Dynamics of cardiomyocyte and muscle stem cell proliferation in pig

Abstract

The cardiac and skeletal muscle tissues are both striated and contractile but their intrinsic cellular properties are distinct. The minimal cardiomyocyte proliferation and the lack of cardiac stem cells directly leads to poor heart repair in adult mammals. But in skeletal muscle, the robust proliferation of widespread muscle stem cells support efficient muscle regeneration. The endogenous cardiomyocyte and muscle stem cell proliferation has been analyzed in common laboratory animals but not in large mammals including pigs, which are more comparable to human. In this study, we rigorously examined the cell cycle dynamics of porcine cardiomyocytes and muscle stem cells through different developmental stages. Proliferative cardiomyocytes and muscle stem cells were broadly observed in the embryonic heart and limb muscle respectively. Muscle stem cells continue to proliferate postnatally but cardiomyocyte proliferation was drastically reduced after birth. However, robust cardiomyocyte cell cycle activity was detected around postnatal day 20, which could be attributed to the binucleation but not cell division. Increased proliferating cells were detected in maternal heart during early pregnancy but they represent non-cardiomyocyte cell types. The islet1 expressing cells were only identified in the embryonic and new born porcine hearts. Furthermore, the accumulated oxidative DNA damage in the cardiac but not skeletal muscle during development could be responsible for the diminished cardiomyocyte proliferation in adult pig. Similarities and differences in the proliferation of heart and skeletal muscle cells are identified in pigs across different developmental stages. Such cellular proliferative features must be taken into account when using porcine models for cardiovascular and muscular research.

Introduction

The mammalian bodies have three types of muscles: skeletal muscle, cardiac muscle and smooth muscle. Both skeletal and cardiac muscle cells show striated sarcomere histologically and are highly contractile. But different types of muscle tissues are inherently different in the developmental dynamics and cellular characteristics [1].

Heart is among the least regenerative organs in mammalian body, whereas the skeletal muscle retains remarkable regenerative capacity throughout life. The major causes of the poor heart repair are the diminished cardiomyocyte proliferation and lack of cardiac progenitors in adult hearts [2]. The inability of heart to regenerate stands in sharp contrast to skeletal muscle, which is capable of considerable tissue repair though the reconstruction of myofibers. The proliferation and differentiation of resident muscle stem cells (satellite cells) are necessary for the new myofiber formation and muscle regeneration [3].

Studies in zebrafish and mice have identified invaluable cues for promoting cardiac regeneration [4]. But the molecular and cellular mechanisms controlling cardiomyocytes proliferation are likely variable across mammalian species including human, thus hindering the potential clinical applications. Pigs provide a clinically relevant large animal model in this respect as the molecular and cellular properties can be examined in detail, which is simply not feasible in human [5]. Recent studies also showed that pig heart could regenerate after myocardial infarction, but only before postnatal day 3 when endogenous cardiomyocytes are still cycling [6,7]. Thus, it is essential to thoroughly examine the cell proliferative behavior during pig heart development to appreciate the full potential of heart regeneration, as well as validate the pig models for pre-clinical trials of new drugs and devices for heart disease. There is also considerable agricultural interest in the improvement of meat production by modulating the muscle stem cell proliferation and differentiation.

In this study, we systematically examined the proliferation of cardiomyocytes and muscle stem cells in pig heart and limb during embryonic development, postnatal and adulthood. A panel of proliferation markers including ki67 (G1 phase to anaphase), aurora B (Aurora-B kinase, G2/M to cytokinesis), MCM2 (Minichromosome Maintenance Complex Component 2, G1 to telophase) and pH3 (Phospho-Histone H3, G2/M to anaphase) were utilized to precisely determine the cell cycle activity of cardiac and skeletal muscle cells. Our data revealed novel cell proliferation dynamics during pig heart and limb muscle development respectively. Furthermore, the presence of Islet1 (Isl⁺) expressing cardiac progenitors in pigs was also confirmed.