20) and 90 % (APD90) repolarisation 84.9 ± 9.4 msec and APD90 303.0 ± 20.8 msec, respectively (n = 17 cells). The pharmacological and clinical relevance of the shipped cardiomyocytes is currently ongoing and here we report data generated with dofetilide. While the application of 0.3 μM dofetilide did not significantly affect RMP and AMP, it markedly increased APD20 and APD90 by 19.0 ± 7.9 % and 40.0 ± 4.5 %, respectively (n = 4 cells). Our study demonstrates that air-shipped human adult primary cardiomyocytes stayed healthy, retained physiological AP signals with a pharmacological response to hERG channel blockade. When fully validated, this human cardiomyocyte model can provide critical data assisting in developing novel non-cardiotoxic therapies.

Late Na + current (INa,L) contributes to cardiac action potential (AP) and helps maintain Na + homeostasis. Contrary to the effect on Nav1.5, hERG and Cav1.2 channels, the effect of novel drugs on INa,L is not routinely studied in safety studies. Yet, INa,L inhibitors can counterbalance hERG blockade and be associated with anti-arrhythmic potential. Hence, evaluating the effects of drugs against Nav1.5, hERG and Cav1.2 channels provides valuable cardiotoxic insights, but does not fully predict changes in the electrophysiological and contractile properties of cardiomyocytes. To address this deficiency, we evaluated the effects of selective and non-selective INa,L inhibitors on cardiomyocyte function. Compounds known to inhibit INa,L (GS-967 specific for INa,L; ranolazine specific for both hERG and INa,L; loperamide which is a hERG, Nav1.5 and Cav1.2 inhibitor) and four preclinical compounds were tested for their effects on cardiac ion channels (peak Nav1.5, hERG, Cav1.2 and INa,L) with automated patch-clamp and multi-electrode array (MEA) in hiPSC-derived cardiomyocytes for electrophysiological properties, and contractility in human primary cardiomyocytes from consented donor hearts with MyoBLAZER™. Each compound was tested separately at multiple concentrations in the presence of ATX-II, a selective enhancer of INa,L. GS-967 and ranolazine reversed ATX-II-induced increases in contractility and field potential duration (FPD) in a concentration-dependent manner providing evidence of a functional INa,L in both hiPSC-derived cardiomyocytes and adult cardiomyocytes. Next, we evaluated the effects of four preclinical compounds. Two out of the four compounds showed similar behavior to GS-967 and ranolazine. For example, compound A inhibited ion channels (hERG, Nav1.5, Cav1.2 and INa,L with IC50 values of 7.9 mM, 12.4 mM, 0.8 mM, respectively) and reversed ATX-II changes on contractility and FPD with IC50 values of 0.99 mM and 2.3 mM, respectively. Here, we developed a protocol for assessing drug interactions with INaL on cardiomyocytes. This assay enhances our ability to predict cardiotoxicity potential and its incorporation into the traditional compound derisking strategy strengthens confidence in advancing molecules into clinical development.

Concomitant inhibition of the late Na+ current (INaL) and/or the L-type Ca2+ current (ICaL) has been hypothesized to mitigate hERG block-mediated QTC prolongation. This hypothesis was tested in a clinical trial using drugs selected based on available patch clamp data at the time. The results showed that hERG block-mediated QTC prolongation with dofetilide was shortened by co-administration of lidocaine or mexiletine–drugs that inhibit INaL. However, diltiazem, selected as the preferential ICaL inhibitor, did not shorten hERG block-mediated QTC prolongation by moxifloxacin. Patch clamp results can be sensitive to experimental differences across laboratories. Therefore, this study reexamined the effects of all drugs on INaL, ICaL, and hERG current using overexpression cell lines and physiologically relevant experimental protocols aimed at producing drug-channel interaction characteristics in humans. Drug effects on ventricular action potentials (APs) from adult human trabeculae were also tested to better understand the nonclinical and clinical findings. Mexiletine and lidocaine showed similar potencies on inhibiting INaL and ICaL in the prior and present patch clamp studies. Both drugs reduced dofetilide-induced AP duration (APD) prolongation, consistent with the clinical data. For diltiazem, the ICaL potency and the separation between ICaL and hERG potencies (ICaL: 1.3 µM; hERG: 8.9 µM; hERG-to-ICaL ratio = 7) is much reduced comparing to the prior results (ICaL: 112.1 nM; hERG: 6.6 µM; ratio = 59). These new findings are consistent with diltiazem-induced APD shortening and AP triangulation caused by greater reductions in the early rather than late repolarization–a signature of multi-ion channel block. Consistent with this interpretation, nifedipine, which preferentially inhibits ICaL over hERG (ICaL: 13.2 nM; hERG: 35 μM; ratio = 2,651) caused APD shortening without AP triangulation. Results from this study thus support the following: 1) diltiazem failed to reduce moxifloxacin-induced QTC prolongation due to its concomitant hERG block at clinical exposure levels; and 2) the importance of using physiologically relevant protocols to generate ion channel pharmacology and obtaining functional recordings from myocytes to provide a better understanding of nonclinical data translation to clinical ECG signals. Data used in this manuscript, including the original electrophysiology records, may be found at: https://osf.io/69ght/.

Cyclin A2 (CCNA2), a master cell cycle regulator silenced in postnatal cardiomyocytes, promotes cardiac repair in animal models. However, its effect on cytokinesis in adult human cardiomyocytes remains unknown. We engineered a replication-deficient adenoviral vector encoding human CCNA2 under the cardiac Troponin T promoter and delivered it to freshly isolated cardiomyocytes from adult human hearts. Time-lapse live imaging revealed induction of complete cytokinesis with preservation of sarcomeres and calcium mobilization in redifferentiated daughter cardiomyocytes. To uncover underlying transcriptional mechanisms, single-nucleus transcriptomics of CCNA2-transgenic versus non-transgenic mouse hearts identified a cardiomyocyte subpopulation enriched for cytokinesis, proliferative, and reprogramming genes. Ultra-deep bulk RNA sequencing of adult and fetal human hearts further highlighted reprogramming pathways relevant to CCNA2-induced effects. Together, these findings demonstrate that CCNA2 can reinitiate cytokinesis in adult human cardiomyocytes and illuminate conserved molecular programs, supporting its promise as a regenerative gene therapy for the heart.

Ibogaine and its main metabolite noribogaine provide important molecular prototypes for markedly different treatment of substance use disorders and co-morbid mental health illnesses. However, these compounds present a cardiac safety risk and a highly complex molecular mechanism. We introduce a class of iboga alkaloids - termed oxa-iboga - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds lack the proarrhythmic adverse effects of ibogaine and noribogaine in primary human cardiomyocytes and show superior efficacy in animal models of opioid use disorder in male rats. They act as potent kappa opioid receptor agonists in vitro and in vivo, but exhibit atypical behavioral features compared to standard kappa opioid agonists. Oxa-noribogaine induces long-lasting suppression of morphine, heroin, and fentanyl intake after a single dose or a short treatment regimen, reversal of persistent opioid-induced hyperalgesia, and suppression of opioid drug seeking in rodent relapse models. As such, oxa-iboga compounds represent mechanistically distinct iboga analogs with therapeutic potential.

Etrasimod (ADP334) is an oral, once‐daily, selective sphingosine 1‐phosphate (S1P)1,4,5 receptor modulator for the treatment of moderately to severely active ulcerative colitis and in development for the treatment of immune‐mediated inflammatory diseases. Interaction between S1P and its five receptor subtypes (S1P1–S1P5) plays a role in several physiologic systems, including the cardiovascular and immune systems. Since differences in S1PR binding and downstream intracellular signaling could contribute to distinct profiles of drug efficacy and safety, we directly compared the S1P1–5 selectivity profile of etrasimod to three marketed S1PR modulators: fingolimod, ozanimod, and siponimod. Using both heterologous expression systems and human umbilical vein endothelial cells that spontaneously express S1P1, we profiled key S1P1 downstream signaling pathways and found that etrasimod had similar potency to the other tested S1PR modulators in promoting β‐arrestin recruitment and S1P1 internalization. However, etrasimod was notably less potent than other S1PR modulators in assays measuring S1P1‐mediated G protein activation (GTPγS binding and cAMP inhibition). Relatively lower potency of etrasimod in inducing G protein signaling corresponded to significantly diminished activation of human cardiac G protein‐coupled inwardly rectifying potassium channels when compared to ozanimod. Together with pharmacokinetic properties, this pharmacologic profile of etrasimod may contribute to the positive benefit risk profile of etrasimod observed during the phase III ELEVATE UC 52 and ELEVATE UC 12 trials in patients with moderately to severely active ulcerative colitis.

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca2+ dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach. We performed a study to evaluate primary human CM and have them compared to freshly isolated dog cardiomyocytes for their basic function and responses to positive inotropes with well-known mechanisms. Our data showed that simultaneous assessment of sarcomere shortening and Ca2+-transient can be performed with both myocytes using the IonOptix system. Amplitude of sarcomere shortening and Ca2+-transient (CaT) were significantly higher in dog compared to human CM in the basic condition (absence of treatment), while longer duration of sarcomere shortening and CaT were observed in human cells. We observed that human and dog CMs have similar pharmacological responses to five inotropes with different mechanisms, including dobutamine and isoproterenol (β-adrenergic stimulation), milrinone (PDE3 inhibition), pimobendan and levosimendan (increase of Ca2+sensitization as well as PDE3 inhibition). In conclusion, our study suggests that myocytes obtained from both human donor hearts and dog hearts can be used to simultaneously assess drug-induced effects on sarcomere shortening and CaT using the IonOptix platform.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentia-tion of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Impor-tantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.