Hypertrophic Cardiomyopathy Mice Research Articles

Carrying both the heterozygous Myh6-R453C and Tnnt2-R92W mutations aggravate the hypertrophic cardiomyopathy phenotype in mice

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart muscle that is dominated by variations in eight genes encoding sarcomere proteins. Although there are clinical or basic research reports that carrying double mutations can lead to more severe HCM phenotypes, there are also research reports that after reanalyzing the reported mutations, the severity of clinical symptoms in patients with double mutations did not significantly increase compared to patients with only one mutation. To determine whether double pathogenic mutations can aggravate the phenotype of hypertrophic cardiomyopathy in mice, we constructed mice carrying single pathogenic heterozygous mutation Myh6-R453C or Tnnt2-R92W and mice carrying both pathogenic heterozygous mutations. Our results showed that mice with double heterozygous mutations exhibited significant hypertrophic cardiomyopathy phenotypes at 4 weeks of age, and the degree of hypertrophy was significantly higher than that of single heterozygous mutant mice of the same age. Our study suggests that carrying the two pathogenic heterozygous mutations simultaneously can aggravate the phenotype of HCM in mice, which provides experimental evidence for the genotype-phenotype relationship of double pathogenic mutations and provides reference significance for clinical risk stratification of HCM patients.

Forskolin is an effective therapeutic small molecule for the treatment of hypertrophic cardiomyopathy through ADCY6/cAMP/PKA pathway

Hypertrophic cardiomyopathy (HCM) arises from a pathogenic variant in the gene responsible for encoding the myocardium-associated protein. Forskolin (FSK), a labdane diterpene isolated from Sphingomonas capillaris, exhibits diverse pharmacological effects, including bronchospasm relief, intraocular pressure reduction, and glaucoma treatment. However, whether FSK could regulate HCM and its associated mechanism remains unclear. Here, we discovered that FSK could mitigate cardiac hypertrophy in two HCM mouse models (Myh6R404Q and Tnnt2R109Q) in vivo. Additionally, FSK could prevent norepinephrine (NE)-induced cardiomyocyte hypertrophy in vitro. It reversed cardiac dysfunction, reduced enlarged cell size, and downregulated the expression of hypertrophy-related genes. We further demonstrated that FSK's mechanism in alleviating HCM relied on the activation of ADCY6. In conclusion, our findings demonstrate that FSK alleviates hypertrophic cardiomyopathy by modulating the ADCY6/cAMP/PKA pathway, suggesting that FSK holds promise as a therapeutic agent for HCM.

Microtubule-induced restriction of nuclear deformability in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq foundation Background Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the left ventricular (LV) wall in conjunction with diastolic dysfunction with a prevalence of ~1:200. HCM pathophysiology is incompletely understood. We hypothesized that misregulated mechanotransduction plays an important role in the disease process. Extensive cytoskeletal remodeling has been observed in HCM patients, with a robust increase in tubulin and desmin levels, with a concomitant increase in stabilizing post-translational modifications, which we previously showed contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex. Little is known about the interaction of the non-sarcomeric cytoskeleton with the nucleus and the role this may have in the development of HCM. Purpose Relate cytoskeleton changes to nuclear and chromatin morphology in HCM patient myocardium and assess nuclear deformability in contracting cardiomyocytes with and without microtubule destabilizing drugs. Methods Nuclear morphology and chromatin organization was assessed using electron microscopy in cardiac septal tissue from 19 patients with obstructive HCM and compared to samples from 4 non-failing donors (NF). Further nuclear changes were studied using wild type (WT) and homozygous Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, KI), which have a HCM phenotype. Nuclear morphology was assessed in slices of 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. To study nuclear deformability during contractions, adult cardiomyocytes were isolated, stained with live cell dyes and imaged live while electrically paced. Lastly, DNA damaged was assessed in 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. Results Cardiomyocyte nuclei of HCM patients had a significantly higher number of invaginations (0.32±0.09 invaginations/µm in HCM versus 0.18±0.03 invaginations/µm in NF hearts), were more irregular of shape and had altered chromatin organization compared to those from NF hearts. Nuclei of Mybpc3c.2373InsG mice were considerably larger (322±75 µm³ versus 221±24 µm³). While sarcomere shortening is similar (7.0±1.4% in Mybpc3c.2373InsG versus 6.5±1.7% in WT), nuclei from Mybpc3c.2373InsG mice are less deformable than WT nuclei (figure). Addition of microtubule destabilizer nocodazole or detyrosination inhibitor EPO-Y restored nuclear deformability to WT levels, indicating a role of detyrosinated microtubules in nuclear deformability. In addition, nuclei from Mybpc3c.2373InsG have more extensive DNA damage (19.7±3.4 foci in Mybpc3c.2373InsG versus 9.1±3.2 foci in WT). Conclusion Extensive nuclear changes are observed in HCM patients and a HCM mouse model. Cytoskeletal remodeling results in less deformable nuclei that might contribute to chromatin alterations and increased DNA damage. Nuclear deformability can be restored by microtubule modifying drugs, specifically those that decrease microtubule detyrosination.

Integrative analysis of transcriptome, DNA methylome and chromatin accessibility reveals candidate therapeutic targets in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM. DNA methylation and chromatin accessibility, as well as gene expression, were assessed by nucleosome occupancy and methylome sequencing (NOMe-seq) and RNA-seq, respectively, using the cardiac tissues of HCM patients. Compared with those of the controls, the transcriptome, DNA methylome and chromatin accessibility of the HCM myocardium showed multifaceted differences. At the transcriptome level, HCM hearts returned to the fetal gene program through decreased sarcomeric and metabolic gene expression and increased extracellular matrix gene expression. In the DNA methylome, hypermethylated and hypomethylated differentially methylated regions (DMRs) were identified in HCM. At the chromatin accessibility level, HCM hearts showed changes in different genome elements. Several transcription factors (TFs), including SP1 and EGR1, exhibited a fetal-like pattern of binding motifs in nucleosome-depleted regions (NDRs) in HCM. In particular, the inhibition of SP1 or EGR1 in an HCM mouse model harboring sarcomere mutations markedly alleviated the HCM phenotype of the mutant mice and reversed fetal gene reprogramming. Overall, this study not only provides a high precision multi-omics map of HCM heart tissue but also sheds light on the therapeutic strategy by intervening the fetal gene reprogramming in HCM.

Reduced Cardiac Beta 1 Receptor Expression and Function in a Mouse Model of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disorder. HCMis characterized by cardiac hypertrophy, fibrosis, and an increased risk of fatalarrhythmias and sudden cardiac death. Previous studies in humans with HCM havedemonstrated increased cardiac norepinephrine spillover and reduced beta-1 receptor(B1R) expression in the left ventricle (LV). We have previously demonstrated thatcardiac sympathetic tone is elevated in an alpha-tropomyosin mutant mouse model ofHCM. We hypothesized that HCM mice would demonstrate reduced B1R expression inthe LV and attenuated heart rate (HR) and dP/dt max responses to ramped infusion of theB1R agonist, dobutamine. First, we utilized western blot to measure B1R proteinexpression in LV tissue from HCM and littermate wild-type (WT) mice (n=4 male micein each group). B1R expression was significantly reduced in the LV of HCM micecompared to WT littermate controls (HCM 60±1% vs. WT 100±4%; paired t-test, p<0.05). Next, we recorded left ventricular pressure in male and female HCM andlittermate WT control (n=5 male mice, each group; n=6 female mice, each group) miceanesthetized with isoflurane (2% in O2). Data were analyzed by 2-way ANOVA withgenotype and sex as factors. Baseline HR and dP/dt max were significantly lower inHCM compared to WT mice (genotype p<0.05), however, there were no sex differencesin either variable (sex p>0.05). Next, in order to assess cardiac B1R function, we firstinfused the ganglionic blocker chlorisondamine (6ug/g) to eliminate autonomic reflexfunction in HCM and littermate WT mice (n=4 mice, each group). Then, we performed adobutamine stress test via ramped infusion of dobutamine starting at 2ng/g/min andincreasing the dose in 2ng/g/min increments every 2 minutes up to 12ng/g/min via aprogrammable syringe pump. Data were analyzed by 2-way ANOVA with dose andgenotype as factors. Contrary to our hypothesis, ramped infusion of dobutamine resultedin a similar, significant (dose p<0.05) increase in HR in both HCM and WT mice(genotype p>0.05). Peak increases in HR occurred during the highest dose ofdobutamine (12ng/g/min; ΔHR HCM 58±19 vs. WT 70±20 beats/min) in both groups.Consistent with our hypothesis, increases in dP/dt max were significantly blunted inHCM mice compared to littermate WT controls (dose x genotype p<0.05). Peakincreases in dP/dt max occurred at a lower dose of dobutamine for HCM (4ng/g/min;ΔdP/dt max 1129±767 mmHg/s) compared to WT (8ng/g/min; ΔdP/dt max 4882±499mmHg/s). In summary, LV B1R expression is reduced in HCM mice. Increases in HR inresponse to ramped infusion of dobutamine were similar between HCM and WT micewhereas increases in dP/dt max were attenuated in HCM mice. We speculate that thedifferential inotropic and chronotropic responses to dobutamine could be due to regionaldifferences in B1R expression or contractile dysfunction in HCM mice. Michigan Technological University Startup. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modeling cardiomyocyte signaling and metabolism predicts genotype-to-phenotype mechanisms in hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is a significant precursor of heart failure and sudden cardiac death, primarily caused by mutations in sarcomeric and structural proteins. Despite the extensive research on the HCM genotype, the complex and context-specific nature of many signaling and metabolic pathways linking the HCM genotype to phenotype has hindered therapeutic advancements for patients. Here, we have developed a computational model of HCM encompassing cardiomyocyte signaling and metabolic networks and their associated interactions. Utilizing a stochastic logic-based ODE approach, we linked cardiomyocyte signaling to the metabolic network through a gene regulatory network and post-translational modifications. We validated the model against published data on activities of signaling species in the HCM context and transcriptomes of two HCM mouse models (i.e., R403Q-αMyHC and R92W-TnT). Our model predicts that HCM mutation induces changes in metabolic functions such as ATP synthase deficiency and a transition from fatty acids to carbohydrate metabolism. The model indicated major shifts in glutamine-related metabolism and increased apoptosis after HCM-induced ATP synthase deficiency. We predicted that the transcription factors STAT, SRF, GATA4, TP53, and FoxO are the key regulators of cardiomyocyte hypertrophy and apoptosis in HCM in alignment with experiments. Moreover, we identified shared (e.g., activation of PGC1α by AMPK, and FHL1 by titin) and context-specific mechanisms (e.g., regulation of Ca2+ sensitivity by titin in HCM patients) that may control genotype-to-phenotype transition in HCM across different species or mutations. We also predicted potential combination drug targets for HCM (e.g., mavacamten plus ROS inhibitors) preventing or reversing HCM phenotype (i.e., hypertrophic growth, apoptosis, and metabolic remodeling) in cardiomyocytes. This study provides new insights into mechanisms linking genotype to phenotype in familial hypertrophic cardiomyopathy and offers a framework for assessing new treatments and exploring variations in HCM experimental models.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling

BackgroundHypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200–500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and resultsUsing high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. ConclusionsAltogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

Three-dimensional (3D) microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and their association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image-processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. Chamber-specific pathological alterations of LyVs were identified, and significant changes were seen in the right atrium (RA). TG hearts had a higher volume percent of ER-TR7+ fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7+ volume percent and both LyV segment density and median diameter. This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. Screening of 157 kinase inhibitors in wild-type (n=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (n=24 HCM and n=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (n=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

Beneficial effects of exercise initiated before development of hypertrophic cardiomyopathy in genotype-positive mice.

The effect of exercise on disease development in hypertrophic cardiomyopathy (HCM) genotype-positive individuals is unresolved. Our objective was to test the effect of exercise training initiated before phenotype development on cardiac fibrosis, morphology, and function in a mouse model of HCM. Genotype-positive Myh6 R403Q mice exposed to cyclosporine A (CsA) for induction of HCM (HCM mice) were allocated to high-intensity interval treadmill running or sedentary behavior for 6 wk. CsA was initiated from week 4 of the protocol. Cardiac imaging and exercise testing were performed at weeks 0, 3, and 6. After protocol completion, arrhythmia provocation was performed in isolated hearts, and left ventricles (LVs) were harvested for molecular biology and histology. Exercised HCM mice ran farther and faster and exhibited attenuated left atrial (LA) dilatation compared with sedentary mice. Exercised HCM mice had no difference in fibrosis compared with sedentary HCM mice despite lower expression of key extracellular matrix (ECM) genes collagen 1 and 3, fibronectin, and lysyl oxidase, accompanied by increased activation of Akt, GSK3b, and p38. Exercise did not have negative effects on LV function in HCM mice. Our findings indicate mild beneficial effects of exercise initiated before HCM phenotype development, specifically lower ECM gene expression and LA dilatation, and importantly, no detrimental effects.NEW & NOTEWORTHY Genotype-positive hypertrophic cardiomyopathy (HCM) mice had beneficial effects of exercise initiated before phenotype development. Exercised HCM mice had increased exercise capacity, smaller left atria, no increase in hypertrophy, or reduction of function, and a similar degree of fibrosis despite reduction of central extracellular matrix (ECM) genes, including collagens, compared with sedentary HCM mice.

Genome editing prevents hypertrophic cardiomyopathy in mice.

Genome editing prevents hypertrophic cardiomyopathy in mice.

Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies—an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9—to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.

In this study, we investigated the rescue potential of two phosphomimetic mutants of the myosin regulatory light chain (RLC, MYL2 gene), S15D, and T160D RLCs. S15D-RLC mimics phosphorylation of the established serine-15 site of the human cardiac RLC. T160D-RLC mimics the phosphorylation of threonine-160, identified by computational analysis as a high-score phosphorylation site of myosin RLC. Cardiac myosin and left ventricular papillary muscle (LVPM) fibers were isolated from a previously generated model of hypertrophic cardiomyopathy (HCM), Tg-R58Q, and Tg-wild-type (WT) mice. Muscle specimens were first depleted of endogenous RLC and then reconstituted with recombinant human cardiac S15D and T160D phosphomimetic RLCs. Preparations reconstituted with recombinant human cardiac WT-RLC and R58Q-RLC served as controls. Mouse myosins were then tested for the actin-activated myosin ATPase activity and LVPM fibers for the steady-state force development and Ca2+-sensitivity of force. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D-RLC or WT-RLC reconstituted preparations. The two S15D and T160D phosphomimetic RLCs were able to rescue Vmax of Tg-R58Q myosin reconstituted with recombinant R58Q-RLC, but the effect of S15D-RLC was more pronounced than T160D-RLC. Low tension observed for R58Q-RLC reconstituted LVPM from Tg-R58Q mice was equally rescued by both phosphomimetic RLCs. In the HCM Tg-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to the disordered relaxed (DRX) state, and the number of heads readily available to interact with actin and produce force was increased. At the same time, T160D-RLC stabilized the SRX state at a level similar to R58Q-RLC reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC vs. C-terminus T160-RLC, with S15D-RLC showing therapeutic potential in mitigating a non-canonical HCM behavior underlined by hypocontractile behavior of Tg-R58Q myocardium.

Abstract P2052: Lowered Pro-fibrotic And Damage Associated Molecular Pattern Gene Expressions After Exercise Training In A Mouse Model Of Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is the most frequent inherited cardiac disease, most often caused by mutations in sarcomere genes. The disease is characterized by left ventricular (LV) hypertrophy, increased risk of arrhythmias and diastolic dysfunction, due to cardiomyocyte hypercontractility, myocyte disarray and fibrosis. Fibrosis is a marker of worse prognosis, adverse remodeling and an increased risk of sudden cardiac death. Hypothesis: We hypothesized that exercise training could attenuate fibrosis development in mice genetically predisposed to HCM. Methods: We allocated male α-Myh6 R403Q/+ to high intensity interval treadmill running or sedentary behavior (N=11 per group) for a total of 6 weeks. HCM was induced in all mice by cyclosporine A given via the feed for the last 3 of the 6 week exercise protocol. All mice underwent echocardiography and an exercise test at weeks 0, 3, and 6, before harvesting LVs for digital droplet PCR (ddPCR) and a qPCR immune array. Results: Exercise training lead to a 2-fold increase in exercise capacity as measured by total distance ran during exercise testing (p<0.0001). In exercised HCM mice, several key fibrosis-related genes were lowered compared to sedentary HCM mice as measured by ddPCR (N=11 per group): Collagen 1 by 51 % (p=0.03), collagen 3 by 54 % (p=0.02), fibronectin by 51 % (p=0.01) and lysyl oxidase by 42 % (p=0.05). An array of 87 selected immune-related genes showed that 39 genes were upregulated >1.5 fold in sedentary HCM mice compared to sedentary wild type controls (N=3 samples, pooled). Twelve upregulated genes belonged to damage associated-molecular patterns (DAMPs). Several of the DAMP genes were lower in exercised HCM mice compared to sedentary HCM mice (N=10-11 per group): Biglycan was lowered by 29 %, versican by 22 % and fibronectin by 55 %. Trends were also found for lowered expression of syndecan 1 (by 26 %), fibromodulin (by 55 %), and tenacin C (by 49 %) (0.07<p<0.08). There was no difference in maximal wall thickness or function measured by echocardiography. Conclusion: Exercise training led to a lower expression of key fibrosis-related genes in an inducible mouse model of HCM. Initial results indicate that attenuation of DAMP signaling might be involved in the mechanism for this effect.

Ablation of lysophosphatidic acid receptor 1 attenuates hypertrophic cardiomyopathy in a mouse model

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

Genotype-Driven Pathogenesis of Atrial Fibrillation in Hypertrophic Cardiomyopathy: The Case of Different TNNT2 Mutations.

Atrial dilation and atrial fibrillation (AF) are common in Hypertrophic CardioMyopathy (HCM) patients and associated with a worsening of prognosis. The pathogenesis of atrial myopathy in HCM remains poorly investigated and no specific association with genotype has been identified. By re-analysis of our cohort of thin-filament HCM patients (Coppini et al. 2014) AF was identified in 10% of patients with sporadic mutations in the cardiac Troponin T gene (TNNT2), while AF occurrence was much higher (25–75%) in patients carrying specific “hot-spot” TNNT2 mutations. To determine the molecular basis of arrhythmia occurrence, two HCM mouse models expressing human TNNT2 variants (a “hot-spot” one, R92Q, and a “sporadic” one, E163R) were selected according to the different pathophysiological pathways previously demonstrated in ventricular tissue. Echocardiography studies showed a significant left atrial dilation in both models, but more pronounced in the R92Q. In E163R atrial trabeculae, in line with what previously observed in ventricular preparations, the energy cost of tension generation was markedly increased. However, no changes of twitch amplitude and kinetics were observed, and there was no atrial arrhythmic propensity. R92Q atrial trabeculae, instead, displayed normal ATP consumption but markedly increased myofilament calcium sensitivity, as previously observed in ventricular preparations. This was associated with reduced inotropic reserve and slower kinetics of twitch contractions and, importantly, with an increased occurrence of spontaneous beats and triggered contractions that represent an intrinsic arrhythmogenic mechanism promoting AF. The association of specific TNNT2 mutations with AF occurrence depends on the mutation-driven pathomechanism (i.e., increased atrial myofilament calcium sensitivity rather than increased myofilament tension cost) and may influence the individual response to treatment.

Abstract 14346: High Intensity Exercise Increases Cardiorespiratory Capacity in a Preclinical Hypertrophic Cardiomyopathy Mouse Model

Introduction: Patients with Hypertrophic Cardiomyopathy (HCM) who possess greater cardiorespiratory capacity (pVO 2 ) are at lower risk for heart transplant and mortality. High intensity exercise (HIE) training used in cardiac rehabilitation for heart failure and cardiovascular disease results in superior increases in pVO 2. The purpose of this investigation was to evaluate the effects of high intensity interval training (HIIT) on pVO 2 , as well as cardiac structural and functional parameters known to be altered by exercise in a transgenic cardiac troponin T (TG) HCM mouse model. Methods: Female and male C57BL/6J non-transgenic nTG (n=27) and TG (n=32) mice underwent a translationally parallel cardiac rehabilitation HIIT protocol. One treadmill training bout included 4-4 minute high intensity intervals (~80% preVO2 max speed) interspersed by 5-3 minute recovery intervals (~50% preVO2 max speed) for 31 total minutes. Bouts were repeated 3 times/wk for 6 wks. Pre and post HIIT pVO 2 and murine echocardiography were measured and analyzed by a blinded technician. Results: Following 6 weeks of the HIIT, nTG female (+13.5 mL/kg/min ± 3.6, p<0.01), nTG male (+13.5 mL/kg/min ± 3.6, p<0.01) and TG female (+7.06 ± 3.3 mL/kg/min, p<0.05) mice demonstrated an average increase in pVO 2 . TG male mice did not reach statistical significance (+4.34 ± p=0.12). Sedentary female (-15.9 mL/kg/min ± 3.3,p<0.001) and male (-13.1 mL/kg/min ± 3.9, p<0.01) TG control mice experienced a significant decrease in pVO 2 following the same period, while no change was observed in nTG sedentary controls. Independent of sex, nTG and TG HIIT mice had a significantly greater change in pVO 2 compared to sedentary counterparts. HIIT training did not result in changes to the following echocardiography measures in either nTG or TG mice: LV mass, LA volume, cardiac output, or stroke volume. Conclusions: Our results indicate that a translationally derived HIIT protocol improves cardiorespiratory capacity in HCM mice, absent of any adverse events or worsening of organ level disease pathology.

Exercise training inhibits left ventricular collagen upregulation in mice with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is estimated to affect 1:500, and is characterised by otherwise unexplained left ventricular (LV) hypertrophy, cardiomyocyte disarray, fibrosis, diastolic dysfunction, and ventricular arrhythmias. Historically, patients with HCM have been discouraged from participation in high intensity sports and exercise. However, the 2020 Sports Cardiology Guidelines recommend that patients with HCM should receive advice about exercise training (ET) based on individual risk assessment. To learn more about the effects of ET in HCM, we exposed mice carrying an HCM-causative sarcomere mutation (Myh6R403Q/+ (R403Q) mice) to high intensity interval training. Purpose To investigate the effect of exercise training on hypertrophic cardiomyopathy in mice. Methods R403Q mice were stratified to treadmill exercise (n=11) or sedentary behaviour (n=11). After 3 weeks, we induced HCM by giving CsA in the feed for 3 weeks, while the ET or sedentary behaviour continued for a total of 6 weeks. Each bout of treadmill running consisted of a 10-minute warm up, followed by 5 intervals of 8 minutes at high intensity (90% of VO2 max speed at week 0) and 2 minutes at medium intensity (60% of VO2 max speed at week 0). Every third day of the ET protocol we increased the high and medium intensity running speeds by 0.6 and 0.4 m/min, respectively. We performed echocardiography after 0, 3, and 6 weeks of the 6-week protocol. After completion of the protocol, we recorded lung and whole heart weight, and harvested LVs for molecular analyses. Results Confirming the expected HCM phenotype, R403Q mice that received CsA (R403Q SED+CsA) had a 1.3-fold increase in whole heart weight (p<0.0001), 1.5-fold increase in lung weight (p<0.001), and 2.4-fold increase in maximal left ventricular posterior wall (LVPW) thickness measured by echocardiography (p<0.0001) compared to sedentary wild type littermates given CsA (WT SED+CsA). Heart weight, lung weight, and maximum LVPW thickness were also increased 1.5- (p<0.0001), 1.6- (p<0.0001), and 2.0-(p<0.0001) fold, respectively, compared to R403Q not given CsA (R403Q SED-CsA). R403Q ET+CsA mice increased their running distance before exhaustion 2.0-fold compared to baseline (p=0.010), and ran 1.6-fold longer than R403Q SED+CsA (p=0.020). In R403Q ET+CsA mice, LV mRNA expression of Col1a2 was 51% (p=0.021), and Col3a1 49% (p=0.013) of R403Q SED+CsA expression levels. Compared to R403Q SED+CsA mice, exercise training did not affect heart weight, maximum LV posterior wall thickness or lung weight in R403Q ET+CsA mice. Conclusion Treadmill ET inhibited upregulation of LV collagen expression in mice with HCM, but did not affect hypertrophy. This could indicate that ET during early development of HCM attenuates development of fibrosis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority (Helse Sør-Øst RHF) and KG Jebsen Cardiac Research Center