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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. Loss in cardiac regeneration coincides with metabolic alterations. Whether cellular metabolism regulates cardiac cells function from young to old age remains unknown. Here we demonstrate that uncoupling protein 2 (UCP2) promotes increased glycolysis, proliferation, and survival in cardiac tissue derived stem‐like cells in neonatal heart. In aging, UCP2 is reduced increasing oxidative phosphorylation, reducing glycolysis parallel with reduced proliferation and survival.

Introduction:The hemodynamic demands on the heart during normal pregnancy induce physiological cardiac hypertrophy. Together with exercise induced cardiac hypertrophy and cardiac enlargement during post-natal growth, these physiological cardiac adaptations are distinct from pathological hypertrophy caused by heart diseases including volume/pressure overload. Exercise-induced cardiac hypertrophy can induce a protective phenotype that leads to resistance from ischemic injury or pressure overload induced pathological remodeling. Whether pregnancy induced cardiac hypertrophy can also protect the heart from post-partum (PP) pathological stimuli remains unknown and is the focus of this study.Methods:We challenged both post-partum and age matched non-pregnant female C57BL/6 mice with 7-day osmotic minipump infusion of Angiotensin II/phenylephrine (AngII/PE) and determined the cardiac alterations.Results:PP mice had more severe cardiac pathological remodeling after AngII/PE infusion compared with non-pregnant counterparts, with greater increases in heart weight, cardiomyocyte hypertrophy and fibrosis. Echocardiography showed both left and right ventricular hypertrophy without overt systolic dysfunction. PP mice had greater elevations in expression levels of fetal genes and fibrotic genes compared with non-pregnant mice after AngII/PE infusion. RNA-sequencing analysis suggested a more robust change in gene expression level in PP mice after AngII/PE treatment compared to non-pregnant counterparts, with a substantial role for extracellular matrix organization genes. We also found a significant alteration of gene expression levels for enriched extracellular structural organization at 1-day PP, which underlies ongoing structural re-organization in the myocardium shortly after deliver. These PP changes may predispose the heart to adverse remodeling when simultaneously challenged with pathological stimuli.Conclusions:Pregnancy induced cardiac remodeling does not protect the heart against PP pathological stimuli. In fact, the rapid remodeling in the PP heart may predispose it to exacerbation of pathological remodeling.

Introduction:Few therapies have produced significant improvement in cardiac function after ischemic cardiac injury (ICI). The activation of local inflammatory responses is critical to cardiac repair after ICI. Our previous studies showed that cortical bone derived stem cells (CBSCs) possess can enhance repair after ICI. Beneficial effects of CBSCs appear to be mediated by paracrine factors including extracellular vesicles (EVs). This study explored if and how these EVs enhance cardiac repair by modulating the ICI immune response.Hypothesis:CBSCs derived extracellular vesicles (CBSC-EV) modulate immune response after ICI.Methods and Results:Both CBSCs and CBSC-EV were transplanted into mice after myocardial infarction (MI). CBSCs and CBSC-EV treated animals had better ICI repair compared with Saline, with reduced scar size, attenuated structural remodeling, improved cardiac function, and reduced cell apoptosis. These effects were linked to alteration of immune response. Plasma analysis of CBSCs and CBSC-EV treated animals showed significantly lower level of pro-inflammatory cytokines such as TNFα 24 hours after MI. CBSCs and CBSC-EV treatment induced significant polarization from CD86+ M1 macrophages towards CD206+ M2 macrophages phenotype 5 days post-MI, with subsequent reduction of CD8+ T cells and increase of CD4+ T cells, especially the FoxP3+ Treg population, from 7 days to 14 days post-MI. RNA sequencing analysis revealed that CBSC-EV contained a distinct transcriptome compared with endothelial progenitor cells and cardiosphere-derived cells. Gene Ontology analysis suggested the differentially expressed genes in CBSC-EV significantly enriched in immune cell receptor binding. MiR-182 and miR-183, which ranked top of the most significantly upregulated genes in CBSC-EV, attenuated M1 macrophage polarization after LPS treatment and promote CD25+ FoxP3+ Treg differentiation in vitro.Conclusions:CBSC-EV enhance cardiac repair by modulating the immune response after injury and highlight the molecular bases of into the beneficial effects of cell-free therapy after ICI.

The neonatal heart holds a remarkable regenerative capacity that is lost within the first week of life coinciding with a profound shift in cardiomyocyte (CM) metabolism, and CM cell cycle arrest. Whether reprogramming metabolism reactivates CM cell cycle enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for the RNA-binding protein LIN28a, a master regulator of cellular metabolism, in cardiac development and repair following injury. LIN28a was found as primarily active during cardiac development and rapidly decreases after birth. Injury to the neonatal heart, but not in adults, reactivates LIN28a leading to regenerative processes while LIN28a inhibition with small molecules attenuated pro-reparative effects of the postnatal heart. LIN28a reintroduction at P1, P3, P5, P7, and P14 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing CM cell cycle activity and number of CMs per hearts in LIN28a transgenic pups compared to WT littermates. Moreover, LIN28a overexpression extended CM cell cycle activity and regenerative processes beyond P7 resulting in increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression promoted formation of new CMs and enhanced cardiac function, and survival in mice 12 weeks after myocardial infarction compared to WT littermate controls. Furthermore, the percentage of mononucleated CMs in the LIN28a overexpressing hearts was positively correlated to better cardiac functional recovery after injury. Mechanistically, CMs overexpressing LIN28a showed increased glycolytic metabolism while its attenuation by 2-DG reverted LIN28a-induced enhancement in CM cell cycle activity in adult hearts 4 days after injury. LIN28a immunoprecipitation followed by RNA sequencing in CMs isolated from LIN28a injured hearts identified lncRNA-H19 as its most significantly altered target. Ablation of lncRNA-H19 blunted LIN28a-induced enhancement on CM metabolism and cell cycle activity. Collectively, LIN28a reprograms CM metabolism enhancing cell cycle activity in the injured heart and pro-reparative processes thereby linking CM metabolism to the regulation of ploidy/nucleation and repair in the heart.

Liver failure represents a serious challenge for cell based therapies. Mesenchymal stem cells (MSCs) possess potential for regeneration of fibrotic liver; however, there is a dire need to improve their hepatic differentiation. This study examines a pretreatment strategy to augment the differentiation potential of MSCs towards hepatic lineage. MSCs were isolated from C57BL/6 wild type mice and were characterized by flow cytometry for CD44 (92.4%), CD90 (96.6%), CD105 (94.7%), CD45 (0.8%) and CD34 (1.4%) markers. To improve the differentiation potential of MSCs towards hepatic lineage, cells were pretreated with injured liver tissue in an in-vitro model, which resulted in high expression of albumin, cytokeratin 8, 18, TAT and HNF1α as compared to untreated MSCs. The efficacy of pretreated MSCs was evaluated by preparing in-vivo mouse model with liver fibrosis by intraperitoneal administration of CCl4. Pretreated MSCs were transplanted in the left lateral lobe of mice with liver fibrosis and showed enhanced localization and differentiation abilities after 1 month. The expression for cytokeratin 8, 18, albumin and Bcl-xl was up-regulated and that of HGF, Bax and Caspase- 3 was down-regulated in animals transplanted with pretreated MSCs. Sirus red staining also confirmed a significant reduction in the fibrotic area in liver tissue transplanted with pretreated MSCs as compared to untreated MSCs and was concomitant with improved serum levels of bilirubin and alkaline phosphatase (ALP). Therefore, it was concluded that pretreatment with injured liver tissue augment homing and hepatic differentiation abilities of MSCs and provides an improved procedure for the treatment of liver fibrosis.

Introduction:Myocardial injury leads to oxidative stress that has a significant impact on the development and progression of disease. Increasing evidence suggests that oxidative stress globally influences chromatin landscape of the cells. Effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including DNA damage, cell death, and accelerated senescence which are all disease-driving mechanisms during the failing heart. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment after myocardial injury. The Polycomb complex protein BMI-1 (Bmi1) an epigenetic regulator, is associated with numerous biological functions including, mediating DNA damage control, ROS signaling and senescence. However, there is currently a lack of understanding on how Bmi1 mediates epigenetic modifications affecting heart function during injury and is a focus of this study.Hypothesis:The epigenetic regulation by Bmi1 mediates reparative process after myocardial injury by regulating ROS production. Specifically, Bmi1 modulates the epigenetic landscape of cardiac cells that mediates various molecular processes during development and stress conditions.Methods and Results:Using a Bmi1 knockout (KO) model through the induction of a tamoxifen Cre recombinase system, cardiac function is determined through echocardiography using adult mice following myocardial infarction. The Bmi1 KO has a significant decrease in heart function after myocardial infarction, which has been associated with increased cellular ROS production, fibrosis, and apoptotic mechanisms which are measured by fluorescent ROS assays, TUNEL assays, western blotting, PCR, and immunohistochemistry. To date, we have found that cardiac cells, including adult cardiomyocytes and adult cardiac fibroblasts, isolated from the Bmi1 KO model have increased cellular ROS production, increased expression of fibrotic genes including Acta2, Vimentin, Collagen, and increased expression of apoptotic pathway regulators including Aldo1, Eno1, NFkB, Ap-1, and HIF1-a.Conclusions:Bmi-1 regulation mediates repair during pathological challenge by regulating ROS production after myocardial injury.

Rationale:The adult mammalian heart has a very limited capacity to regenerate cardiomyocytes that die from pathological damage. Several studies suggest that those few new myocytes found after injury are derived from pre-existing cardiomyocytes. A recent study suggests that systemic hypoxemia in adult mice can induce their cardiac myocytes to reenter the cell cycle and form two daughter cells. The goal of the present experiments was to determine if hypoxemia can induce adult murine cardiomyocytes to reenter the cell cycle and divide.Hypothesis:Hypoxia induces adult cardiomyocytes to complete the cell cycle and form new cardiac myocytes.Methods and Results:EdU mini pumps were implanted in 2-month-old C57BL/6 mice. Mice were then placed in a hypoxia chamber and the oxygen was lowered by 1% every day for 14 days to reach a final concentration of 7% oxygen. The animals remained in hypoxia (7% Oxygen) for 2 weeks and then were euthanized. Hypoxic mice had an increase in heart weight and echocardiography documented an increase in cardiac pump function and LV wall thickness when compared to control mice. Analysis of isolated cardiac myocytes in C57BL/6 animals showed an increase in EdU+ cardiomyocytes in hypoxemia and DAPI fluorescence intensity was also increased in these EdU+ myocytes which may indicate DNA synthesis. In fixed heart tissue sections, hypoxic mice contained more EdU+ myocytes and had an increase in the total amount of EdU+ cells in the heart. To further evaluate cardiomyocyte proliferation, we employed a mosaic mouse model with double markers (MADM) in which myocytes entering the cell cycle express both red fluorescent protein (RFP) and green fluorescent protein (GFP) and therefore are yellow in color. When cells complete the cell cycle and divide they express only RFP or GFP. MADM mice were exposed to hypoxia at 7% Oxygen as described above. MADM mice subjected to hypoxemia had increased numbers of yellow myocytes versus controls. There was a much smaller number of hypoxic myocytes with just RFP or GFP.Conclusion:Systemic hypoxia induces cardiac myocyte cell cycle re-entry and DNA synthesis, but it seems that very few of these myocytes complete the cell cycle and divide into two daughter cells.

Introduction:Neonatal heart can regenerate after myocardial injury unlike adult heart. Its capacity diminishes and disappears by post neonatal day 7 (P7). Recent studies have shown that immune cells especially T cells plays an important role to regulate inflammation, however, their role in cardiac regeneration/repair after injury is understudied. Therefore, in the present study we will characterize the temporal dynamics of T cells in neonatal and adult heart after injury and will study how temporal regulation is essential to maintain repair processes.Hypothesis:Regulatory T cells plays an important role in mediating reparative processes after ischemic cardiac injury.Methods and Results:Myocardial infarction (MI) was induced in neonatal mice at P2 (n=30) and in 8-week-old (n=10) C57BL/6 mice by ligating the left anterior descending artery (LAD). CD4+, CD8+ and Foxp3+ T cells were sorted by FACS at post MI day 2, 3, 4, 5 and 7 in neonatal mice and post MI day 7 in adult mice and analyzed for the differences in transcriptome by ultra-low input RNA sequencing. Flow cytometry analysis showed CD4+ T cells peaked at post MI day 4 compared to CD8+ T cells that peaked at post MI day 7. FoxP3+ T cells increased at post MI day 2 and diminished by post MI day 7 which coincides with the non-regenerative window of the heart. CD4+ and Foxp3+ T subsets showed increased expression of transcript in anatomical structure morphogenesis and regulation of response to stimulus. Our data showed 11.2%, 12.8% and 13.4% genes in CD4+, CD8+ and Foxp3+ T cells subsets were upregulated in neonatal mice versus adult animals. Enrichment analysis resulted that cellular component morphogenesis and cell development were activated in CD4+ T cells, and cellular component biogenesis and organelle organization were elevated in Foxp3+ T cells in neonatal mice. In addition, chromosome organization and cell cycle were upregulated in CD8+ T cells. Additionally, Foxp3+ T cells showed 198 genes uniquely expressed in neonatal hearts which are involved in cardiomyocytes proliferation and myofibroblasts phenotype transition.Conclusions:CD4+/CD8+/Foxp3+ regulate inflammation and mediate reparative processes in cardiac cells including myocytes and fibroblast after myocardial ischemic injury.

Histone deacetylase inhibition counteracts adverse left ventricular remodeling, improves pulmonary function, and reverses diastolic dysfunction.

Rationale:Cell therapy has produced modest improvement in cardiac function, for reasons not yet clear. One possibility is that activation of local inflammatory response and immune cell infiltration along with release of pro-inflammatory cytokines following ischemic injury affects cardiac repair process. Cortical bone derived stem cells (CBSCs) have gained prominence due to their reparative properties. Salutary effects of CBSCs in large are mediated by paracrine secretions. Since exosomes represent active component of released factors whether CBSC derived exosomes (CBSCs-Exo) are a viable therapy for heart repair after injury is presently unknown.Objective:Determine the therapeutic value of CBSCs exosomes in mediating immune response after cardiac injury.Methods and Results:Exosomes isolated from murine CBSCs by ultracentrifugation showed typical exosome size (30-100nm) validated by electron microscopy and dynamic light scattering. To determine cardiac therapeutic value, CBSC exosomes (120?g) were injected in mice after myocardial infarction (MI) demonstrated reduced infarct size. Interestingly, serum levels of pro-inflammatory cytokines were significantly reduced along with decreased expression of CD86+ cells in animals receiving CBSCs-Exo versus control animals. Concurrently, CBSCs-Exo can mediate T cell response with increase in CD4+ cells. CBSC-Exo animals also showed improved cardiac function and contractility compared to saline treated animals. Salutary effects of CBSC-Exo were confirmed in vitro and showed increased cardiac protection in NRVMs after hypoxic challenge. Simultaneously, treatment of bone marrow-derived macrophages and T cells stimulated and treated with CBSCs-Exo showed increased polarization towards pro reparative phenotype demonstrating immunomodulation capacity of CBSC-Exo. The underlying mechanism for beneficial effects was tied to increased packaging of cardioprotective/ immunomodulatory factors in CBSCs-Exo confirmed by iRNA array analysis.Conclusion:Exosomes derived from CBSCs can augment cardiac function by increased packaging of cardioprotective and immune-modulatory miRs highlight a potential new insight into the salutary effects of CBSCs after injury.