Hypertrophic Phenotype Research Articles

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.

Methotrexate inhibits BMP4 and abrogates the hypertrophic chondrocyte phenotype of synovial fibroblasts in juvenile idiopathic arthritis

BackgroundJuvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS.MethodsWe selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4.ResultsColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells.ConclusionsMethotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation.

TARGETING HYPERTROPHIC CHONDROCYTES IN OSTEOARTHRITIS: THE ROLE OF INFLAMMATION AND THE USE OF A MULTI-MODEL APPROACH IN SEARCH OF PHARMACOLOGICAL TREATMENTS

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780).In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis.Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants.In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis.

Collagen type X expression and chondrocyte hypertrophic differentiation during OA and OS development.

Chondrocyte hypertrophy and the expression of its specific marker, the collagen type X gene (COL10A1), constitute key terminal differentiation stages during endochondral ossification in long bone development. Mutations in the COL10A1 gene are known to cause schmid type metaphyseal chondrodysplasia (SMCD) and spondyloepiphyseal dyschondrodysplasia (SMD). Moreover, abnormal COL10A1 expression and aberrant chondrocyte hypertrophy are strongly correlated with skeletal diseases, notably osteoarthritis (OA) and osteosarcoma (OS). Throughout the progression of OA, articular chondrocytes undergo substantial changes in gene expression and phenotype, including a transition to a hypertrophic-like state characterized by the expression of collagen type X, matrix metalloproteinase-13, and alkaline phosphatase. This state is similar to the process of endochondral ossification during cartilage development. OS, the most common pediatric bone cancer, exhibits characteristics of abnormal bone formation alongside the presence of tumor tissue containing cartilaginous components. This observation suggests a potential role for chondrogenesis in the development of OS. A deeper understanding of the shifts in collagen X expression and chondrocyte hypertrophy phenotypes in OA or OS may offer novel insights into their pathogenesis, thereby paving the way for potential therapeutic interventions. This review systematically summarizes the findings from multiple OA models (e.g., transgenic, surgically-induced, mechanically-loaded, and chemically-induced OA models), with a particular focus on their chondrogenic and/or hypertrophic phenotypes and possible signaling pathways. The OS phenotypes and pathogenesis in relation to chondrogenesis, collagen X expression, chondrocyte (hypertrophic) differentiation, and their regulatory mechanisms were also discussed. Together, this review provides novel insights into OA and OS therapeutics, possibly by intervening the process of abnormal endochondral-like pathway with altered collagen type X expression.

Spatiotemporal Modulated Scaffold for Endogenous Bone Regeneration via Harnessing Sequentially Released Guiding Signals.

The design of a scaffold that can regulate the sequential differentiation of bone marrow mesenchymal stromal cells (BMSCs) according to the endochondral ossification (ECO) mechanism is highly desirable for effective bone regeneration. In this study, we successfully fabricated a dual-networked composite hydrogel composed of gelatin and hyaluronic acid (termed GCDH-M), which can sequentially release chondroitin sulfate (CS) and magnesium/silicon (Mg/Si) ions to provide spatiotemporal guidance for chondrogenesis, angiogenesis, and osteogenesis. The fast release of CS is from the GCDH hydrogel, and the sustained releases of Mg/Si ions are from poly(lactide-co-glycolide) microspheres embedded in the hydrogel. There is a difference in the release rates between CS and ions, resulting in the ability for the fast release of CS and sustained release of ions. The dual networks between the modified gelatin and hyaluronic acid via covalent bonding and host-guest interactions render the hydrogel with some dynamic feature to meet the differentiation development of BMSCs laden inside the hydrogel, i.e., transforming into a chondrogenic phenotype, further to a hypertrophic phenotype and eventually to an osteogenic phenotype. As evidenced by the results of in vitro and in vivo evaluations, this GCDH-M composite hydrogel was proved to be able to create an optimal microenvironment for embedded BMSCs responding to the sequential guiding signals, which aligns with the rhythm of the ECO process and ultimately boosts bone regeneration. The promising outcome achieved with this innovative hydrogel system sheds light on novel scaffold design targeting bone tissue engineering.

Electrophysiological mechanisms underlying T wave pseudonormalisation on stress ECGs in hypertrophic cardiomyopathy

BackgroundPseudonormal T waves may be detected on stress electrocardiograms (ECGs) in hypertrophic cardiomyopathy (HCM). Either myocardial ischaemia or purely exercise-induced changes have been hypothesised to contribute to this phenomenon, but the precise electrophysiological mechanisms remain unknown. MethodsComputational models of human HCM ventricles (n = 20) with apical and asymmetric septal hypertrophy phenotypes with variable severities of repolarisation impairment were used to investigate the effects of acute myocardial ischaemia on ECGs with T wave inversions at baseline. Virtual 12-lead ECGs were derived from a total of 520 biventricular simulations, for cases with regionally ischaemic K+ accumulation in hypertrophied segments, global exercise-induced serum K+ increases, and/or increased pacing frequency, to analyse effects on ECG biomarkers including ST segments, T wave amplitudes, and QT intervals. ResultsRegional ischaemic K+ accumulation had a greater impact on T wave pseudonormalisation than exercise-induced serum K+ increases, due to larger reductions in repolarisation gradients. Increases in serum K+ and pacing rate partially corrected T waves in some anatomical and electrophysiological phenotypes. T wave morphology was more sensitive than ST segment elevation to regional K+ increases, suggesting that T wave pseudonormalisation may sometimes be an early, or the only, ECG feature of myocardial ischaemia in HCM. ConclusionsIschaemia-induced T wave pseudonormalisation can occur on stress ECG testing in HCM before significant ST segment changes. Some anatomical and electrophysiological phenotypes may enable T wave pseudonormalisation due to exercise-induced increased serum K+ and pacing rate. Consideration of dynamic T wave abnormalities could improve the detection of myocardial ischaemia in HCM.

In Vitro Chondrogenesis Induction by Short Peptides of the Carboxy-Terminal Domain of Transforming Growth Factor β1.

Τransforming growth factor β1 (TGF-β1) comprises a key regulator protein in many cellular processes, including in vivo chondrogenesis. The treatment of human dental pulp stem cells, separately, with Leu83-Ser112 (C-terminal domain of TGF-β1), as well as two very short peptides, namely, 90-YYVGRKPK-97 (peptide 8) and 91-YVGRKP-96 (peptide 6) remarkably enhanced the chondrogenic differentiation capacity in comparison to their full-length mature TGF-β1 counterpart either in monolayer cultures or 3D scaffolds. In 3D scaffolds, the reduction of the elastic modulus and viscous modulus verified the production of different amounts and types of ECM components. Molecular dynamics simulations suggested a mode of the peptides' binding to the receptor complex TβRII-ALK5 and provided a possible structural explanation for their role in inducing chondrogenesis, along with endogenous TGF-β1. Further experiments clearly verified the aforementioned hypothesis, indicating the signal transduction pathway and the involvement of TβRII-ALK5 receptor complex. Real-time PCR experiments and Western blot analysis showed that peptides favor the ERK1/2 and Smad2 pathways, leading to an articular, extracellular matrix formation, while TGF-β1 also favors the Smad1/5/8 pathway which leads to the expression of the metalloproteinases ADAMTS-5 and MMP13 and, therefore, to a hypertrophic chondrocyte phenotype. Taken together, the two short peptides, and, mainly, peptide 8, could be delivered with a scaffold to induce in vivo chondrogenesis in damaged articular cartilage, constituting, thus, an alternative therapeutic approach for osteoarthritis.

Inflammatory and Metabolic Signaling Interfaces of the Hypertrophic and Senescent Chondrocyte Phenotypes Associated with Osteoarthritis.

Osteoarthritis (OA) is a complex disease of whole joints with progressive cartilage matrix degradation and chondrocyte transformation. The inflammatory features of OA are reflected in increased synovial levels of IL-1β, IL-6 and VEGF, higher levels of TLR-4 binding plasma proteins and increased expression of IL-15, IL-18, IL-10 and Cox2, in cartilage. Chondrocytes in OA undergo hypertrophic and senescent transition; in these states, the expression of Sox-9, Acan and Col2a1 is suppressed, whereas the expression of RunX2, HIF-2α and MMP-13 is significantly increased. NF-kB, which triggers many pro-inflammatory cytokines, works with BMP, Wnt and HIF-2α to link hypertrophy and inflammation. Altered carbohydrate metabolism and the upregulation of GLUT-1 contribute to the formation of end-glycation products that trigger inflammation via the RAGE pathway. In addition, a glycolytic shift, increased rates of oxidative phosphorylation and mitochondrial dysfunction generate reactive oxygen species with deleterious effects. An important surveyor mechanism, the YAP/TAZ signaling system, controls chondrocyte differentiation, inhibits ageing by protecting the nuclear envelope and suppressing NF-kB, MMP-13 and aggrecanases. The inflammatory microenvironment and synthesis of key matrix components are also controlled by SIRT1 and mTORc. Senescent chondrocytes represent the functional end stage of hypertrophic differentiation and characteristically upregulate p16 and p21, but also a variety of inflammatory cytokines, chemokines and metalloproteinases, developing the senescence-associated secretory phenotype. Senolysis with dendrobin, miR29b-5p and other agents has been shown to be efficient under experimental conditions, and appears to be a promising tool for the treatment of OA, as it restores COL2A1 and aggrecan synthesis, suppressing NF-kB and destructive metalloproteinases.

Right ventricular longitudinal strain in transthyretin cardiac amyloidosis (ATTR-CA)

Abstract Transthyretin cardiac amyloidosis (ATTR-CA) is a life-threatening condition characterized by a restrictive phisiology. This condition can be hereditary (ATTRv) or acquired (ATTRwt). Left ventricular global longitudinal strain (LVGLS) measured with speckle-tracking echocardiography (STE) has been well described and it appears to be significantly lower in ATTR-CA than in other hypertrophic phenotypes, presenting with a typical "apical sparing" pattern. Furthermore, LVGLS can predict prognosis among patients with ATTR-CA beyond LVEF. Few studies investigated the role of right ventricular longitudinal strain in ATTR-CA. The aim of this study is to describe right ventricular (RV) involvement in patients with ATTR-CA with the use of STE and to explore its prognostic implications. We retrospectively analyzed echocardiographic images of 221 patients with ATTR-CA evaluated between 1995 and 2019 at our Centre. Since the right ventricular free wall strain (RVFWSL) is the current standard parameter being evaluated when performing STE, we chose it as variable of interest among the others collected. Finally, univariate and multivariate linear regression analyses were used to analyze variables related with clinical events such as heart failure and death. The median age of our cohort was 76,7 years old and they were predominantly males. Among these, most patients had a diagnosis of ATTR-CA wild-type (only 32,1% of study population was affected by ATTRv). At STE, LVGLS was abnormal in both ATTRwt patients (-12%; IQR: -13,75% -10,15%) and ATTRv patients (-12,5%; IQR: -15,5% -10,73%) with no significant difference between groups (p= 0,1715). Furthermore, STE showed impaired right ventricular longitudinal function in both aetiologies, but with a different grade of involvement as the median RVFWLS was -19,8% (IQR: -25,1% -13,9% ) in the ATTRv group and -16,4% in the ATTRwt group (IQR: -21,65% -12,2%) (p=0,043). In our study, a right ventricular apical sparing pattern was not observed. In the univariate analysis, RVFWLS and other parameters such as age, eGFR, tricuspid regurgitation and LVGLS were significantly associated with heart failure, so they were forced into multivariable analysis, but RVFWLS did not show an independent correlation with the endpoint, although a certain trend toward significance could be observed (HR 1,037 95%CI 0.998 - 1.077; p=0,063). Finally, we explored the association between our collected variables and death. At univariate analysis, RVFWLS showed a strong correlation with death (HR 1,034; 95%CI 1.005 - 1.064; p= 0,021), together with NYHA class, eGFR, LVEF and LVGLS. Notably, RVFWLS predicted death at our multivariable regression analysis (HR 1.041 95%CI 1.010 - 1.072; p= 0,008). RVFWLS is an emerging parameter to evaluate RV dysfunction which appears significantly impaired in patients with ATTR-CA. In this setting, it might have not only a diagnostic role, but also a prognostic power, although further data are needed.

Betaine therapy of cardiac hypertrophy through inhibition of Hydroxy-carboxylic Acid Receptor 1 signaling

Abstract Background Hydroxy-carboxylic Acid Receptor 1 (HCAR1), formerly known as G protein-coupled Receptor 81 (GPR81), is a cell membrane receptor for lactate, and essential for activation of a variety of intracellular signaling pathways. We found a correlation between the activation of lactate-HCAR1 signaling and occurrence of cardiac hypertrophy. However, the regulatory mechanism remains unclear. Purpose Our purpose is to investigate the regulatory mechanism between the activation of lactate-HCAR1 signaling and occurrence of cardiac hypertrophy. Methods Neonatal rat cardiomyocytes (NRCMs) were used for preparing cardiac hypertrophy model in vitro by phenylephrine (PE) stimulation, and C57 BL/6 mice were used for preparing pressure overloaded-induced cardiac hypertrophy model in vivo by TAC (transverse aortic constriction) surgery. Adenovirus containing shRNA-HCAR1 (ADV-shHCAR1) were constructed to knockdown HCAR1 in NRCMs. Binding interaction between HCAR1 and Betaine was predicted by AUTODOCK 4 and further validated with RNA-seq analysis. Results Lactate concentration and HCAR1 expression were both increased in the NRCMs upon PE treatment. Activation of HCAR1 by its agonist 3-Chloro-5-hydroxybenzoic acid (3Cl-HBA) specially induced hypertrophy in NRCMs. Furthermore, the hypertrophy phenotypes in NRCMs showed alleviation by transfection of ADV-shHCAR1. An AI-based molecule-docking screening analysis identified Betaine as a potential molecule to bind HCAR1. Treatment with Betaine significantly attenuated PE- or 3Cl-HBA- induced hypertrophy in NRCMs in vitro. In addition, Betaine administration of TAC mice (i.g. 50 mg/kg/day) for 4 weeks in vivo led to increase of ejection fraction (EF) and decreases of left ventricular (LV) posterior wall thickness, heart weight/tibia length ((HW/TL), cross-sectional area and cardiac fibrosis area as well. RNA-seq analysis and functional assays demonstrated that Betaine could bind to HCAR1, activated Hippo signaling pathway, and thereby blocked the HCAR1-mediated hypertrophy in cardiomyocytes. Conclusions In summary, the current study not only identified HCAR1 as a therapeutic target of cardiac hypertrophy, but also demonstrated Betaine as a therapeutic candidate molecule holding potential to benefit cardiac hypertrophy patients.

Characterisation of hypertrophic response and the effect of a GATA4-targeting compound on the phenotype of hypertrophic cardiomyopathy patient-derived hiPSC-cardiomyocytes

Abstract Introduction Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an informative platform to study disease pathomechanisms. Transcription factor GATA4 serves a significant role in cardiomyocyte hypertrophy, and GATA4-targeted compounds inhibit hypertrophy [1]. Purpose This study aims at identifying phenotypic differences between patient-derived hiPSC-CMs and control hiPSC-CMs and at investigating the effect of a novel GATA4-targeting compound C-2021 on the patient-derived hiPSC-CMs phenotype. Methods hiPSC-CMs were differentiated from a healthy, control line (iPS(IMR90)-4; here, control hiPSC-CMs) and from HCM patient-derived hiPSCs (HCM hiPSC-CMs) that carry a mutation in the MYBPC3 gene. Two types of hypertrophic stimuli were applied to hiPSC-CMs: mechanical stretch and endothelin-1 (ET-1) treatment. For pharmacological assays, compound C-2021 (at 30 µM) or vehicle (0.1 % DMSO) were introduced to hiPSC-CMs one hour prior to the stimuli. hiPSC-CMs were exposed to cyclic equibiaxial mechanical stretching for 24 h or 72 h using Flexcell FX-5000 Tension System (Flexcell International Corporation) [2]. The expression of hypertrophic markers (such as NPPB, NPPA) and structural proteins (including α-myosin and β-myosin heavy chains; MYH6 and MYH7, respectively) was investigated with qPCR. The relative gene expression was analysed using the 2-ΔΔCt method using 18S and ACTB as reference genes. Furthermore, hiPSC-CMs were subjected to ET-1 at 100 nM for 24 h and proBNP expression was quantified using high-content analysis (HCA). Results HCM hiPSC-CMs displayed increased basal expression of NPPB (Fig. 1A-B), lower expression of MYH6 (Fig. 1C) and higher expression of MYH7 (Fig. 1D) compared to control hiPSC-CMs, which is in line with the hypertrophic disease phenotype. Control and HCM hiPSC-CMs responded to 24 h and 72 h mechanical stretching by increased NPPB expression (Fig. 1A-B). At 72 h, the increase in NPPB expression was more pronounced in the HCM hiPSC-CMs (Fig. 1B). The compound C-2021 inhibited stretch-induced NPPB expression after 72 h stretching. HCA results indicate that ET-1 induced a 17-fold increase in proBNP-positive control hiPSC-CMs, and this effect was attenuated by C-2021 treatment. Interestingly, ET-1 did not enhance proBNP expression in HCM hiPSC-CMs (Fig. 2). Conclusions Our results show that the HCM hiPSC-CMs differ from the control hiPSC-CMs in response to hypertrophic stimuli and in myosin heavy chain isoform expression. Additionally, C-2021 may have anti-hypertrophic effects in both in control and HCM hiPSC-CMs based on the downregulation of NPPB expression, and the decrease in proBNP-positive hiPSC-CMs. Further studies are needed to elucidate the exact mechanisms underlying HCM and the action of C-2021 on the HCM hiPSC-CM phenotype.

Enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction in patient-specific induced pluripotent stem cell-derived cardiomyocytes with MYH7 mutation

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the β-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca2+ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Abstract 15036: Identification of Fabry Disease Cardiac Phenotype in Hypertrophic Cardiomyopathy With Cardiac Magnetic Resonance Imaging Technology

Introduction: Cardiac magnetic resonance(CMR), especially T1 mapping, has an important role in the diagnosis of Fabry disease, but the differential value in myocardial diseases with hypertrophic phenotype needs further study. More importantly, there has been no systematic study of the difference between different CMR techniques indistinguishing Fabry cardiomyopathy from hypertrophic cardiomyopathy (HCM). Hypothesis: Myomapping technology can be a parameter in distinguishing Fabry cardiomyopathy from HCM. Methods: We continuously included patients with clinically diagnosed hypertrophic cardiomyopathy, completed systematic magnetic resonance imaging, and used genetic testing as the diagnostic criteria. We reported the prevalence rate in the population of HCM, and deeply conducted comparative studies on the differences in magnetic resonance characteristics between Fabry disease and HCM. We also analyzed the diagnostic value of magnetic resonance imaging in distinguishing Fabry cardiomyopathy from HCM. Results: We continuously included patients diagnosed with hypertrophic cardiomyopathy at West China Hospital from 2012 to 2022 and completed systematic magnetic resonance imaging examinations. We reported the prevalence of Fabry cardiomyopathy in the population using genetic testing as the diagnostic criteria, and deeply conducted comparative studies on the differences in CMR features between Fabry disease and HCM. We also analyzed the diagnostic value of CMR in distinguishing Fabry cardiomyopathy from HCM. Conclusions: We found a prevalence of 13 (2.6%) cases of Fabry disease in the hypertrophic cardiomyopathy cohort (494 cases). Our further analysis found that there were no significant differences in the degree of hypertrophy, overall cardiac function and size of Fabry patients compared with patients with HCM, as well as strain analysis. NativeT1 was significantly reduced in Fabry disease, and segmental analysis found that the basal and middle ventricular septum and anterior wall were the most obvious. ECV differed significantly between the two groups. There was no difference in T2 between the two groups. ROC analysis showed that T1 had the most discriminating value

Abstract 16562: Subclinical Flow Inefficiencies in Non-Obstructive Hypertrophic Cardiomyopathy Subgroups Revealed by 4D Flow Cardiovascular Magnetic Resonance

Background Patients with non-obstructive hypertrophic cardiomyopathy (nHCM) may exhibit flow inefficiencies despite normal flow profiles on transthoracic echocardiogram. We investigated whether left ventricular (LV) kinetic energy (KE) and hemodynamic forces (HDF) on 4D flow CMR are abnormal in nHCM, and examined the impact of genotype and hypertrophy phenotype on flow profiles. Methods 90 participants (70 HCM without echocardiographic outflow tract obstruction and 20 healthy controls) underwent 4D flow CMR. Patients were categorized as phenotype positive (P+) if they had LV hypertrophy (≥15mm or ≥13mm with an HCM family history), or phenotype negative (P-) referring to pre-hypertrophic pathogenic sarcomeric variant carriers. LV KE, HDF and transversal/longitudinal HDF ratio were computed from 4D flow CMR. Results P+ patients and controls were of similar age, but P- were younger. P+, P- and controls had comparable LV ejection fraction on CMR, and LV outflow tract pressure gradients and diastolic velocities on echocardiography (Table 1). Systolic KE was higher in P+ compared to controls (Table 1, Figure 1). Early diastolic KE was higher in P- than P+, reflecting younger P-, but did not differ between P+ and controls. Late diastolic KE was higher in P+ versus P- and controls. Systolic HDF ratio was higher in P+ than controls (Table 1, Figure 1). Diastolic HDF and HDF ratio were similar between P+ and controls. Conclusion Despite comparable flow velocities on echocardiography, nHCM exhibited higher systolic KE and HDF ratio, and late diastolic KE on 4D flow CMR relative to controls. Both the extent and pattern of hypertrophy are important determinants of abnormalities in KE and HDF.

Abstract 15426: Stress-Induced Transcriptomic Shift in Human iPSC-Derived Cardiomyocytes Carrying Hypertrophic Cardiomyopathy Associated MYH7 G256E Mutation

Introduction: Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as MYH7. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, it remains unclear whether the cellular phenotypes of low penetrant mutations such as MYH7 G256E and others, can be adequately elucidated. Hypothesis: We hypothesize that the incorporation of cellular stressors can enhance the manifestation of pathogenic HCM phenotypes in low penetrant MYH7 mutations. Methods: We generated HET and homozygous (HOM) hiPSC-lines for mutation G256E in the MYH7 locus, which is associated with HCM. We performed single-cell RNA sequencing (scRNAseq) with CellPlex technology on wildtype (WT), HET, and HOM iPSC-CMs at day 30 and 60 of cardiac differentiation to establish baseline cellular transcriptional phenotype. We then treated day 30 hiPSC-CMs with 1μM isoproterenol treatment for 5 days or glucose starvation for 10 days and performed scRNAseq to assess their transcriptional shift. Additional Digital Droplet PCR studies were performed to investigate the MYH7 mutant allele expression. Results: In day 30 iPSC-CMs we detected a large clone-to-clone and batch-to-batch variability transcriptionally that obscured the identification of a consistent mutant phenotype in HET MYH G256E vs WT hiPSC lines. These phenotypes correlated with a 1:1 ratio of mutant vs WT allele expression. Implementation of long-term culture or treatment with isoproterenol or glucose starvation led to enhanced hypertrophic and oxidative phosphorylation gene expression in HET hiPSC-CMs. Intriguingly, the expression of MYH6 increased in MYH7-mutant hiPSC-CMs. Conclusions: The application of cellular stressors can enhance the manifestation of hypertrophic phenotypes in HET MYH G256E hiPSC-CMs and suggests a potential mechanism for the variable penetrance of MYH7 mutations in HCM patients.

Effects of Different Generations and Sex on Physiological, Biochemical, and Growth Parameters of Crossbred Beef Cattle by Myostatin Gene-Edited Luxi Bulls and Simmental Cows.

(1) Background: Myostatin (MSTN) is a protein that regulates skeletal muscle development and plays a crucial role in maintaining animal body composition and muscle structure. The loss-of-function mutation of MSTN gene can induce the muscle hypertrophic phenotype. (2) Methods: Growth indexes and blood parameters of the cattle of different months were analyzed via multiple linear regression. (3) Results: Compared with the control group, the body shape parameters of F2 cattle were improved, especially the body weight, cross height, and hip height, representing significant development of hindquarters, and the coat color of the F2 generation returned to the yellow of Luxi cattle. As adults, MSTN gene-edited bulls have a tall, wide acromion and a deep, wide chest. Both the forequarters and hindquarters are double-muscled with clear muscle masses. The multiple linear regression demonstrates that MSTN gene-edited hybrid beef cattle gained weight due to the higher height of the hindquarters. Significant differences in blood glucose, calcium, and low-density lipoprotein. Serum insulin levels decreased significantly at 24 months of age. MSTN gene editing improves the adaptability of cattle. (4) Conclusions: Our findings suggest that breeding with MSTN gene-edited Luxi bulls can improve the growth and performance of hybrid cattle, with potential benefits for both farmers and consumers.

A rare case of Noonan syndrome associated with biallelic variants 
 in the LZTR1

Introduction. Noonan syndrome is a clinically and genetically heterogeneous disease with multiple organ involvement associated with mutations in the genes of the RAS/MAPK signalling pathway. Most patients with Noonan syndrom (up to 50–80%) have disorders of the cardiovascular system, presented by a wide range of congenital heart defects and/or cardiomyopathy, predominantly hypertrophic phenotype. Thanks to the introduction of high-throughput sequencing, knowledge of the genetic causes of Noonan syndrome has expanded significantly, so since 2014, the LZTR1 gene (OMIM 601247) has been included in the list of genes responsible for the development of Noonan syndrome. The nucleotide variants of this gene are known to be inherited both in an autosomal dominant and autosomal recessive manner. However, the number of reports describing the clinical and genetic characteristics of patients with LZTR1 gene mutations is scarce in the world scientific literature.
 Objective. To describe the clinical features of Noonan syndrome with an autosomal recessive type of inheritance caused by biallelic variants c.1259A>G (p.Q420R) and c.2051T>C (p.I684T) in the LZTR1 gene.
 Materials and methods. A detailed analysis of the history data, the results of clinical, laboratory, and instrumental studies, a molecular genetic study using high-throughput sequencing technology and direct Sanger sequencing was carried out. After verifying the biallelic variants in the proband, a search was made for the identified nucleotide substitutions in the venous blood samples of the parents and sibs.
 Results. The article presents the data of a clinical observation of a rare case of Noonan syndrome caused by pathogenic variants in the LZTR1 gene with an autosomal recessive type of inheritance by the Department of Cardiology of the National Medical Research Center for Children’s Health of the Ministry of Health of Russia.
 Conclusion. The diversity of clinical manifestations makes it difficult to diagnose Noonan syndrome based on phenotype alone. The possibility of using high-throughput sequencing improves the quality of diagnostics, contributes to the replenishment of data on new pathogenic variants and the establishment of genotype-phenotypic correlations.

ERRγ agonist under mechanical stretching manifests hypertrophic cardiomyopathy phenotypes of engineered cardiac tissue through maturation

Engineered cardiac tissue (ECT) using human induced pluripotent stem cell-derived cardiomyocytes is a promising tool for modeling heart disease. However, tissue immaturity makes robust disease modeling difficult. Here, we established a method for modeling hypertrophic cardiomyopathy (HCM) malignant (MYH7 R719Q) and nonmalignant (MYBPC3 G115∗) pathogenic sarcomere gene mutations by accelerating ECT maturation using an ERRγ agonist, T112, and mechanical stretching. ECTs treated with T112 under 10% elongation stimulation exhibited more organized and mature characteristics. Whereas matured ECTs with the MYH7 R719Q mutation showed broad HCM phenotypes, including hypertrophy, hypercontraction, diastolic dysfunction, myofibril misalignment, fibrotic change, and glycolytic activation, matured MYBPC3 G115∗ ECTs displayed limited phenotypes, which were primarily observed only under our new maturation protocol (i.e., hypertrophy). Altogether, ERRγ activation combined with mechanical stimulation enhanced ECT maturation, leading to a more accurate manifestation of HCM phenotypes, including non-cardiomyocyte activation, consistent with clinical observations.

SIRT2 counteracts primate cardiac aging via deacetylation of STAT3 that silences CDKN2B.

Aging is a major risk factor contributing to pathophysiological changes in the heart, yet its intrinsic mechanisms have been largely unexplored in primates. In this study, we investigated the hypertrophic and senescence phenotypes in the hearts of aged cynomolgus monkeys as well as the transcriptomic and proteomic landscapes of young and aged primate hearts. SIRT2 was identified as a key protein decreased in aged monkey hearts, and engineered SIRT2 deficiency in human pluripotent stem cell-derived cardiomyocytes recapitulated key senescence features of primate heart aging. Further investigations revealed that loss of SIRT2 in human cardiomyocytes led to the hyperacetylation of STAT3, which transcriptionally activated CDKN2B and, in turn, triggered cardiomyocyte degeneration. Intra-myocardial injection of lentiviruses expressing SIRT2 ameliorated age-related cardiac dysfunction in mice. Taken together, our study provides valuable resources for decoding primate cardiac aging and identifies the SIRT2-STAT3-CDKN2B regulatory axis as a potential therapeutic target against human cardiac aging and aging-related cardiovascular diseases.

Human engineered cardiac tissue model of hypertrophic cardiomyopathy recapitulates key hallmarks of the disease and the effect of chronic mavacamten treatment.

Introduction: The development of patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an opportunity to study genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM), one of the most common inherited cardiac diseases. However, immaturity of the iPSC-CMs and the lack of a multicellular composition pose concerns over its faithfulness in disease modeling and its utility in developing mechanism-specific treatment. Methods: The Biowire platform was used to generate 3D engineered cardiac tissues (ECTs) using HCM patient-derived iPSC-CMs carrying a β-myosin mutation (MYH7-R403Q) and its isogenic control (WT), withal ECTs contained healthy human cardiac fibroblasts. ECTs were subjected to electro-mechanical maturation for 6weeks before being used in HCM phenotype studies. Results: Both WT and R403Q ECTs exhibited mature cardiac phenotypes, including a lack of automaticity and a ventricular-like action potential (AP) with a resting membrane potential < -75mV. Compared to WT, R403Q ECTs demonstrated many HCM-associated pathological changes including increased tissue size and cell volume, shortened sarcomere length and disorganized sarcomere structure. In functional assays, R403Q ECTs showed increased twitch amplitude, slower contractile kinetics, a less pronounced force-frequency relationship, a smaller post-rest potentiation, prolonged AP durations, and slower Ca2+ transient decay time. Finally, we observed downregulation of calcium handling genes and upregulation of NPPB in R403Q vs. WT ECTs. In an HCM phenotype prevention experiment, ECTs were treated for 5-weeks with 250nM mavacamten or a vehicle control. We found that chronic mavacamten treatment of R403Q ECTs: (i) shortened relaxation time, (ii) reduced APD90 prolongation, (iii) upregulated ADRB2, ATP2A2, RYR2, and CACNA1C, (iv) decreased B-type natriuretic peptide (BNP) mRNA and protein expression levels, and (v) increased sarcomere length and reduced sarcomere disarray. Discussion: Taken together, we demonstrated R403Q ECTs generated in the Biowire platform recapitulated many cardiac hypertrophy phenotypes and that chronic mavacamten treatment prevented much of the pathology. This demonstrates that the Biowire ECTs are well-suited to phenotypic-based drug discovery in a human-relevant disease model.