Background Variants of unknown significance (VUS) pose a barrier to cascade genetic screening in families affected by inherited cardiomyopathies. Here, we investigate scalable computational methods for assessing pathogenicity of TPM1 VUS in the context of cardiomyopathy and test them against in vitro data. Methods Twenty TPM1 VUS (all missense) from clinical databases were computationally evaluated by inserting each into atomistic representations of tropomyosin. Molecular dynamic simulations were used to assess changes in tropomyosin flexibility, and a structural model of tropomyosin on actin allowed calculation of altered electrostatic interaction energies. Four variants representing diverse changes in these properties were carried forward for functional studies that included in vitro motility, contractility of engineered heart tissues (EHTs), and morphology of iPSC-derived cardiomyocytes. Results The TPM1 variants A102D, D258E, K233N, and A239T all showed at least one significantly altered facet of contractile function. A102D was associated with increased thin filament Ca2+ sensitivity, increased twitch contraction force, and slowed relaxation. D258E was shown to cause a slowing of twitch relaxation and reduced ability to block myosin activity at low Ca2+. K233N and A239T both reduced in vitro thin filament motility, drastically decreased twitch contraction force, and triggered pronounced increases in cardiomyocyte aspect ratio. Conclusions By showing that large molecular aberrations predicted by thin filament structural models translate into meaningful functional changes (as benchmarked against clinically pathogenic mutations), this study supports the feasibility of TPM1 variant classification by means of a scalable computational pipeline.

Background Evaluating risk in first-degree relatives of patients diagnosed with hypertrophic cardiomyopathy (HCM) is a critical, ongoing clinical challenge, particularly when genetic testing yields a variant of unknown significance (VUS) in an HCM-related gene. Our goal is to develop a pipeline for efficient and scalable evaluation of VUS pathogenicity. Methods Molecular dynamics simulations were used to screen twenty TPM1 VUS and identify four for further study: A102D, D258E, A239T, and K233N. Each VUS was engineered into an adenoviral TPM1 vector which was in turn applied to human engineered heart tissues (hEHTs) made from iPSC-derived cardiomyocytes. One week after viral exposure, isometric contractile behavior of hEHTs was measured. Results hEHTs expressing A102D were markedly hypercontractile, with a significant increase in contractile force and slowing of relaxation. D258E expression caused a mild phenotype, showing only a mildly increased relaxation rate relative to control. A238T and K233N both caused a dramatic decrease in contractile force. These same four VUS were studied in regulated in vitro motility assays and showed patterns of increased or decreased myofilament activity that were entirely consistent with findings in hEHT. Conclusions A102D displayed a hypercontractile phenotype that resembled that of our benchmark HCM mutation (E62Q), while A238T and K233N produced contractile weakness that closely mimicked that of our benchmark DCM mutation (E54K). D258E had a minimal effect that could be interpreted as mildly hypertrophic. We conclude that hEHTs have the potential to serve as a scalable platform for evaluating pathogenicity of TPM1 VUS. Evaluating risk in first-degree relatives of patients diagnosed with hypertrophic cardiomyopathy (HCM) is a critical, ongoing clinical challenge, particularly when genetic testing yields a variant of unknown significance (VUS) in an HCM-related gene. Our goal is to develop a pipeline for efficient and scalable evaluation of VUS pathogenicity. Molecular dynamics simulations were used to screen twenty TPM1 VUS and identify four for further study: A102D, D258E, A239T, and K233N. Each VUS was engineered into an adenoviral TPM1 vector which was in turn applied to human engineered heart tissues (hEHTs) made from iPSC-derived cardiomyocytes. One week after viral exposure, isometric contractile behavior of hEHTs was measured. hEHTs expressing A102D were markedly hypercontractile, with a significant increase in contractile force and slowing of relaxation. D258E expression caused a mild phenotype, showing only a mildly increased relaxation rate relative to control. A238T and K233N both caused a dramatic decrease in contractile force. These same four VUS were studied in regulated in vitro motility assays and showed patterns of increased or decreased myofilament activity that were entirely consistent with findings in hEHT. A102D displayed a hypercontractile phenotype that resembled that of our benchmark HCM mutation (E62Q), while A238T and K233N produced contractile weakness that closely mimicked that of our benchmark DCM mutation (E54K). D258E had a minimal effect that could be interpreted as mildly hypertrophic. We conclude that hEHTs have the potential to serve as a scalable platform for evaluating pathogenicity of TPM1 VUS.

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) has been shown to be an independent risk factor for heart failure and is associated with increased cardiomyopathy and mortality in cancer patients. Given that CHIP is associated with increased inflammatory cytokines, we sought to characterize the CHIP secretome in cancer patients and its impact on human engineered heart tissue (EHT) function. Methods: We used machine learning to identify top cytokine features associated with CHIP in a cardio oncology cohort of 173 patients. Serum from breast cancer patients who were age, co-morbidity and medication matched between CHIP and non CHIP were used in the experiments. We used human EHTs to assess whether CHIP serum treatment affects contractility and used RNA sequencing to identify significantly differentially expressed pathways. Cytokine profiling of serum samples was done using a Luminex platform for 71 human cytokines. Results: Machine learning identified IL-1B and CD40L as two top features of CHIP. Given DNMT3A CHIP was the most common in the cardio onc cohort, we focused on evaluating the effect of DNMT3A CHIP serum on EHT function. Compared to non CHIP serum, DNMT3A CHIP serum significantly reduced EHT contractility (p= 0.03). To evaluate whether the top cytokines identified by our machine learning model could contribute to the impairment in contractility, we co-incubated CHIP and Non-CHIP serum treated EHTs with antagonists of IL-1B or CD40L. Treatment with either IL-1B or CD40L antagonists rescued contractility defects in CHIP serum treated EHTs (p=0.015, 0.0095, respectively) but had no significant impact on non CHIP serum treated EHTs. RNA sequencing of EHTs and pathway analysis revealed differential expression of genes and pathways in CHIP-treated and Non-CHIP serum-treated EHTs as well as in IL-1B antagonist and CD40L antagonist treated EHTs. Inflammation, metabolism pathways were top pathways differentially regulated in CHIP vs non CHIP serum treated EHTs and antagonist treatment showed modulation of these pathways. Conclusion: Identification of IL-1B as a top feature of CHIP is consistent with prior work. CD40L, however, has not previously been implicated in CHIP and may be more unique to cancer patients with CHIP. We show that antagonizing this pathway was able to rescue the contractility defect in CHIP serum treated EHTs, which could be another therapeutic target in patients with CHIP. The effect of other CHIP variants are underway.

Introduction: Cascade genetic screening for risk stratification is standard practice in the family of a patient diagnosed with hypertrophic cardiomyopathy (HCM), provided that a pathogenic mutation is present. We sought an alternative to genetic testing for cases where the patient lacks any suspicious variants (genotype-negative). Hypothesis: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes from HCM patients with pathogenic mutations exhibit one or more altered contractile parameters when formed into engineered heart tissues (EHTs). These include elevated peak force, delayed relaxation, or blunted length-dependent activation (LDA). We hypothesized that EHTs derived from genotype-negative HCM patients would show similar contractile aberrations. If so, aberrant contractile phenotype could serve as a marker of HCM risk in screening family members when genetic testing is inconclusive. Methods: A total of 18 iPSC lines were obtained from a combination of public biobanks, institutional sources, and blood samples from consenting patients. These included lines from apparently healthy individuals (n=11) and from genotype-unknown or genotype-negative HCM patients (n=7). Four of the healthy lines were randomly selected as reference controls and the remaining 14 lines formed a blinded cohort. iPSCs were differentiated into cardiomyocytes and formed into EHTs using MyoPod tissue scaffolds. Following three weeks of culture, EHT contractility and LDA were assessed. Bootstrapping analysis was used to compare blinded EHT behavior against the reference group to mimic a diagnostic test. Results: None of the genotype-negative EHT groups showed statistically significant contractile differences compared to reference panels. One apparently healthy control EHT showed a significant (p<0.05) increase in contractile force; exome sequencing revealed a rare FHOD3 mutation (D284N). Only one HCM patient line showed statistically significant change in contractility relative to reference (31% drop in normalized LDA, p<0.05). Sequencing revealed a known pathogenic HCM mutation ( MYH7 K351E) in that patient. Conclusions: EHTs derived from genotype-negative HCM patients do not exhibit detectable alterations in contractile behavior, but patient EHTs harboring suspected or known pathogenic variants reliably do. This method has the potential to determine mutation penetrance in specific patients or pathogenicity of novel variants.

Dozens of mutations to genes encoding proteins of the cardiac sarcomere have been associated with familial forms of hypertrophic cardiomyopathy (HCM). Patients carrying sarcomeric mutations constitute a large fraction of the total HCM population, but a significant portion of individuals with unexplained left-ventricular hypertrophy are not found to have rare sarcomeric variants. The etiology of disease in these so-called genotype-negative phenotype-positive (G-P+) patients remains essentially unknown. We and others have repeatedly demonstrated aberrant contractile behavior in cells or tissues expressing known HCM-causing sarcomeric mutations. Typically, this involves elevated contractile force, slowed relaxation, blunted length-dependent activation, or some combination of these factors. We hypothesized that G-P+ tissues would also display some subset of these same aberrant behaviors. In order to test our hypothesis, induced pluripotent stem cell (iPSC) lines were developed from three G-P+ HCM patients as well as male and female control subjects. iPSCs were differentiated into cardiomyocytes and seeded into laser-cut decellularized cryosections of porcine myocardium to form ribbon-like engineered heart tissues (EHTs). EHTs were cultured for three weeks before being subjected to mechanical testing to assess key dimensions of contractile performance. Preliminary results indicate both disease- and sex-dependent changes to EHT contractile behavior. Ultimately, data produced in this study are anticipated to provide early insights into potential sources of cardiac hypertrophy in patients that lack recognizable variations in sarcomeric genes.

Hypertrophic (HCM) and dilated cardiomyopathy (DCM) can both be caused by missense mutations in alpha-tropomyosin (TPM1). Previous studies on TPM1 mutations E62Q (HCM) and E54K (DCM) have shown divergent changes in phenotype including changes in cardiomyocyte morphology, altered calcium sensitivities, changes in isometric force production and impaired length dependent activation. However, there is a lack of understanding regarding the precise mechanisms through which these point mutations bring about observed phenotypic diversity.

Heritable forms of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) represent starkly diverging clinical phenotypes, yet may be caused by mutations to the same sarcomeric protein. The precise mechanisms by which point mutations within the same gene bring about phenotypic diversity remain unclear. Our objective was to develop a mechanistic explanation of diverging phenotypes in two TPM1 mutations, E62Q (HCM) and E54K (DCM). Drawing on data from the literature and experiments with stem cell-derived cardiomyocytes expressing the TPM1 mutations of interest, we constructed computational simulations that provide plausible explanations of the distinct muscle contractility caused by each variant. In E62Q, increased calcium sensitivity and hypercontractility was explained most accurately by a reduction in effective molecular stiffness of tropomyosin and alterations in its interactions with the actin thin filament that favor the "closed" regulatory state. By contrast, the E54K mutation appeared to act via long-range allosteric interactions to increase the association rate of the C-terminal troponin I mobile domain to tropomyosin/actin. These mutation-linked molecular events produced diverging alterations in gene expression that can be observed in human engineered heart tissues. Modulators of myosin activity confirmed our proposed mechanisms by rescuing normal contractile behavior in accordance with predictions.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder often caused by mutations to sarcomeric genes. Many different HCM-associated TPM1 mutations have been identified but they vary in their degrees of severity, prevalence, and rate of disease progression. The pathogenicity of many TPM1 variants detected in the clinical population remains unknown. Our objective was to employ a computational modeling pipeline to assess pathogenicity of one such variant of unknown significance, TPM1 S215L, and validate predictions using experimental methods. Molecular dynamic simulations of tropomyosin on actin suggest that the S215L significantly destabilizes the blocked regulatory state while increasing flexibility of the tropomyosin chain. These changes were quantitatively represented in a Markov model of thin-filament activation to infer the impacts of S215L on myofilament function. Simulations of in vitro motility and isometric twitch force predicted that the mutation would increase Ca2+ sensitivity and twitch force while slowing twitch relaxation. In vitro motility experiments with thin filaments containing TPM1 S215L revealed higher Ca2+ sensitivity compared with wild type. Three-dimensional genetically engineered heart tissues expressing TPM1 S215L exhibited hypercontractility, upregulation of hypertrophic gene markers, and diastolic dysfunction. These data form a mechanistic description of TPM1 S215L pathogenicity that starts with disruption of the mechanical and regulatory properties of tropomyosin, leading thereafter to hypercontractility and finally induction of a hypertrophic phenotype. These simulations and experiments support the classification of S215L as a pathogenic mutation and support the hypothesis that an inability to adequately inhibit actomyosin interactions is the mechanism whereby thin-filament mutations cause HCM.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have wide potential application in basic research, drug discovery, and regenerative medicine, but functional maturation remains challenging. Here, we present a method whereby maturation of hiPSC-CMs can be accelerated by simultaneous application of physiological Ca2+ and frequency-ramped electrical pacing in culture. This combination produces positive force-frequency behavior, physiological twitch kinetics, robust β-adrenergic response, improved Ca2+ handling, and cardiac troponin I expression within 25 days. This study provides insights into the role of Ca2+ in hiPSC-CM maturation and offers a scalable platform for translational and clinical research.

Hypertrophic (HCM) or dilated cardiomyopathy (DCM) can be caused by missense mutations in alpha-tropomyosin (TPM1). In this study, we focus on the TPM1 mutations E62Q (HCM) and E54K (DCM), which were introduced homozygously into separate induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9. iPSC-derived cardiomyocytes were seeded into decellularized myocardial sections to form human engineered heart tissues (EHT) that are suitable for biomechanical evaluation of muscle phenotypes. After two weeks in culture, E62Q EHTs displayed a nearly 3-fold increase in peak isometric twitch force relative to isogenic control with no change in relative slope of length dependent activation.

Nanomaterials (NMs) have revolutionized multiple aspects of medicine by enabling novel sensing, diagnostic, and therapeutic approaches. Advancements in processing and fabrication have also allowed significant expansion in the applications of the major classes of NMs based on polymer, metal/metal oxide, carbon, liposome, or multi-scale macro-nano bulk materials. Concomitantly, concerns regarding the nanotoxicity and overall biocompatibility of NMs have been raised. These involve putative negative effects on both patients and those subjected to occupational exposure during manufacturing. In this review, we describe the current state of testing of NMs including those that are in clinical use, in clinical trials, or under development. We also discuss the cellular and molecular interactions that dictate their toxicity and biocompatibility. Specifically, we focus on the reciprocal interactions between NMs and host proteins, lipids, and sugars and how these induce responses in immune and other cell types leading to topical and/or systemic effects.

Hypertrophic Cardiomyopathy (HCM) is an inherited disorder often caused by mutations in sarcomeric proteins. The disease is characterized by thickening of the left ventricular wall, hypercontractility, and microstructural tissue abnormalities. Although HCM causing mutations in the thick filaments are more prevalent, thin filament mutations such as those in alpha-tropomyosin (TPM1) often exhibit severe HCM phenotypes. Approximately twenty HCM associated TPM1 mutations have been identified which all vary in their degrees of severity, prevalence and disease progression. Using isogenic mutant human induced pluripotent stem cell lines, we attempt to shed light on the pathogenicity of a tropomyosin variant of unknown significance S215L. Co-sedimentation assays and ATPase assays using purified S215L TPM1 protein variants have previously shown significantly reduced actin affinity of TPM1 and increased calcium sensitivity respectively. Molecular dynamics simulations of key tropomyosin regulatory states reveal the altered atomic level interactions in tropomyosin caused by the S215L mutation that potentially lead to the hypertrophic phenotype. Preliminary kinetic viscosity measurements suggest that the end-to-end bond strength of the mutant protein is higher than that of the wild-type. To assess the impact of the mutation in a more native system, we utilized three-dimensional engineered heart tissues (EHT) generated by seeding iPSC-derived cardiomyocytes into decellularized porcine myocardial scaffolds. Initial characterization of TPM1 S215L in isometrically contracting EHTs shows a hypercontractile phenotype compared to its isogenic wild-type line, with an increased time to reach peak force as well as an increased relaxation time. Preliminary data from gene expression analysis on S215L EHTs seem to suggest that the expression of certain hypertrophic markers are upregulated. These data lend support to the idea that TPM1 mutations that are associated with hypertrophy act by reducing the ability of the thin filament to inhibit actomyosin interactions.

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.