Rationale Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. Objective To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. Methods and Results High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical MSC and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. Whilst genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, Tbx20, caused marked myocardial dysmorphology and perturbations in scar formation upon myocardial infarction. Conclusions The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs and direct contribution to cardiac development and repair provokes alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes 1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest 3 . Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 doubleknockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin

Background: The adult mammalian heart is incapable of meaningful functional recovery after injury, and thus promoting heart regeneration is one of the most important therapeutic targets in cardiovascular medicine. In contrast to the adult mammalian heart, the neonatal mammalian heart is capable of regeneration after various types of injury. Since the first report in 2011, a number of groups have reported their findings on neonatal heart regeneration. The current review provides a comprehensive analysis of heart regeneration studies in neonatal mammals conducted to date, outlines lessons learned and poses unanswered questions. Methods: We performed a PubMed search using the keywords “neonatal” and “heart” and “regeneration.” In addition, we assessed all publications that cited the first neonatal heart regeneration reports: Porrello et al, Science, Feb 2011 for apical resection injury; Porrello et al, PNAS, Dec 2012 for coronary ligation injury; and Mahmoud et al, Nature Methods, Jan 2014 for surgical methodology. Publications were examined for surgical models used, timing of surgery, and postinjury assessment including anatomical, histological and functional assessment, as well as conclusions drawn. Results: We found 30 publications that performed neonatal apical resection, 19 publications that performed neonatal myocardial infarction by coronary artery ligation, and 6 publications that performed cryoinjury using liquid nitrogen-cooled metal probes. Both apical resection and ischemic infarction injury in neonatal mice result in a robust regenerative response, mediated by cardiomyocyte proliferation. On the other hand, several reports have demonstrated that cryoinjury is associated with incomplete heart regeneration in neonatal mice. Not surprisingly, several studies suggest that injury size, as well as surgical and histological techniques can strongly influence the observed regenerative response and final conclusions. Studies have utilized these neonatal cardiac injury models to identify factors that either inhibit or stimulate heart regeneration. Conclusions: Overall, there is consensus that both apical resection and coronary ligation injuries during the first two days of life result in heart regeneration in neonatal mammals, whereas cryoinjury was not associated with a similar regenerative response. This regenerative response is mediated by proliferation of pre-existing cardiomyocytes, and is modifiable by injury size and surgical technique, as well as metabolic, immunologic, genetic and environmental factors.

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