Impact of the cardiomyocyte transcription factor GATA4 for cardiac regeneration

心肌梗死（MI）是德国死亡的主要原因之一。由于急性事件结局的改善，作为MI长期后遗症的心力衰竭的患病率目前正在上升。这主要是由于成年哺乳动物心肌的再生能力缺陷，导致心肌细胞死亡后瘢痕形成，而不是功能性肌肉组织的恢复。最近的研究表明，新生小鼠在心肌尖部切除或（冷冻）梗死后，与成年小鼠相比，可以完全再生心肌。这使得能够检查哺乳动物出生后心肌的再生机制，以及鉴定成人心脏再生缺陷的原因。在新生儿心脏中鉴定出促进再生的基因或调节RNA可能在未来被用作改善患者MI后心脏再生的疗法。 在这项提议的初步工作中，我们发现心肌细胞转录因子GATA 4的心脏表达在出生时非常高，但从出生后第7天开始急剧下降，此时心肌的再生能力也强烈减弱。由于GATA 4在胚胎发育过程中促进心肌细胞增殖，我们推测它可能支持损伤后的心肌再生。因此，我们将分析新生心肌细胞特异性GATA 4基因敲除（GATA 4-CKO）小鼠和对照小鼠心脏冷冻损伤后的心脏再生。我们将在两组小鼠中诱导损伤后的多个时间点探测心肌瘢痕的大小、心肌细胞增殖和肥大、血管生成、心脏炎症以及心脏转录组（通过深度测序）。第一个结果显示，心肌冷冻损伤后7天，GATA 4-CKO小鼠的瘢痕明显更大，心肌细胞增殖减少。在深度测序分析中，我们的目标是确定以前未知的促进心肌再生的GATA 4依赖因子。将在心脏外植体培养物以及分离的胎儿和新生儿心肌细胞中检查鉴定的候选基因的再生潜能。 此外，我们将使用腺病毒和AAV 9载体在7日龄以及成年野生型小鼠的心脏中过表达GATA 4，其中内源性GATA 4表达显著下调，并将分析这种治疗是否可以改善心肌再生。以类似的方式，我们的目标是过表达选择的候选基因，并确定它们在心脏冷冻损伤后的再生潜力。

Myocardial infarction (MI) is one of the main reasons for mortality in Germany. Because of improved outcome in the acute event, the prevalence of heart failure as long-term sequel of MI is currently rising. This is mainly due to the defective regeneration capacity of the adult mammalian myocardium, which leads to scar formation after cardiomyocyte demise rather than the restitution of functional muscle tissue. It was recently demonstrated that newborn mice, in contrast to adult mice, can completely regenerate the myocardium after myocardial apex resection or (cryo)-infarction. This enables examination of regenerative mechanisms in the mammalian myocardium right after birth as well as the identification of reasons for defective regeneration in the adult heart. Genes or regulatory RNAs identified to promote regeneration in the neonatal heart could potentially be used as therapy to improve cardiac regeneration after MI in patients in the future. In preliminary work for this proposal we found that cardiac expression of the cardiomyocyte transcription factor GATA4 is very high at birth, but sharply declines starting at postnatal day 7, when also the regenerative capacity of the myocardium becomes strongly diminished. Because GATA4 promotes cardiomyocyte proliferation during embryonic development, we hypothesize that it might support myocardial regeneration after injury. Therefore, we will analyze heart regeneration after cardiac cryoinjury in neonatal cardiomyocyte specific GATA4 knock-out (GATA4-CKO) and control mice. We will probe the size of the myocardial scar, cardiomyocyte proliferation and hypertrophy, angiogenesis, cardiac inflammation as well as the cardiac transcriptome (by deep-sequencing) at multiple time-points after the induction of injury in both groups of mice. First results revealed a significantly larger scar and reduced cardiomyocyte proliferation in GATA4-CKO mice 7 days after myocardial cryoinjury. In the deep-sequencing analysis, we aim to identify previously unknown GATA4 dependent factors that promote myocardial regeneration. The regenerative potency of identified candidate genes will be examined in cardiac explant culture as well as in isolated fetal and neonatal cardiomyocytes. In addition, we will use adenoviral and AAV9 vectors to overexpress GATA4 in the heart of 7 day old as well as adult wild-type mice, in which endogenous GATA4 expression is dramatically downregulated and will analyze whether this treatment can improve myocardial regeneration. In a similar manner, we aim to overexpress select candidate genes and determine their regenerative potential after cardiac cryoinjury.