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Pressure overload induces cardiac remodeling involving both the contractile machinery and intercalated disks (IDs). Filamin C (FlnC) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adapters localizing in IDs of higher vertebrates. Knockout of the gene encoding Xin (Xirp1) in mice leads to a mild cardiac phenotype with ID mislocalization. In order to amplify this phenotype, we performed transverse aortic constriction (TAC) on control and Xirp1-deficient mice. TAC induced similar left ventricular hypertrophy in both genotypes, suggesting that the lack of Xin does not lead to higher susceptibility to cardiac overload. However, in both genotypes, FlnC appeared in "streaming" localizations across multiple sarcomeres proximal to the IDs, suggesting a remodeling response. Furthermore, FlnC-positive areas of remodeling, reminiscent of sarcomeric lesions previously described for skeletal muscles (but so far unreported in the heart), were also observed. These adaptations reflect a similarly strong effect of the pressure induced by TAC in both genotypes. However, 2 weeks post-operation TAC-treated knockout hearts had reduced levels of connexin43 and slightly increased incidents of ventricular tachycardia compared to their wild-type (WT) counterparts. Our findings highlight the FlnC-positive sarcomeric lesions and ID-proximal streaming as general remodeling responses in cardiac overload-induced hypertrophy.

Background and goal of the study Pulmonary inflammation, increased vascular permeability, and pulmonary edema, occur in response to primary pulmonary infections like pneumonia but are also evident in endotoxemia or sepsis. Mechanical ventilation augments pre-existing lung injury and inflammation resulting from exposure to microbial products. The objective of this study was to test the hypothesis that low-tidal-volume prevent ventilation induced lung injury in sepsis. Materials and methods 10–12-week-old male C57BL/6N-mice received an intraperitoneal (i.p.) injection with equipotent dosages of LPS, 1668-thioate, 1612-thioate, or PBS. 120 min after injection, mice were randomized to low- (LV, 7 ± 1 ml/kg) or high-tidal-volume (HV, 25 ± 1 ml/kg) ventilation. Hemodynamic and ventilatory parameters were recorded and inflammatory markers were analyzed form BAL that was generated after 90 minute ventilation. Results and discussion Arterial blood pressures declined during mechanical ventilation in all groups. pO2 decreased in LPS injected and CO2 increased in sham, LPS, and 1612-thioate administered mice at 45 min and in 1668-thioate injected mice after 90 minute LV ventilation compared to respective HV groups. BAL protein concentrations increased in HV ventilated and 1668- or 1612-thioat pre-treated mice. BAL TNF-α protein concentrations increased in both LPS- and 1668-thioate-injected and IL-1β protein concentrations only in LPS-injected and HV ventilated mice. Most notably, no increased protein concentrations were observed in any of the LV ventilated groups. Conclusion We conclude that low-tidal-volume ventilation may be a potential strategy for the prevention of ventilator induced lung injury in a murine model of systemic TLR agonist induced lung injury.

Aims The incidence of secondary systemic fungal infections has sharply increased in bacterial septic patients. Antimycotics exhibit immunomodulatory properties, yet these effects are incompletely understood in secondary systemic fungal infections following bacterial sepsis. We investigated a model of systemic inflammation to determine whether antimycotics (liposomal amphotericin B (L-AMB), itraconazol (ITC), and anidulafungin (ANI)) modulate the gene and protein expression as well as the phagocytic activity of lipopolysaccharide (LPS)-stimulated human monocytes. Main methods THP-1 monocytes were incubated with L-AMB, ITC or ANI and LPS. Gene expression levels of cytokines (TNF- , IL-1 , IL-6, and IL-10) were measured after 2 h, 6 h, and 24 h. Cytokine protein levels were evaluated after 24 h and phagocytic activity was determined following co-incubation with Escherichia coli. Key findings All antimycotics differentially modulated the gene and protein expression of cytokines in sepsis-like conditions. In the presence of LPS, we identified L-AMB as immunosuppressive, whereas ITC demonstrated pro-inflammatory properties. Both compounds induced remarkably less phagocytosis. Significance Our study suggests that antimycotics routinely used in septic patients alter the immune response in sepsis-like conditions by modulating cytokine gene and protein expression levels and phagocytic activity. Future treatment strategies should consider the immune status of the host and apply antimycotics accordingly in bacterial septic patients with secondary fungal infections.

Conventional antibiotics exhibit immunomodulatory properties beneficial in the treatment of sepsis. Antibiotic-resistant Gram-positive bacteria have become a problem in sepsis therapy, giving rise to increased use of last-resort antibiotics; for example, linezolid (LIN), vancomycin (VAN) and daptomycin (DAP). As the immunomodulatory properties of these antibiotics in treating sepsis are unknown, this study examined the effect of VAN, LIN and DAP on the immune response under sepsis-like conditions in vitro. Lipopolysaccharide (LPS)-activated THP-1 monocytes were incubated with LIN, VAN or DAP. Gene expression of cytokines (TNFα, IL-1β, IL-6, IL-10) and Toll-like receptors (TLR1, 2, 4, 6, 7 and 9) was monitored and phagocytosis was determined following coincubation with E. coli. The antibiotics differentially modulated the gene expression of the investigated cytokines. While LIN and VAN upregulated the expression of all TLRs, DAP downregulated mRNA levels of TLR1, TLR2 and TLR6, which recognize pathogen-associated molecular patterns from Gram-positive bacteria. In addition, LIN inhibited, whereas VAN promoted the phagocytic activity of monocytes. Our results suggest that LIN and VAN possess pro-inflammatory properties, whereas DAP might reduce the immune response to Gram-positive bacteria in sepsis. Furthermore, VAN might be beneficial in the prevention of Gram-negative infections by increasing the phagocytosis of E. coli.

In this study we investigated the responsiveness of near-infrared spectroscopy (NIRS) recordings measuring regional cerebral tissue oxygenation (rSO2) during hypoxia in apneic divers. The goal was to mimic dynamic hypoxia as present during cardiopulmonary resuscitation, laryngospasm, airway obstruction, or the "cannot ventilate cannot intubate" situation. Ten experienced apneic divers performed maximal breath hold maneuvers under dry conditions. SpO2 was measured by Masimo™ pulse oximetry on the forefinger of the left hand. NIRS was measured by NONIN Medical's EQUANOX™ on the forehead or above the musculus quadriceps femoris. Following apnea median cerebral rSO2 and SpO2 values decreased significantly from 71 to 54 and from 100 to 65%, respectively. As soon as cerebral rSO2 and SpO2 values decreased monotonically the correlation between normalized cerebral rSO2 and SpO2 values was highly significant (Pearson correlation coefficient = 0.893). Prior to correlation analyses, the values were normalized by dividing them by the individual means of stable pre-apneic measurements. Cerebral rSO2 measured re-saturation after termination of apnea significantly earlier (10 s, SD = 3.6 s) compared to SpO2 monitoring (21 s, SD = 4.4 s) [t(9) = 7.703, p < 0.001, r(2) = 0.868]. Our data demonstrate that NIRS monitoring reliably measures dynamic changes in cerebral tissue oxygen saturation, and identifies successful re-saturation faster than SpO2. Measuring cerebral rSO2 may prove beneficial in case of respiratory emergencies and during pulseless situations where SpO2 monitoring is impossible.

BACKGROUND: Most antiepileptic drugs (AEDs) inhibit seizure generation by acting on voltage-dependent ion channels. Voltage-dependent sodium and calcium channels are commonly expressed in brain and heart, suggesting that AEDs may have considerable cardiodepressive effects, thereby facilitating sudden cardiac death as a potential cause of sudden unexpected death in epilepsy (SUDEP). Here, we investigated the effects of carbamazepine (CBZ), lamotrigine (LTG) and levetiracetam (LEV) alone and in combination on the shortening properties of isolated ventricular cardiomyocytes of wild type mice. METHODS: Properties of murine cardiomyocytes were determined by recording the sarcomere shortening with a video imaging system before, during and after administration of AEDs in different concentrations and combinations. We assessed (i) the number of successful shortenings during continuous electrical stimulation (electromechanical coupling) and (ii) the shortening amplitude as well as other shortening-related properties upon repetitive electrical stimulation at 4 Hz. Data are given as mean±SEM. RESULTS: At 100 µM, CBZ (10 cells), LTG (11 cells) and LEV (11 cells) alone had no effect on the electromechanical coupling, but reversibly reduced shortening amplitudes by 15±4 %, 24±3 % and 11±3 %, respectively. Increasing the LTG concentration to 250 (21 cells) and 500 µM (4 cells) reversibly inhibited the electromechanical coupling in 62 % and 100 % of the experiments. Importantly, simultaneous application of CBZ, LTG and LEV at 100 µM also impaired the electromechanical coupling in 8 of 19 cardiomyocytes (42 %) and reduced the shortening amplitude by 21±4 %. CONCLUSION: Our data show that AEDs reversibly impair cardiac excitation and contraction. Importantly, the blocking effect on electromechanical coupling appears to be additive when different AEDs are simultaneously applied. The translational value of these experimental findings into clinical practice is limited. Our results, however, suggest that rationale AED-therapy may be important with respect to cardiac side-effects and potential facilitation of serious cardiac dysfunction especially when AEDs are used in combination or at very high doses.

Aims Toll-like receptor (TLR)9 ligand CpG-oligodeoxynucleotide (CpG-ODN) exerts preconditioning in myocardial ischemia/reperfusion. We hypothesized a postconditioning effect of CpG-ODN in a murine closed-chest model of myocardial infarction. Materials and Methods C57BL/6 (12 weeks, male, WT) mice were instrumented at the left anterior descending artery, then allowed 5d of recovery before 30 min ischemia. Treatments comprised: 1) PBS: 250 μl phosphate buffer solution intraperitoneally 5 min before reperfusion and 2) IPC (ischemic postconditioning): 3 twenty-second reperfusion and occlusion episodes at the end of ischemia 3) CpG-ODN: 1668 thioate 0.2 μmol/kg BW intraperitoneally 5 min before reperfusion. Infarct size was assessed via triphenyltetrazolium chloride (TTC) staining after 2 and 24 h reperfusion. Myocardial mRNA-expression of cytokines was measured using real-time PCR after 2 h reperfusion. Phosphatidylinositol-3 kinase (PI3K)-inhibitor wortmannin was injected intraperitoneally in WT 15 min before postconditioning and PBS in each group. Cardiac function in WT was assessed with a left-ventricular pressure-volume catheter at 24 h reperfusion. Key findings Following 30 min ischemia and 2 h reperfusion, infarct size was diminished by 90% in WT postconditioned with CpG-ODN (2.4 ± 1.55 IS/AAR%) and IPC (1.98 ± 1.03 IS/AAR%) compared to PBS mice (23.2 ± 3.97 IS/AAR%). Infarct size increased following 24 h reperfusion but the differences remained robust. Expression of TNF-α and IL-10 was increased in CpG-ODN. Wortmannin abolished the postconditioning effect of CpG-ODN and IPC. Ejection fraction and preload-recruitable stroke work were significantly greater in CpG-ODN mice. Significance CpG-ODN confers postconditioning via activation of TLR9. Cardiac function is preserved following CpG-ODN postconditioning. The PI3K -inhibitor wortmannin attenuates CpG-ODN postconditioning.

Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive and sporadic forms of protein aggregation myopathies and cardiomyopathies. We generated R349P desmin knock-in mice, which harbor the ortholog of the most frequently occurring human desmin missense mutation R350P. These mice develop age-dependent desmin-positive protein aggregation pathology, skeletal muscle weakness, dilated cardiomyopathy, as well as cardiac arrhythmias and conduction defects. For the first time, we report the expression level and subcellular distribution of mutant versus wild-type desmin in our mouse model as well as in skeletal muscle specimens derived from human R350P desminopathies. Furthermore, we demonstrate that the missense-mutant desmin inflicts changes of the subcellular localization and turnover of desmin itself and of direct desmin-binding partners. Our findings unveil a novel principle of pathogenesis, in which not the presence of protein aggregates, but disruption of the extrasarcomeric intermediate filament network leads to increased mechanical vulnerability of muscle fibers. These structural defects elicited at the myofiber level finally impact the entire organ and subsequently cause myopathy and cardiomyopathy. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1363-2) contains supplementary material, which is available to authorized users.

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Background: Toll-like receptors (TLRs) are involved in a variety of cardiovascular disorders, including septic cardiomyopathy, ischemia/reperfusion, heart failure, and cardiac hypertrophy. Previous research revealed that TLR4 promotes cardiac hypertrophy in vivo. Therefore, we investigated whether TLR2 is also involved in the development of cardiac hypertrophy. Methods: Tlr2 deficient and wild type mice were subjected to transverse aortic constriction (TAC) or sham operation procedure. Left ventricular, heart and lung weights as well as hemodynamic parameters were determined after 3, 14 or 28 days. Real-time RT PCR was used to evaluate left ventricular gene expression. Protein content was determined via ELISA. Results: TAC increased systolic left ventricular pressure, contraction and relaxations velocities as well as the heart weight in both genotypes. Tlr2 deficiency significantly enhanced cardiac hypertrophy after 14 and 28 days of TAC. Left ventricular end-diastolic pressure and heart rate increased in Tlr2 ⁻/⁻ TAC mice only. Fourteen days of TAC led to a significant elevation of ANP, BNP, TGFβ and TLR4 mRNA levels in Tlr2 ⁻/⁻ left ventricular tissue. Conclusion: These data suggest that Tlr2 deficiency may promote the development of cardiac hypertrophy and ventricular remodeling after transverse aortic constriction.

Severe sepsis and septic shock are often accompanied by acute cardiovascular depression. Lipo- polysaccharide (LPS) signaling via Toll-like receptor 4 (TLR4) can induce septic organ dysfunction. The aim of this study was to elucidate the in vivo impact of pharmacological TLR4 antagonism on LPS-induced cardiovascular depression using eritoran tetrasodium (E5564). To simulate sepsis, C3H/HeN mice were challenged i.p. with 2 mg/kg body weight LPS. With the intent to antagonize the LPS effects, eritoran was administered i.v. (4 mg/kg body weight). Physical activity, peripheral blood pressure, and heart frequency were recorded before and after LPS and eritoran injection. In addition, intracardiac hemodynamic parameters were analyzed with a pressure conductance catheter. After 2 and 6 h of LPS stimulation T eritoran treatment, the hearts and aortae were harvested, and TLR as well as inflammatory mediator expression was measured using reverse transcriptionYquantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Lipopolysaccharide significantly decreased arterial blood pressure over time. Administration of eritoran partially prevented the LPS-dependent reduction in blood pressure and preserved cardiac function. In addition, LPS increased the expression of CD14 and TLR2 in cardiac and aortic tissue. In aortic tissue, eritoran attenuated this increase, whereas no significant reduction was observed in the heart. Furthermore, cardiac and aortic inducible nitric oxide synthetase mRNA levels were significantly increased 6 h after LPS application. This effect was reduced in the presence of eritoran. In summary, the beneficial influence of eritoran on cardiovascular function in vivo seems to rely mainly on reduction of LPS-induced inducible nitric oxide synthetase expression as well as on attenuated cytokine expression in the vascular wall. KEYWORDS—TLR4 antagonism, sepsis, cardiac depression, endotoxemia, eritoran

Sarcoidosis is a systematic granulomatous disorder. Genetic susceptibility could play a central role in the disease development and progression. In this study, we investigated whether caspase recruitment domain 15 (CARD15) gene haplotypes are associated with the onset or the clinical course of sarcoidosis. Three hundred Caucasian sarcoidosis patients and 381 matched controls were included. Eight haplotype-tagging single nucleotide polymorphisms (SNPs) in the CARD15 gene were examined by mass spectrometry-based SNP genotyping. By haplotype analysis, mutations located in between tested SNPs can also be identified. Therefore, we can conclude that there is no association between the CARD15 gene and the development or a special phenotype of sarcoidosis in our cohort.

The aetiology of sarcoidosis is unclear. Single nucleotide polymorphisms (SNPs) in transforming growth factor (TGF)-β2 and -β3 have been reported to be associated with the development of lung fibrosis in patients with sarcoidosis. SNPs in TGF-β2 (rs1891467) and TGF-β3 (rs3917200) were investigated in 296 patients with sarcoidosis (acute/self remitting, n = 70 (including 62 patients with Löfgren's syndrome); chronic, n = 168; acute/chronic, n = 58) by real-time PCR. 32 patients showed radiological signs of lung fibrosis. The genotype frequencies were compared among the sarcoidosis groups as well as to 377 healthy controls. We found a significant association with the G-allele in rs1891467 in TGF-β2 and an acute/self remitting course of sarcoidosis compared to a chronic course (p = 0.001). The results were even more evident for patients with Löfgren's syndrome (p0.001). Moreover, we could demonstrate a borderline significance between TGF-β3 (rs3917200) and lung fibrosis (p = 0.050). Carriers of the G-allele in rs1891467 might be protected from developing a chronic course. Moreover, there is evidence that rs3917200 is involved in the development of lung fibrosis in sarcoidosis. This study is the first in sarcoidosis patients to suggest a genetic implication of TGF-β2 as a protective factor in the course of sarcoidosis.

Aims Xin is a striated muscle-specific F-actin binding protein that has been implicated in cardiomyopathies. In cardiomyocytes, Xin is localized at intercalated discs (IDs). Mice lacking only two of the three Xin isoforms (XinAB−/− mice) develop severe cardiac hypertrophy. To further investigate the function of Xin variants in the mammalian heart, we generated XinABC−/− mice deficient in all Xin isoforms. Methods and results XinABC−/− mice showed a very mild phenotype: heart weight, heart weight to tibia length ratios, and cardiac dimensions were not altered. Increased perivascular fibrosis was only observed in hearts of young XinABC−/− mice. Striking differences were revealed in isolated cardiomyocytes: XinABC−/− cells demonstrated a significantly increased number of non-terminally localized ID-like structures. Furthermore, resting sarcomere length was increased, sarcomere shortening, peak shortening at 0.5–1 Hz, and the duration of shortening were decreased, and shortening and relengthening velocities were accelerated at frequencies above 4 Hz in XinABC−/− cardiomyocytes. ECG showed a significantly shorter HV interval and a trend towards shorter QRS interval in XinABC−/− mice, suggesting a faster conduction velocity of the ventricular-specific conduction system. In human cardiac tissue, expression of XinC protein was detected solely in samples from patients with cardiac hypertrophy. Conclusion Total Xin deficiency leads to topographical ID alterations, premature fibrosis and subtle changes in contractile behaviour; this is a milder cardiac phenotype than that observed in XinAB−/− mice, which still can express XinC. Together with the finding that XinC is detected solely in cardiomyopathic human tissues, this suggests that its expression is responsible for the stronger dominant phenotype in XinAB−/− mice. Furthermore, it indicates that XinC may be involved in the development of human cardiac hypertrophy.

Gelsolin (gsn) is involved in the reorganization of the cytoskeleton, thereby modulating cardiomyocytal L-type Ca(2+) channels. We investigated global cardiac electrophysiological characteristics in a gsn-deficient (gsn(-/-)) mouse strain.Using transvenous catheterization, atrial and ventricular stimulation were performed in 15 male mice [eight gsn(-/-), seven wild-type (gsn(+/+))]. Surface ECG, standard electrophysiological parameters, and inducibility of atrial fibrillation (AF) were evaluated.The surface ECG showed shorter PQ (37.8 +/- 4.6 versus 42.9 +/- 2.7 ms; P = 0.02), but longer QRS (16.5 +/- 1.8 versus 13.9 +/- 1.2 ms; P = 0.005) and QT intervals (38.5 +/- 2.2 versus 35.6 +/- 2.4 ms, P = 0.03) in gsn(-/-). Gsn(-/-) exhibited significantly higher susceptibility to induction of prolonged AF episodesor =60 s [six of eight gsn(-/-) versus one of seven gsn(+/+); P = 0.04]. Sustained AF episodesor =10 min were observed in 50% of the gsn-deficient animals.Gsn deficiency results in perpetuation of inducible episodes of atrial fibrillation. Altered L-type Ca(2+) currents and disturbed Ca(2+) handling known to be associated to gsn deficiency likely contribute to this effect.

Background Pressure overload induces the cardiac expression of parathyroid hormone-related protein (PTHrP). Plasma levels are elevated in patients with heart disease. It is unknown whether this represents an epiphenomenon or suggests involvement in hypertrophy. Aim To identify a potential role of PTHrP in pressure induced hypertrophy and heart failure. Methods and results Pressure load was produced via thoracic aortic constriction (TAC) and application of a PTHrP antagonist (PTHrP(7–34)) via osmotic minipumps in mice. Main findings were confirmed in vitro by exposing isolated adult ventricular mice cardiomyocytes to PTHrP(1–34) (100 nmol/l). TAC treated animals developed myocardial hypertrophy within 2 weeks. The heart weight to body weight ratio increased from 5.02±0.14 mg/g (sham/vehicle) and 5.16±0.19 mg/g (sham/antagonist) to 6.59±0.85 mg/g (TAC/vehicle) and 7.07±0.80 mg/g (TAC/antagonist) (each n=6–8; p

Objectives: Annexin A7 is involved in cardiomyocyte membrane organization and Ca 2+ -dependent signalling processes. We investigated theimpact of annexin A7 on cardiac electrophysiological properties using an annexin A7-deficient mouse strain (annexin A7 −/ ).Methods: Nineteen adult annexin A7 −/ and 14 wild-type mice were examined electrophysiologically in vivo by transvenous catheterization.Hearts were additionally perfused by the Langendorff method and epicardial activation mapping was performed.Results: The susceptibility to induction of atrial fibrillation was elevated in annexin A7 −/− mice. Ten deficient animals showed atrialfibrillation (AF) episodes ≥1 min and sustained AF ≥30 min was observed in 4 annexin A7 −/ mice, but in none of the wild-type mice. Theincidence of ventricular tachycardia (VT) was higher in annexin A7 −/ mice and VT duration was prolonged. Epicardial mapping showedelevated anisotropy and inhomogeneity of conduction, leading to conduction blocks in the deficient mice. Besides alterations of intracellularcalcium homeostasis, electron microscopy showed a homogeneous, electron-dense material that filled the myocardial intercellularcompartments and accumulated at the basement membranes. This led to expansion of the extracellular spaces, which was the most probablesubstrate factor responsible for the disturbances of electrical communication.Conclusions: Annexin A7 deficiency causes severe electrical instability in the murine heart, including conduction disturbances andanisotropy of impulse propagation, which is accompanied by disturbed calcium handling and intercellular deposits.© 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Objective—Aim was to assess whether lipopolysaccharide (LPS)-induced decrease of total peripheral resistance depends on Toll-like receptor (TLR)4 signaling and whether it is sensitive to NO-synthase or TLR4 antagonists.Methods and Results—C3H/HeN mice (control), expressing a functional, and C3H/HeJ mice, expressing a nonfunctional TLR4, were compared. LPS (20 mg/kg) was injected i.p. 6 hours before hemodynamic measurements. L-NAME and SMT, inhibitors of NO production, and Eritoran, a TLR4 antagonist, were tested for their impact on vascular contractility. Aortic rings were incubated for 6 hours with or without LPS (1 μg/mL), or with LPS+Eritoran (2 μg/mL) and their phenylephrine-induced contractility was measured using a myograph. The expression of cytokines in aortic tissue was examined by real-time polymerase chain reaction. In control mice LPS induced a significant decrease of blood pressure and an increase of heart rate, whereas C3H/HeJ remained unaffected. LPS induced an increase of cytokine expression and a depression of vascular contractility only in control mice but not in C3H/HeJ. L-NAME and SMT increased contractility in all rings and restored LPS-dependent depression of contractility. Eritoran prevented LPS-induced loss of contractility.Conclusions—LPS upregulates cytokine expression via TLR4 and induces attenuation of smooth muscle contractility which can be effectively antagonized.

Summary The pathogenesis of myocardial ischemia-reperfusion injury (MI/R) involves an inflammatory response in the myocardium undergoing reperfusion. Modulation of this response by splenectomy constitutes an option to protect the heart from MI/R. To mimic the effect of splenectomy in a pharmacological approach, the sphingosine-1-phosphate agonist FTY720 was applied at the onset of reperfusion. In a closed chest model of MI/R, infarct size was assessed by triphenyltetrazolium chloride staining after 1 h of ischemia and 24 h of reperfusion, and by Masson trichrome staining 21 days after reperfusion in splenectomised mice, mice post-conditioned with FTY720 IP (1 mg/kg), and controls. In addition, hemodynamic parameters were recorded after 24 h and 21 days by catheterization. Infarct size, and immune cell invasion of phagocytic monocytes investigated by FACS after 24 h of reperfusion were significantly reduced by both splenectomy, and FTY720 treatment. Evaluation after 21 days of reperfusion revealed that FTY720 treated animals had an improved hemodynamic outcome compared to placebo treated as well as splenectomised animals. FTY720 treatment reduced cell injury as effectively as splenectomy by lowering the number of phagocytic monocytes invading the myocardium and ameliorated hemodynamic outcome within the first 21 days.

Transverse aortic constriction (TAC) results in a transient increase of proinflammatory cytokines, which return to baseline levels within 3 d. In contrast to cytokine baseline levels, the myocardium remains capable to respond even stronger to a new stimulus. As the molecular mechanisms for this phenomenon are unknown, we tested whether TAC modulates the innate immune system in mice and changes the inflammatory reaction to a new stimulus.Following 3 d of TAC or sham-operation procedure (SOP), LPS (20 mg/kg) or PBS (control) were administered intraperitoneal for 10 min as well as for 6 h. Hemodynamic parameters were recorded to measure the effects of TAC and LPS. After TAC/SOP alone CD14 expression was monitored and after additional 6 h of LPS/PBS the expression of CD14, TLR4 and proinflammatory cytokines were determined by western-blot, ELISA and RNase protection assay, respectively. Following TAC/SOP and 10 min of LPS/PBS, NFkappaB activation was investigated by EMSA.TAC induced cardiac hypertrophy and elevated blood pressure. LPS application led to hypotension and other symptoms of sepsis. CD14 expression increased after TAC alone and even further after additional LPS challenge. However, we did not detect changes of TLR4 expression. Also NFkappaB activation increased after LPS challenge higher in the TAC than in the SOP group. LPS-stimulation induced also higher cytokine expression in the TAC than in the SOP group.TAC modulates innate immunity by regulating the expression of CD14 and changes the myocardial tissue to respond more powerful to LPS.

BACKGROUND ACE inhibitors are widely used to antagonize the biological activity of angiotensin II in hypertensive heart disease. Oestrogen reduces angiotensin type 1 receptor expression, and thereby modifies angiotensin signalling. AIM To investigate the interaction of oestrogen status and ACE inhibition on the development of left ventricular hypertrophy and expression of transforming growth factor (TGF)-beta(1) in female spontaneously hypertensive rats (SHR). METHODS AND RESULTS Intact female SHR, ovariectomised SHR, and ovariectomised SHR with 17beta-oestradiol (E2) replacement therapy were either treated with placebo or the ACE inhibitor moexiprilat. Blood pressure, left ventricular hypertrophy, and expression of TGF-beta(1) and TGF-beta(1)-regulated genes were investigated. ACE inhibition reduced blood pressure in all groups. When normalised to blood pressure, a significant reduction in hypertrophy was found in ovariectomised animals receiving E2. Expression of TGF-beta(1) was increased in all three groups treated with the ACE inhibitor, with top levels in ovariectomised animals. Moreover, expression of ornithine decarboxylase (ODC), an adrenoceptor dependent gene, downstream of TGF-beta(1), was up-regulated upon ACE inhibition, except in animals which were ovariectomised and oestrogen supplemented. Parathyroid hormone-related peptide, a growth factor negatively regulated by TGF-beta(1), was down-regulated in all animals receiving the ACE inhibitor. CONCLUSION ACE inhibition modulated TGF-beta(1) and TGF-beta(1) dependent genes. Oestrogen deficiency alone did not influence the progression of cardiac hypertrophy in this model of female SHR.

Objective: Atrial fibrillation (AF) as an “indicator arrhythmia” for enhanced atrial vulnerability in mouse hearts has not yet been systematically examined. We therefore evaluated a transesophageal rapid atrial stimulation protocol for the induction of AF in C57Bl/6 mice. Methods: 40 C57Bl/6 mice (19 female and 21 male; 5.2 ± 2.1 months; 18 – 27 g) were examined by closed chest transesophageal atrial stimulation. Baseline ECG and electrophysiological parameters, AF-inducing stimulation cycle length (CL) and AF duration were analyzed. Results: The surface ECG demonstrated a significantly faster heart rate in female mice (R-R: 138.7 ± 19.9 ms versus 150.5 ± 15.7 ms, P < 0.05). AF was inducible in 90 % of the population and not inducible in 4 mice, all female (21 % in this subgroup). Mean induction CL was 27.4 ± 7.3 ms. Mean AF duration was 26.9 ± 42.6 s before spontaneous termination. In a subgroup of 4 female and 4 male mice (mean age 7.5 months), successive testing of AF induction showed a range of higher susceptibility to AF at stimulus amplitudes of 3.0 – 4.0 mA and stimulation CLs between 15 – 25 ms. AF induction was observed to be constantly reproducible in the individual animals. No correlation to pacing stimulus length and amplitude was found. Conclusions: This study demonstrates that it is possible to reproducibly induce self-terminating AF and supraventricular arrhythmias in mice by transesophageal atrial burst stimulation. The presented method allowing serial testings of the same animal can be a useful tool in further investigations with transgenic mice and might be helpful in the characterization of underlying genetic or molecular mechanisms of AF.

The aim of the study was to investigate whether pre-conditioning with CpG-oligodeoxynucleotides (CpG-ODN) may change cardiac ischemia/reperfusion (I/R)-dependent inflammation and modulates infarct size and cardiac performance. WT and TLR9-deficient mice were pre-treated with 1668-, 1612- and H154-thioate or D-Gal as control. Priming with 1668-thioate significantly induced inflammatory mediators in the serum and a concomitant increase of immune cells in the blood and spleen of WT mice. Furthermore, it induced myocardial pattern recognition receptors and pro-inflammatory cytokines peaking 2 h after priming and a continuous increase of IL-10. 16 h after pre-conditioning, myocardial ischemia was induced for 1 h. Infarct size determined after 24 h of I/R was reduced by 75 % due to pre-conditioning with 1668-thioate but not in the other groups. During reperfusion, cytokine expression in 1668-thioate primed mice increased further with IL-10 exceeding the other mediators by far. These changes were observed neither in animals pre-treated with 1612- or H154-thioate nor in TLR9-deficient mice. The 1668-thioate-dependent increase of IL-10 was further supported by results of a micro-array analysis 3 h after begin of reperfusion. Block of IL-10 signaling increased I/R size and prevented influence of priming. In the group pre-treated with 1668-thioate, cardiac function was preserved 24 h, 14 days and 28 days after I/R, whereas animals without pre-conditioning exhibited impaired heart function 24 h and 14 days after I/R. The excessive 1668-thioate-dependent IL-10 up-regulation during pre-conditioning and after I/R seems to be the key factor for reducing infarct size and improving cardiac function. This is in agreement with the finding that IL-10 block prevents cardioprotection by pre-conditioning. Electronic supplementary material The online version of this article (doi:10.1007/s00395-013-0376-7) contains supplementary material, which is available to authorized users.

The involvement of toll-like receptor 9 (TLR9), a receptor for bacterial DNA, in septic cardiac depression has not been clarifiedin vivo. Thus, the aim of the study was to test possible TLR9 inhibitors (H154-thioate, IRS954-thioate, and chloroquine) for their ability to protect the cardiovascular system in a murine model of CpG oligodeoxynucleotide- (ODN-) dependent systemic inflammation. Sepsis was induced by i.p. application of the TLR9 agonist 1668-thioate in C57BL/6 wild type (WT) and TLR9-deficient (TLR9-D) mice. Thirty minutes after stimulation TLR9 antagonists were applied i.v. Survival was monitored up to 18 h after stimulation. Cardiac mRNA expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point. Stimulation with 1668-thioate induced a severe sepsis-like state with significant drop of body temperature and significantly increased mortality in WT animals. Additionally, there was a time-dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure. These effects were not observed in TLR9-D mice. Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival. This suppressive ODN was the most efficient inhibitor of the tested substances.

Background: Bacteria such as Staphylococcus aureus induce myocardial dysfunction in vivo. To rectify conflicting evidence about the role of TLR2 signaling and cardiac dysfunction, we hypothesized that the specific TLR2 agonist purified lipoteichoic acid (LTA) from S. aureus contributes to cardiac dysfunction in vitro and in vivo. Methods: Wildtype (WT-) and TLR2-deficient (TLR2-D) mice were challenged with LTA and in comparison with equivalent doses of lipopolysaccharide (LPS) and CpG-oligodeoxynucleotide (CpG-ODN). TLR2-expression, NFκB as well as cytokine response were determined. Sarcomere shortening of isolated cardiomyocytes was analyzed in vitro and cardiac function in vivo after stimulation with LTA. Results: LTA induced up-regulation of TLR2 mRNA, activation of NFκB and cytokine expression within 2–6 h in WT-, but not in TLR2-D hearts. Cytokines were also elevated in the serum. LPS and CpG-ODN induced a more severe cardiac inflammation. In vitro incubation of cardiomyocytes with LTA reduced sarcomere shortening via NO at stimulation frequencies ≤ 8 Hz only in WT cells. However, hemodynamic parameters in vivo were not affected by LTA challenge. Conclusions: LTA induced cardiac inflammation was relatively weak and sarcomere shortening was reduced only below physiological heart rates. This may explain the apparent contradiction between the in vivo and in vitro LTA effects. Keywords: LTA, TLR2, Sepsis, Cardiac dysfunction, Cardiac contractility, JRC.I.5-Systems Toxicology

Background. Aim was to elucidate the role of toll-like receptor 9 (TLR9) in cardiac inflammation and septic heart failure in a murine model of polymicrobial sepsis.Methods. Sepsis was induced via colon ascendens stent peritonitis (CASP) in C57BL/6 wild-type (WT) and TLR9-deficient (TLR9-D) mice. Bacterial load in the peritoneal cavity and cardiac expression of inflammatory mediators were determined at 6, 12, 18, 24, and 36 h. Eighteen hours after CASP cardiac function was monitoredin vivo. Sarcomere length of isolated cardiomyocytes was measured at 0.5 to 10 Hz after incubation with heat-inactivated bacteria.Results. CASP led to continuous release of bacteria into the peritoneal cavity, an increase of cytokines, and differential regulation of receptors of innate immunity in the heart. Eighteen hours after CASP WT mice developed septic heart failure characterised by reduction of end-systolic pressure, stroke volume, cardiac output, and parameters of contractility. This coincided with reduced cardiomyocyte sarcomere shortening. TLR9 deficiency resulted in significant reduction of cardiac inflammation and a sustained heart function. This was consistent with reduced mortality in TLR9-D compared to WT mice.Conclusions. In polymicrobial sepsis TLR9 signalling is pivotal to cardiac inflammation and septic heart failure.

Left ventricular hypertrophy is an independent risk factor for morbidity and mortality in patients with hypertensive heart disease. Cardiac hypertrophy, associated with increased cardiac fibrosis and myocardial relaxation impairment, shows gender-based differences with significantly higher mortality in men. The role of estrogen in the pathogenesis of this process is poorly understood. After our previous demonstration that cardiac myocytes and fibroblasts contain functional estrogen receptors, we therefore investigated: 1) the influence of different estrogen metabolites on cardiac fibroblast growth; 2) the influence of different estrogen metabolites on the expression of the immediate early gene c-Fos; 3) the influence of estrogen on the L-type calcium channel in cardiomyocytes. Methods: 1) Neonatal rat cardiac fibroblasts were incubated with 17β-estradiol, estrone, 2-hydroxyestrone, and 2-methoxyestradiol (all 10−9 M). Bromodeoxyuridine incorporation was measured after 24 h. 2) c-Fos expression was demonstrated by immunoblotting. 3) L-type (Ca2+) current with and without 17β-estradiol was assessed in adult guinea pig cardiomyocytes by whole cell patch clamp. Results: Cardiac fibroblast growth was stimulated by estrogen metabolites with 2-hydroxyestrone as the most potent activator; in addition, 10−5 M 17β-estradiol reduced the L-type Ca2+ current by about 20% in cardiomyocytes. Conclusions: Estrogen induces both short term effects (non-genomic) and long term effects (genomic) on the heart and may therefore account for gender- and age-based differences in hypertensive heart disease.

Aims Inflammation and Toll-like receptor (TLR) signalling have been linked to the development of cardiac hypertrophy following transverse aortic constriction (TAC). In the present study, we investigated whether pre-treatment with the synthetic TLR9 ligands 1668-thioate or 1612-thioate modulates the progression of TAC-induced cardiac inflammation and hypertrophy. Methods and results C57BL/6N-mice were pre-treated with 1668-thioate, 1612-thioate (0.25 nmol/g, i.p.), or phosphate-buffered saline 16 h prior to TAC or sham surgery. Heart-weight/body-weight ratio (HW/BW), cardiomyocyte cell size, cellular macrophage accumulation, myofibroblast differentiation, and collagen deposition were investigated for up to 28 days. Cardiac function was monitored using a pressure–volume catheter and M-mode echocardiography. Inflammatory gene expression in the heart was analysed via gene array, while the time course of mRNA expression of key inflammatory mediators was assessed via RT-qPCR. TAC increased the HW/BW ratio and cardiomyocyte cell size and induced macrophage accumulation, myofibroblast differentiation, and collagen deposition. These changes were accompanied by cardiac inflammation and a significant loss of left ventricular function. Pre-treatment with cytosine-phosphate-guanine (CpG)-containing 1668-thioate attenuated the inflammatory response, the progression of cardiac hypertrophy, and cardiac remodelling, which resulted in a prolonged preservation of left ventricular function. These changes were induced to a smaller extent by the use of the non-CG-containing oligodeoxynucleotide 1612-thioate. Conclusion Pre-treatment with 1668-thioate attenuated cardiac hypertrophy following pressure overload, possibly by modifying the hypertrophy-induced inflammatory response, thereby reducing cardiac growth and fibrosis as well as delaying loss of cardiac function.

AIMS: Aim was to elucidate the specific role of pattern recognition receptors in vascular dysfunction during polymicrobial sepsis (colon ascendens stent peritonitis, CASP). METHODS AND RESULTS: Vascular contractility of C57BL/6 (wildtype) mice and mice deficient for Toll-like receptor 2/4/9 (TLR2-D, TLR4-D, TLR9-D) or CD14 (CD14-D) was measured 18 h following CASP. mRNA expression of pro- (Tumor Necrosis Factor-α (TNFα), Interleukin (IL)-1β, IL-6) and anti-inflammatory cytokines (IL-10) and of vascular inducible NO-Synthase (iNOS) was determined using RT-qPCR. Wildtype mice exhibited a significant loss of vascular contractility after CASP. This was aggravated in TLR2-D mice, blunted in TLR4-D animals and abolished in TLR9-D and CD14-D animals. TNF-α expression was significantly up-regulated after CASP in wildtype and TLR2-D animals, but not in mice deficient for TLR4, -9 or CD14. iNOS was significantly up-regulated in TLR2-D animals only. TLR2-D animals showed significantly higher levels of TLR4, -9 and CD14. Application of H154-ODN, a TLR9 antagonist, attenuated CASP-induced cytokine release and vascular dysfunction in wildtype mice. CONCLUSIONS: Within our model, CD14 and TLR9 play a decisive role for the development of vascular dysfunction and thus can be effectively antagonized using H154-ODN. TLR2-D animals are more prone to polymicrobial sepsis, presumably due to up-regulation of TLR4, 9 and CD14.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia. Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model. For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control. At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

The aetiology of sarcoidosis is unclear. Single nucleotide polymorphisms (SNPs) in transforming growth factor (TGF)-b2 and -b3 have been reported to be associated with the development of lung fibrosis in patients with sarcoidosis. SNPs in TGF-b2 (rs1891467) and TGF-b3 (rs3917200) were investigated in 296 patients with sarcoidosis (acute/self remitting, n570 (including 62 patients with Lofgren's syndrome); chronic, n5168; acute/chronic, n558) by real-time PCR. 32 patients showed radiological signs of lung fibrosis. The genotype frequencies were compared among the sarcoidosis groups as well as to 377 healthy controls. We found a significant association with the G-allele in rs1891467 in TGF-b2 and an acute/self remitting course of sarcoidosis compared to a chronic course (p50.001). The results were even more evident for patients with Lofgren's syndrome (p,0.001). Moreover, we could demonstrate a borderline significance between TGF-b3 (rs3917200) and lung fibrosis (p50.050). Carriers of the G-allele in rs1891467 might be protected from developing a chronic course. Moreover, there is evidence that rs3917200 is involved in the development of lung fibrosis in sarcoidosis. This study is the first in sarcoidosis patients to suggest a genetic implication of TGF- b2 as a protective factor in the course of sarcoidosis.

Backgound It has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT). Results Infarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R. Conclusion These results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.

Toll-like receptor 4 (TLR4) mediates innate immune responses following endotoxemia and myocardial ischaemia–reperfusion (I/R) injury. Pre-treatment with the major TLR4 ligand lipopolysaccharide (LPS) reduces infarct size. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play a crucial role in endotoxemia possibly also determining I/R injury. Aims: We investigated the influence of TLR4 on infarct size and assessed the influence of MMP and TIMP regulation on I/R injury. Methods: Left anterior descending artery (LAD) occlusion was performed on wild-type (C3H/HeN) and TLR4-deficient (C3H/HeJ) mice. Animals were stimulated with LPS (1mg/kg) or PBS 16h ahead of 60min LAD ligation. After 24h of reperfusion, triphenyltetrazolium chloride staining was performed and infarct size was measured by planimetry. MMP- and TIMP-mRNA expression were determined by RPA after 3h of reperfusion. MMP zymographic activity was monitored 6h after occlusion. Results: TLR4-deficient mice and LPS-treated wild-type mice showed significantly reduced infarct areas. LPS-stimulation significantly increased the overall MMP/TIMP mRNA expression ratio due to elevated MMP-3, −8, −9, and TIMP-1 in wild-type mice. I/R overall reduced the MMP/TIMP ratio due to increased MMP-1, TIMP-1, and −3 mRNA expression. Conclusions: LPS pre-treatment and TLR4-deficiency led to a decreased infarct size. However, infarct area and MMP/TIMP ratio were not correlated. This means that in TLR4-deficient mice MMP/TIMP ratios are not determining the infarct size.

Background— Major gender-based differences in the incidence of ventricular tachyarrhythmia after myocardial infarction have been shown in humans. Although the underlying mechanisms are unclear, earlier studies suggest that estrogen receptor–mediated effects play a major role in this process. Methods and Results— We examined the effect of estrogen receptor α (ERα) and estrogen receptor β (ERβ) on the electrophysiological phenotype in female mice with and without chronic anterior myocardial infarction. There was no significant difference in overall mortality, infarct size, and parameters of left ventricular remodeling when we compared infarcted ERα-deficient and ERβ-deficient mice with infarcted wild-type animals. In the 12-hour telemetric ECG recording 6 weeks after myocardial infarction, surface ECG parameters did not show significant differences in comparisons of ERα-deficient mice versus wild-type controls, infarcted versus noninfarcted ERα-deficient mice, and infarcted ERα-deficient versus infarcted wild-type mice. However, infarcted ERβ-deficient versus noninfarcted ERβ-deficient mice showed a significant prolongation of the QT (61±6 versus 48±8 ms; P P P P P P P P P I to ) in ERβ-deficient mice ( P Conclusions— Estrogen receptor β deficiency results in prolonged ventricular repolarization and decreased ventricular automaticity in female mice with chronic myocardial infarction.

Cardiovascular diseases are the leading cause of death in the industrialised countries and display significant gender-based differences. Estrogen plays an important role in the pathogenesis of heart disease and is able to modulate the progression of cardiovascular disease. The focus on the beneficial influence of estrogen is gradually shifting from the vascular system to the myocardium. The presence of functional estrogen receptors in the myocardium has been demonstrated. Estrogen is important for cardiovascular baseline physiology and modulates the myocardial response under pathological conditions. Here we summarise the current knowledge of the regulatory network of estrogenic action in the myocardium and its effects on cardiovascular function.

Cardiac tissue remodeling in the course of chronic left ventricular hypertrophy requires phagocytes which degrade cellular debris, initiate and maintain tissue inflammation and reorganization. The dynamics of phagocytes in left ventricular hypertrophy have not been systematically studied. Here, we characterized the temporal accumulation of leukocytes in the cardiac immune response by flow cytometry and fluorescence microscopy at day 3, 6 and 21 following transverse aortic constriction (TAC). Cardiac hypertrophy due to chronic pressure overload causes cardiac immune response and inflammation represented by an increase of immune cells at all three time points among which neutrophils reached their maximum at day 3 and macrophages at day 6. The cardiac macrophage population consisted of both Ly6C(low) and Ly6C(high) macrophages. Ly6C(low) macrophages were more abundant peaking at day 6 in response to pressure overload. During the development of cardiac hypertrophy the expression pattern of adhesion molecules was investigated by qRT-PCR and flow cytometry. CD11b, CX3CR1 and ICAM-1 determined by qRT-PCR in whole cardiac tissue were up-regulated in response to pressure overload at day 3 and 6. CD11b and CX3CR1 were significantly increased by TAC on the surface of Ly6C(low) but not on Ly6C(high) macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6C(low) macrophages could be observed attached to the intra- and extra-vascular vessel-wall. Taken together, TAC induced the expression of adhesion molecules, which may explain the accumulation of Ly6C(low) macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.

A large array of cardiac diseases such as hypertensive heart disease and cardiac remodeling after myocardial infarction display significant gender-based differences (1, 2, 3). In this context it has been shown that during the process of aging cardiac number and diameter vary significantly between men and women. While cardiac myocytes of male patients tend to develop hypertrophy and polyploidy, cardiac myocytes of female patients remain consistent over time in terms of size and number of nuclei (4). The underlying mechanisms of this process remain to be elucidated. However, it is remarkable that the incidence of cardiac disease in female gender reveals a significant increase after the onset of menopause (5). Therefore it has been hypothesized that the decline of ovarian sex hormones after the onset of menopause, in particular estrogens, play an important role in the pathogenesis of cardiac disease in women. The role of estrogen in the pathogenesis of this process is currently under investigation (6–11). The influence of estrogen on the development of cardiac diseases can be divided in systemic and direct effects on the cardiovascular system. Systemic effects include the influence of sex hormones on lipid and insulin metabolism (12, 13). Furthermore, it has become evident that estrogens display a variety of genomic and non-genomic effects on cardiovascular tissues that may modulate the respective phenotype of these tissues. Two different estrogen receptors have been identified so far, estrogen receptor α and estrogen receptor β. These two subtypes differ in ligand binding as well as in DNA binding properties (14-18).

The inflammatory response to pathogen-associated molecular patterns such as lipopolysaccharide (LPS) in sepsis is mediated via Toll-like receptors (TLRs). Since TLRs also trigger various immune functions, including phagocytosis, their modulation is a promising strategy in the treatment of sepsis. As antibiotics have immunomodulatory properties, this study examined the effect of commonly used classes of antibiotics on i) the expression of TLRs and cytokines and ii) the phagocytic activity under sepsis-like conditions in vitro. This was achieved by incubating THP-1 monocytes and peripheral blood mononuclear cells (PBMCs) obtained from patients after open-heart surgery with the addition of LPS and six key antibiotics (piperacillin, doxycycline, erythromycin, moxifloxacin or gentamicin). After 24h, mRNA levels of both cytokines (IL-1β, IL-6) and TLRs (1, 2, 4, and 6) were monitored and phagocytosis was determined following coincubation with Escherichia coli. Each antibiotic differentially regulated the gene expression of the investigated TLRs and cytokines in monocytes. Erythromycin, moxifloxacin and doxycyclin displayed the strongest effects and changed mRNA-levels of the investigated genes up to 5.6-fold. Consistent with this, antibiotics and, in particular, moxifloxacin, regulated the TLR-and cytokine expression in activated PBMCs obtained from patients after open-heart surgery. Furthermore, piperacillin, doxycyclin and moxifloxacin inhibited the phagocytic activity of monocytes. Our results suggest that antibiotics regulate the immune response by modulating TLR- and cytokine expression as well as phagocytosis under septic conditions. Moxifloxacin, doxycycline and erythromycin were shown to possess the strongest immunomodulatory effects and these antibiotic classes should be considered for future immunomodulatory studies in sepsis.