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.

中文翻译：

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出生后猪的多核化引起的心肌细胞循环、成熟和生长。

背景

啮齿动物心肌细胞 (CM) 在出生后经历有丝分裂停滞和单核二倍体种群的下降，这与新生儿心脏再生潜力的丧失有关。然而，尽管新生儿心脏再生能力与啮齿动物相似，但猪的出生后 CM 成熟的动力学在很大程度上是未知的。在这里，我们提供了猪出生后心脏成熟的综合分析，包括 CM 细胞循环、多核和肥大生长，以及非 CM 心脏因素，如细胞外基质 (ECM)、免疫细胞、毛细血管和神经元。我们的研究揭示了与啮齿动物相比，出生后猪心脏成熟事件的不一致。

方法和结果

分析了产后第 0 至 6 个月（6 个月）白约克夏-长白猪的左心室心肌。成熟的心脏肌节特征，如胎儿TNNI1抑制和 Cx43 共定位到细胞连接，直到 P30 在猪中才明显。在 CM 中，P7 观察到明显的双核，在 P15 之后具有广泛的多核（每个 CM 4-16 个核）。各个 CM 细胞核在所有年龄段都以二倍体为主。在 P7-P15 观察到约 50% 发生率的 CM 单核化，并且 CM 有丝分裂活动可测量长达 2 个月。尽管在 P15 之后与多核成正比的纵向 CM 生长发生在猪中，CM 横截面积直到 2mo-6mo 才会增加。新生猪左心室的 RNAseq 分析显示 ECM 成熟、免疫信号、神经元重塑和活性氧反应基因的表达增加，突出了非 CM 环境在出生后哺乳动物心脏成熟中的重要性。

结论

CM 成熟事件，例如单核细胞减少和细胞周期停滞在猪出生后 2 个月内发生，尽管据报道 P3 会丧失心脏再生潜力。此外，CMs 主要通过老年猪 CMs 的多核和纵向肥大生长，与小鼠和人类不同。这些差异对于使用猪进行心血管治疗的临床前测试很重要，并且可能为研究其他模型中不可用的心脏再生方面提供机会。