Cardiac muscle disease and therapeutic targets

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ∼50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, SMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, SMN). Gene mutations are most often present in genes encoding the sarcomere proteins myosin heavy chain, myosin-binding protein C, and troponin T. Studies in cardiac tissue samples from patients with obstructive HCM that were obtained during myectomy surgery showed increased myofilament calcium sensitivity, increased kinetics and tension cost, and a reduction of the super-relaxed state of myosin, which is associated with an energy-conserving status of the crossbridges. The increase in myofilament calcium sensitivity is observed at a low dose of mutant protein, while the magnitude of the increase in calcium sensitivity depends on the specific mutation location. These mutation-mediated myofilament changes may underlie inefficient in vivo cardiac performance in mutation carriers. Reduced cardiac efficiency has been observed before onset of cardiac hypertrophy and at advanced disease stages. In addition, impaired diastolic function is an early disease characteristic of HCM. Our recent proteomics studies revealed increased detyrosination of microtubules, which may be a cause of diastolic dysfunction. Recent treatments that target inefficient cardiac performance, such as myosin inhibitors and metabolic drug therapies, may have the potential to prevent, delay, or even reverse disease in HCM-mutation carriers. Treatment response may depend on the specific gene mutation in SMP individuals and may explain diverse response of HCM patients to therapy. While mutation-mediated cardiomyocyte defects have become clear in past years, more research is warranted to define the cellular pathomechanisms of cardiac dysfunction in SMN patients.

Generative adversarial networks for construction of virtual populations of mechanistic models: simulations to study Omecamtiv Mecarbil action

Biophysical models are increasingly used to gain mechanistic insights by fitting and reproducing experimental and clinical data. The inherent variability in the recorded datasets, however, presents a key challenge. In this study, we present a novel approach, which integrates mechanistic modeling and machine learning to analyze in vitro cardiac mechanics data and solve the inverse problem of model parameter inference. We designed a novel generative adversarial network (GAN) and employed it to construct virtual populations of cardiac ventricular myocyte models in order to study the action of Omecamtiv Mecarbil (OM), a positive cardiac inotrope. Populations of models were calibrated from mechanically unloaded myocyte shortening recordings obtained in experiments on rat myocytes in the presence and absence of OM. The GAN was able to infer model parameters while incorporating prior information about which model parameters OM targets. The generated populations of models reproduced variations in myocyte contraction recorded during in vitro experiments and provided improved understanding of OM’s mechanism of action. Inverse mapping of the experimental data using our approach suggests a novel action of OM, whereby it modifies interactions between myosin and tropomyosin proteins. To validate our approach, the inferred model parameters were used to replicate other in vitro experimental protocols, such as skinned preparations demonstrating an increase in calcium sensitivity and a decrease in the Hill coefficient of the force–calcium (F–Ca) curve under OM action. Our approach thereby facilitated the identification of the mechanistic underpinnings of experimental observations and the exploration of different hypotheses regarding variability in this complex biological system.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

Abstract MP211: NAD Redox Imbalance Exacerbates Diabetic Cardiomyopathy

Diabetes and heart failure are linked to NAD redox imbalance, whose role in diabetic cardiomyopathy has not been directly tested. Streptozotocin-induced diabetes in WT mice for 16 weeks promoted declines in systolic and diastolic function, which associated with lowered cardiac NAD/NADH ratio (NAD redox imbalance). To test the hypothesis that , we employed mouse models with cardiac-specific manipulations of NAD redox states. Cardiac-specific Ndufs4-KO mice (cKO) exhibit lowered cardiac NAD/NADH ratio with normal baseline function, geometry and energetics. Control and cKO mice were challenged with 8-week diabetic stress. Metabolomic analyses of plasma collected after the diabetic stress showed similar hyperglycemia and dyslipidemia stresses in diabetic control and diabetic cKO mice. Chronic diabetic stress promoted systolic and diastolic dysfunctions in control mice, which were further exacerbated in diabetic cKO mice in both male and female cohorts. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways showed no change in diabetic cKO hearts, suggesting that cardiomyocyte dysfunction is a likely culprit for the exacerbated dysfunction. Increased protein acetylation, including SOD2-K68Ac, was observed in diabetic cKO hearts. Inhibited antioxidant function by SOD2-K68Ac promoted protein oxidation in diabetic cKO hearts, suggesting oxidative stress as a pathogenic mechanism. We next examined phosphorylation status of myofilament proteins in these diabetic hearts. MyBPC-S282Pi levels are suppressed in failing hearts and remained unchanged in diabetic cKO hearts. TnI-S150Pi increases myofilament calcium sensitivity and prolongs calcium dissociation, while TnI-S23/24Pi imposes the opposite effects. TnI-S150Pi levels were elevated in diabetic cKO hearts, while TnI-S23/24Pi levels unchanged. Therefore, exacerbated diastolic dysfunction in diabetic cKO hearts is due to the selective phosphorylation at TnI-S150. AMPK is activated by energetic stress and phosphorylates TnI-S150. ATP levels decreased, and AMP/ATP ratio increased in diabetic cKO hearts, implicating impaired energetics to promote TnI-S150Pi and dysfunction. Elevation of NAD levels normalized cardiac NAD redox balance in diabetic cKO hearts. Elevated levels of SOD2-K68Ac and TnI-S150Pi, exacerbated systolic and diastolic dysfunction in diabetic cKO hearts were all reversed by elevation of NAD levels. Dysfunction in diabetic control hearts was also ameliorated by elevation of NAD levels. These data collectively conclude that NAD redox imbalance is a positive mediator of the progression of diabetic cardiomyopathy.

Reconnoitering the Role of Long-Noncoding RNAs in Hypertrophic Cardiomyopathy: A Descriptive Review

Hypertrophic cardiomyopathy (HCM) is the most common form of hereditary cardiomyopathy. It is characterized by an unexplained non-dilated hypertrophy of the left ventricle with a conserved or elevated ejection fraction. It is a genetically heterogeneous disease largely caused by variants of genes encoding for cardiac sarcomere proteins, including MYH7, MYBPC3, ACTC1, TPM1, MYL2, MYL3, TNNI3, and TNNT23. Preclinical evidence indicates that the enhanced calcium sensitivity of the myofilaments plays a key role in the pathophysiology of HCM. Notably, this is not always a direct consequence of sarcomeric variations but may also result from secondary mutation-driven alterations. Long non-coding RNAs (lncRNAs) are a large class of transcripts ≥200 nucleotides in length that do not encode proteins. Compared to coding mRNAs, most lncRNAs are not as well-annotated and their functions are greatly unexplored. Nevertheless, increasing evidence shows that lncRNAs are involved in a variety of biological processes and diseases including HCM. Accumulating evidence has indicated that lncRNAs are dysregulated in HCM, and closely related to sarcomere construction, calcium channeling and homeostasis of mitochondria. In this review, we have summarized the known regulatory and functional roles of lncRNAs in HCM.

The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium

Rationale: Myofilament length dependent activation (LDA) is the key underlying mechanism of cardiac heterometric autoregulation, commonly referred as the Frank-Starling law of the heart. Although alterations in LDA are common in cardiomyopathic states, the precise structural and biochemical mechanisms underlying LDA remain unknown. Objective: Here, we examine the role of structural changes in the thick filament during diastole, in particular changes in the availability of myosin heads, in determining both calcium sensitivity and maximum contractile force during systole in permeabilized porcine cardiac fibers. Methods and Results: Permeabilized porcine fibers from ventricular myocardium were studied under relaxing conditions at short and long sarcomere length (SL) using muscle mechanics, biochemical measurements, and X-ray diffraction. Upon stretch, porcine myocardium showed the increased calcium sensitivity and maximum calcium activated force characteristic of LDA. Stretch increased diastolic ATP turnover, recruiting reserve myosin heads from the super-relaxed state (SRX) at longer SL. Structurally, X-ray diffraction studies in the relaxed-muscle confirmed a departure from the helical ordering of the thick-filament upon stretch which occurred concomitantly with a displacement of myosin heads towards actin, facilitating cross-bridge formation upon systolic activation. Mavacamten, a selective myosin-motor inhibitor known to weaken the transition to actin-bound power-generating states and to enrich the ordered SRX myosin population, reversed the structural effects of stretch on the thick-filament, blunting the mechanical consequences of stretch; mavacamten did not, however, prevent other structural changes associated with LDA in the sarcomere, such as decreased lattice spacing or troponin-displacement. Conclusions: Our findings strongly indicate that in ventricular muscle, LDA and its systolic consequences are dependent on the population of myosin heads competent to form cross-bridges and involves the recruitment of myosin heads from the reserve SRX pool during diastole.

Mechanisms in Tridax procumbens leaf extract reversal of paroxetine-induced erectile dysfunction in corpus cavernosum of male Wistar rats

Introduction: Aqueous leave extract of Tridax procumbens (AETPL) is reported to improve erectile functions; however, the mechanism is unclear. This study investigates the mechanism involved in the contractile activity of the corpus cavernosum after AETPL treatment of paroxetine-induced erectile dysfunctional adult male Wistar rats. Methods: A total of 20 male Wistar rats were categorized into four groups of five and treated orally for four weeks: Group 1 (distilled water), Group 2 (paroxetine 10 mg/kg), Group 3 (paroxetine + AETPL 100 mg/kg), and Group 4 (paroxetine + Viagra 0.5 mg/kg). Contractile responses of excised corpus cavernosum strips (CS) were determined in response to acetylcholine (ACh), phenylephrine (PHE), potassium chloride (KCl), and calcium chloride (CaCl2), and after incubation in L-NAME, indomethacin, nifedipine, adenosine, caffeine, nicorandil, and acetovanillone. Results: The relaxation response (%) of CS to ACh was significantly inhibited in the paroxetine group compared to the AETPL- and the Viagra-co-treated group. Pre-incubation in L-NAME considerably enhanced the percentage relaxation in groups co-treated with AETPL and Viagra. Groups co-treated with AETPL and Viagra significantly inhibited contraction in response to cumulative doses of CaCl2. Contractile responses of CS to cumulative doses of PHE after incubation in caffeine and adenosine were considerably inhibited in groups co-treated with AETPL and Viagra. Similarly, nicorandil (10-4 M) enhanced the percentage relaxation to cumulative doses of ACh (10-9 — 10-5 M) in groups co-treated with AETPL and Viagra. The pre-incubation of CS with acetovanillone (10-4 M) enhanced the percentage relaxation to ACh across groups. Conclusion: Erectile dysfunction was reversed by AETPL-induced antioxidant/NADPH oxidase inhibitor activity, reduced calcium sensitivity, activation of ATP-sensitive K+ channel, and endothelial Nitric Oxide (NO) release.

A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.