Comprehensive phosphoproteomics analysis of the STRIPAK associated kinase MST4 in cardiomyocytes reveals perspectives for precision medicine in heart failure

Deciphering the molecular mechanisms of heart failure remains an important challenge to brighten the perspective of cardiovascular precision medicine.

We recently identified mammalian STE20-like kinase 4 (MST4) as a novel factor in heart failure. MST4 is upregulated in human end-stage failing hearts (both DCM and ICM) and in genetic animal models (Calsarcin-1-KO and MLP-KO). In isolated cardiomyocytes, MST4 overexpression inhibited apoptosis, enhanced contractility, and induced hypertrophy. Although pathological hypertrophy often corraltes with activation of fetal genes like NPPA, NPPB, and RCAN1.4, MST4 overexpression did not affect these genes but instead we saw increased phosphorylation of protein kinase B (PKB/Akt). This suggests that MST4 may activate protective pathways, as Akt is typically associated with physiological hypertrophy.

To identify direct or indirect targets of MST4 in cardiomyocytes, we conducted an in-depth phosphoproteomic analysis of neonatal rat ventricular cardiomyocytes (NRVCMs). Cells infected with either MST4- or control-adenovirus were collected at two different time points, with an additional group in which MST4 was overexpressed and treated with an MST4 inhibitor. Mass spectrometry analysis identified over 70,000 peptides, including more than 20,000 phosphopeptides across more than 4,000 protein groups.

We applied linear modeling to a refined dataset containing regulated features selected based on p-value and fold-change thresholds for principal component analysis. With minimal variance within each group, the two sets of MST4 overexpression samples clustered closely together, indicating a consistent effect of MST4 that is further underscored by its inverse correlation with the inhibitor-treated set. To improve the quality of the results, we compared untreated cells to the control cells to filter out noise.

Enriched gene ontology terms at peptide, phosphopeptide and protein level in the early overexpression group (MST4 vs. LacZ 48h) relate to the cytoskeleton (i.e. cardiac muscle contraction, Z disc, actin filament, cytoskeleton, stress fiber), cell-cell-interaction (i.e. cell junction, intercalated disc, adherens junction, focal adhesion, gap junction activity) and kinase activity (i. e. protein kinase C binding, protein phosphorylation). While fewer GO terms were enriched in the later overexpression group (MST4 vs. LacZ 72h), they emphasized the same categories, reinforcing MST4’s functional role. No enriched GO terms at the phosphopeptide level were observed in the MST4 + inhibitor vs. LacZ comparison, suggesting effective MST4 inhibition—a first demonstration in cardiomyocytes.

Furthermore, we identified a number of potential MST4 substrates including known substrates such as MST4 itself or PP14C. In cardiac context, especially interesting novel candidates are i.e. Phospholemman (controls cardiac Na+/K+-ATPase), Connexin 43 (important intercalated disc protein), elongation factor 2 (controls cardiac protein synthesis under stress) and NHE1 (regulates intracellular pH and its cardiac modulation is already in early clinical trials).

In summary, our data suggest that MST4 upregulation in human heart failure may play a protective role. Many newly identified potential MST4 substrates highlight perspectives for further investigation into their translational potential towards cardiovascular precision medicine.