Abstract Body: Background: The LMNA gene encodes Lamin A/C, and mutations can disrupt protein structure, leading to disorganization of the nuclear membrane and chromatin architecture. The E203K LMNA mutation has previously been associated with Arrhythmogenic Cardiomyopathy (ACM).

Research Question/Hypothesis: Although all LMNA mutations exhibit increased sensitivity to mechanical stress compared to normal cardiomyocytes, the degree of sensitivity likely varies among different LMNA mutations.

Goals/Aims: We compared a LMNA mutation with its isogenic control under both normal and mechanically stressed conditions to assess how cellular morphology responds to mechanical stress.

Methods: We used a healthy induced pluripotent stem cell (iPSC) line (DT19-9-11) and an isogenic CRISPR-edited heterozygous LMNA mutant E203K (LMNA^-/+E203K), both differentiated into iPSC-derived cardiomyocytes (iPSC-CMs). Two culture platforms mimicked the in vivo mechanical environment: 10 kPa PDMS representing a healthy adult human heart and 50 kPa PDMS representing the stiffer environment associated with ACM. Immunofluorescence via high resolution confocal microscopy was used to identify changes in cytoskeletal organization, nuclear shape, and membrane protein architecture, with custom MATLAB software employed to quantify differences.

Results: Micropatterned Control iPSC-CMs showed stiffness-dependent morphological changes between 10 kPa and 50 kPa conditions, including an 81.4% increase in nuclear area from 10 to 50 kPa. In contrast, LMNA^-/+E203K iPSC-CMs were less responsive to substrate stiffness alterations, as their nuclear area decreased by 15.4% from 10 to 50 kPa, suggesting impaired mechanical sensing. Morphological shifts were present but not significant and did not alter the overall conclusion that the LMNA^-/+E203K mutation impairs mechanosensing.

Conclusion: The reduced responsiveness of LMNA^-/+E203K iPSC-CMs to the 50 kPa PDMS stiffness indicates a loss of normal nuclear mechanotransduction, likely due to Lamin A/C dysfunction. This model brings us closer to replicating the complexity of the diseased human heart and improves our understanding of LMNA-related ACM.