Hypertrophic cardiomyopathy-associated mutations in myosin heavy chain drive pathologic expression of TGF-beta in cardiomyocytes within weeks of developmental specification

Introduction: Hypertrophic cardiomyopathy (HCM) is linked to over 1500 mutations, mainly in genes encoding sarcomeric proteins. These mutations disrupt cardiac function and are associated with sudden cardiac death in young individuals and athletes. Despite the need for effective treatments, progress is hampered by a lack of understanding of early-stage deficiencies that drive disease progression. We previously demonstrated that monolayers of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) with MYH7 R723C and MYH6 R725C mutations show altered expression of extracellular matrix (ECM)-related genes with associated defects in ECM attachment. To further investigate the cell-ECM interface and pathological ECM dynamics in HCM, we adopted a 3D engineered heart tissue (EHT) model containing cardiomyocytes and fibroblasts, the primary contributor to ECM remodeling. Comparing mutant EHTs with mutant hiPSC-CMs to the controls, we observed aberrant cardiomyocyte distribution and alignment with augmented calcium handling and force generation. Investigation into mutation-induced ECM dynamics revealed altered proteoglycan deposition and heightened phosphorylated focal adhesion kinase (pFAK) levels in mutant EHTs, indicating changes in matrix composition and connectivity. Additionally, mutant EHTs exhibited higher levels of transforming growth factor beta-1 (TGF-beta1), more activated fibroblasts, and sustained TGF-beta1 transcription in cardiomyocytes. Remarkably, blocking TGF-beta1 signaling reduced fibroblast activation and restored the contraction force of mutant EHTs to control levels. This study underscores the early interplay of mutant hiPSC-CMs with fibroblasts, wherein the mutant CM initiate early dysfunction partly through TGF-beta1 overexpression. These findings provide a promising foundation for developing novel HCM treatments before the manifestation of clinically detectable cardiac dysfunction.

Materials and

Methods: EHT model with supporting cells was utilized to better replicate the pathological changes associated with HCM mutations. Mutant and control cardiomyocytes were obtained by directly differentiating via modulation of Wnt/-catenin signaling. To study early-stage HCM pathology associated with myosin heavy chain (MHC) mutations and to avoid the effect of wild-type-MHC expressed by immature hiPSC-CMs, the mutant hiPSCs were CRISPR/Cas9-edited to have a homozygous c.2167C>T mutation in the MYH7 locus and an analogous heterozygous c.2173C>T mutation in the MYH6 locus. Healthy neonatal human dermal fibroblasts were incorporated into the EHTs to assist the tissue remodeling, allowing the in vitro tissue model to mimic in vivo cardiac tissues soon after EHT fabrication. A PDMS culture well with two fixed posts was designed to secure the EHTs and provide passive strain during cultivation. This passive strain, combined with spontaneous contraction of cardiomyocytes, enabled the recapitulation of heart tissue stretching by blood filling in vivo. Functional assessments, including calcium transient and force generation measurements, immunostaining, western blot, ELISA, and RNA fluorescent in situ hybridization (FISH) were performed at day 15 and 30 after EHT fabrication.

Results, Conclusions, and Discussions: In this study, we utilized a multicellular tissue model to investigate the early interplay between mutant hiPSC-CMs and fibroblasts, uncovering the pathological features associated with HCM.
Calcium transient analysis showed that mutant EHTs displayed HCM-associated phenotypes, such as arrhythmia and calcium accumulation. Increased downstroke and upstroke velocity observed at a later time point in both mutant and control EHTs indicated improved tissue development over the 2-week culture, reflecting enhanced maturation in this 3D EHT model. By Day 30, mutant EHTs exhibited reduced downstroke velocity, suggesting increased relaxation, a hallmark of diastolic dysfunction in HCM patients. Additionally, calcium signals in mutant EHTs were localized within smaller domains, likely due to fibroblast infiltration, mirroring the separation of cardiomyocytes in HCM tissues with interstitial fibrosis clinically.
Force generation measurements revealed significantly higher forces in mutant EHTs, indicative of hypercontractility. Mutant EHTs exhibited faster contraction rate during spontaneous contraction, while 1Hz pacing synchronized contractions and led to increased force generation. This suggests fibroblast infiltration into cardiomyocyte domains, where multiple beating sub-domains could offset each other. Considering the suspected activation of fibroblasts to support cardiomyocytes in the EHT system, TGF-beta1 signaling, a key pathway for fibroblast activation, was examined. ELISA analysis showed that mutant EHTs secreted significantly higher TGF-beta1 starting from Day 23. Further, RNA FISH revealed that even though fibroblasts generated more TGF-beta1 mRNA, the quantity remained unchanged over time and unaffected in the mutant case. Additionally, the number of TGF-beta1 mRNA per cardiomyocyte showed no difference between mutant and control on Day 15, while by Day 30, only mutant hiPSC-CMs maintained TGF-beta1 mRNA expression.
To investigate whether blocking intercellular communication could mitigate pathological alterations, SB431542 was added into the culture medium 7 days before assessments. Correspondingly, force of contraction in mutant EHTs was reduced to levels comparable to untreated control EHTs.
Overall, our EHT system provides a comprehensive model for studying HCM at the tissue level at early developmental time points, underscoring the role of TGF-beta1 signaling in disease progression. These findings suggest that early therapeutic intervention targeting TGF-beta1 signaling could delay or prevent HCM development, offering a new avenue for treatment strategies.

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