The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key β-cell transcription factors necessary for β-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance β-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain β-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key β-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature β-cell gene expression profile.

Adult mammalian cardiomyocytes have limited proliferative potential, and after myocardial infarction (MI), injured cardiac tissue is replaced with fibrotic scar rather than with functioning myocardium. In contrast, the neonatal mouse heart possesses a regenerative capacity governed by cardiomyocyte proliferation; however, a metabolic switch from glycolysis to fatty acid oxidation during postnatal development results in loss of this regenerative capacity. Interestingly, a sarcomere isoform switch also takes place during postnatal development where slow skeletal troponin I (ssTnI) is replaced with cardiac troponin I (cTnI). In this study, we first employ integrated quantitative bottom-up and top-down proteomics to comprehensively define the proteomic and sarcomeric landscape during postnatal heart maturation. Utilizing a cardiomyocyte-specific ssTnI transgenic mouse model, we found that ssTnI overexpression increased cardiomyocyte proliferation and the cardiac regenerative capacity of the postnatal heart following MI compared to control mice by histological analysis. Our global proteomic analysis of ssTnI transgenic mice following MI reveals that ssTnI overexpression induces a significant shift in the cardiac proteomic landscape. This shift is characterized by an upregulation of key proteins involved in glycolytic metabolism. Collectively, our data suggest that the postnatal TnI isoform switch may play a role in the metabolic shift from glycolysis to fatty acid oxidation during postnatal maturation. This underscores the significance of a sarcomere-metabolism axis during cardiomyocyte proliferation and heart regeneration.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Leucine-rich repeat containing 10 (LRRC10) is a cardiomyocyte-specific protein, but its role in cardiac biology is little understood. Recently Lrrc10 was identified as required for endogenous cardiac regeneration in zebrafish; however, whether LRRC10 plays a role in mammalian heart regeneration remains unclear. In this study, we demonstrate that Lrrc10-/- knockout mice exhibit a loss of the neonatal mouse regenerative response, marked by reduced cardiomyocyte cytokinesis and increased cardiomyocyte binucleation. Interestingly, LRRC10 deletion disrupts the regenerative transcriptional landscape of the regenerating neonatal mouse heart. Remarkably, cardiac overexpression of LRRC10 restores cardiomyocyte cytokinesis, increases cardiomyocyte mononucleation, and the cardiac regenerative capacity of Lrrc10-/- mice. Our results are consistent with a model in which LRRC10 is required for cardiomyocyte cytokinesis as well as regulation of the transcriptional landscape during mammalian heart regeneration.

The metabolic switch from glycolysis to fatty acid oxidation in postnatal cardiomyocytes contributes to the loss of the cardiac regenerative potential of the mammalian heart. However, the mechanisms that regulate this metabolic switch remain unclear. The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) is a central signaling hub that regulates cellular metabolism and protein synthesis, yet its role during mammalian heart regeneration and postnatal metabolic maturation is undefined. Here, we use immunoblotting, rapamycin treatment, myocardial infarction, and global proteomics to define the role of mTORC1 in postnatal heart development and regeneration. Our results demonstrate that the activity of mTORC1 is dynamically regulated between the regenerating and the non-regenerating hearts. Acute inhibition of mTORC1 by rapamycin or everolimus reduces cardiomyocyte proliferation and inhibits neonatal heart regeneration following injury. Our quantitative proteomic analysis demonstrates that transient inhibition of mTORC1 during neonatal heart injury did not reduce protein synthesis, but rather shifts the cardiac proteome of the neonatal injured heart from glycolysis towards fatty acid oxidation. This indicates that mTORC1 inhibition following injury accelerates the postnatal metabolic switch, which promotes metabolic maturation and impedes cardiomyocyte proliferation and heart regeneration. Taken together, our results define an important role for mTORC1 in regulating postnatal cardiac metabolism and may represent a novel target to modulate cardiac metabolism and promote heart regeneration.

Sympathetic innervation influences homeostasis, repair, and pathology in the cardiac ventricles; in contrast, parasympathetic innervation is considered to have minimal contribution and influence in the ventricles. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling to define cardiac nerve architecture during development, disease, and regeneration. Our approach reveals that parasympathetic nerves extensively innervate the cardiac ventricles. Furthermore, we identify that parasympathetic and sympathetic axons develop synchronously and are bundled throughout the ventricles. We further investigate cardiac nerve remodeling in the regenerative neonatal and the non-regenerative postnatal mouse heart. Our results show that the regenerating myocardium undergoes a unique process of physiological reinnervation, where proper nerve distribution and architecture is reestablished, in stark contrast to the non-regenerating heart. Mechanistically, we demonstrate that physiological reinnervation during regeneration is dependent on collateral artery formation. Our results reveal clinically significant insights into cardiac nerve plasticity which can identify new therapies for cardiac disease.

Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration.