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.