Abstract 4369891: Modeling the Early Effects of a Novel Intronic <i>LMNA</i> mutation in iPSC-Derived 3D Cardiac Organoids

Introduction: The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which are essential for nuclear structure and function. Mutations in LMNA are commonly associated with dilated cardiomyopathy (DCM), the second leading cause of heart failure in the U.S. and one of the most lethal inherited cardiomyopathies, with a five-year mortality rate of 55.9%. To investigate the morphological and functional consequences of a patient-specific intronic LMNA mutation (c.937-1G&gt;A), we employed an iPSC-derived 3D cardiac organoid model. Hypothesis: The (c.937-1G&gt;A) intronic LMNA mutation reduces the differentiation efficiency into cardiomyocytes and leads to impaired cardiomyocyte function in 3D-cardiac organoids. Methods: Peripheral blood mononuclear cells of a patient with dilated cardiomyopathy carrying a uncharacterized novel LMNA (c.937-1G&gt;A) mutation were reprogrammed into iPSCs. We then utilized CRISPR-Cas9 to generate an isogenic corrected control. Both mutant and corrected iPSCs were differentiated into 3D-cardiac organoids that recapitulate key features of human heart tissue. Morphology, protein expression and calcium handling differences were evaluated using immunocytochemistry (ICC) and calcium imaging in an IonOptix system. Results: Western blot analysis showed reduced Lamin A protein expression in mutant organoids, with restored expression in CRISPR-corrected controls. Immunocytochemistry revealed marked reduction in cTnT-positive (cardiomyocytes) and disrupted nuclear morphology in mutant organoids compared to corrected controls. Calcium imaging showed altered calcium handling in LMNA mutants, including depressed systolic Ca 2+ levels, prolonged time to peak (n = 3, p &lt; 0.05) and slow departure velocity (d[Ca 2+ ]/dt max ), which were improved with the correction of the mutation. Conclusions: These findings suggest that the early effects of the novel intronic LMNA mutation (c.937-1G&gt;A) that occur during development can be modeled in 3D-cardiac organoids. The mutation impairs cardiomyocyte differentiation and disrupts calcium dynamics, underscoring its pathogenic potential during cardiac development.