Molecular characterization of the sarcomeric Z-line proteins nebulette and palladin in the heart: roles in cardiac structure, function, and disease.

The sarcomeres are the smallest contractile units of striated muscle. It is formed by actin (thin) and myosin (thick) filaments that slide over each other during contraction and is stabilized by titin filaments, which acts as a spring during contraction. In the Z-line at the boundary between sarcomeres, actin and titin filaments are cross-linked and the contractile apparatus is connected with the cytoskeleton and extracellular matrix. The Z-line is important for efficient force generation and maintenance of cardiac structure, and is a focal point for mechanosensing and signal transduction. Consistently, mutations in many Z-line associated proteins have been associated with cardiomyopathies, including nebulette and its interaction partner myopalladin. On the other hand, the role of palladin (PALLD), a homologue of myopalladin (MYPN) associated with nebulette, in the heart has remained unknown. The aim of the present project was to investigate the function of nebulette and palladin in the heart. Nebulette is a 109 kDa modular protein composed of nebulin repeats and a C-terminal SH3 domain. In vitro studies have suggested a role of nebulette in stabilizing the thin filament, and missense mutations in the nebulette gene have been found to be causative for dilated cardiomyopathy and endocardial fibroelastosis in human and mice. However, the role of nebulette in vivo has remained unknown. To study the functional role of nebulette in the heart in vivo, we generated and studied nebulette knockout (nebl-/-) mice. Functional analyses revealed normal cardiac function of nebl-/- mice both at basal conditions and in response to transaortic constriction (TAC). Furthermore, histological, immunofluorescence, and Western blot analyses showed no cardiac abnormalities in nebl-/- mice. On the other hand, Z-line widening was observed from 5 months of age and the presence of chronic cardiac stress was suggested by the upregulation of cardiac stress responsive genes. Thus, nebulette may play an important role for Z-line integrity, while the absence of a functional phenotype of nebl-/- mice suggests that nebulette cardiomyopathy mutations have gain-of-function effects. PALLD, MYPN, and myotilin (MYOT) make up a small protein family of immunoglobulin-containing proteins in the sarcomeric Z-line associated with α-actinin and filamentous actin. MYPN and MYOT are expressed as single isoforms in striated muscle, while PALLD is ubiquitously expressed in several isoforms. The longest 200 kDa PALLD isoform is expressed predominantly in striated muscle and is highly homologous in structure to MYPN, suggesting a similar role of the two proteins in striated muscle. However, while mutations in the MYPN gene have been associated with dilated, hypertrophic, and restrictive cardiomyopathy, the role of PALLD in the heart has remained unknown. Since PALLD knockout mice are embryonic lethal, to study the role of PALLD in heart, we generated constitutive (cPKO) and inducible (cPKOi) cardiac specific PALLD knockout mice. cPKO mice showed normal cardiac function both at basal conditions and following TAC, while inducible knockout of PALLD in adult cPKOi mice resulted in progressive cardiac dilation and systolic dysfunction, associated with fibrosis, upregulation of markers of cardiac pathological remodeling, and ERK activation. Furthermore, PALLD was found to activate serum response factor (SRF) signaling and strongly increase MRTF-A-mediated activation of SRF, likely through its direct interaction MRTF-A and role in modulating actin dynamics. The development of a dilated cardiomyopathy in cPKOi mice induced at adult stage demonstrates that PALLD is essential for normal cardiac function and suggests that PALLD gene mutations may be causative for cardiomyopathy. The absence of a cardiac phenotype of cPKO mice may be due to compensatory mechanisms.