Determining the Molecular Mechanisms that Link a Titin Mutation to Cardiomyopathy

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A mutation in the giant elastic protein titin has recently been linked to arrhythmogenic cardiomyopathy (AC), a disease primarily characterized by fibrofatty infiltration of the myocardium. Titin is the first sarcomeric protein linked to AC. In addition to largely unstructured segments, the extensible I-band region of titin contains ∼40 immunoglobulin(Ig)-like domains that are natively folded as highly stable beta barrels. One of these domains, Ig10, contains a single Thr→Ile mutation that leads to AC. The first step in determining how this mutation leads to severe cardiac disease was to study the mutation at the single molecule level using atomic force microscopy (AFM) and in vitro degradation assays. With AFM individual proteins are mechanically stretched, and it was found that the mutation significantly reduces the force needed to unfold Ig10. Although no difference in refolding rate was found, by comparing the experimental AFM unfolding forces with computer simulated Ig unfolding, it was found that the mutation increases Ig10's unfolding rate fourfold. This effect predicts increased Ig10 unfolding (i.e. transition into a non-native state) under physiological conditions. It was also found that mutant Ig10 is more prone to protease degradation due to compromised local structure at the point mutation. This suggests that accelerated titin turnover in cardiomyocytes instigates the cardiac remodeling process in patients with titin-linked AC. This is the first time an Ig domain in titin's elastic I-band region has been linked to cardiac disease, and this study proposes a novel disease mechanism contingent on Ig domain unfolding.