Cardiac dysfunction in StrepTSS: interplay of SLO and MMPs

描述（由申请人提供）： A组链球菌（GAS）引起一种特别严重的败血性休克，称为链球菌中毒性休克综合征（StrepTSS），尽管现代医学实践，死亡率仍在30-70%之间。显著的低血压和早期多器官衰竭是显著特征，最近在一些StrepTSS患者中记录了心脏功能严重受损的证据，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产生的强效成孔毒素，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处理的显著损害直接相关。我们推测SLO通过其成孔能力促进钙从外部环境进入心肌细胞。此外，钙不能从心肌细胞内部充分去除，尽管存在用于此目的的复杂途径。这表明从细胞质中去除钙的ATP驱动的酶受到SLO的不利影响。基质金属蛋白酶（MMP）是一个需要锌的酶家族，它能分解包括心脏在内的各种组织中的结构蛋白，如胶原蛋白。来自患有GAS菌血症的实验动物的心脏仅表达高水平的MMP-13。虽然MMP-2和MMP-9已明确涉及急性革兰氏阴性脓毒性心肌病和在各种应激如高血压和心脏病发作下的心脏细胞外重塑，但对于MMP-13不存在这样的证据。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，MMP-13，诱导细菌毒力因子，如SLO，可能是唯一驱动急性心功能不全的StrepTSS。因此，关于StrepTSS中的心功能障碍的最可能的假设是，在感染过程的早期，SLO对心脏功能具有直接影响，并且在感染过程的后期，心肌细胞、浸润性炎性细胞和/或心脏成纤维细胞产生MMP-13，其进一步导致心肌细胞功能的恶化。因此，第一个具体目标是确定SLO如何引起心肌细胞内Ca 2+的增加和储存池中钙重摄取的减少。研究将利用单个分离的鼠心肌细胞来测量响应于SLO和可以结合细胞但不能形成孔的独特SLO构建体的细胞内Ca 2+瞬变的动力学。第二个目的是确定是什么驱动GAS菌血症动物心脏中心脏MMP-13的上调，并阐明这种上调的功能后果。为此，将研究来自野生型小鼠和MMP-13缺陷小鼠的整个心脏和分离的心脏细胞。分别通过PCR、ELISA和酶谱法测量MMP-13基因表达、蛋白质产生和功能性酶活性。将使用为此目的设计的IonOptix系统评估单个心肌细胞的机械功能。长期目标是为由产生与SLO同源的毒素的生物体引起的严重感染中的心功能不全提供分子基础，从而可以开发新的靶向治疗以限制感染严重程度并提高生存率。

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.