Roles of MAPK Signaling Pathway in Cardiomyocyte Proliferation and Function in Engineered Cardiac Tissues

Abstract

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte proliferation have been identified as potential candidates for functional heart repair following myocardial infarction (MI). Mature adult CMs are highly resistant to mitogenic stimuli, with many mitogens only slightly raising adult CM proliferation rates in vivo. Conversely, immature neonatal CMs are more proliferative, permitting early neonatal rodent hearts to partially regenerate following injury. It is therefore of importance to improve our understanding of the mechanisms behind CM maturation and cell cycle regulation, which could be aided by the development and utilization of more relevant in vitro systems to study CM proliferation. Of particular interest, CMs in vivo undergo polyploidization, an increase in cellular DNA content without division, during postnatal maturation. This has been shown to correlate with a decline in their proliferative capacity and an increase in cell size and maturation. The goals of this dissertation have been to utilize in vitro cardiac tissue models to: 1) identify potent mitogenic stimuli capable of inducing CM proliferation, 2) describe the underlying mechanisms behind how this stimulus promotes CM proliferation, and 3) investigate the link between CM proliferation, maturation, and polyploidy.First, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues (ECTs) generated from NRVMs, a constitutively active mutant form of the membrane receptor of mitogen activated protein kinase (MAPK) signaling pathway, human Erbb2 (cahErbb2), was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM cell cycle entry and mitosis, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of MEK or ERK signaling. These results suggested transient activation of Erbb2/ERK axis in cardiomyocytes as a potential strategy for regenerative heart repair.We then explored whether activating ERK via a constitutively active mutant of a more downstream effector of MAPK pathway, BRAF (BRAF-V600E, caBRAF), can induce pro-proliferative effects in NRVM ECTs. Sustained CM-specific caBRAF expression induced chronic ERK activation, significant tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. CaBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a pro-migratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid, precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase was sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.In the aforementioned in vitro studies, we relied on use of the more mature and less-proliferative NRVM vs. hiPSC ECT model system for identifying cardiac mitogens. Unlike predominantly polyploid adult CMs, immature hiPSC-CMs are primarily mononuclear and diploid, which makes them permissive to proliferation and therefore potentially less suitable for mitogen screens. We therefore studied whether induction of polyploidy alone in hiPSC-CMs would be sufficient to promote their maturation in vitro. Lentiviral overexpression of dominant negative ECT2 (dnECT2), an important cytokinesis gene, was sufficient to induce polyploidy in hiPSC-CMs and NRVMs. Upon screening of small molecules for polyploidy induction, we identified transient small molecule inhibition of AURKB as an even more effective approach than dnECT2 expression in producing polyploid hiPSC-CMs. Compared to diploid hiPSC-CMs, small molecule-induced polyploid hiPSC-CMs exhibited increased cell size, mitochondrial density, and rates of transcription and translation. Polyploid hiPSC-CMs were also less proliferative than diploid hiPSC-CMs, suggesting an improved potential for use in screening for cardiac mitogens in vitro.In summary, this dissertation describes the effects of MAPK activation on CMs and shows a causative relationship between polyploidy and some aspects of CM maturation. We produced multiple RNAseq datasets of NRVM ECTs overexpressing different constitutively active MAPK genes, which could be of interest to research beyond cardiac biology. Further investigation of MAPK pathway activation in CMs may show that some downstream ERK effectors mediate the pro-proliferative effects in CMs, while others coordinate CM functional changes or changes in cell hypertrophy and maturation. Dissecting these mechanisms could enable the design of more targeted MAPK pathway modifiers with utility in cardiac regenerative biology and anticancer therapeutics.