Deshayes, L. | Search bioRxiv

bioRxiv2025

Mavacamten improves energy balance in a pre-clinical model of RASopathy-associated hypertrophic cardiomyopathy

Background and aimDevelopmental disorders caused by activating mutations in the RAS-MAPK pathway account for nearly 20% of hypertrophic cardiomyopathy (HCM) cases in paediatric patients. Compared to sarcomeric HCM, RAS-HCM presents a higher risk of obstruction and hospitalisation. The myosin inhibitor mavacamten has been approved in the European Union for treating adults with obstructive HCM; however, clinical trials have excluded syndromic HCM. Consequently, this study aimed to characterise the functional and energetic disturbances induced by the RASopathy mutation BRAF p.Thr599Arg in cardiomyocytes and to evaluate the effects of mavacamten treatment. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a CRISPR-induced BRAF T599R mutation and their isogenic control were employed to model RAS-HCM in vitro. The cell size, contractility, and transcriptomic profile were assessed to determine the phenotype of the cardiomyocytes. Energetics were evaluated using the Mito Stress assay, live ATP levels, and NAD(P)H and FAD+ autofluorescence. ResultsBRAF-mutant cardiomyocytes demonstrated hypertrophy and hypercontractility. Furthermore, energetic profiling revealed increased mitochondrial NAD(P)H and FAD+ pools and an enhanced energetic state in the Mito Stress assay with increased maximal respiratory capacity. However, they also exhibited a significant ATP drop during rapid pacing compared to the control, suggesting that mitochondrial capacity remains insufficient to meet the ATP demand. Mavacamten treatment normalised excessive ATP consumption during acute pacing, suggesting reduced mitochondrial overactivity. ConclusionsBRAF-mutant cardiomyocytes recapitulate the characteristics of HCM in vitro. Mavacamten mitigates dysfunctions and restores energetic balance under stress conditions, indicating it holds potential as a therapeutic option for RASopathy-associated HCM.

physiology↗

bioRxiv2024

The p.H222P lamin A/C mutation induces heart failure via impaired mitochondrial calcium uptake in human cardiac laminopathy

BackgroundMutations in the LMNA gene, which encodes lamin A/C, cause a variety of diseases known as laminopathies. Some mutations are particularly associated with the occurrence of dilated cardiomyopathy and heart failure, but the genotype-phenotype relationship and underlying mechanisms are unclear. Here, we used induced pluripotent stem cells (hiPSCs) from a patient carrying a LMNA point mutation (c.665A>C, p.His222Pro) to investigate the mechanisms leading to contractile dysfunction. MethodsLMNA p.H222P mutant and a CRISPR/Cas9 corrected isogenic control hiPSCs clones were differentiated into cardiomyocytes (hiPSC-CMs). Immunofluorescence staining was performed on hiPSC-CMs to quantify their sarcomere organization (SarcOrgScore) using a Matlab code. Ring-shaped cardiac 3D organoids were generated to compare the contractile properties of the two clones. Calcium transients in mutant and corrected hiPSC-CMs were measured by live confocal imaging. Mitochondrial respiration parameters were measured by Seahorse. ResultshiPSC-CMs were generated from the LMNA mutant and the corrected hiPSCs with no difference in the differentiation yield (proportion of troponin-positive cells: 95.0% for LMNA p.H222P vs. 95.1% for Ctrl-iso1, p=0.726). hiPSC-CMs displayed well-formed sarcomeres and their organization was similar between the two cell lines. However, cardiac 3D organoids generated with LMNA p.H222P hiPSC-CMs showed an impaired contractility compared to control organoids. Calcium transient recordings in LMNA p.H222P mutant cardiomyocytes showed a significantly higher calcium transient amplitude with a significantly slower calcium re-uptake. Transcriptomic analyses suggested a global mitochondrial dysfunction and in particular an impaired mitochondrial calcium uptake with a significantly decreased expression of the mitochondrial calcium uniporter (MCU). This decrease in MCU expression was confirmed by western blot and was accompanied by an increased MICU1:MCU, as well as an increased PDH Ser232 and PDH Ser300 phosphorylation, indicating a decreased mitochondrial calcium uptake in the LMNA mutant hiPSC-CMs. Measurement of mitochondrial respiration showed lower basal and maximal respiration in LMNA p.H222P hiPSC-CMs. Consistently, the ATP levels were significantly lower in LMNA p.H222P hiPSC-CMs as compared to isogenic controls. ConclusionsLMNA p.H222P mutant hiPSC-CMs exhibit contractile dysfunction associated with mitochondrial dysfunction with impaired MCU complex activity, decreased mitochondrial calcium homeostasis and reduced mitochondrial energy production. NOVELTY AND SIGNIFICANCEO_ST_ABSWhat is known?C_ST_ABS- Mutations in LMNA, which encodes the nuclear lamins A/C, cause a variety of diseases (called laminopathies), which can involve the cardiac muscle leading to dilated cardiomyopathy and systolic heart failure. - The pathological mechanisms linking the nuclear envelope abnormalities induced by LMNA mutations to the development of a reduced cardiac muscle contractility are not well understood. What new information does this article contribute?- LMNA mutant cardiomyocytes have a profound mitochondrial dysfunction with impaired MCU complex activity, decreased mitochondrial calcium homeostasis, and reduced mitochondrial energy production. - Our study uncovers an unappreciated pathophysiological mechanism and opens new possibilities by suggesting MCU activators as a novel therapeutic for patients with LMNA cardiomyopathy.

pathology↗