Background. Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.Methods. Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.Results. Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.Conclusions. CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

Background. Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.Methods. Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.Results. Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.Conclusions. CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

OBJECTIVE: To determine the role of Toll-like receptor 3 in cardiac dysfunction during polymicrobial sepsis. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Male C57BL/6, wild-type, Toll-like receptor 3. INTERVENTION: Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. Toll-like receptors (TLRs) play a critical role in the pathophysiology of sepsis/septic shock. TLR3 is located in intracellular endosomes, and recognizes double-stranded RNA. This study examined the role of TLR3 in cardiac dysfunction following cecal ligation and puncture (CLP)-induced sepsis. TLR3 knockout (TLR3, n = 12) and age-matched wild-type (n = 12) mice were subjected to CLP. Cardiac function was measured by echocardiography before and 6 hrs after CLP. MEASUREMENTS AND MAIN RESULTS: CLP resulted in significant cardiac dysfunction as evidenced by decreased ejection fraction by 25.7% and fractional shortening by 29.8%, respectively. However, TLR3 mice showed a maintenance of cardiac function at pre-CLP levels. Wild-type mice showed 50% mortality at 58 hrs and 100% mortality at 154 hrs after CLP. In striking contrast, 70% of TLR3 mice survived indefinitely, that is, >200 hrs. TLR3 deficiency significantly decreased CLP-induced cardiac-myocyte apoptosis and attenuated CLP-induced Fas and Fas ligand expression in the myocardium. CLP-activation of TLR4-mediated nuclear factor-κB and Toll/IL-1 receptor-domain-containing adapter-inducing interferon-β-dependant interferon signaling pathways was prevented by TLR3 deficiency. In addition, CLP-increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression, and neutrophil and macrophage sequestration in the myocardium were also attenuated in septic TLR3 mice. More significantly, adoptive transfer of wild-type bone-marrow stromal cells to TLR3 mice abolished the cardioprotective effect in sepsis. CONCLUSIONS: These data indicate that TLR3 plays a deleterious role in mediating cardiac dysfunction in sepsis. Thus, modulation of the TLR3 activity may be useful in preventing cardiac dysfunction in sepsis.

Cardiovascular collapse is the major factor contributing to the mortality of trauma-hemorrhage (T-H) patients. Toll-like receptors (TLRs) play a critical role in T-H-induced cardiac dysfunction. This study evaluated the role of TLR9 agonist, CpG-oligodeoxynucleotide (ODN) 1826, in cardiac functional recovery after T-H. Trauma-hemorrhage was induced in a murine model by soft tissue injury and blood withdrawals from the jugular vein to a mean arterial pressure of 35 ± 5 mmHg. Mice were treated with CpG-ODN 1826 (10 μg/30 g body weight) by intraperitoneal injection 1 h before T-H (n = 5-8/group). Hemodynamic parameters were measured before, during hemorrhage, and at 60 min after T-H. Trauma-hemorrhage significantly decreased the mean arterial pressure and left ventricular pressure compared with sham controls. In contrast, CpG-ODN administration significantly attenuated the decrease in arterial pressure and left ventricular pressure due to T-H. Trauma-hemorrhage markedly decreased myocardial levels of phosphorylated Akt by 57.9%. However, CpG-ODN treatment significantly blunted the decrement in phospho-Akt by activating the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY294002 partially abolished CpG-induced cardioprotection, indicating that additional signaling pathways are involved in the protective effect of CpG-ODN after T-H. We observed that CpG-ODN treatment also significantly attenuated the decrease in myocardial phospho-ERK levels after T-H. Inhibition of ERK by U0126 also partially abolished the cardioprotective effect of CpG-ODN after T-H. Our data suggest that CpG-ODN significantly attenuates T-H-induced cardiac dysfunction. The mechanisms involve activation of both PI3K/Akt and ERK signaling pathways. The TLR9 agonist, CpG-ODN 1826, may provide a novel treatment strategy for preventing or managing cardiac dysfunction and enhancing recovery in T-H patients.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with shamoperated control. In contrast, PGN administration attenuated CLPinduced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3β in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGNinduced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H2O2, which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H2O2. In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H2O2 generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. We have previously reported that glucan phosphate (GP) significantly increased survival in a murine model of cecal ligation and puncture (CLP)-induced sepsis. In the present study, we examined the effect of GP on cardiac dysfunction in CLP-induced septic mice. GP was administered to ICR/HSD mice 1 h before induction of CLP. Sham surgically operated mice served as control. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham control. In contrast, GP administration prevented CLP-induced cardiac dysfunction. Macrophage migration inhibitory factor (MIF) has been implicated as a major factor in cardiomyocyte apoptosis and cardiac dysfunction during septic shock. CLP increased myocardial MIF expression by 88.3% (P < 0.05) and cardiomyocyte apoptosis by 7.8-fold (P < 0.05) compared with sham control. GP administration, however, prevented CLP-increased MIF expression and decreased cardiomyocyte apoptosis by 51.2% (P < 0.05) compared with untreated CLP mice. GP also prevented sepsis-caused decreases in phospho-Akt, phospho-GSK-3β, and Bcl-2 levels in the myocardium of septic mice. These data suggest that GP treatment attenuates cardiovascular dysfunction in fulminating sepsis. GP administration also activates the phosphoinositide 3-kinase/Akt pathway, decreases myocardial MIF expression, and reduces cardiomyocyte apoptosis.

The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.

Indiana University-Purdue University Indianapolis (IUPUI) / Congenital heart defects occur in approximately one percent of births every year, which makes it the most frequently occurring congenital defect in patients. The aim of this project was to use two mutant neural crest (NC) mouse models to study the mechanisms underlying congenital heart failure in utero. The first mouse model was a Pax3 systemic knockout, which was lethal by mouse gestational day 14, and had appreciably reduced numbers of migratory NC cells. The second mouse model was a Wnt1Cre-mediated NC genetic cell ablation model, which was surprisingly viable and survived to birth, despite an apparent lack of migratory NC cells. The resultant data indicated that both mouse models had similar heart structural defects including persistent truncus arteriosus, which was due to fewer or no migratory cardiac NC cells. However, in utero heart function was appreciably perturbed in Pax3 mutants when compared to that of the ablated mutant model. The loss of embryonic cardiac function in Pax3 mutants was directly attributed to a substantial decrease in the activity of the beta-adrenergic pathway. This was due to a lack of proper specification of trunk NC cells, leading to diminished levels of circulating catecholamine levels in the embryo. To definitively confirm this conclusion, poor cardiac function was successfully restored by pharmacological stimulation of the beta-adrenergic pathway via administration of isoproterenol and forskolin to pregnant dams, which led to embryonic survival of Pax3 mutants to birth. By comparison of these two mutant mouse models, perturbation in the beta-adrenergic pathway was identified as the underlying mechanism responsible for in utero heart failure and lethality in Pax3 mutant embryos. The results of this study are expected to be significant in developing future therapeutic targets for congenital heart failure in prenatal and newborn patients.

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