Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart’s cellularity and maturation during development, contributing novel information about caspase biology and heart development.

效应半胱天冬酶（executioner caspase）-3与-7是介导细胞死亡的蛋白酶，但目前学界逐渐发现其存在非凋亡相关的生物学功能。心脏仅在发育阶段表达半胱天冬酶，提示其可能参与心脏器官的成熟过程。基于此，本研究旨在阐明半胱天冬酶在心脏发育过程中的全新功能。我们在小鼠心肌组织中同时敲除效应半胱天冬酶-3与-7，并对其表型效应进行分析。半胱天冬酶敲除的小鼠心脏在出生时发育不全，后续通过心肌细胞肥大逐步恢复至正常重量。为明确介导上述表型的分子通路，我们采用基于微阵列的转录组学（microarray-based transcriptomics）与多重定量蛋白质组学（multiplexed quantitative proteomics）技术，对比不同发育阶段野生型与效应半胱天冬酶缺陷型小鼠心肌组织的表达谱差异。转录组学分析显示，新生期半胱天冬酶缺陷型心肌组织中，促进DNA复制与细胞周期进展的基因表达显著下调，提示心肌细胞增殖能力受损；而成年敲除小鼠心肌组织中则出现结构蛋白非心脏亚型的异常表达。蛋白质组学分析发现，氧化磷酸化相关蛋白的丰度显著降低，而糖酵解酶的丰度显著升高，这提示半胱天冬酶敲除型心肌组织的代谢成熟过程出现阻滞。我们验证了相关基因的mRNA表达水平与蛋白质丰度之间的相关性，但转录组学与蛋白质组学分别鉴定出了心脏发育过程中受半胱天冬酶调控的互补分子通路。在培养的心肌细胞中过表达野生型或蛋白水解活性缺失的半胱天冬酶，均可上调促进细胞分裂的基因的表达。本研究结果表明，效应半胱天冬酶可在心脏发育过程中调控心肌细胞组成与器官成熟进程，为半胱天冬酶生物学及心脏发育研究提供了全新的认知。