Purpose: 

Muscle LIM protein (MLP) promotes myogenesis and cell differentiation and is downregulated in heart failure. MLP-null mice show signs of cardiac hypertrophy and develop a cardiomyopathy 4–8 weeks after birth. MLP may thus be involved in cardiac hypertrophic signaling, but specific mechanisms are unknown. The aim of this study was to identify post-translational modifications of cardiac MLP and possible links to hypertrophic gene activation.

Methods: 

Two dimensional difference gel electrophoresis and mass spectrometry were used to detect posttranslational modifications of MLP, comparing normal mouse hearts to those dephosphorylated by alkaline phosphatase. MLP localization was studied in cultured neonatal rat ventricular myocytes (NRVM) by immunofluorescence and in subcellular fractions of mouse hearts by Western blotting. Recombinant MLP and mutation constructs were expressed in E. coli and antibodies to MLP were generated in rabbits. Signaling mechanisms involving MLP were tested in NRVM and in mouse hearts with homozygous knockout of MLP or calcineurin-A-beta (CnAb). Disease-related alterations in cardiac MLP properties were studied in a hypertensive old dog model with cardiac hypertrophy and diastolic heart failure (DHF).

Results: 

A conserved phosphorylation site was identified on cardiac MLP at S95, prompting the generation of MLP-S95phospho-specific antibodies. In search for kinases (PKCs, FAK, ERK2) phosphorylating recombinant human MLP, we established ERK2 as the specific mediator of MLP phosphorylation at S95. Treatment of NRVM with Raf-1 inhibitor GW5074 to block ERK2 activity decreased both ERK2 and MLP-S95 phosphorylation. The opposite effect was seen in NRVM treated with endothelin-1. As the phosphatase CnA is a direct interaction partner of MLP, we tested whether CnA can dephosphorylate MLP-S95. Incubating ERK2-phosphorylated recombinant MLP with CnA decreased MLP-S95 phosphorylation by 80%. Compared to wildtype mouse hearts, MLP^-/- hearts showed a 9-fold increase in CnAb expression and CnAb^-/- hearts a 2-fold increase in MLP-S95 phosphorylation. In old dog hearts with hypertrophy and DHF, MLP-S95 phosphorylation was decreased by 40% compared to normal dog hearts. Moreover, CnAb was 2.7-fold increased in DHF, whereas ERK2 was unchanged. Therefore, the decreased MLP-S95 phosphorylation is due to increased CnA expression/activity, previously noted in hypertrophic hearts.

Conclusions: 

ERK2 specifically phosphorylates, and CnA dephosphorylates, MLP-S95. MLP-S95 phosphorylation is linked to the MAPK and CnA-NFAT signaling pathways. Diminished MLP-S95 phosphorylation is associated with cardiac hypertrophy and failure.