Mouse Model Of Hypertrophic Cardiomyopathy Research Articles

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.

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.

Abstract 12927: Impact of AAV- MYBPC3 Gene Transfer on Heart Structure and Function in Human and Mouse Models of Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a life-threatening inherited heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. The most common cause of HCM is genetic variants in MYBPC3 , encoding cardiac myosin binding protein C (cMyBP-C), a sarcomeric protein with structural and regulatory roles. The majority of MYBPC3 gene variants are truncating leading to protein haploinsufficiency. Hypothesis: Transfer of a functional copy of MYBPC3 to heart muscle deficient in cMyBP-C will lead to sustained improvements in cardiac function. Aims: To determine whether BMN 293 (AAV-hMYBPC3) can restore cMyBP-C levels in the sarcomere and halt and/or reverse disease progression in non-clinical models of genetic HCM due to MYPBC3 deficiency. Methods: BMN 293 is an adeno-associated virus (AAV) vector that encodes wild-type human MYBPC3 under the control of a cardiomyocyte-selective promoter. We transduced human iPSC-derived cardiomyocytes and engineered heart tissues (EHTs) carrying a compound heterozygous truncating MYBPC3 mutation (MYBPC3 -/- ) with BMN 293 and assessed cMyBP-C levels and contractile parameters. We also systemically administered BMN 293 to MYBPC3 -/- mice and assessed cardiac distribution of human cMyBP-C by molecular and histological methods and determined its impact on left ventricular hypertrophy and function by echocardiography and other imaging techniques. Results: BMN 293 transduction of human iPSC MYBPC3 -/- cardiomyocytes and EHTs resulted in high levels of human MYBPC3 mRNA and cMyBP-C protein, correct incorporation of cMyBP-C into the sarcomere, and complete normalization of contractile kinetics. BMN 293 was well tolerated in MYBPC3 -/- mice and resulted in uniform restoration of cMyBP-C expression throughout the heart and significant correction of structural and functional cardiac abnormalities. Conclusions: A single IV infusion of BMN 293 to MYBPC3 -/- mice resulted in early and sustained reduction in left ventricular hypertrophy and durable improvements in diastolic function.

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.

Improved Cardiac Performance and Decreased Arrhythmia in Hypertrophic Cardiomyopathy With Non-β-Blocking R-Enantiomer Carvedilol.

Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. β-Adrenergic receptor antagonists (β-blockers) are the first-line therapy for HCM. However, β-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown. We screened 21 β-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM (MYH7R403Q/+). We identified that carvedilol, a β-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a β1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of β-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ iPSC-derived cardiomyocytes. R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.

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.

Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice.

The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by variants in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia and sudden cardiac death. The dominant-negative c.1208G>A (p.R403Q) pathogenic variant (PV) in β-myosin (MYH7) is a common and well-studied PV that leads to increased cardiac contractility and HCM onset. In this study we identify an adenine base editor and single-guide RNA system that can efficiently correct this human PV with minimal bystander editing and off-target editing at selected sites. We show that delivery of base editing components rescues pathological manifestations of HCM in induced pluripotent stem cell cardiomyocytes derived from patients with HCM and in a humanized mouse model of HCM. Our findings demonstrate the potential of base editing to treat inherited cardiac diseases and prompt the further development of adenine base editor-based therapies to correct monogenic variants causing cardiac disease.

Lumican accumulates with fibrillar collagen in fibrosis in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in-depth assessment of lumican in clinical and experimental HCM. Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n=15), and controls from hearts deemed unsuitable for transplantation (n=8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild-type littermates (n=8-10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid-ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n=3) and examined using confocal microscopy. Lumican mRNA was increased threefold in HCM patients (P<0.05) and correlated strongly with expression of collagen I (R2 =0.60, P<0.01) and III (R2 =0.58, P<0.01). Lumican protein was increased by 40% in patients with HCM (P<0.01) and correlated with total (R2 =0.28, P=0.05) and interstitial (R2 =0.30, P<0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P<0.001), and lumican protein increased 20-fold (P<0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co-localization measured in patients and mice with HCM (patients: +19%, P<0.01; mice: +13%, P<0.01). dSTORM super-resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to (-15nm, P<0.05), overlapped more frequently with (+7.3%, P<0.05) and to a larger degree with (+5.6%, P<0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8nm, P<0.001), longer (+345.9nm, P<0.001), and fewer (-8.9%, P<0.001) collagen fibres. The ECM proteoglycan lumican is increased in HCM and co-localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM.

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&lt;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 &gt;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&lt;p&lt;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.

BRG1 is a biomarker of hypertrophic cardiomyopathy in human heart specimens

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the sarcomere that causes otherwise unexplained cardiac hypertrophy and is associated with sudden death. While previous studies showed the role of the epigenetic modifier Brg1 in mouse models of HCM, additional work is needed to identify its role in humans. We tested the hypothesis that BRG1 expression is increased in periods of cardiac remodeling during fetal growth and in development of HCM. We employed immunohistochemical staining to evaluate protein expression of BRG1 in 796 human cardiac specimens (81 from patients with HCM) and describe elevated BRG1 expression in human fetal hearts in early development. In addition, we not only demonstrate increased expression of BRG1 in HCM, but we also show that other diseases that lead to heart failure have similar BRG1 expression to healthy controls. Inhibition of BRG1 in human induced pluripotent stem cell-derived cardiomyocytes significantly decreases MYH7 and increases MYH6, suggesting a regulatory role for BRG1 in the pathological imbalance of the two myosin heavy chain isoforms in human HCM. These data are the first demonstration of BRG1 as a specific biomarker for human HCM and provide foundation for future studies of epigenetics in human cardiac disease.

High Intensity Exercise Improves Cardiorespiratory Capacity in a Mouse Model of Hypertrophic Cardiomyopathy

IntroductionCardiorespiratory capacity (pVO2) is an important prognostic indicator in patients with HCM. Individuals with HCM that have a lower pVO2 possess a greater risk for heart failure (HF) and mortality. High intensity exercise (HIE) can increase pVO2 to a greater degree compared to other training modalities, including when used for cardiac rehabilitation for in cardiac disease states (e.g., HF and Cardiovascular disease). The effects of HIE on HCM, however, are largely unknown. Our study sought to evaluate the effects of High Intensity Interval Training (HIIT) on pVO2, as well as cardiac structural and functional parameters, in a preclinical mouse model of HCM.MethodsFemale and male C57BL/6J non‐transgenic nTG (n=27) and TG (n=32) mice were subjected to HIIT protocol that mirrored methods used clinically in cardiac rehabilitation. A single treadmill training session 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. Exercise training sessions were repeated 3 times per week for 6 weeks. Pre and post pVO2 and murine echocardiography were measured and analyzed by a blinded technician.ResultsFollowing 6 weeks of the HIIT, nTG female (+13.5 mL/kg/min ± 3.6, p&lt;0.01), nTG male (+13.5 mL/kg/min ± 3.6, p&lt;0.01) and TG female (+7.06 ± 3.3 mL/kg/min, p&lt;0.05) mice demonstrated an average increase in pVO2. A change in pVO2 observed in TG male mice did not reach statistical significance (+4.34 ± p=0.12). Sedentary female (‐15.9 mL/kg/min ± 3.3, p&lt;0.001) and male (‐13.1 mL/kg/min ± 3.9, p&lt;0.01) TG control mice experienced a significant decrease in pVO2 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 pVO2 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.ConclusionOur results indicate that a translationally derived HIIT protocol improved cardiorespiratory capacity in a preclinical HCM mouse model, absent of adverse events or worsening of organ level disease pathology.

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.

CMYA5 is a novel interaction partner of FHL2 in cardiac myocytes.

Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.

Abstract 12856: Novel Four-Dimensional Ultrasound Metric Improves Detection of Heart Failure Progression in Hypertrophic Cardiomyopathy

Introduction: Progression to heart failure is a known consequence of longstanding hypertrophic cardiomyopathy (HCM); however, routine function markers such as ejection fraction (EF) are often inadequate to predict dysfunction progression. The objective of this abstract was to create a novel metric, the Hybrid Strain Index (HSI), that can provide better characterization of heart failure progression. We hypothesized that longitudinal changes in a mouse model with known cardiac dysfunction could be more accurately differentiated with HSI than EF. Methods: Longitudinal 4D ultrasound (4DUS) data from mice with hypertrophic cardiomyopathy secondary to cardiac deletion of the carnitine palmitoyltransferase 2 enzyme ( Cpt2 M-/- ; n=12) and littermate controls (n=14) were analyzed using a custom MATLAB toolbox that derived both global volumetric (e.g., EF) and regional kinematic (e.g., circumferential and longitudinal strain) measurements. The circumferential and longitudinal regions most sensitive to disease progression were identified as the base (E θθ ) and posterior-wall (E LL ), respectively. The HSI metric was then calculated as the L2 norm of those peak-strain values. Results: To identify the earliest age of deviation between Cpt2 M-/- and controls, 95% confidence intervals from linear regression through HSI values intersected at 5.7 weeks old, compared to 7.1 weeks old using EF, suggesting HSI provides earlier sensitivity to cardiac dysfunction. Additionally, area-under-curve (AUC) measurements from ROC analysis of each metric, regardless of age, showed greater differentiation between cohorts using HSI (AUC = 0.91) than EF (AUC = 0.84). This further suggests enhanced diagnostic value of HSI compared to EF in the setting of cardiac dysfunction. Conclusions: The proposed HSI metric demonstrated greater sensitivity to detecting cardiac dysfunction and disease progression, compared to EF, in a mouse model of hypertrophic cardiomyopathy ( Cpt2 M-/- ).

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&lt;0.01), nTG male (+13.5 mL/kg/min ± 3.6, p&lt;0.01) and TG female (+7.06 ± 3.3 mL/kg/min, p&lt;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&lt;0.001) and male (-13.1 mL/kg/min ± 3.9, p&lt;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.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].