1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Universitätsklinikum Schleswig-Holstein Innere Medizin III mit den Schwerpunkten Kardiologie, Angiologie und internistische Intensivmedizin Kiel, Deutschland; 3Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland; 4Universitätsklinikum Gießen und Marburg GmbH Medizinische Klinik I - Kardiologie und Angiologie Gießen, Deutschland
Heart failure still poses substantial challenges in understanding its underlying molecular pathways. We recently found the mammalian STE20-like kinase 4 (MST4) significantly upregulated in hearts of patients suffering from end stage heart failure (both DCM and ICM). These findings were consistent with an upregulation of MST4 in several genetic animal models of heart failure (i.e. Calsarcin-1-KO and MLP-KO). In order to understand its potential role in heart failure pathogenesis we conducted a series of in vitro experiments.
Adenoviral overexpression of MST4 in neonatal rat ventricular cardiomyocytes (NRVCM) results in a significant increase in cell size. While pathological hypertrophy often is accompanied by the activation of fetal genes such as NPPA, NPPB or RCAN1.4, these genes were not regulated upon induction of MST4. In contrast, we observed an increase in phosphorylation of Protein kinase B (PKB/Akt). Akt is thought to be especially active in physiological hypertrophy, indicating that MST4 may be involved in protective pathways in cardiomyocytes.
In support of this notion, we found a reduction of apoptotic activity: The cleavage of key factors of apoptosis such as Caspases 3 (-61% p<0.001) and 7 (-71%, p<0.001) or PARP1 (-22%, p<0.05) was reduced. Furthermore, MST4 overexpression in isolated cardiomyocytes improved both contractility and relaxation of the whole cell as well as on sarcomere level as measured by the IonOptix system.
In order to identify direct or indirect targets of MST4 in cardiomyocytes, we performed a comprehensive analysis of the phosphoproteome of NRVCM. We harvested cells infected with either MST4- or control-adenovirus at two different time points. An additional group featured MST4 overexpression as well as a treatment with an MST4 inhibitor. Using mass spectrometry, we identified more than 70,000 peptides including more than 20,000 phosphopeptides in more than 4,000 protein groups.
We used linear modelling with a reduced data set containing regulated features selected by thresholds for p-value and fold change for principal component analysis. In addition to little variance within each set, the two sets of MST4 overexpression samples grouped very closely together suggesting a consistent MST4 effect that is further emphasised by its anti-correlation with the inhibitor set. Enriched gene ontology terms were connected to cell cell interaction, cytoskeleton and protein kinase C.
When filtering the phosphosites for significant upregulation upon MST4 overexpression at both time points and a downregulation after inhibitor treatment, we found a number of potential MST4 targets. These include proteins that have previously been described as MST4 targets such as MST4 itself or Protein phosphatase 1 regulatory subunit 14C (PPP1R14C) as well as novel candidates such as Phospholemman, Actin filament associated protein 1 like 1, Desmin or Connexin 43. On protein level, we also found a connection to intercalated disc such as regulation of Xin actin-binding repeat-containing protein 2 (XIRP2, Xinβ) or Frizzled 1. Interestingly, in human heart samples MST4 was also localized to the intercalated disc region by immunostaining.
Taken together, our data imply that MST4 upregulation in human heart failure serves a protective role. Analyses of the downstream targets of MST4 revealed novel pathways that are currently being analysed for their translational potential.