Mst4, a novel member of the intercalated disc and the crucial kinase of cardiac STRIPAK complex in the heart

Objective: Cardiomyopathies are major causes of heart failure and sudden cardiac death. Analyzing the underlying genes, encoded proteins and signalling mechanisms involved to understand the interplay of pathogenic and protective signalling pathways is a major goal of current research. We recently discovered a protein called Strip2/Myoscape that controls cardiac contractility and is part of the STRIPAK (striatin-interacting phosphatase and kinase) complexes. STRIPAK complexes are highly expressed in many tissues and involved in a variety of cellular processes, including signalling, apoptosis and cell polarity. In contrast to the phosphatases of the STRIPAK complex, like protein phosphatase 2a (Pp2a), kinases such as Mst4 and the interplay within STRIPAK complexes remain poorly characterized in the heart.

This study aimed to gain a deeper understanding of Mst4’s functions, localizations and interactions, to assess whether Mst4’s kinase activity controls binding to partners, and to determine compositions and connections of STRIPAK complexes within cardiomyocytes.

Methods: Native Co-immunoprecipitation (Co-IP) experiments were performed using neonatal rat ventricular cardiomyocytes (NRVCMs) and adult rat heart tissue, followed by immunoblotting to examine STRIPAK complexes containing Mst4 in cardiomyocytes. To investigate the interplay between Mst4 and other cardiac STRIPAK members, we used a Pp2a inhibitor to assess its effect on Mst4 phosphorylation at T178, a key site for dimerization and kinase activity. Further, LacZ and Mst4 were adenovirally overexpressed in NRVCMs combined with hesperadin (an Mst4 kinase inhibitor) followed by Co-IP and mass spectrometry (MS) of the co-eluting proteins to identify changes in the Mst4 interactions linked to its kinase activity. Immunofluorescence in human heart tissue further assessed Mst4´s localization. 

Results: Co-IP results confirmed the presence of STRIPAK complexes containing Mst4 in cardiomyocytes. Fishing with Mst4, we co-immunoprecipitated core STRIPAK members Striatin and Striatin 3. Other STRIPAK members identified in cardiomyocytes were Strip1, Strip2 and Slmap. Pp2a inhibition significantly increase Mst4 phosphorylation at T178, indicating enhanced Mst4 activity. MS analysis of the co-eluted proteins confirmed interactions between Mst4 and STRIPAK proteins, including Strip1, intercalated disc (ID) members Striatin and Striatin3, as well as with Serca2a and Desmin, independent of the inhibitor. In the presence of kinase inhibitor, Mst4 interacts with Slmap, desmoglein and junctional plakophilin. In addition, proteins such as Myh6 and Myh7 showed interactions with Mst4, particularly in the absence of Mst4 inhibition. Immunofluorescence confirmed colocalization of Mst4 with ID markers such as β-catenin, indicating Mst4 localization at the ID.

Conclusions: This study presents the first evidence that cardiac Mst4 is part of a cardiac STRIPAK complex. We show that MST4 phosphorylation and activity are regulated by Pp2a and that interactions with Mst4 are dependent on its kinase activity. Given the significant role of ID dysfunctions in dilated and arrhythmogenic right ventricular cardiomyopathy further investigations are warranted to clarify the precise Mst4-dependent mechanisms at the ID and to gain a deeper understanding of the intricate interplay between stripak-dependent kinases and phosphatases in these diseases, which may point to future structural therapeutic options.