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

https://doi.org/10.1016/j.yjmcc.2020.07.004Get rights and content

Highlights

  • Pig cardiomyocyte (CM) maturation and mitotic arrest occur over 2–6 months.

  • Nearly 50% mononucleated-diploid CMs persist at 2 weeks after birth in swine.

  • Adolescent pig CMs exhibit progressive multinucleation and longitudinal growth.

  • Pig CM terminal maturation does not coincide with reported P3 regenerative loss.

  • RNAseq suggests critical role for non-CM factors in postnatal pig heart maturation.

Abstract

Background

Rodent cardiomyocytes (CM) undergo mitotic arrest and decline of mononucleated-diploid population post-birth, which are implicated in neonatal loss of heart regenerative potential. However, the dynamics of postnatal CM maturation are largely unknown in swine, despite a similar neonatal cardiac regenerative capacity as rodents. Here, we provide a comprehensive analysis of postnatal cardiac maturation in swine, including CM cell cycling, multinucleation and hypertrophic growth, as well as non-CM cardiac factors such as extracellular matrix (ECM), immune cells, capillaries, and neurons. Our study reveals discordance in postnatal pig heart maturational events compared to rodents.

Methods and results

Left-ventricular myocardium from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed. Mature cardiac sarcomeric characteristics, such as fetal TNNI1 repression and Cx43 co-localization to cell junctions, were not evident until P30 in pigs. In CMs, appreciable binucleation is observed by P7, with extensive multinucleation (4–16 nuclei per CM) beyond P15. Individual CM nuclei remain predominantly diploid at all ages. CM mononucleation at ~50% incidence is observed at P7–P15, and CM mitotic activity is measurable up to 2mo. CM cross-sectional area does not increase until 2mo–6mo in pigs, though longitudinal CM growth proportional to multinucleation occurs after P15. RNAseq analysis of neonatal pig left ventricles showed increased expression of ECM maturation, immune signaling, neuronal remodeling, and reactive oxygen species response genes, highlighting significance of the non-CM milieu in postnatal mammalian heart maturation.

Conclusions

CM maturational events such as decline of mononucleation and cell cycle arrest occur over a 2-month postnatal period in pigs, despite reported loss of heart regenerative potential by P3. Moreover, CMs grow primarily by multinucleation and longitudinal hypertrophy in older pig CMs, distinct from mice and humans. These differences are important to consider for preclinical testing of cardiovascular therapies using swine, and may offer opportunities to study aspects of heart regeneration unavailable in other models.

Introduction

Cardiovascular diseases are the leading cause of mortality worldwide, but clinical strategies for effecting regenerative repair in diseased adult human hearts remain elusive [1]. Mouse hearts have been extensively utilized in laboratory research as a mammalian cardiac disease model, but for clinical translation, studies in large mammals such as pigs, dogs, and sheep are mandated. Juvenile pigs, in particular, are often used as a cardiac preclinical model, due to their hemodynamic and anatomic similarity to the human heart [2]. However, fundamental developmental characteristics of the porcine heart, including cardiomyocyte (CM) nucleation, hypertrophic growth, and mitotic arrest after birth, are still minimally characterized.

In the first postnatal week, rodent CMs undergo rapid binucleation and cell cycle arrest, characterized by diminishing expression of mitotic markers, repression of cell cycle-promoting cyclins and cyclin-dependent kinases (CDKs), and cytokinesis failure [3,4]. At the same time, CMs switch from hyperplastic to hypertrophic mode of growth, identified by increased cross-sectional area [5]. There is also concurrent decline of mononucleated-diploid CMs with a corresponding increase in binucleation [5,6]. Further, sarcomeric maturation and induction of oxidative metabolic pathways also occur in the first two postnatal weeks in rodent hearts. Alongside these CM maturational processes, there is also increased extracellular matrix (ECM) deposition and stiffness, vascular capillary growth, sympathetic neuronal maturation, and immune cell signaling [[7], [8], [9], [10], [11], [12]]. Thus, by 2–3 weeks after birth, rodent hearts are terminally mature, growing primarily by CM hypertrophy and forming fibrotic scar upon injury [13]. Each of these maturational events have also been identified in rodents as targets for regulation of neonatal regenerative potential after birth.

Recent reports show that swine, similar to rodents, have a transient heart regenerative capacity in the first few days after birth [14,15]. However, the timing and sequence of cardiac maturational events after birth are unknown in swine. Also, though pigs are widely used in preclinical studies for cardiac therapeutics [2], there is a dearth of knowledge in pig CM growth dynamics, particularly in the postnatal period. Direct comparison of postnatal growth mechanisms between swine and rodents could thus help establish the conserved processes underlying CM terminal maturation and loss of cardiac regenerative potential in large and small mammals after birth, facilitating clinical translation to humans.

In this study, we provide a comprehensive characterization of heart development up to 6 months after birth in swine. Our results describe the sequence of events involved in postnatal cardiac terminal maturation, in this important large mammalian model. We investigated CM characteristics including cell cycle arrest, nucleation, and hypertrophy in postnatal pigs. Our data show discordance in the timing of CM terminal maturation, compared to reported loss of heart regenerative potential [14,15], in swine. Also, the CM growth dynamics involved in increased nucleation and transition to hypertrophic growth are distinctive in pigs from rodents and humans. Moreover, RNA sequencing (RNAseq) for myocardial gene expression profiling in postnatal pigs showed upregulation of genes involved in ECM, immune, and neuronal maturation as well as reactive oxygen species (ROS) response. These data in swine add support to the importance of physiological and local microenvironment cues in regulating cardiac terminal maturation and regenerative capacity after birth in mammals.

Section snippets

Methods

An abbreviated methods section with key experimental details is provided below. The expanded methods section can be found in the online supplementary data file. Details on antibodies and reagents, including dilutions, working concentrations, and manufacturer information, are provided in Supplementary Table 1. Primer sequences used for RT-qPCR are listed in Supplementary Table 2. RNAseq differential expression analyses are available in Supplementary Table 3.

Cardiac sarcomeric and gap junctional maturation occur beyond P30 in pig hearts

To assess cardiac maturational transitions in the weeks after birth, pig hearts were collected at P0 (postnatal day 0), P7, P15, P30, 2mo (2 months post-birth) and 6mo (6 months post-birth), to span birth, weaning, and adolescent ages (Fig. 1A), similar to the 1-month period of cardiac terminal maturation in mice [22] (Fig. S1A–B). Both cardiac and total body weights significantly increased beyond P30 in pigs (Fig. S2A–C), and heart weight-to-body weight ratios were only decreased by 6mo (Fig. 1

Discussion

Here we report a comprehensive analysis of cardiac myocyte and non-myocyte maturational dynamics in postnatal swine. Our results show that CM maturation, including mitotic arrest, decline of mononucleated-diploid CMs, and sarcomeric isoform switching, occurs over a 2 to 6-month postnatal period in pigs, despite reported loss of regenerative potential by P3 [14,15]. This contrasts with mice where CM mitotic arrest and maturation occurs at the same time as loss of regenerative potential (Fig. 8).

Author contributions

Conceptualization of study was by NV, CMA, and KEY. Data curation, formal analysis, and validation were done by NV, CMA, EJA, and SRP. Contributions to methodology, resources, and project administration were made by NV, CMA, KWR, RSB, and FZ. Visualization of figures and writing of the original manuscript draft were performed by NV and KEY. Review and editing of the manuscript was performed by all authors.

Funding

This work was supported by the National Institutes of Health [R01HL135848, R01HL142217 to KEY]; the American Heart Association Predoctoral Fellowship [19PRE34380046 to NV]; and the Cincinnati Children's Hospital Research Foundation.

Declaration of Competing Interest

None declared.

Acknowledgements

We thank Dr. Bernhard Kühn (UPMC Children's Hospital, Pittsburgh) for providing cardiomyocyte dissociation protocols, and Dr. Michaela Patterson (Medical College of Wisconsin, Milwaukee) for critical suggestions in optimizing ploidy assessment in pig cardiomyocytes. We also thank past and current members of the Yutzey Lab for assistance in harvest of pig cardiac tissues as well as valuable discussions.

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