Bacterial RNA Activates PKR-JAK-STAT Signaling and Inflammasome-Associated Apoptosis in Human Cardiac and Stromal Cell Lines: Implications for Sepsis-Induced Cardiac Pathophysiology

Abstract

Background: Sepsis-induced myocardial dysfunction (SIMD) is common and highly predictive of poor outcomes, however the proximal molecular events linking bloodstream infection to myocardial injury remain poorly characterized. Bacterial RNA is an immunostimulatory molecule that could activate the double-stranded RNA-dependent protein kinase (PKR), however, its impact on signaling networks in human cardiac myocytes and non-immune stromal cells has not been explored. Methods: Adult human cardiac myocytes and human fibrosarcoma 2fTGH cells, as well as JAK-STAT-IFN pathway-deficient 2fTGH mutants, were treated with purified Escherichia coli RNA without transfection reagents. PKR activation was monitored by autophosphorylation and eIF2 α phosphorylation. JAK1, JAK2, TYK2, IFNAR2, STAT1 and STAT2 (IRF9-dependent) were investigated using signalling-defective cell lines and in vitro kinase assays. STAT1, STAT2, IRF-1 and NF-kB DNA binding were studied by electrophoretic mobility shift assays. Immunoblots for IL-1 β induction and caspase activation were performed, and the structural requirements probed using enzymatic or 5′-dephosphorylation of RNA. Results: Exogenous bacterial RNA itself was able to induce substantial PKR autophosphorylation and eIF2 α phosphorylation in cardiac myocyte and 2fTGH cells. PKR activation was dependent on JAK1, JAK2, TYK2, IFNAR2, STAT1, STAT2 and IRF9, suggesting their involvement in amplification through an autocrine type I interferon-JAK-STAT signaling. Bacterial RNA induced increased nuclear translocation and DNA binding of STAT1, STAT2, IRF-1, and NF- κ B; an up-regulation of IL-1 β ; as well as activation of caspase-1 and the executioner caspases with mild involvement of casapase-8 consistent with inflammasome-driven apoptosis. These signaling responses were inhibited by RNase digestion RNA dephosphorylation. Conclusion: Extracellular bacterial RNA is a potent danger signal for human cardiac myocytes and fibroblast-like cells, which induces PKR-JAK-STAT activation, pro-inflammatory transcription factor recruitment, IL-1 β upregulation and caspase-mediated apoptosis. These findings suggest a PKR-mediated mechanism for driving circulating bacterial RNA into pro-inflammatory and pro-apoptotic responses in cardiac myocytes, thereby priming the myocardium for contractile dysfunction and injury in sepsis.