EFFECTS OF CYCLIC STRAIN ON NUCLEAR MORPHOLOGY OF FIBROBLAST WITH LMNA MUTATION

Nuclear morphology is defined by the nuclear wall, nuclear lamina, which is a meshwork of different proteins attached to the inner nuclear membrane, providing structural support and integrity to the nucleus. The nuclear wall can have defects such as ‘blebs’, protrusions of the nuclear membrane, and invaginations. Mutations in the LMNA gene can increase incidents of these structural abnormalities because the corresponding proteins, Lamin A and C, make up the structural network of the nuclear lamin. Some LMNA mutations lead to systemically devastating phenotype, while others are more targeted. For example, there are LMNA mutations that result exclusively in cardiac pathologies. Here we aim to elucidate if cyclic strain, which is present in the heart but not in the skin, can potentiate a change in nuclear shape in cells with and without a cardiac specific LMNA mutation. Glass coverslips coated with 184-polydimethylsiloxane (PDMS) were patterned with fibronectin through microcontact-printing. Coverslips were seeded either with skin fibroblasts of patients possessing the cardiac specific LMNA mutation or those of age-matched controls. Cells were separated into two groups: one subjected to 20% uniaxial strain relative to their un-stretched length for 24 hours, using a custom built stretcher, and one not exposed to mechanical stretching. After 48 hours, cells were fixed using paraformaldehyde and Triton X-100. The cells were then immunostained with DAPI and Phalloidin. Fibroblasts were visualized using a fluorescent inverted microscope, and then the nuclei shapes were analyzed using custom MATLAB codes, thus quantifying the percent of defective nuclei for each condition. Preliminary we have found that in the no-strain condition, no differences in nuclear deformity were found between patients affected by the LMNA gene mutation when compared to aged-matched controls. In the fibroblast with the LMNA mutation subjugated to cyclic strain, there was an increase in nuclei deformity. These findings are in line with patient symptoms, who do not experience any skin pathologies (no cyclic strain), but do tend to present a variety of heart diseases.