mTORC1 regulates the metabolic switch of postnatal cardiomyocytes during 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.

mTORC1 Regulates the Metabolic Switch of Postnatal Cardiomyocytes During 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.

LRRC10 regulates mammalian cardiomyocyte cell cycle during heart regeneration.

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.

Valproic acid promotes SOD2 acetylation: a potential mechanism of valproic acid-induced oxidative stress in developing systems.

Valproic acid (VPA) is an antiepileptic, bipolar, and migraine medication, which is associated with embryonic dysmorphology, more specifically neural tube defects (NTDs), if taken while pregnant. One mechanism by which VPA may cause NTDs is through oxidative stress that cause disruption of cell signaling. However, mechanisms of VPA-induced oxidative stress are not fully understood. Since VPA is a deacetylase inhibitor, we propose that VPA promotes mitochondrial superoxide dismutase-2 (SOD2) acetylation, decreasing SOD2 activity and increasing oxidant levels. Using the pluripotent embryonal carcinoma cell line, P19, VPA effects were evaluated in undifferentiated and neurodifferentiated cells. VPA treatments increased oxidant levels, oxidized the glutathione (GSH)/glutathione disulfide (GSSG) redox couple, and decreased total SOD and SOD2 activity in undifferentiated P19 cells but not in differentiated P19 cells. VPA caused a specific increase in mitochondrial oxidants in undifferentiated P19 cells, VPA did not alter respirometry measurements. Immunoblot analyses demonstrated that VPA increased acetylation of SOD2 at lysine68 (AcK68 SOD2) in undifferentiated P19 cells but not in differentiated P19 cells. Pretreatments with the Nrf2 inducer, dithiol-3-thione (D3T), in undifferentiated P19 cells prevented increased oxidant levels, GSH/GSSG redox oxidation and restored total SOD and SOD2 activity, correlating with a decrease in AcK68 SOD2 levels. In embryos, VPA decreased total SOD and SOD2 activity and increased levels of AcK68 SOD2, and D3T pretreatments prevented VPA effects, increasing total SOD and SOD2 activity and lowering levels of AcK68 SOD2. These data demonstrate a potential, contributing oxidizing mechanism by which VPA incites teratogenesis in developing systems. Moreover, these data also suggest that Nrf2 interventions may serve as a means to protect developmental signaling and inhibit VPA-induced malformations.