Cardiomyocyte CaM kinase II as a driver of cardiac inflammation and remodeling

摘要 心脏通过心肌细胞的肥大生长对压力作出反应，并进展为心力衰竭。 当压力持续时。我们之前的研究表明，肥大对各种刺激的反应 不依赖于通过钙/钙调蛋白依赖的蛋白激酶II(CaMKII)的信号发生，但 当CaMKII被删除时，进展为心力衰竭的程度会减弱。炎症是致病的关键因素 与心力衰竭相关的不良重塑。这项建议的长期目标是 证明心肌细胞内的CaMKII信号启动心脏炎症反应 非缺血干预，如压力超负荷(TAC)，这在 心力衰竭的发展。Aim 1中提出的研究确定心肌细胞是否定位于CaMKII 使用心脏特异的CaMKII基因敲除小鼠(CKO)演示信号驱动心脏炎症 TAC诱导的炎性基因表达缺失和炎症体激活。我们确定这些是否 从成年小鼠的CTL和CTL中分离出成年小鼠的心室肌细胞，在心肌细胞中发生特异性的反应 采用组织切片原位杂交和酶分析方法。CaMKIIC转基因和 NFkB p65亚单位心脏特异性KO的小鼠被用来进一步证明 心肌细胞在点燃炎症中的作用目标2询问心肌细胞CaMKII信号是否参与 TAC引起的炎症/免疫细胞积聚。这项工作使用CKO小鼠来 证明TAC通过心肌细胞CaMKII启动的信号促进免疫细胞反应。 研究集中在巨噬细胞和T细胞，使用免疫组织化学和流式细胞术以及单个 细胞RNA序列，以全面定义聚集在心脏中的特定巨噬细胞群。 心脏特异性KO或抑制趋化因子/细胞因子被用来证明这些细胞的产生 心肌细胞中的介体可触发特定免疫细胞类型的反应。目标3确定封锁是否 心肌细胞CaMKII启动的炎症减轻不利的重塑 需要完成的任务。建议的研究使用心脏特异性KO或抑制选定的炎症 以证明它们在心肌细胞中的形成对纤维化的发展和 TAC后的心功能不全。使用AAV9 Cre的条件基因缺失来确定时间 从而实现了抑制CaMKII的最大益处。我们的发现应该会对未来产生重大影响 由于心肌细胞以前没有被认为是炎症信号的生成者， 在没有“警报”的情况下，炎症反应被激活的机制还没有 到目前为止已经确定，最有效的预防心力衰竭发展的概念可以 通过早期心肌细胞靶向抗炎干预实现这一目标是新颖的。

ABSTRACT The heart responds to stress through hypertrophic growth of cardiomyocytes and progresses to heart failure when stress is sustained. Our previous studies showed that hypertrophy in response to a variety of stimuli occurs independent of signaling through the calcium/calmodulin dependent protein kinase II (CaMKII) but that progression to heart failure is attenuated when CaMKII is deleted. Inflammation is a key contributor to the adverse remodeling associated with heart failure. The long term objective of this proposal is to demonstrate that CaMKII signaling within cardiomyocytes initiates cardiac inflammation in response to non-ischemic interventions such as pressure overload (TAC) and that this plays a significant role in the development of heart failure. Studies proposed in Aim 1 determine if cardiomyocyte localized CaMKII signaling drives cardiac inflammation using cardiac specific CaMKII knockout mice (CKO) to demonstrate loss of TAC-induced inflammatory gene expression and inflammasome activation. We determine if these responses occur specifically in cardiomyocytes by isolation of adult mouse ventricular myocytes from CTL and CKO mice, and by in situ hybridization and enzymatic assays in tissue sections. CaMKIIC transgenics and mice with cardiac specific KO of the p65 subunit of NFkB are used to further demonstrate involvement of the cardiomyocyte in igniting inflammation. Aim 2 asks whether cardiomyocyte CaMKII signaling contributes to accumulation of inflammatory/immune cells in response to TAC. Work in this aim uses CKO mice to demonstrate that TAC promotes immune cell responses through cardiomyocyte CaMKII initiated signals. Studies focus on macrophages and T-cells, using immunohistochemistry and flow cytometry as well as single cell RNA seq to comprehensively define specific populations of macrophages that accumulate in the heart. Cardiac specific KOs or knockdown of chemokines/cytokines is used to demonstrate that generation of these mediators in cardiomyocytes triggers responses of specific immune cell types. Aim 3 determines if blockade of cardiomyocyte CaMKII-initiated inflammation attenuates adverse remodeling and at what point this needs to be accomplished. Proposed studies use cardiac specific KO or inhibition of selected inflammatory mediators to demonstrate that their formation in cardiomyocytes is critical for development of fibrosis and ventricular dysfunction following TAC. Conditional gene deletion with AAV9 Cre is used to establish the time at which maximal benefit from CaMKII inhibition is achieved. Our findings should significantly impact future research since the cardiomyocyte has not previously been considered as a generator of inflammatory signals, the mechanisms by which inflammatory responses are activated in the absence of “alarmins” has not heretofore been determined, and the concept that most effective prevention of heart failure development could be achieved by early cardiomyocyte-targeted anti-inflammatory interventions is novel.