Abstract 1423: MLP, Novel MLP Interacting Proteins and Mechanosensation
Introduction: Mutations in genes involved in cardiac mechanosensation (mec) have been shown to cause cardiomyopathies.
Hypothesis: Identification of novel mec genes by searching for new muscle LIM protein (MLP) interacting proteins (MiP) and consecutive functional analysis may provide novel insights into cardiomyopathy causing molecular mechanisms.
Methods and Results: A yeast 2 hybrid heart cDNA library has been screened and novel MiPs (MiP1, 2) have been identified. MiP1 consists of an N-terminal POZ domain and additional 13 carboxyterminal zinc fingers, whereas MiP2 belongs to the MYM domain family of proteins. Interestingly, both proteins interact with each other. Confocal microscopy localized both proteins to the cytoskeleton (Z-discs, intercalated-discs) and MiP1 in addition to the nucleus. Western blot analysis revealed downregulation of MiP1 in human ischemic and dilated cardiomyopathy (DCM) samples versus control tissue (n=12, p=0.01), whereas MiP2 is transcriptionally induced in DCM (n=12, p=0.001). We screened as well 95 DCM patients for MiP1 mutations and identified one human MiP1 missense mutation, not identified in 1600 control chromosomes. Furthermore, no additional mutation in 12 known cardiomyopathy disease genes was identified - supporting our hypothesis that MiP1 is a novel gene involved in the etiology of DCM. MiP1 and MiP2 conventional knockout animals are embryonically lethal. Therefore, we designed a myocardial conditional MiP1 knockout mouse model (CKO) using the MLC2V promotor driven Cre line, as well as a POZ domain floxed allele. CKO animals do not develop any spontaneous phenotype, however male CKO mice failed to respond with an increase in cardiac function upon biomechanical stress in form of transaortic constriction (TAC). Four weeks after surgery, %FS was significantly decreased (46.5±6.3% versus 52.0±3.7%, P<0.04) and LVEDD (3.74±0.19% versus 3.44±0.16%, P<0.003) as well as LVESD (2.00±0.28% versus 1.65±0.14%, P<0.005) was significantly increased in CKO mice (n=10) compared with WT littermates (n=8).
Conclusions: MiP1 and 2 are novel MLP interacting proteins with potentially important implications in cardiac mechanosensation. In addition, MiP1 mutations might be a novel cause of human cardiomyopathy.