STRIPAK kinase MST4 as a novel mediator in heart failure

Background/Introduction Key molecular circuits in the heart include kinase–phosphatase networks that regulate cardiac remodelling. The STRIPAK (striatin-interacting phosphatase and kinase) complex represents an emerging signalling hub, yet its cardiac role remains poorly defined. We identified mammalian STE20-like kinase 4 (MST4), a STRIPAK component, as a potential mediator in heart failure. MST4 is upregulated in human end-stage failing hearts (dilated and ischaemic cardiomyopathy) and in genetic mouse models of heart failure. Purpose The aim of this study was to futher characterise the molecular and functional role of MST4 in cardiomyocytes, possible targets and to define its contribution to cardiac stress signalling and heart failure. Methods Adenoviral overexpression of MST4 was performed in neonatal rat ventricular cardiomyocytes (NRVCMs), with and without pharmacological inhibition using hesperidin. Apoptosis, hypertrophy markers and signalling pathways were analysed. MST4 interactors were identified by co-immunoprecipitation and mass spectrometry. Phosphoproteomics was conducted 48 h and 72 h after overexpression and compared with untreated, LacZ- and inhibitor-treated cells. Kinase-substrate prediction was performed using the Kinase Library platform. Regulated phosphosites were integrated with datasets from transverse aortic constriction (TAC) mice and human cardiomyopathy. Results MST4 overexpression reduced apoptosis, enhanced contractility and induced hypertrophy without activation of foetal gene programmes (NPPA, NPPB, RCAN1.4), but with increased PKB/Akt phosphorylation, consistent with protective hypertrophic signalling. Co-immunoprecipitation confirmed interaction with core STRIPAK proteins and additional binding to intercalated disc proteins. Immunofluorescence demonstrated a localisation at the intercalated disc in human myocardium, including co-localisation with β-catenin. Phosphoproteomics revealed a distinct MST4-dependent phosphorylation signature. Kinase prediction ranked MST4 as the top candidate driving the observed phosphosite regulation, suggesting that many effects are direct consequences of MST4 activity. Integration with TAC and human datasets identified conserved phosphosites associated with cardiac stress, defining an MST4-specific cardiac fingerprint. Conclusion In summary, MST4 is a component of cardiac STRIPAK complexes localised to the intercalated disc and generates a distinct phosphorylation programme associated with cardiac stress. Conserved phosphosites may help with finding mechanistic explanations for the observed biological effects. These findings position MST4 as a novel mediator within molecular circuits in heart failure and a potential target for therapeutic modulation.