image: (A) The incidence data of MYBPC3 in hypertrophic cardiomyopathy were summarized, and the results were obtained from the ClinVar database. (B) The structure of the MYBPC3 gene and the location of gRNA for epiCRISPR/Cas9 editing. (C) Sequencing chromatograms verified a homozygous MYBPC3-KO hiPSC line, characterized by a deletion of 2 nucleotides in each allele. (D) Immunostaining of MYBPC3-KO colonies for the pluripotency markers OCT4 and SSEA4. Scale bar, 20 μm. (E) Molecule-based methods were used to induce cardiac differentiation. (F) Analysis for cTnT from WT and MYBPC3-KO differentiation protocols before purification at day 10. (G, H) Quantitative real-time PCR and western blotting analysis of MYBPC3 gene in both WT and MYBPC3-KO hiPSC-CMs at day 20. (I) Immunostaining for expression of MYL2 in WT and MYBPC3 KO CMs at day 40. Scale bar, 50 μm. The results were presented as mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01; n.s. means no significance. MYBPC3, myosin binding protein C3; KO, knockout; WT, wild type; hiPSC, human induced-pluripotent stem cell; CM, cardiomyocyte; SSEA4, stage-specific embryonic antigen 4; OCT4, octamer-binding protein 4; cTnT, cardiac troponin T; MYL2, myosin light chain 2.
Credit: Jinhua Cao, Yafei Zhai, Ke Li, Jiajv Li, Xiaoxu Tian, Jianchao Zhang, Shuang Li, Mengduan Liu, Xiaowei Li, Jianzeng Dong, Xiaofang Wang
Hypertrophic cardiomyopathy (HCM) is an inherited disease characterized by diastolic dysfunction, fibrosis, and asymmetric thickening of the left ventricle, which ultimately leads to heart failure. The lack of effective treatment to mitigate HCM progression and the rise in adverse clinical outcomes as the disease advances emphasize the need to develop therapeutic strategies that can slow disease progression.
A recent study published in the Genes & Diseases journal, conducted by researchers from the First Affiliated Hospital of Zhengzhou University and the Henan Key Laboratory of Hereditary Cardiovascular Diseases, demonstrated that reactive oxygen species (ROS) play a crucial role in the progression of HCM.
Previous studies have shown that mutations in the myosin binding protein C3 (MYBPC3) account for approximately 40%–50% of HCM. To investigate how ROS-induced oxidative stress relates to the course of HCM to heart failure, the authors generated MYBPC3 knockout-human induced pluripotent stem cell-derived cardiomyocytes (MYBPC3-KO hiPSCs). They showed that these knockout cells can differentiate into cardiomyocytes similar to their wild-type counterparts (WT hiPSC-CMs).
Experiments with MYBPC3-KO hiPSC-CMs inserted with a calcium sensor green fluorescent calcium-modulated protein 6 fast type (GCaMP6f) initially showed increased Ca2+ transient amplitude, Ca2+ reuptake rate, and release rate compared with WT. However, the calcium release and decay time were significantly prolonged during the later stages (day 40), which indicates calcium transport disruption in these cells. Similarly, the contractility amplitude of MYBPC3-KO hiPSC-CMs decreased with prolonged myocardial diastolic time during the later stages. These disruptions collectively lead to increased Ca2+ entering the mitochondria, which in turn causes mitochondrial calcium overload, increased ROS production, and oxidative stress.
Furthermore, the authors observed that the MYBPC3-KO hiPSC-CMs recapitulated the HCM phenotypes in vitro, as evidenced by disordered sarcomere arrangement, enlarged cell area, and an increased number of binuclear CMs. Transcriptome analysis revealed an increase in hypertrophy-related genes and pathways, including up-regulation of calcium channels (RYR2 & CACNA1C) and down-regulation of oxidative stress-related genes (SOD, CAT & GPX1). Additionally, MYBPC3 knockout was associated with activation of the PI3K/AKT signaling pathway and inhibition of forkhead box O3a (FOXO3a), indicating that MYBPC3 knockdown promotes oxidative stress.
Moreover, mitochondria in the MYBPC3-KO hiPSC-CMs displayed a unique, fragmented, and punctate pattern and were irregularly distributed throughout the cytoplasm. These changes were accompanied by a decrease in mitochondrial membrane potential and increased pAMPK levels, which highlight that the knockdown of MYBPC3 causes mitochondrial dysfunction.
Addition of melatonin, a potent antioxidant, was shown to improve i) cellular ROS and mitoSOX levels, ii) calcium transport, and iii) contractility amplitude of MYBPC3-KO hiPSC-CMs. Melatonin also decreased phosphorylation of PI3K, AKT, and FOXO3a, suggesting that melatonin alleviates HCM by inhibiting the PI3K/AKT/FOXO3a signaling pathways.
In conclusion, this study elucidates how ROS accelerates HCM progression via activation of the PI3K/AKT/FOXO3a signaling pathways. It also highlights the protective effect of melatonin in HCM, suggesting that understanding ROS-mediated mechanisms may have potential therapeutic implications in cardiac diseases.
Reference
Title of the original paper: ROS accelerates the progression of hypertrophic cardiomyopathy
Journal: Genes & Diseases
Genes & Diseases is a journal for molecular and translational medicine. The journal primarily focuses on publishing investigations on the molecular bases and experimental therapeutics of human diseases. Publication formats include full length research article, review article, short communication, correspondence, perspectives, commentary, views on news, and research watch.
DOI: https://doi.org/10.1016/j.gendis.2025.101741
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