SUMMARY Cardiac development requires coordinated biphasic regulation of the WNT/β-catenin signaling pathway. By intersecting gene expression and loss-of-function siRNA screens we identified Alpha Protein Kinase 2 (ALPK2) as a candidate negative regulator of WNT/β-catenin signaling in cardiogenesis. In differentiating human embryonic stem cells (hESCs), ALPK2 is highly induced as hESCs transition from mesoderm to cardiac progenitors. Using antisense knockdown and CRISPR/Cas9 mutagenesis in hESCs and zebrafish, we demonstrate that ALPK2 promotes cardiac function and cardiomyocyte differentiation. Quantitative phosphoproteomics, protein expression profiling, and β-catenin reporter assays demonstrate that loss of ALPK2 led to stabilization of β-catenin and increased WNT signaling. Furthermore, cardiac defects attributed to ALPK2 depletion can be rescued in a dose-dependent manner by direct inhibition of WNT signaling through the small molecule XAV939. Together, these results demonstrate that ALPK2 regulates β-catenin-dependent signaling during developmental commitment of cardiomyocytes.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, 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 generated from NRVMs, a constitutively-active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in cardiomyocytes as a potential strategy for regenerative heart repair.

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Mitogens capable of promoting cardiomyocyte proliferation represent important targets for functional heart regeneration following myocardial infarction. We previously described an ERK-dependent pro-proliferative tissue phenotype following overexpression of constitutively-active (ca) human ERBB2 in both neonatal rat ventricular myocytes (NRVMs) and human iPSC-derived cardiomyocytes (hiPSC-CMs). Since ERBB2 canonically regulates multiple other pathways in addition to ERK, it is unclear whether ERK activation alone can drive CM proliferation. Here, we activated ERK in a targeted fashion by CM-specific lentiviral expression of a constitutively active mutant of BRAF, BRAF-V600E (caBRAF), in cultured NRVMs and examined the effects on engineered NRVM tissue proliferation, morphology, and function. caBRAF expression induced ERK activation, tissue growth, loss of contractile function, and increased tissue stiffness, all of which were sustained for at least 4 weeks in vitro. From bulk RNA-sequencing analysis of engineered tissues, we found that caBRAF had broad transcriptomic effects on CMs and induced a shift to glycolytic metabolism. Together, this work shows that direct ERK activation is sufficient to modulate CM cycling and functional maturation in a cell-autonomous fashion and could offer a potential target for cardiac regenerative therapies.