Cardiac dysfunction in StrepTSS: interplay of SLO and MMPs

描述(由申请人提供)： A组链球菌(GAS)可引起一种特别严重的感染性休克，称为链球菌中毒性休克综合征(Streipdical中毒性休克综合征)，尽管有现代医学实践，死亡率仍在30%-70%之间。明显的低血压和早期多器官衰竭是其显著特征，最近有证据表明部分StrepTSS患者心功能严重受损，这些患者的死亡率最高。我们的初步结果表明，极少量的链球菌溶血素O，一种由GAS产生的有效的成孔毒素，会导致单个心肌细胞对电刺激的反应能力发生快速而深刻的变化(1-5分钟)。这些变化与这些细胞对钙离子处理的显著损害直接相关。我们假设SLO通过其成孔能力促进钙离子从外部环境进入心肌细胞。此外，尽管存在用于此目的的复杂途径，但钙不能从心肌细胞内部充分去除。这表明，由ATP驱动的从细胞质中移除钙的酶受到SLO的不利影响。基质金属蛋白酶(MMPs)是一类需要锌的酶，能分解包括心脏在内的各种组织中的胶原等结构蛋白。患有GAS菌血症的实验动物的心脏只表达高水平的基质金属蛋白酶-13。尽管MMPs-2和MMPs-9与急性革兰氏阴性感染性心肌病以及高血压和心脏病发作等各种应激状态下的心脏细胞外重构明显相关，但对于MMPs-13则没有这样的证据。然而，分解的胶原支架不能有效地作为肌原纤维的结构支撑。因此，在细菌毒力因子如SLO的诱导下，基质金属蛋白酶-13可能是导致StrepTSS急性心功能不全的唯一原因。因此，关于StrepTSS心功能障碍的最有可能的假设是，在感染过程的早期，SLO对心脏功能有直接影响，而在感染过程的后期，心肌细胞、浸润性炎症细胞和/或心脏成纤维细胞产生基质金属蛋白酶-13，从而进一步促进心肌细胞功能的恶化。因此，第一个特定的目标是确定SLO如何导致心肌细胞内钙离子的增加和储存池中钙再摄取的减少。研究将利用单个分离的小鼠心肌细胞来测量细胞内钙瞬变对SLO和一种独特的SLO结构的响应，这种结构可以结合细胞，但不能形成毛孔。第二个目的是确定是什么驱动了GAS菌血症动物心脏中心脏基质金属蛋白酶-13的上调，并阐明了这种上调的功能后果。为此，将对野生型小鼠和那些缺乏基质金属蛋白酶-13的小鼠的完整心脏和分离的心肌细胞进行研究。分别用聚合酶链式反应、酶联免疫吸附试验和酶谱法检测基质金属蛋白酶-13的基因表达、蛋白产量和功能酶活性。为此目的设计的IonOptix系统将评估单个心肌细胞的机械功能。长期目标是为由产生与SLO同源的毒素的微生物引起的严重感染中的心脏功能障碍提供分子基础，以便可以开发新的靶向治疗来限制感染严重程度并提高存活率。

DESCRIPTION (provided by applicant): Group A streptococcus (GAS) causes a particularly severe form of septic shock termed Streptococcal Toxic Shock Syndrome (StrepTSS) and mortalities range from 30-70% despite modern medical practices. Marked hypotension and early multi-organ failure are distinctive features and recently evidence of severely impaired cardiac function has been documented in some patients with StrepTSS and these patients have the highest mortality. Our preliminary results suggest that extremely small amounts of streptolysin O, a potent pore-forming toxin produced by GAS, causes rapid and profound changes (1-5 min) in the ability of individual heart muscle cells to contract in response to electrical stimuli. These changes were directly correlated with marked impairment of calcium handling by these cells. We hypothesize that SLO facilitates calcium entry into the cardiac cell from the external environment via its pore-forming ability. In addition, calcium is not adequately removed from the inside of the cardiac myocyte despite the presence of elaborate pathways for this purpose. This suggests that the ATP-driven enzyme that removes calcium from the cytoplasm is adversely affected by SLO. Matrix metalloproteinases (MMP) are a family of zinc-requiring enzymes that break down structural proteins such as collagen in various types of tissue including the heart. Hearts from experimental animals with GAS bacteremia express high levels of only MMP-13. While MMPs-2 and -9 have been clearly implicated in acute Gram negative septic cardiomyopathy and in the cardiac extracellular remodeling under various stresses such as hypertension and heart attacks, no such evidence exists for MMP-13. Yet disassembled collagen scaffolding cannot function efficiently as structural support for myofibrils. Thus, MMP-13, induced by bacterial virulence factors such as SLO, may uniquely drive acute cardiac dysfunction in StrepTSS. Thus, the most likely hypothesis regarding cardiac dysfunction in StrepTSS is that early in the course of infection, SLO has direct effects on heart function and that later in the course of infection, cardiomyocytes, infiltrating inflammatory cells and/or cardiac fibroblasts produce MMP-13 that further contributes to the deterioration of cardiac myocyte function. Thus the first specific aim i to determine how SLO causes an increase in intracellular Ca2+ in cardiac myocytes and a decrease in calcium reuptake into storage pools. Studies will utilize single isolated murine cardiac myocytes to measure the dynamics of intracellular Ca2+ transients in response to SLO and to a unique SLO construct that can bind cells but cannot form pores. The second aim is to determine what drives upregulation of cardiac MMP-13 in hearts of animals with GAS bacteremia and to elucidate the functional consequences of this upregulation. For this, whole hearts and isolated cardiac cells from both wild-type mice and those deficient in MMP-13 will be studied. MMP-13 gene expression, protein production and functional enzymatic activity will be measured by PCR, ELISA, and zymography, respectively. Mechanical functioning of individual cardiomyocytes will be assessed with an IonOptix system designed for this purpose. The long-term objective is to provide a molecular basis for cardiac dysfunction in severe infections caused by organisms that produce toxins homologous to SLO such that new targeted therapies can be developed to limit infection severity and improve survival.