Abstract 186: Heart Disease Associated Lamin A/C Mutations and Resultant Defects in Cell and Nuclei Structure

Abstract

Changes in cell organelle structure or shape, which can be associated with a variety of diseases, can be caused by inherited genetic mutations. For example, the progeria disease is associated with a mutation to the LMNA gene, and it leads to a devastating early aging and death. However, other LMNA gene mutations do not cause early aging, but instead have a more subtle effect with patients presenting only with heart disease symptoms. One way to study the mechanisms that lead to these differences is in vitro with patient specific cell-lines. Here we will present a study of 10 cell-lines negative for mutations or with four different types of LMNA mutations: 1) Hutchinson-Gilford progeria syndrome LMNA gene mutation, 2) LMNA splice-site mutation (c.357-2A>G , p.N120Lfs*5), 3) LMNA nonsense mutation (c.736 C>T, p.Q246X), and 4) LMNA missense mutation (c.1003C>T, p.R335W) in exon 6. Fibroblasts from each of these cells lines were cultured on either isotropic fibronectin or fibronectin patterned into anisotropic lines. The cells were then fixed and stained to visualize the nuclei, actin fibrils, and the underline remodeled fibronectin. To analyze this data, we applied existing algorithms to quantify the re-organization of actin in response to extracellular matrix cues by calculating both the actin and fibronectin orientational order parameter. Additionally, we have created a new algorithm to automatically identify nuclei defects, which is a known property of the progeria LMNA mutation. Using the new algorithm, it is possible to separately characterize the shape of normal and defective nuclei for each cell-line. Through this algorithm, we show the varied effects of the different mutations on the number of fibroblasts that present with nuclear defects and their properties. The current data is consistent with the known patient symptoms providing further avenues of study into the mechanisms triggered by these mutations. Furthermore, we probe whether the unique mechanical conditions present in the myocardium are responsible for the heart disease symptoms of some of the tested mutations.