Heart Regeneration in the Leopard Gecko ( Eublepharis macularius ): a Comparison of Spontaneous Repair and Regrowth Following Two Different Injury Models

Abstract

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.