Wound healing is a fundamental evolutionary adaptation with two possible outcomes: scar formation or reparative regeneration. Scars participate in re-forming the barrier with the external environment and restoring homeostasis to injured tissues, but are well understood to represent dysfunctional replacements. In contrast, reparative regeneration is a tissue-specific program that near-perfectly replicates that which was lost or damaged. Although regeneration is best known from salamanders (including newts and axolotls) and zebrafish, it is unexpectedly widespread among vertebrates. For example, mice and humans can replace their digit tips, while many lizards can spontaneously regenerate almost their entire tail. Whereas the phenomenon of lizard tail regeneration has long been recognized, many details of this process remain poorly understood. All of this is beginning to change. This Review provides a comparative perspective on mechanisms of wound healing and regeneration, with a focus on lizards as an emerging model. Not only are lizards able to regrow cartilage and the spinal cord following tail loss, some species can also regenerate tissues after full-thickness skin wounds to the body, transections of the optic nerve and even lesions to parts of the brain. Current investigations are advancing our understanding of the biological requirements for successful tissue and organ repair, with obvious implications for biomedical sciences and regenerative medicine.

Although the contractile function of the heart is universally conserved, the organ itself varies in structure across species. This variation includes the number of ventricular chambers (one, two, or an incompletely divided chamber), the structure of the myocardial wall (compact or trabeculated), and the proliferative capacity of the resident cardiomyocytes. Whereas zebrafish are capable of comparatively high rates of constitutive cardiomyocyte proliferation, humans and rodents are not. However, for most species, the capacity to generate new cardiomyocytes under homeostatic conditions remains unclear. Here, we investigate cardiomyocyte proliferation in the lizard Eublepharis macularius, the leopard gecko. As for other lizards, the leopard gecko heart has a partially septated ventricular lumen with a trabeculated myocardial wall. To test our hypothesis that leopard gecko cardiomyocytes routinely proliferate, we performed 5-bromo-2'-deoxyuridine incorporation and immunostained for the mitotic marker phosphorylated histone H3 (pHH3) and the DNA synthesis phase (S phase) marker proliferating cell nuclear antigen (PCNA). Using double immunofluorescence, we co-localized pHH3 or PCNA with the cardiomyocyte marker myosin heavy chain (MHC). We found that ~0.5% of cardiomyocytes were mitotically active (pHH3+/MHC+), while ~10% were in S phase (PCNA+/MHC+). We also determined that cell cycling by gecko cardiomyocytes is not impacted by caudal autotomy (tail loss), a dramatic form of self-amputation. Finally, we show that populations of cardiac cells are slow cycling. Overall, our findings provide predictive evidence that geckos may be capable of spontaneous cardiac self-repair and regeneration following a direct injury.

Survivable injuries to the heart resolve via one of two outcomes: scar formation or tissue regeneration. Among mammals, injury to the heart typically results in the loss of contractile muscle cells (cardiomyocytes) and the formation of a non‐contractile fibrous scar. In contrast, some teleost fish and salamanders are capable of tissue‐specific regeneration, thus replacing lost or damaged cardiomyocytes. Here, we investigate the regenerative capacity of the reptilian heart, focusing on the lizard Eublepharis macularius, the leopard gecko. Unlike mammals, but similar to teleost fish, the gecko heart has a single ventricle. To determine if the ventricle was capable of regeneration, we performed a cardiac puncture (cardiocentesis) – a penetrating wound that passes through the thoracic cavity and into the heart. Cardiac punctures are readily tolerated by geckos, and normal (pre‐injury) behaviours are observed within hours of the procedure. To characterize the reparative events, we used serial histology and immunostaining for markers of cell proliferation (proliferating cell nuclear antigen, PCNA) or cell death (Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]), each with markers for cardiomyocytes (myosin heavy chain [MHC], alpha‐smooth muscle actin [α‐SMA]), and fibroblasts/endocardial cells (Vimentin). Prior to injury, we observed proliferating populations of both cardiomyocytes and non‐cardiomyocytes. One day post‐cardiac puncture (dpc), the wound site is characterized by cell death, a localized loss of cardiomyocytes, and the formation of a blood clot capping the puncture. Between 5 and 10 dpc, there is an increase in the number of proliferating cardiomyocytes bordering the lesion, simultaneous with an increase in proliferating non‐cardiomyocytes (cardiac fibroblasts and endothelial cells) within the wound bed itself. By 14dpc, mature cardiomyocytes have repopulated the wound site, restoring the original architecture of the myocardium. Overall, our findings reveal that regeneration of the gecko heart closely resembles that of zebrafish, and is distinct from the scar‐forming response of mammals. Support or Funding Information Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants 400358

Relative rates of cardiomyocyte proliferation vary among and within species. Whereas embryonic and fetal mammals demonstrate robust rates of cardiomyocyte proliferation, this capacity sharply declines within days following birth. Continued growth of the heart in mammals primarily involves cardiomyocyte hypertrophy. In contrast, cardiomyocytes in some teleost fish and salamanders retain a relative high proliferative capacity, even as adults. In these species, continued growth involves increasing the relative population of cardiomyocytes (cardiomyocyte hyperplasia). Previously, we have demonstrated that subadult leopard geckos (Eublepharis macularius, hereafter ‘geckos’) demonstrate relatively high rates of cardiomyocyte proliferation. As subadult geckos are actively undergoing somatic growth, we sought to determine if the rates of cardiomyocyte proliferation changed in adult geckos. We hypothesized that the elevated rate of cardiomyocyte proliferation observed in subadults is a function of ontogeny, and contributes to overall heart growth through cardiomyocyte hyperplasia. To document cardiomyocyte proliferation, we performed double immunofluorescence on heart ventricles from subadult and adult geckos. To identify proliferating cardiomyocytes we immunostained for the motor protein marker myosin heavy chain with either the DNA synthesis (S) phase marker proliferating cell nuclear antigen or the mitotic (M) phase marker phosphorylated histone H3. We found that there were significantly fewer cardiomyocytes in S phase (~0.1%) and M phase (~0%) in adult geckos as compared to subadult geckos (~11% and ~0.5%, respectively). This decline in proliferation likely reflects a shift from tissue growth to maintenance and remodeling. We also determined that the ventricle of adult geckos had more than twice as many cardiac cells when compared to subadult geckos. These data indicate that, similar to zebrafish (and unlike mammals), ontogenetic growth of the gecko heart involves continued cardiomyocyte proliferation and hyperplasia.

Erythropoietin (EPO) is widely recognized as the principle regulator of erythropoiesis, however, extra‐erythropoietic functions have been identified including cytoprotection, cardiac inotropy, cellular proliferation, and embryonic development. Whole body deletion of either EPO or the EPO receptor is embryonic lethal with impaired cardiogenesis leading to ventricular hypoplasia. While multiple extra‐renal tissue and cell types produce EPO, whether the heart is a direct source remains unclear. Human recombinant EPO increases myocardial contractility and confers cytoprotection against cardiac injury, which suggests a role for EPO signalling in the heart. Our objectives were to (1) confirm whether the heart produces EPO and (2) determine if there is a role for paracrine EPO signalling during cardiogenesis. We generated constitutive, cardiomyocyte‐specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v‐cre+/−; EPOΔ/Δ‐CM). We confirmed that the heart is a source of EPO expression with a distinct circadian rhythm in adult hearts and increased expression during embryonic development. During cardiogenesis, cardiac EPO expression was reduced, but not eliminated, in EPOΔ/Δ‐CM hearts with decreased cardiac cell proliferation. This suggests EPO signalling is partially compensated by an alternate cardiac cell type during cardiac development. In adult EPOΔ/Δ‐CM mice, global cardiac mass was preserved while cardiomyocyte cross‐sectional area was increased. Taken together, cellular cardiomyocyte hypertrophy in the absence of gross organ hypertrophy, and the observed reduction in cardiac cell proliferation during cardiogenesis, points towards a reduction in the overall number of cardiomyocytes in EPOΔ/Δ‐CM mice. Collectively, these data identify the first physiological roles of extra‐renal EPO by confirming that the heart is a source of EPO and that paracrine expression is required for cardiogenesis. Further, cardiac EPO expression is a complex interplay of multiple cell types where loss of cardiomyocyte production results in compensation from other cardiac cell lineages. Support or Funding Information This work was funded in part by the Canadian Institutes of Health Research (JAS), the Natural Sciences and Engineering Research Council of Canada (KRB, MKV, and JAS), the Canadian Glycomics Network (JAS), and the Heart and Stroke Foundation of Canada (KRB and JAS). JAS is also a new investigator with the Heart and Stroke Foundation of Ontario.