Cardiac regeneration in situ: Is it possible?

描述(申请人提供)：修复受损心脏的策略主要集中在输注、直接注射或移植各种类型的干细胞/祖细胞。我们最近报道了心肌细胞特异性GSK-3b的条件性缺失导致心肌细胞增殖。此外，我们发现，在胸主动脉收缩(导致左心室收缩压显著增加)或心肌梗死(MI)引起的应激状态下，增殖程度被放大。事实上，我们已经发现GSK-3b的缺失(或抑制)会导致小鼠胚胎干细胞来源的类胚体中的心肌细胞、培养中的新生大鼠心肌细胞、发育中的心脏中的心肌细胞、正常心脏中的心肌细胞以及如上所述，特别是在应激心脏中的心肌细胞中。如果这种增殖足够强劲，就可以在不需要移植的情况下诱导有意义的再生(即原位再生)。我们将以三个具体目标来解决这一假设。我们将首先通过量化正常心脏和应激心脏的增殖率和心肌细胞质量来确定由于GSK-3b缺失而导致的心肌细胞增殖的强健程度。然后，我们将通过确定是否对左心功能有有益的影响来检查这种增殖的生理学后果。最后，我们将确定GSK-3b的缺失是否会对患有中度心力衰竭的小鼠产生有益影响--重述患有晚期心力衰竭的患者的临床情景，这些患者除了心脏移植或靶向左冠状动脉成形术治疗外，几乎没有其他治疗选择。在“未来方向”一节中，我们还讨论了GSK-3b在推动未成熟心肌细胞分化为成熟心肌细胞中所起的关键作用。我们建议在增殖反应产生新的心肌细胞后，通过腺相关病毒基因转移到心脏恢复GSK-3b的表达。因此，任何因GSK-3b缺失而形成的未成熟心肌细胞都将分化为成熟的、功能齐全的心肌细胞，从而最大限度地改善左心功能。我们相信，这一应用与R21机制的目标完美匹配，R21机制支持开创性研究，有可能大幅推进心力衰竭患者的治疗选择--这种疾病多年来没有新的重要类别的药物进入市场。 公共卫生相关性：在这里，我们建议确定是否有可能充分促进心肌细胞的增殖，从而导致原位心脏再生(即，不需要使用外源干细胞/祖细胞)。基于我们在小鼠模型上的广泛研究，我们假设，通过在体内操纵单一的信号通路-糖原合成酶-3b(GSK-3b)信号通路，我们确实能够实现再生。如果我们能够做到这一点，这将是在治疗多种病因的终末期心脏病患者方面向前迈出的重要一步。

DESCRIPTION (provided by applicant): Strategies to repair injured hearts have predominantly focused on infusion, direct injection, or transplantation of any of a variety of types of stem/progenitor cells. We have recently reported that conditional deletion of GSK-3b specifically in cardiomyocytes leads to proliferation of cardiomyocytes. Furthermore, we find that the degree of proliferation is amplified in the setting of stress induced by thoracic aortic constriction (producing marked increases in left ventricular systolic pressure) or myocardial infarction (MI). In fact, we have found that deletion (or inhibition) of GSK-3b leads to proliferation of cardiomyocytes in mouse embryonic stem cell-derived embryoid bodies, in neonatal rat ventricular myocytes in culture, in cardiomyocytes in the developing heart, in cardiomyocytes in the normal heart, and, as noted above, particularly in cardiomyocytes in the stressed heart. If this proliferation is sufficiently robust, one could induce meaningful regeneration without the need for transplantation (i.e. regeneration in situ). We will address this hypothesis with three Specific Aims. We will first determine how robust is the cardiomyocyte proliferation resulting from deletion of GSK-3b by quantifying proliferation rates and cardiomyocyte mass in both the normal heart and in the stressed heart. We will then examine the physiologic consequences of this proliferation by determining whether there is a beneficial effect on left ventricular function. Finally, we will determine whether deletion of GSK-3b can lead to beneficial effects in mice with moderately advanced heart failure- recapitulating the clinical scenario of patients with advanced heart failure for whom there are very few treatment options besides heart transplant or destination LVAD therapy. In a "future directions" section, we also discuss the critical role played by GSK-3b in driving differentiation of immature cardiomyocytes into mature cardiomyocytes. We propose studies to restore expression of GSK-3b via adeno-associated virus gene transfer to the heart after the proliferative response has created new myocytes. Thus any immature myocytes formed in response to the deletion of GSK-3b will be differentiated into mature, fully functional myocytes, maximizing the chances of improving LV function. We believe that this application perfectly matches the goals of the R21 mechanism of supporting groundbreaking research with the potential to significantly advance therapeutic options for heart failure patients- a disease that has had no new important classes of agents come to market in many years. PUBLIC HEALTH RELEVANCE: Herein we propose to determine whether it is possible to drive cardiomyocyte proliferation sufficiently to result in cardiac regeneration in situ (i.e. without the need to utilize exogenous stem/progenitor cells). Based on our extensive studies in mouse models, we hypothesize that by manipulating a single signaling pathway- the glycogen synthase kinase-3b (GSK-3b) signaling pathway- in vivo, we will indeed be able to achieve regeneration. If we can achieve this, it would be a significant step forward in the treatment of patients with end- stage heart diseases of many etiologies.