Cardiomyocyte cell cycling, maturation, and growth by multinucleation in postnatal swine

Abstract

words)Aims: Cardiomyocyte (CM) cell cycle arrest, decline of mononucleated-diploid CMs, sarcomeric maturation, and extracellular matrix remodeling are implicated in loss of cardiac regenerative potential in mice after birth. Recent studies show a 3-day neonatal regenerative capacity in pig hearts similar to mice, but postnatal pig CM growth dynamics are unknown. We examined cardiac maturation in postnatal pigs and mice, to determine the relative timing of developmental events underlying heart growth and regenerative potential in large and small mammals. Methods and Results:Left ventricular tissue from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed to span birth, weaning, and adolescence in pigs, compared to similar physiological timepoints in mice. Collagen remodeling increases by P7 in postnatal pigs, but sarcomeric and gap junctional maturation only occur at 2mo. Also, there is no postnatal transition to beta-oxidation metabolism in pig hearts. Mononucleated CMs, predominant at birth, persist to 2mo in swine, with over 50% incidence of mononucleated-diploid CMs at P7-P15. Extensive multinucleation with 4-16 nuclei per CM occurs beyond P30. Pigs also exhibit increased CM length relative to multinucleation, preceding increase in CM width at 2mo-6mo. Further, robust CM mitotic nuclear pHH3 activity and cardiac cell cycle gene expression is apparent in pig left ventricles up to 2mo. By contrast, in mice, these maturational events occur concurrently in the first two postnatal weeks alongside loss of cardiac regenerative capacity. Conclusions:Cardiac maturation occurs over a 6mo postnatal period in pigs, despite a similar early-neonatal heart regenerative window as mice. Postnatal pig CM growth includes increase in CM length alongside multinucleation, with CM cell cycle arrest and loss of mononucleated-diploid CMs occurring at 2mo-6mo. These CM characteristics are important to consider for pig preclinical studies and may offer opportunities to study aspects of heart regeneration unavailable in other models.

Scar Formation with Decreased Cardiac Function Following Ischemia/Reperfusion Injury in 1 Month Old Swine

“…When assessed at 2 months of age, sham-operated and IR pigs showed evidence of mitotic activity (pHH3 expression) and ongoing multinucleation in approximately 0.35% of CMs. This confirms findings from our lab that show low, but detectable, CM mitotic activity in a subset of CMs at 2 months of age [12]. In mice, CMs undergo binucleation, cell cycle arrest, and lose their capacity to regenerate approximately 1 week after birth [2,7,29].…”

“…Here, we report cardiac repair related to scar formation following transient ischemia/reperfusion injury in P30 pigs. We demonstrate that P30 pigs lack the capacity of cardiac regeneration through production of new CMs following IR, despite detectable mitotic activity in a subset of CMs at this stage [12]. Myocardial damage is evident immediately following IR injury, with a decrease in cardiac function detected 2 h after injury, that does not Scar formation and collagen remodeling were assessed after IR injury.…”

“…In unoperated pigs at 2 months of age, mitotic activity as identified by phospho-histone H3 (pHH3)-positive nuclei, is apparent in CMs as they become multinucleated [12]. Similar cell cycling in multinucleated CMs was observed in sham-operated or IR hearts, as measured by immunohistochemical staining of pHH3 with CMs identified by cardiac troponin I ( Figure 4A).…”

“…Since CM mitotic rates are similar at P0 and P30 in young swine [12], cardiac repair following temporary injury was assessed in P30 pigs. Blood collection and echocardiography were performed at baseline and subsequent time points over the course of the 4 week study ( Figure 1A).…”