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With the rapid development of single-cell sequencing technology, the Langendorff perfusion system has emerged as a common approach to decompose cardiac tissue and obtain living cardiomyocytes to study cardiovascular disease with the mechanism of cardiomyocyte biology. However, the traditional Langendorff perfusion system is difficult to master, and further, the viability and purity of cardiomyocytes are frequently unable to meet sequencing requirements due to complicated devices and manipulate processes. Here, we provide an optimized Langendorff perfusion system with a simplified and standardized operating protocol which utilizes gravity as the perfusion pressure, includes a novel method for bubbles removing and standardizes the criteria for termination of digestion. We obtained stable cardiomyocyte with high viability and purity after multiple natural gravity sedimentation. The combination of the optimized Langendorff perfusion system and the multiple natural gravity sedimentation provides a stable system for isolating adult mouse heart, which will provide higher-quality cardiomyocytes for further experiments., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: A key cause of the high mortality of cardiovascular diseases is the cardiomyocyte inability to renew after cardiac injury. As a promising strategy to supplement functional myocytes for cardiac repair, there is a pressing need to understand the cellular and molecular mechanisms of heart regeneration., Methods: Seven genetic mouse lines were used: global OSM (oncostatin M) knockout, monocyte-/macrophage-specific OSM deletion, cardiomyocyte-specific lines, including OSM receptor deletion, gp130 (glycoprotein 130) deletion, gp130 activation, and Yap (yes-associated protein) ablation with gp130 activation mice. A series of molecular signaling experiments, including RNA sequencing, immunostaining, coimmunoprecipitation, and imaging flow cytometry, were conducted. Two models of cardiac injury, apical resection and myocardial infarction operation, were performed in neonatal, juvenile, and adult mice. Heart regeneration and cardiac function were evaluated by Masson staining and echocardiography, respectively. Gene recombinant adenovirus-associated virus was constructed and infected myocardial-infarcted mice as a gene therapy., Results: OSM was identified by RNA sequencing as a key upstream regulator of cardiomyocyte proliferation during neonatal heart regeneration in mice. Cardiomyocyte proliferation and heart regeneration were suspended in neonatal mice after cardiac injury when OSM was conditionally knockout in macrophages. The cardiomyocyte-specific deficiency of the OSM receptor heterodimers, OSM receptor and gp130, individually in cardiomyocytes reduced myocyte proliferation and neonatal heart regeneration. Conditional activation of gp130 in cardiomyocytes promoted cardiomyocyte proliferation and heart regeneration in juvenile and adult mice. Using RNA sequencing and functional screening, we found that Src mediated gp130-triggered cardiomyocyte proliferation by activating Yap (yes-associated protein) with Y357 phosphorylation independently of the Hippo pathway. Cardiomyocyte-specific deletion of Yap in Myh6-gp130 ACT mice blocked the effect of gp130 activation-induced heart regeneration in juvenile mice. Gene therapy with adenovirus-associated virus encoding constitutively activated gp130 promoted cardiomyocyte proliferation and heart regeneration in adult mice after myocardial infarction., Conclusions: Macrophage recruitment is essential for heart regeneration through the secretion of OSM, which promotes cardiomyocyte proliferation. As the coreceptor of OSM, gp130 activation is sufficient to promote cardiomyocyte proliferation by activating Yap through Src during heart regeneration. gp130 is a potential therapeutic target to improve heart regeneration after cardiac injury.