Abstract 393 MST4, a novel STRIPAK associated kinase is upregulated in human cardiomyopathy and regulates cardiomyocyte hypertrophy, cardiac contractility and apoptosis

Heart failure is one of the leading causes of morbidity and death worldwide. Myocardial failure is associated with loss of cardiomyocytes due to apoptosis, cardiac fibrosis, alterations in cardiomyocyte calcium cycling and altered hypertrophic signalling. Mammalian striatin-interacting phosphatase and kinase (STRIPAK) complexes have recently been intensely studied in cellular processes and in the development of different diseases, except the heart. To date, the role of STRIPAK complexes in cardiomyocytes is not well understood.

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 stabilize LTCC surface expression. Mice with knockout of Myoscape develop progressive heart failure.

Here, we demonstrate that STRIPAK kinase MST4 interacts with Myoscape as well as SLMAP in neonatal rat ventricular cardiomyocytes (NRVCM) indicating that STRIPAK is present in the heart. To elucidate potential MST4 targets in cardiomyocytes, we used an MST4-inhibitor and performed a phosphoproteomics analysis in NRVCM under adenoviral overexpression with or without inhibitor compared with LacZ-controls. Results strongly suggest the involvement of MST4 in cytoskeleton and membrane organisation, cell-cell-interaction, ion channels function and apoptosis.

Multi tissue immunoblot experiments of rat and mouse show highly enriched MST4-expression in the heart. Analyses of human biopsy samples from patients suffering from dilated (DCM, n=10) or ischemic cardiomyopathy (ICM, n=9) revealed that MST4 is upregulated in DCM (5.71-fold, p<0.001) and ICM (3.83-fold, p<0.05) compared with non-failing controls (n=6). In MLP-KO mice (n=5) MST4-mRNA was upregulated 5.57-fold (p<0.001) compared with wildtypes (n=5). Immunoblot analyses of Calsarcin 1-KO mice’s hearts (n=14) show MST4-levels 2.74-fold (p<0.01) higher than in wildtype littermates (n=11).

In contrast, transgenic mice with permanent calcineurin-activation (CnA-TG, n=5) show reduced MST4-levels of 55% (p<0.001) compared with wildtype (n=5). Moreover, experimental aortic constriction (n=5) decreases MST4-expression to 39% (p<0.001) compared to controls (n=5). In-vitro, biomechanical stretch (n=6) downregulates MST4-mRNA-levels by about 23% (p<0.05) compared to controls (n=6). Interestingly, 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). This effect is even stronger when focussing on the active form of MST4, autophosphorylated at T178, revealing a strong reduction at lower cell density by 79% (p<0.001).

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.

Clin Res Cardiol (2021) DOI DOI https://doi.org/10.1007/s00392-021-01843-w
MST4, a novel STRIPAK associated kinase is upregulated in human cardiomyopathy and regulates cardiomyocyte hypertrophy, cardiac contractility and apoptosis
M. Leye¹, C. Tannert¹, B. Völschow¹, N. Frey², S. Martini¹, S. T. Sossalla³, M. Eden¹
¹Klinik für Innere Medizin III, Schwerpunkt Kardiologie und Angiologie, Universitätsklinikum Schleswig-Holstein, Kiel; ²Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie, Universitätsklinikum Heidelberg, Heidelberg; ³Klinik und Poliklinik für Innere Med. II, Kardiologie, Universitätsklinikum Regensburg, Regensburg;
Heart failure is one of the leading causes of morbidity and death worldwide. Myocardial failure is associated with loss of cardiomyocytes due to apoptosis, cardiac fibrosis, alterations in cardiomyocyte calcium cycling and altered hypertrophic signalling. Mammalian striatin-interacting phosphatase and kinase (STRIPAK) complexes have recently been intensely studied in cellular processes and in the development of different diseases, except the heart. To date, the role of STRIPAK complexes in cardiomyocytes is not well understood. 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 stabilize LTCC surface expression. Mice with knockout of Myoscape develop progressive heart failure. Here, we demonstrate that STRIPAK kinase MST4 interacts with Myoscape as well as SLMAP in neonatal rat ventricular cardiomyocytes (NRVCM) indicating that STRIPAK is present in the heart. To elucidate potential MST4 targets in cardiomyocytes, we used an MST4-inhibitor and performed a phosphoproteomics analysis in NRVCM under adenoviral overexpression with or without inhibitor compared with LacZ-controls. Results strongly suggest the involvement of MST4 in cytoskeleton and membrane organisation, cell-cell-interaction, ion channels function and apoptosis. Multi tissue immunoblot experiments of rat and mouse show highly enriched MST4-expression in the heart. Analyses of human biopsy samples from patients suffering from dilated (DCM, n=10) or ischemic cardiomyopathy (ICM, n=9) revealed that MST4 is upregulated in DCM (5.71-fold, p<0.001) and ICM (3.83-fold, p<0.05) compared with non-failing controls (n=6). In MLP-KO mice (n=5) MST4-mRNA was upregulated 5.57-fold (p<0.001) compared with wildtypes (n=5). Immunoblot analyses of Calsarcin 1-KO mice’s hearts (n=14) show MST4-levels 2.74-fold (p<0.01) higher than in wildtype littermates (n=11). In contrast, transgenic mice with permanent calcineurin-activation (CnA-TG, n=5) show reduced MST4-levels of 55% (p<0.001) compared with wildtype (n=5). Moreover, experimental aortic constriction (n=5) decreases MST4-expression to 39% (p<0.001) compared to controls (n=5). In-vitro, biomechanical stretch (n=6) downregulates MST4-mRNA-levels by about 23% (p<0.05) compared to controls (n=6). Interestingly, 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). This effect is even stronger when focussing on the active form of MST4, autophosphorylated at T178, revealing a strong reduction at lower cell density by 79% (p<0.001). 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.
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