Cellular replacement in the heart is restricted to postnatal stages with the adult heart largely postmitotic. Studies show that loss of regenerative properties in cardiac cells seems to coincide with alterations in metabolism during postnatal development and maturation. Nevertheless, whether changes in cellular metabolism are linked to functional alternations in cardiac cells is not well studied. We report here a novel role for uncoupling protein 2 (UCP2) in regulation of functional properties in cardiac tissue derived stem‐like cells (CTSCs). CTSC were isolated from C57BL/6 mice aged 2 days (nCTSC), 2 month (CTSC), and 2 years old (aCTSC), subjected to bulk‐RNA sequencing that identifies unique transcriptome significantly different between CTSC populations from young and old heart. Moreover, results show that UCP2 is highly expressed in CTSCs from the neonatal heart and is linked to maintenance of glycolysis, proliferation, and survival. With age, UCP2 is reduced shifting energy metabolism to oxidative phosphorylation inversely affecting cellular proliferation and survival in aged CTSCs. Loss of UCP2 in neonatal CTSCs reduces extracellular acidification rate and glycolysis together with reduced cellular proliferation and survival. Mechanistically, UCP2 silencing is linked to significant alteration of mitochondrial genes together with cell cycle and survival signaling pathways as identified by RNA‐sequencing and STRING bioinformatic analysis. Hence, our study shows UCP2‐mediated metabolic profile regulates functional properties of cardiac cells during transition from neonatal to aging cardiac states.

Rationale Cell-based therapeutics have been extensively used for cardiac repair yet underperform due to inability of the donated cells to survive in near anoxia after cardiac injury. Cellular metabolism is linked to maintenance of cardiac stem cell (CSC) renewal, proliferation and survival. Ex vivo expansion alters (CSC) metabolism increasing reliance on oxygen dependent respiration. Whether promoting ‘metabolic flexibility’ in CSCs augments their ability to survive in near anoxia and repair the heart after injury remains untested. Objective Determine the effect of LIN28a induced metabolic flexibility on cardiac tissue derived stem like cell (CTSC) survival and repair after cardiac injury. Methods and results LIN28a expression coincides during heart development but is lost in adult CTSCs. Reintroduction of LIN28a in adult CTSC (CTSC- LIN ) increased proliferation, survival, expression of pluripotency genes and reduced senescence compared to control (CTSC- GFP ). Metabolomic analysis show glycolytic intermediates upregulated in CTSC- LIN together with increased lactate production, pyruvate kinase activity, glucose uptake, ECAR and expression of glycolytic enzymes compared to CTSC- GFP . Additionally, CTSC- LIN showed significantly reduced ROS generation and increase antioxidant markers. In response to H2O2 induced oxidative stress, CTSC- LIN showed increased survival and expression of glycolytic genes. LIN28a salutary effects on CTSCs were linked to PDK1/let-7 signaling pathway with loss of PDK1 or alteration of let-7 abrogating LIN28a effects. Following transplantation in the heart after myocardial infarction (MI), CTSC- LIN showed 6% survival rate at day 7 after injection compared to control cells together with increased proliferation and significant increase in cardiac structure and function 8 weeks after MI. Finally, CSTC-LIN showed enhanced ability to secrete paracrine factors under hypoxic conditions and ability to promote cardiomyocyte proliferation following ischemic cardiac injury. Conclusions LIN28a modification promotes metabolic flexibility in CTSCs enhancing proliferation and survival post transplantation including ability to repair the heart after myocardial injury.

Rationale: The adult Heart is largely a dormant organ supporting limited cellular turnover. In contrast, neonatal cardiac tissue proliferates and is capable of regeneration while operating under a specialized metabolic state. During transition to adulthood, cardiac metabolism undergoes a rapid shift that coincides with termination of regenerative processes. Whether altering cardiac metabolism recapitulates regenerative potential remains untested. Recently, introduction of Lin28, a metabolic regulator of pluripotency, enhances tissue repair after injury. Nevertheless, there are no studies characterizing the effect of Lin28 on cardiac repair. Objective: Determine the effect of Lin28 on cardiac progenitor cell function and cardiac repair after injury. Methods and Results: Lin28 expression coincides during heart development with c-kit and declines postnatal with complete abrogation in 3-week-old adult heart as measured by qRT-PCR and immunohistochemistry. CPCs were engineered with Lin28-GFP (CPCLin) lentivirus GFP expressing CPCs were used as controls (CPC-G). CPCLin demonstrated increased proliferation measured by CyQuant compared to CPC-G, concurrent with decreased apoptosis. Interestingly, CPCLin demonstrated a significant increase in lactate production, pyruvate kinase activity, glucose uptake together with enhancement of glycolysis and expression of glycolytic enzymes compared to CPC-G. Oxidative metabolism was also upregulated together with increased intracellular ATP in CPCLin compared to controls. Additionally, CPCLin demonstrated significantly reduced ROS generation as measured by CM-H 2 TMROS based FACS analysis compared to CPC-G. To determine in vivo efficacy, CPCLin and CPC-G were transplanted in the heart after myocardial infarction. CPC-Lin hearts showed significant increase in cardiac structure and function 8 weeks after MI. Increased persistence and proliferation of transplanted CPC-Lin cells together with reduced apoptosis were observed in CPCLin hearts compared to control CPC-G hearts 2 days after transplantation. Conclusions: Lin28 modification of CPCs reprograms cellular metabolism in CPCs enhancing proliferation and survival including ability to repair the heart after myocardial injury.

Rationale: The developmental cardiac tissue is a proliferative organ capable of regeneration that extends briefly into the postnatal period. Studies show that the heart undergoes alterations in metabolism that coincide with cessation of regenerative processes during postnatal development. Whether cardiac cells in the early postnatal heart exhibit metabolic properties that favor regeneration remains unknown. Objective: To determine whether postnatal cardiac tissue harbors CPCs with unique metabolic profile. Methods and Results: CPCs were isolated from C57BL/6 mice aged 2-day (NCPC), 2-month (adult CPC) and 2-year old (ACPC). Morphological assessment showed NCPCs were smaller and rounded compared to CPCs and ACPCs and expressed putative stem cell markers and were negative for hematopoietic marker CD45. Interestingly, NCPCs expressed elevated levels of pluripotency markers such as LIN28 compared to ACPCs and CPCs. Increased viability, proliferation rate, metabolic activity and reduced doubling times were observed in NCPCs compared to CPCs and ACPCs measured by CyQuant, MTT, and Resazurin assays. NCPCs demonstrated a youthful phenotype and were resistant to H2O2 induced stress compared to ACPCs and CPCs as measured by β-Galactosidase and TUNEL labeling respectively together with a unique expression of paracrine factors as assessed by proteome profiler array. Interestingly, NCPCs demonstrated increased ATP generation, glycolytic activity and reduced oxidative phosphorylation compared to CPCs and ACPCs measured by seahorse assay parallel with enhanced expression of glycolytic enzymes measured by qRT-PCR and western blot. NCPCs showed increased lactate production and pyruvate kinase activity and low mitochondrial membrane potential. Interestingly, mitochondrial fuel dependency assay showed increased glutamine dependency as the fuel for mitochondrial oxidative phosphorylation. RNA-sequencing analysis demonstrated increased expression of metabolic signaling in NCPCs compared to CPCs and ACPCs. Conclusions: Postnatal cardiac tissue possesses progenitor cell population with unique metabolic profile that coincides with enhanced functional properties of NCPCs suggesting their potential therapeutic value for cardiac repair.