Mechanistic Basis of Cardiac Laminopathy

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PROJECT SUMMARY Mutations in the lamin A/C gene (LMNA) encoding structural proteins of the nuclear lamina are responsible for up to ten percent of cases of inherited dilated cardiomyopathy. The disease is often referred to as cardiac laminopathy. Experimental evidence partially supports various pathogenic mechanisms of how defects in nuclear structural proteins cause cardiomyopathy, including that they lead to abnormalities in cell mechanical stability, dysregulation of gene expression and altered cell signaling. However, there is no unifying hypothesis integrating these defective processes and explaining exactly how they lead to cardiomyocyte damage and dysfunction. We recently found a surprising relationship between aberrant extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and altered nuclear positioning in cardiac laminopathy. This has led us to hypothesize the existence of a mechanic checkpoint in which alterations in the nuclear lamina upregulate ERK1/2 activity, which causes mispositioning of the nucleus by phosphorylating and inactivating the actin bundling activity of the formin homology domain-containing protein (FHOD). Inactivation of FHOD prevents the linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the inner and outer nuclear membranes and connects to actin filaments, to mediate nuclear positioning. Normally, the mechanical checkpoint acts to prevent excessive force from being applied to the nucleus in contracting cardiomyocytes. However, with permanent alterations in nuclear structure resulting from LMNA mutations, the persistently activated checkpoint becomes maladaptive, resulting in abnormal nuclear positioning, nuclear envelope rupture, DNA damage and defects in sarcomere function. This Project is designed to prove the nuclear mechanical checkpoint hypothesis and determine its role in the pathogenesis of cardiac laminopathy. In Aim 1, we will examine how activation of the mechanical checkpoint for nuclear positioning alters cardiomyocyte biology. We will directly measure force on the nucleus using a nesprin- 2 actin tension sensor. As recent data suggest that the nucleus contributes to normal sarcomere, we will test the hypothesis that persistent mechanical checkpoint activation and nuclear mispositioning leads to defective sarcomere assembly and function in cardiomyocytes. In Aim 3, we will determine how altering the mechanical checkpoint affects the heart in vivo. We will test if expressing a phosphomimetic FOHD variant (checkpoint activation) in the heart induces cardiomyopathy in wild type mouse hearts and if a non-phosphorylatable variant (checkpoint inactivation) ameliorates pathology in a mouse model of cardiac laminopathy. Proving the existence of a novel nuclear mechanical checkpoint and establishing its role in the pathogenesis of cardiomyopathy caused by LMNA mutations will shift research directions in the field and potentially lead to new treatments for this life- threatening inherited heart disease.