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.
Across vertebrates, non‐lethal injuries to the heart are resolved in one of two ways: scar formation or regeneration. Injury to the mammalian heart results in the permanent loss of contractile muscle ...cells (cardiomyocytes) and formation of a non‐contractile fibrous scar. In contrast, species such as zebrafish and salamander are capable regenerating damaged or lost cardiomyocytes, thus restoring heart function. Previous work has shown that the cellular processes involved in heart regeneration, such as cell death and proliferation, appear to be conserved across teleost fish and salamanders. Whether similar the cellular processes are involved in heart regeneration in reptiles remains largely unexplored. Here, we characterize heart regeneration in the lizard Eublepharis macularius (the leopard gecko, hereafter ‘gecko’), following two different injury models: a physical puncture (cardiocentesis) and a cryolesion. Cardiocentesis is a penetrating wound that creates a small‐scale lesion as a needle passed through the body wall into the heart ventricle. A cryolesion requires opening the body cavity to expose the heart, and then placing a pre‐cooled probe directly on the ventricle. This results in the destruction of ~30% of the ventricle. Both injury models were tolerated by geckos, and normal movement and feeding behaviours were resumed within days. To characterize the cellular events involved in heart repair, we used serial histology and immunostaining for markers of cell death (Terminal deoxynucleotidyl transferase dUTP nick end labeling) or cell proliferation (proliferating cell nuclear antigen), each with markers for cardiomyocytes (alpha‐smooth muscle actin, myosin heavy chain), and fibroblasts/endocardial cells (Vimentin). Within the first 1‐3 days, both injury models are characterized by localized cell death and a loss in cardiomyocytes at the wound site. Over the next two weeks, injured hearts no longer show evidence of cell death. Simultaneously, there is an increase in cell proliferation by populations of cardiomyocytes bordering the wound site and by non‐cardiomyocytes within the wound bed itself. Cardiocentesis injuries were resolved within 14 days, while cryoinjuries were resolved within 60 days. Ultimately, both injury models demonstrate the return of mature cardiomyocytes and the near‐perfect restoration of the original architecture of the ventricular wall. Overall, our findings reveal that the cellular responses involved in gecko heart regeneration are conserved across different injury models but the duration of repair varies with the magnitude of the injury.
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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.
Tissue homeostasis is a dynamic process involved in the maintenance of tissue structure and function in response to physiological fluctuations in biochemistry, metabolism, and physical conditions. ...Despite the profound role that growth factors play in homeostasis of organs such as the heart, constitutive patterns of expression are only known for a handful of species. Here, we investigated growth factor expression in the heart of a representative reptile, the leopard gecko (Eublepharis macularius; hereafter, ‘gecko’). We focused our investigation on three major classes known to be expressed by mammalian cardiac tissues: transforming growth factor β (TGFβ); vascular endothelial growth factor A (VEGF); and basic fibroblast growth factor (FGF‐2). Similar to other squamates (snakes and lizards), geckos have a three‐chambered heart with two atria and one ventricle. The ventricular myocardium includes a trabeculated or spongy‐like inner compartment, and a thin, compact cortical layer. We determined that cardiac cells in both compartments of the gecko myocardium (trabeculated and compact) constitutively express a diverse panel of growth factor ligands and receptors commonly associated with wound healing and repair, including: TGFβ1, activin‐βA, and phosphorylated SMAD2; FGF‐2; and VEGF, VEGF receptor 1 (VEFGR1), VEGFR2, and phosphorylated VEGFR2. Using double immunofluorescence, we observed co‐localization of VEGF/VEGFR2 and VEGFR1/VEGFR2, indicating that – similar to mammals – reptilian cardiomyocytes may use paracrine and autocrine signalling. Taken together, these findings indicate a homeostatic role for these growth factors in the heart that is conserved across reptiles and mammals.
Support or Funding Information
Natural Sciences and Engineering Research Council (NSERC) Discovery Grant 400358 (to MV); Ontario Veterinary College Scholarship (to KJ)
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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Although historically considered to be mitotically inactive, mammalian cardiac muscle cells (cardiomyocytes) are now widely viewed as being capable of at least low levels of homeostatic ...renewal throughout adulthood. Emerging evidence indicates that the extent to which cardiomyocytes can spontaneously re‐enter the cell cycle correlates with the capacity for cardiac self‐repair and regeneration. Therefore, proliferation rates may offer a predictive tool for investigating the regenerative potential of a species. Here we explore homeostatic cardiomyocyte proliferation in a representative reptile, the leopard gecko (
Eublepharis macularius
)
.
The gecko heart is characterized by a trabeculated, spongy‐like ventricular lumen. This architecture is also seen in species capable of cardiac regeneration (such as zebrafish), as it provides an increased surface area for diffusion with a decreased reliance on coronary circulation. To assess cardiac cell proliferation, we employed a short duration bromodeoxyurdine (BrdU) pulse‐chase experiment (2 day pulse, 7 day chase), and immunostained for the S phase marker proliferating cell nuclear antigen (PCNA) and the mitotic marker phosphorylated histone H3 (pHH3). We determined that cardiac cells of the gecko heart continually proliferate, even into adulthood. Using double immunofluorescence, we then co‐localized the cardiomyocyte marker myosin heavy chain (MHC) with each of PCNA and pHH3. We found that ~10% of cardiomyocytes have entered the synthesis phase (MHC+/PCNA+), while ~0.5% are mitotically active (MHC+/pHH3+). Next, we performed a long duration BrdU pulse‐chase experiment (7 day pulse, 140 day chase). Unexpectedly, we observed long‐term label‐retaining (= slow cycling) cells throughout the ventricular myocardium. These data suggest that the gecko heart contains populations of both constitutively active and comparatively quiescent cells. Our results combined with a trabeculated ventricular architecture point towards the gecko as an excellent candidate to study cardiac self‐repair and regeneration.
Support or Funding Information
Natural Sciences and Engineering Research Council (NSERC) Discovery Grant 400358
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.
We determined that leopard gecko cardiomyocytes routinely proliferate under homeostatic conditions. Cardiomyocyte proliferation is not altered in response to tail loss, suggesting geckos are resilient to injury‐mediate systemic activation.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection damages the heart, increasing the risk of adverse cardiovascular events. Female sex protects against complications of infection; ...females are less likely to experience severe illness or death, although their risk for postacute sequelae of COVID-19 ("long COVID") is higher than in males. Despite the important role of the heart in COVID-19 outcomes, molecular elements in the heart impacted by SARS-CoV-2 are poorly understood. Similarly, the role sex has on the myocardial effects of SARS-CoV-2 infection has not been investigated at a molecular level. We intranasally inoculated female and male ferrets with SARS-CoV-2 and assessed myocardial stress signals, inflammation, and the innate immune response for 14 days. Myocardial phosphorylated GSK3α/β decreased at
postinfection (pi) in male ferrets, whereas females showed no changes. Myocardial levels of p62/SQSTM1 decreased in male ferrets at
,
, and
pi while lower baseline levels in females increased on
. Phosphorylated ERK1/2 increased in cardiomyocyte nuclei in females on
and
pi, whereas male ferrets had no changes. Only hearts from females increased fibrosis on
pi. Immune and inflammation markers increased in hearts, with some sex differences. These results are the first to identify myocardial stress responses following SARS-CoV-2 infection and reveal sex differences that may contribute to differential outcomes. Future research is required to define the pathways involving these stress signals to fully understand the myocardial effects of COVID-19 and identify targets that mitigate cardiac injury following SARS-CoV-2 infection.
Cardiovascular disease is a leading risk factor for severe COVID-19, and cardiovascular pathologies are among the most common adverse outcomes following SARS-CoV-2 infection. Females and males have different outcomes and adverse cardiovascular events following SARS-CoV-2 infection. This study shows sex differences in stress proteins p62/SQSTM1, ERK1/2, and GSK3α/β, along with innate immunity and inflammation in hearts of ferrets infected with SARS-CoV-2, identifying mechanisms of COVID-19 cardiac injury and cardiac complications of long COVID.
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Many lizards are capable of spontaneous tail regeneration following tail loss. Although the regenerate tail superficially resembles the original, it is not identical. One of the main ...differences is that a cartilaginous cone replaces the original bony vertebrae of the axial skeleton. Here we characterize spontaneous chondrogenesis during tail regeneration in the leopard gecko. Following tail loss, the first sign of regenerative outgrowth is the formation of a blastema, an aggregation of proliferating mesenchymal‐like cells localized at the site of tail loss. Cartilage regeneration begins within the blastema 8–14 days post‐injury. Presumptive cartilage first appears as a glycosaminoglycan (GAG) and type II collagen (Col2) positive condensation that surrounds the newly regenerating spinal cord. With continued regenerative outgrowth, this condensation forms a hollow cone‐like structure. Presumptive cartilage cells express phosphorylated Smad2 (pSmad2), indicating activation of the canonical transforming growth factor beta (TGFβ) signalling pathway. Curiously, early pSmad2 expression is not matched by TGFβ1 or TGFβ3 expression, indicating that a different member of the TFGβ pathway is involved in early chondrogenesis. Furthermore, the transcription factor Sox9, often described as the master regulator of chondrogenesis, is not expressed until several days after the first appearance of Col2 and GAG in the newly forming tail. Chondrogenesis is complete within ~30 days, yielding a distinctly cell‐rich/matrix‐poor structure. Our findings indicate that regeneration‐mediated chondrogenesis in the lizard tail is not a simple recapitulation of embryonic cartilage development.
Support or Funding Information
Natural Sciences and Engineering Research Council (NSERC) Discovery Grant 400358
Regeneration is the ability to regrow injured or missing body parts. While you might think that regeneration is science fiction or a superpower, it is surprisingly common to many types of animals, ...including lizards. So why are lizards superhealers? What organs can lizards repair, and what can we learn from lizards to help people? To answer these and other questions, we take a closer look at our scaly neighbors. Our research reveals new ways to look at old problems, such as how to repair skin, the heart and even the brain. Lizards and other species offer important lessons for how to heal, and may one day offer new methods for advancing human health.
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