The H222P-Lamin mutation induces heart failure via impaired mitochondrial calcium uptake in human cardiac laminopathy

Abstract Background Mutations in LMNA gene, which encodes lamins A/C, cause a variety of diseases, called laminopathies. Some mutations are particularly associated with the occurrence of dilated cardiomyopathy. The pathological mechanisms are still unclear limiting the development of specific therapies. Purpose Here, we focused on a mutation in the LMNA gene (c.665A>C, p.His222Pro), associated with the Emery-Dreyfuss Muscular dystrophy. We used LMNA H222P mutant human induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 corrected isogenic control hiPSCs to better characterize the cardiac phenotype associated with the mutation. Methods LMNA H222P mutant and the isogenic control hiPSCs clones were differentiated into cardiomyocytes (CMs). Immunofluorescence staining was performed on hiPSC-CMs to quantify their sarcomere organization (SarcOrgScore) using a Matlab code. Ring-shaped cardiac 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 was measured by Seahorse. Results hiPSC-CMs were generated from the LMNA H222P mutant and the corrected hiPSCs with no difference in the differentiation yield (proportion of troponin-positive cells: 92.74% for LMNA H222P vs. 89.38% for Ctrl-iso1, p=0.1038). The nuclei of LMNA H222P mutant hiPSC-CMs showed morphological abnormalities, typically observed with Lamin A/C mutations. hiPSC-CMs displayed well-formed sarcomeres and their organization was similar between the two cell lines. However, 3D cardiac organoids generated with LMNA H222P hiPSC-CMs showed an impaired contractility compared to control organoids. Calcium transient recordings in LMNA H222P mutant hiPSC-CMs 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 Ser300 phosphorylation, indicating a decreased mitochondrial calcium uptake in the LMNA H222P mutant hiPSC-CMs. Finally, measurement of mitochondrial respiration using Seahorse showed lower basal and maximal respiration in LMNA H222P hiPSC-CMs. Conclusions LMNA 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.