Heart failure is one of the leading causes of morbidity and death worldwide. However, its molecular causes are still only partially understood.

Striatin interacting phosphatases and kinases complexes (STRIPAK) have been studied mainly in oncological contexts where they have been identified as signalling hubs in diverse molecular pathways. Our group has recently demonstrated that the STRIPAK member Myoscape/STRIP2 interacts with the L-type calcium-channel (LTCC) and cardiac alpha-actinin 2 to stabilise LTCC surface expression. Myoscape-KO mice develop progressive heart failure, emphasising the possible role for cardiac STRIPAK in the progression of the disease.

Here, we provide the first characterisation of the STRIPAK kinase MST4 in the heart. Patients suffering from end-stage dilated (DCM, n=10) or ischemic cardiomyopathy (ICM, n=9) have higher cardiac MST4 levels (5.71-fold, p<0.001 and 3.83-fold, p<0.05) compared with non-failing controls (n=6). Upregulation could also be observed in experimental heart failure models, both in MLP-KO (n=14, 2.493-fold, p<0.05) and in Calsarcin-1-KO mice (n=14, 2.491-fold, p<0.01) compared to wildtype animals (n=14 and n=11).

In contrast, in models of cardiac hypertrophy reduced cardiac MST4-levels were observed: Mice with persistently active calcineurin (CnA-TG, n=5) reveal MST4-levels of about 55% (p<0.001) compared with wildtype (n=5). Likewise, experimental aortic constriction (n=5) decreases MST4-expression to 39% (p<0.001) compared with controls (n=5). Decreased cell density of NRVCM also results in a decreased MST4-expression: Cells plated at a density of 250,000/9.6cm² (n=9) express 63% (p<0.001) less MST4 than cells plated at a density of 1,000,000/9.6cm² (n=9).

Immunofluorescence imaging of isolated rat cardiomyocytes and human heart tissue reveal different subcellular localisations, primarily at the intercalated discs. Co-immunoprecipitation experiments demonstrated interaction of MST4 with STRIPAK members Myoscape/STRIP2, Striatin and SLMAP, indicating that the STRIPAK complex is also present in the heart.

On a functional level, cell size measurement of NRVCM indicates that overexpression of MST4 (8,145 cells, 485µm²) results in cardiomyocyte hypertrophy compared with LacZ (6,888 cells, 421µm², p<0.001). Overexpression of MST4 also inhibits apoptotic cell death shown by reduction of cleaved Caspase-3 (17kDa) (-61%, p<0.001, n=6 per group) as well as cleaved Caspase-7 (-71%, p<0.001, n=6 per group) and cleaved PARP (-22%, p<0.05, n=3 per group). Concurrently, knockdown of MST4 via siRNA (n=3) increases cleaved Caspase-7 3.47-fold (p<0.01) compared with siRNA-control (n=3). In isolated adult rat cardiomyocytes, MST4-overexpression (n=52) compared with LacZ-controls (n=46) increases cellular and sarcomeric fractional shortening (p<0.05) indicating enhanced contractility.

Finally, to elucidate potential MST4 targets in cardiomyocytes, we performed a phosphoproteomics analysis in NRVCM under adenoviral overexpression with or without inhibitor compared with LacZ-controls. Results suggest the involvement of MST4 in cytoskeleton and membrane organisation, cell-cell-interaction, ion channels function and apoptosis. Potential targets include Phospholemman and Connexin-43.

In summary, we provide the first evidence of relevance of the STRIPAK complex and the MST4 kinase in heart. Future investigations will concentrate on its potential as a target in heart failure.