Abstract
Mitogen-activated protein kinases (MAPKs) are implicated in cardiac hypertrophy, cardiomyocyte apoptosis, and heart failure, but little is known of the proximal MAP4Ks that selectively couple cardiac stress signals to downstream effector pathways. Mitogenactivated protein kinase kinase kinase kinase-4 (MAP4K4, ZC1, HGK) is a mammalian orthologue of yeast Ste20 that is implicated in coupling TNFα and other inflammatory cytokines to activation of the terminal MAPK, Jun N-terminal kinase (JNK). We discovered activation of MAP4K4 and its target MAP3K7 in >20 failing human hearts from diverse settings, including dilated, hypertrophic, ischemic and nthracyclineinduced cardiomyopathies. In mouse myocardium, we observed activation of MAP4K4 using four defined triggers for cardiomyocyte death with relevance to human heart failure: increased biomechanical load (partial occlusion of the transverse aorta), ischemia/reperfusion injury, and cardiomyocyte-restricted Myh6-driven expression of TNFα or Gαq (a G protein sub-unit that mediates the cardiac effects of angiotensin II, endothelin, and mechanical stress). In transgenic mice, forced expression of MAP4K4 in myocardium evoked little or no baseline phenotype, but conferred marked sensitivity to Gαq: all mice inheriting both genes died by 3 months of age, with dilated cardiomyopathy, fibrosis, apoptosis (caspase activation, TUNEL), and severely diminished contractile function (peak aortic ejection velocity by Doppler echocardiography). Thus, MAP4K4 potentiates the lethality of Gαq. In preliminary experiments, transgenic MAP4K4 also exacerbates the response to aortic banding. At the levels of over-expression obtained acutely in culture by adenoviral gene transfer, MAP4K4 was sufficient to trigger apoptosis in cultured cardiomyocytes, as measured by loss of mitochondrial membrane potential, hypodiploid (sub-G1) DNA, and caspase activation. To suppress MAP4K4 expression and activity, we engineered recombinant adenovirus expressing MAP4K4 shRNA. MAP4K4 shRNA produced a complete loss of epitope-tagged MAP4K4 when transfected together, with no reduction of the family member TNIK/ZC2. In virally transduced cardiomyocytes, MAP4K4 shRNA successfully suppressed endogenous MAP4K4 protein levels and activity. In the MAP4K4-deficient myocytes, ceramide-induced apoptosis was reduced by 54% and H2O2- induced apoptosis by 60% (P < 0.001). In summary, developing small-molecule inhibitors of MAP4K4 is a wellposed strategy, for which extensive target validation has already taken place. Currently on-going studies are focused on proof of MAP4K4 function in a human cardiac context, by RNA interference in human iPSC-derived cardiomyocytes. Use of human cardiomyocytes created from pluripotent stem cells provides unprecedented opportunity for cardiac pathway dissection and drug discovery in a human cardiac muscle cell.