MLP interacting protein 1 (MIP1) is important for the cardiac stress response, regulates autophagy and is a novel candidate gene for cardiomyopathy

R Knöll, S Kostin, C Mansfield, O Choi, R Isaacson, K-L Poon, G Knöll, S Mcsweeney, A Perrot, MR Toliat, J Chen, I Gould, E Lara-Pezzi, P Luther, E Petretto, H Milting, T Brand, P Nürnberg, B Buyandelger. Karolinska Institute, Stockholm, Sweden

Mutations in genes involved in cardiac mechanosensation (mec), such as the muscle LIM protein (MLP) have been shown to cause various forms of heart failure. Identification of novel mec genes by searching for new MLP interacting proteins (MIP) may provide novel insights into underlying mechanisms.

A yeast two hybrid screen identified a novel MIP, located at a chromosomal region recently implicated via genome-wide association studies in the pathogenesis of dilated cardiomypathy (DCM). The interaction was confirmed by coimmunoprecipitation, colocalization and cross linking experiments.

We found that MIP1 is a stress inducible transcription factor and chromatin immunoprecipitation experiments revealed that MIP1 binds to the calcineurin promoter and activates NFAT, a known mediator of hypertrophy. Also MIP1 promotes autophagy and activates expression of anti-apoptotic genes when overexpressed in vitro in cardiac myoctyes.

Mip1 conventional knockout animals die during gastrulation at embryonic day 4 and Mip1 conditional knockout (cKO) animals failed to adapt to transverse aortic constriction. Four weeks after intervention, fractional shortening (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) were significantly increased in cKO mice (n=10) compared to control littermates (n=8).

Analysis of the underlying molecular mechanisms revealed that autophagic vesicles were less present in cKO as judged by electron microscopy, which was associated with significantly less expression of pro-autophagic genes such as Atg5, Atg7, LC3a, LC3b, and Beclin 1. TUNEL assays, combined with activated caspase, whole gene expression arrays and RT-PCRs provide strong evidence that Mip1 deficient cardiac myocytes are unable to undergo autophagy and as a result activate apoptotic processes which are responsible for the observed heart failure phenotype. We also performed the reverse experiment, i.e. overexpression of MIP1 in vivo, which resulted in marked cardiac hypertrophy at the organ and single cell level. In addition, MIP1 is downregulated in human DCM (P<0.05).

MIP1 contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. We sequenced this gene in 575 unrelated individuals affected by cardiomyopathy and heart failure and identified four MIP1 missense mutations (T106M, G179S, A188V, Q531R) of which two co-segregate in two small families with DCM and arrhythmia. The presence of additional mutations in the index patients was excluded by whole exome sequencing. All four variants show abnormalities when analysed in regards to their ability to interact with MLP, localization, structure, stability and potential to drive the calcineurin promoter. Wildtype MIP1 is able to efficiently protect cells from apoptosis, a property missing in all mutants. At least G179S and Q531R cause heart failure in the zebrafish.

MIP1 is important for the initiation of survival pathways upon biomechanical stress and represents a novel cardiomyopathy candidate gene.