The role of metabolism in cardiomyocyte maturation, disease and regeneration

Abstract

Cardiovascular disease, broadly encompassing both genetic and environmental cardiac pathology, remains the leading cause of death. As such, there is a great need to better model cardiac diseases in an attempt to find novel therapeutics as well as discover novel ways to regenerate the damaged heart. One such disease that is the leading fatty acid oxidation disorder with no known cure is mitochondrial tri-functional protein (MTP) deficiency. With the advent of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs), it is now possible to study human cardiac diseases in vitro. However, stem cell derived cardiomyocytes are more representative of fetal cardiomyocytes instead of a more mature adult like cardiomyocyte. Consequently, in order to utilize this cell type to model disease, strategies to mature hPSC-CMs must first be discovered. In order to mature hPSC-CMs we developed a microRNA maturation cocktail, termed MiMaC, that rapidly shifts the fetal like transcriptome in hPSC-CMs to a more adult like transcriptome to mature the cell. MiMaC consists of four microRNAs, the overexpression of Let7 and miR-452, and the knockout of miRs-122 and -200a. MiMaC treated hPSC-CMs displayed larger cell size, greater twitch force, greater metabolic activity and the ability to utilize long chain fatty acids for ATP production as compared to control hPSC-CMs. To better understand the way in which MiMaC brought about maturation in the hPSC-CMs we examined the predicted targets of the MiMaC microRNAs. We found that both of the knockout microRNAs had homeodomain-only protein (HOPX) as a common predicted target. To study the role of HOPX in hiPSC-CM maturation, we generated a doxycycline inducible HOPX overexpression hiPSC line that overexpressed HOPX in hiPSC-CMs. We found that HOPX overexpression led to a robust increase in cell size and the repression of cell cycle via the repression of nuclear kinetochore genes. Furthermore, we identified that HOPX overexpression led to the repression of serum response factor gene loci. These data suggest that MiMaC, in part, acts to mature hiPSC-CMs via the stimulation of CM growth and cell cycle repression. Utilizing MiMaC we were now able to study human cardiac diseases in vitro. MTP deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome (SIDS) and cardiomyopathies. MTP deficiency results in impaired fatty acid β-oxidation, however, immature hPSC-CMs can not use fatty acids. This is why it was essential to develop a tool like MiMaC to mature hPSC-CMs so they could utilize fatty acids and make it possible to study this disease. Since MTP deficiency results in sudden death, it is difficult to study MTP deficiency using in vivo model organisms. Consequently, we generated the first human in vitro model of MTP deficiency utilizing HADHA deficient hiPSC-CMs. Mutations in exon 1 of HADHA were generated using CRISPR/cas9. We found HADHA mutant (Mut) CMs challenged with fatty acids displayed sarcomere dissolution, abnormal calcium handling, elongated repolarization, erratic beating, an inability to utilize fatty acids as compared to wild type (WT) CMs and swollen and poorly functioning mitochondria. We discovered that many of the cardiac pathologies were a result of the mitochondrial dysfunction. We continued to probe the mitochondrial disfunction in HADHA Mut CMs and discovered there was abnormal cardiolipin remodeling occurring. Consequently, we elucidated the potential underlying etiology of MTP deficiency to defective cardiolipin remodeling that may be a direct or indirect cause of the mutation in HADHA. The other aspect of cardiac disease is due to non-genetic cardiac insults such as myocardial infarctions (MI). After a MI, the heart loses a large amount of muscle tissue and is replaced by non-contractile scar tissue. As a result, patients have reduced contractility resulting in reduced blood ejection and eventual heart failure. One way to remuscularize the heart is to promote cell proliferation of existing cardiomyocytes. However, adult mammalian cardiomyocytes are unable to re-enter the cell cycle yet, neonatal mice can regenerate their heart and some fish like the zebrafish, are able to regenerate their heart at both the neonatal and adult state after injury. Consequently, these two model organisms provide a means to study the molecular mechanisms governing cardiomyocyte proliferation. We discovered one of the earliest signaling pathways that are engaged during zebrafish heart regeneration was Wnt/β-catenin signaling. Utilizing a cell ablation model of cardiomyocyte injury, we elucidated that Wnt/β-catenin signaling was active as downstream targets showed high protein abundance 3-12 days post injury (dpi). Furthermore, we showed that Wnt/β-catenin signaling directly regulated the expression of Myc, electron transport chain protein complex I and cell proliferation during heart regeneration. Since many Myc targets are oxidative phosphorylation genes and mitochondrial biogenesis genes we examined how the metabolic state of the heart changes during regeneration. We identified that 7 dpi there was a dramatic increase of many metabolic pathways including oxidative phosphorylation, citric acid cycle, glycolysis and fatty acid metabolism. This led to the discovery of a unique metabolite profile found in the adult uninjured heart, high glutamine levels which depleted during the first week of regeneration while an essential amino acid, leucine, became enriched during the first week of regeneration. We showed that this dynamic amino acid profile was stimulating the mammalian target of rapamycin complex 1 (mTORC1) and that mTORC1 signaling was necessary for heart regeneration. This led to the realization that the primed state for a pro-regenerative cardiomyocyte was one with an abundance of glutamine. Finally, we showed that a primed pro-regenerative cardiomyocyte with high glutamine was conserved in the young mammalian heart. These data together provide novel insight into how a unique metabolic state primes a cardiomyocyte to enter the cell cycle which, could potentially be leveraged to develop novel therapeutics.