The main characteristics of septic cardiomyopathy are depressed contractility, reduced LV ejection fraction and bi-ventricular dilation 
. Recent studies have indicated that intrinsic
alterations within cardiac myocytes are key mechanisms of this acute heart failure, which may be mediated by cardiac TLRs 
. Cardiac myocytes express several TLRs 
, and several TLR ligands -LPS, peptidoglycan and bacterial DNA- activate NF-κB in these cells, respectively through activation of TLR4, TLR2 and TLR9, leading to the production of inflammatory mediators and alterations in normal cardiac physiology 
. In the present study, we extend further this emerging concept, by showing that bacterial flagellin, the ligand of mammalian TLR5 
, is a potent inducer of cardiac innate immune responses and contractile dysfunction.
Our results are the first to formally demonstrate that the heart expresses TLR5. Although the presence of TLR5 mRNA had been previously reported in the HL-1 cardiac cell line 
, we now show that TLR5 protein is expressed in vitro both by H9c2 cells and primary ventricular myocytes, and that it is also strongly expressed in cardiac tissue. Cardiac TLR5 expression was not restricted to mice and rats, but was also observed in humans, indicating that the ability of the heart to detect flagellin is not species-related.
In vitro, minute concentrations of flagellin activated NF-κB in H9c2 cells and primary cardiomyocytes, substantiating the presence of an intact TLR5-dependent signaling system in cardiac myocytes. The essential role of TLR5 in the process of NF-κB activation elicited by flagellin was clearly evidenced by the lack of NF-κB activation in primary murine cardiomyocytes isolated from neonatal TLR5 deficient mice. Flagellin also activated two major stress activated kinases, p38 and JNK, which may play a role in septic cardiomyopathy, notably by activating the production of TNFα within the myocardium 
. Taken together, these data indicate that recognition of extracellular flagellin by cells from cardiac origin triggers signaling cascades intimately linked with the development of cardiac dysfunction in sepsis.
In line with these in vitro results, flagellin also provoked major inflammatory changes in the heart in vivo, characterized by a robust activation of NF-κB, and the upregulation of the inflammatory cytokines TNFα, IL-1β, IL-6, MIP-2 and MCP-1 within the cardiac tissue. In accordance with the roles of MIP-2 and MCP-1 as chemokines attracting polymorphonuclear cells (PMN), respectively monocytes/macrophages, we found some recruitment of poly- and mononuclear cells in the heart after flagellin administration. The degree of activation of these infiltrating cells appeared, however, limited, given the modest increase in myeloperoxydase activity, and the lack of increase of cardiac TREM-1, a specific cytokine derived from myeloid cells 
, suggesting that the cardiac inflammatory phenotype triggered by flagellin did not result primarily from the activation of infiltrating inflammatory cells.
It must be underscored that NF-κB was activated extremely early upon the injection of flagellin, as shown by stimulated DNA-binding activity starting 10 minutes after the injection of flagellin. Such immediate response supports the hypothesis that cardiac inflammation was a direct consequence of flagellin-heart interactions rather than an indirect effect due to the remote generation of inflammatory mediators. Furthermore, NF-κB activation occurred at very low concentrations of flagellin, as indicated by a threshold-inducing dose of 10 ng/mouse (0.4 µg/kg), which are clinically relevant, given that plasma levels of free circulating flagellin between 2 and 16 µg/l have been detected in humans with septic shock 
. Overall, these findings indicate that the heart is equipped to immediately recognize circulating flagellin with high sensitivity, implying that this bacterial protein may represent a particularly critical danger signal in this organ.
It is currently assumed that the local upregulation, within the heart itself, of inflammatory cytokines with negative inotropic properties, including TNFα, IL-1β 
, and IL-6, 
, as well as the cardiac recruitment of inflammatory cells, mostly polymorphonuclear cells 
, are key mechanisms leading to cardiac dysfunction in sepsis. The strong immune response of the heart in response to flagellin prompted us therefore to evaluate whether it would also affect cardiac physiology. To address this question, a microtip pressure volume (PV) conductance catheter was inserted into the left ventricle (LV) of mice, 4, 24 or 48 hours after an iv challenge with 1 µg (40 µg/kg) flagellin. In contrast to other methods, which provide indirect, load-dependent, indices of contractility, PV catheters allow the direct assessment of contractility by computing the slope of the LV end-systolic PV relationship (ESPVR), known as end-systolic elastance (Ees), which is independent from preload and afterload 
We could thus demonstrate that flagellin promotes the depression of cardiac contractility, as evidenced by cardiac dilation and reduced ejection fraction. These alterations were related to a direct negative inotropic effect, attested by significant reductions of both Ees and time-varying elastance, Emax. An important aspect of the cardiac dysfunction provoked by flagellin was its reversibility, as indicated by maximal systolic depression at 4 hours, followed by progressive recovery over a 48 hours period, a pattern which is highly consistent with the transient nature of septic myocardial dysfunction reported both in humans and in large aninal models of sepsis 
. Such reversibility argues against the development of significant cardiac damage upon flagellin administration. Indeed, we could not detect caspase activation in the heart and we did not notice any increase in plasma troponin I after flagellin, while systolic function was already significantly altered. This is in agreement with the lack of significant myocardial necrosis or apoptosis reported in animal models and in postmortem studies in septic shock patients, indicating that myocardial cell death is insufficient to account for the functional depression observed, as recently reviewed 
Although flagellin clearly depressed myocardial contractile function, our findings do not allow the conclusion that flagellin may be the causal factor in the development of cardiac dysfunction in sepsis. As stated earlier, many other factors released by bacteria may also promote myocardial inflammation and cardiac dysfunction, including bacterial DNA, lipopolysacharride and peptidoglycan. It is therefore highly likely that the depression of contractile function during sepsis is the result of the concerted action of multiple microbial virulence factors, each promoting intracellular innate immune defense mechanisms by interacting with distinct TLRs. To further precise the role of flagellin in the cardiac depression of bacterial sepsis would therefore require additional experiments using whole cell bacteria in TLR5 deficient mice, which should be performed in future studies.
Several limitations of our study deserve further discussion. Firstly, we cannot rule out that cells from non myocyte origin, mostly vascular endothelial and smooth muscle cells, as well as cardiac fibroblasts, may have contributed to the observed effects of flagellin in vivo. Secondly, our study may be criticized due to the lack of experiments in TLR5-deficient mice. However, these animals being unresponsive to flagellin 
, they would not allow to get further insights into the effects of flagellin on the heart. Cardiomyocyte-specific TLR5-knockout mice should therefore be developed in the future to further precise the actions of flagellin on this subset of cardiac cells. Thirdly, our findings do not prove that the depression of cardiac contractility observed after flagellin was a direct consequence of the cardiac immune response to flagellin. Indeed, the innate immune response to flagellin was not restricted to the heart, but instead was a widespread phenomenon, as shown by the rise of cytokines in other organs such as the lung and liver as well as the increase of circulating inflammatory mediators. Therefore, the contractile dysfunction observed upon flagellin administration cannot be simply attributed to a purely localized, cardiac restricted inflammation, but should be viewed as one component of the systemic innate immune response to this bacterial protein. In conclusion, our results collectively indicate that bacterial flagellin induces a prototypical innate immune response in cells from cardiac origin in vitro and in the whole heart in vivo, and that this response is associated with the development of an acute, transient and reversible, contractile dysfunction, which resembles the clinical picture of septic myocardial dysfunction.