A highly penetrant LMNA R541C variant associated with dilated cardiomyopathy leads to dysregulation in metabolism and proliferation pathways in stem cell-derived cardiomyocytes

LMNA p.H222P mutation causes contractile dysfunction via impaired mitochondrial calcium uptake in human cardiac laminopathy.

AIMS Mutations 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. METHODS AND RESULTS Induced pluripotent stem cells (hiPSCs) from a patient carrying a LMNA point mutation (c.665A>C, p.His222Pro) and a CRISPR/Cas9 corrected isogenic control hiPSCs clones were differentiated into cardiomyocytes (hiPSC-CMs), with no difference in the differentiation yield and in sarcomere organisation between the two cell lines. However, 3D cardiac 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 ratio, as well as an increased PDH Ser232 and PDH Ser300 phosphorylation, indicating an altered mitochondrial calcium uptake in the LMNA mutant hiPSC-CMs. Consistently, lower mitochondrial respiration and ATP levels were found in LMNA p.H222P hiPSC-CMs as compared to isogenic controls. Strikingly, treatment with the MCU activator amorolfine restored mitochondrial calcium uptake and improved contractility in LMNA mutant hiPSC-CMs. CONCLUSIONS Our results establish a direct mechanistic link between nuclear envelope dysfunction and impaired mitochondrial function, and highlight the MCU complex as a potential therapeutic target in LMNA-related cardiomyopathy. More broadly, this work provides a paradigm for connecting gene-specific nuclear defects to mitochondrial dysfunction in inherited cardiomyopathies.

CRISPR/dCas9-mediated tuning of DMPK transcription reveals a quantitative relationship between toxic repeat RNA expression and MBNL1 activity in myotonic dystrophy.

It is demonstrated that expanded DMPK transcript levels modulate free MBNL1 concentration and alternative splicing in a dose-dependent manner, underscoring the central role of repeat RNA expression in DM1 pathogenesis.

Epilepsy-associated SCN2A-L1342P mutation drives network hyperexcitability and widespread transcriptomic changes in human cortical organoids.

These findings demonstrate that the Nav1.2-L1342P mutation drives a multifaceted disease phenotype, including network hyperexcitability and disruption of pathways related to neuronal and synaptic functions, which advances understanding of SCN2A-related developmental and epileptic encephalopathy (DEE).

Berberine Attenuates Hypertrophic Phenotype in RAF1-Mutant Induced Pluripotent Stem Cell-Derived Cardiomyocytes through Suppression of ERK5-Cyclin D1 Signaling.

RBM20 variants disrupt Ca2+ handling and metabolism in dilated and non-compaction cardiomyopathy stem cell models

Mutations in the splice-regulator RBM20 cause heart failure with reduced ejection fraction (HFrEF), typically manifesting as dilated cardiomyopathy (DCM). Mutations at position 634 in the RS-domain cause DCM with (R634L) or without (R634W) left ventricular non-compaction (LVNC). However, the mechanisms underlying phenotype variability and personalized therapy beyond HFrEF remain unclear. We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), 3D-cardiospheres and engineered myocardial tissues from patients with RBM20 mutations R634L (LVNC) or R634W (DCM). Using CRISPR/Cas9, we created isogenic rescue and mutation-insertion lines, identifying RBM20 mis-localization, splicing errors in TTN and RYR2, and sarcomere irregularities in both. DCM-CM showed increased resting Ca2+ leak and reduced Ca2+ transient amplitude, typical of HFrEF, and spatial disorganization of sarcoplasmic reticulum and mitochondria. In contrast, LVNC-CM exhibited elevated Ca2+ transient amplitude with faster kinetics, driven by elevated cAMP and mis-spliced, hyperactive CAMK2D, leading to PLN-hyperphosphorylation and increased metabolic respiration. Further, LVNC showed desmosomal derangement potentially from mis-splicing of Junction plakoglobin and reduced 3D cardiosphere compaction. Despite distinct mechanisms, contractile force was reduced in both. Isogenic controls confirm mutation causality. Drug intervention with verapamil partially improved selected abnormal Ca2+ handling and contractile phenotypes in LVNC- and DCM-CM, whereas the CAMK2D inhibitor AIP improved systolic Ca2+ handling predominantly in LVNC-CM. In conclusion, different amino acid substitutions at the same RBM20-residue induce opposing Ca2+-handling and structural phenotypes. While DCM features impaired Ca2+ handling, LVNC shows defective cell-cell coupling and activated Ca2+ handling and metabolism, yet insufficient to compensate for organ-level dysfunction. This supports personalized pharmacological therapies in early HF, and potential CRISPR/Cas9 repair for RBM20 cardiomyopathy.

Advanced iPSC-based modelling of LMNA-related congenital muscular dystrophy enables development of genetic therapies for muscle laminopathies.

The selection of L-CMD iPSCs is expanded, disease-associated readouts are validated using a transgene-free differentiation protocol and gene editing strategies are assessed using 2D and 3D cultures, providing an advanced, humanised platform for translational research and precision medicine in laminopathies.