Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for studying the role of MAP4K4 kinase in cell death

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Kinases comprise universal signalling cascades potentially pivotal to cardiac cell death pathways. Cell death is divided into several modes, chiefly apoptosis and necrosis. Both contribute to the pathophysiology of heart disease, with overlapping signalling pathways of caspase activation and apoptogen release from the mitochondria. This project focuses on the genetic validation of the stress-activated protein kinase, MAP4K4 / HGK, which functions as a control point integrating diverse signals for cardiomyocyte cell death. MAP4K4 and its reported target MAP3K7 / TAK1 appear to operate upstream of JNK in driving cardiac muscle cell death. MAP4K4 is activated in diseased mouse and human myocardium and in cultured rat cardiomyocytes subjected to death signals. Its overexpression in the rodent cardiomyocytes is sufficient to induce cardiomyocyte apoptosis, whereas knockdown of endogenous MAP4K4 using shRNA suppresses it. However, there is no direct or concrete evidence that MAP4K4 has functional importance in a human cardiac muscle cell. Given this unmet need, we deployed shRNAmir-based technology in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to knockdown MAP4K4 and investigate inhibition of cardiac muscle cell death, in response to death-inducing agents (i.e. H2O2 and c2-ceramide). First, we characterized the purity of the proposed human system by cardiac-specific antibodies and confirmed the cells’ cardiac identity, with a purity of 95% for the most prevalent marker tested. Then, via a series of optimization efforts using assays for caspase-3 and caspase-8 activities, diminished mitochondrial membrane potential and loss of surface membrane integrity, we developed a short-term maintenance protocol for hiPSC-CMs entailing 2 days in defined (transition) medium prior to death induction. Using this method, the cells were refractory to serum-induced proliferation, thus providing us with conditions under which the cell death assays could be carried out without confounding effects on purity. Prior to phenotypic assessment of MAP4K4 knockdown in hiPSC-CMs, we validated the efficacy of multiple shRNAmirs in a heterologous system. Three potent shRNAmirs were selected for virus production and proven to inhibit MAP4K4 (approximately 60-70%) in hiPSC-CMs. We then defined the conditions for provoking cell death with H2O2 and c2-ceramide, and identified the biochemical responses to each of these complementary death signals. Cell death with various H2O2 concentrations at different times was characterized by diminished mitochondrial potential, decreased caspase-8 activity and lack of caspase-3 activation. Cell death with 20μM c2-ceramide was evident at 16h associated with increased caspase-3 activity. Suppression of MAP4K4 in hiPSC-CMs with the most potent shRNAmir resulted in a 2-fold reduction of death (p<0.001) measured by DRAQ7 and 2-fold reduction (p<0.05) of cells with diminished mitochondrial potential, compared to the non-silencing control (NSC) upon H2O2 treatment. Interestingly, the reduction of cell death was associated with increased caspase-8 activity. Thus, we prove that MAP4K4 is pro-apoptotic in human cardiac muscle cells, as its knockdown leads to salvage from cell death. This proof-of-concept study will serve as a technical and scientific springboard for later, “higher bandwidth” studies of the entire human kinome or other essential mediators of cardiomyocyte demise.