Jan Mersmann

High-Density Lipoproteins and Their Constituent, Sphingosine-1-Phosphate, Directly Protect the Heart Against Ischemia/Reperfusion Injury In Vivo via the S1P3 Lysophospholipid Receptor

Background— All treatments of acute myocardial infarction are aimed at rapid revascularization of the occluded vessel; however, no clinical strategies are currently available to protect the heart from ischemia/reperfusion injury after restitution of blood flow. We hypothesized that some of the cholesterol transport–independent biological properties of high-density lipoprotein (HDL) implied in atheroprotection may also be beneficial in settings of acute myocardial reperfusion injury. Methods and Results— In an in vivo mouse model of myocardial ischemia/reperfusion, we observed that HDL and its sphingolipid component, sphingosine-1-phosphate (S1P), dramatically attenuated infarction size by ≈20% and 40%, respectively. The underlying mechanism was an inhibition of inflammatory neutrophil recruitment and cardiomyocyte apoptosis in the infarcted area. In vitro, HDL and S1P potently suppressed leukocyte adhesion to activated endothelium under flow and protected rat neonatal cardiomyocytes against apoptosis. In vivo, HDL- and S1P-mediated cardioprotection was dependent on nitric oxide (NO) and the S1P3 lysophospholipid receptor, because it was abolished by pharmacological NO synthase inhibition and was completely absent in S1P3-deficient mice. Conclusions— Our data demonstrate that HDL and its constituent, S1P, acutely protect the heart against ischemia/reperfusion injury in vivo via an S1P3-mediated and NO-dependent pathway. A rapid therapeutic elevation of S1P-containing HDL plasma levels may be beneficial in patients at high risk of acute myocardial ischemia.

Biglycan Is Required for Adaptive Remodeling After Myocardial Infarction

Background— After myocardial infarction (MI), extensive remodeling of extracellular matrix contributes to scar formation and preservation of hemodynamic function. On the other hand, adverse and excessive extracellular matrix remodeling leads to fibrosis and impaired function. The present study investigates the role of the small leucine-rich proteoglycan biglycan during cardiac extracellular matrix remodeling and cardiac hemodynamics after MI. Methods and Results— Experimental MI was induced in wild-type (WT) and bgn−/0 mice by permanent ligation of the left anterior descending coronary artery. Biglycan expression was strongly increased at 3, 7, and 14 days after MI in WT mice. bgn−/0 mice showed increased mortality rates after MI as a result of frequent left ventricular (LV) ruptures. Furthermore, tensile strength of the LV derived from bgn−/0 mice 21 days after MI was reduced as measured ex vivo. Collagen matrix organization was severely impaired in bgn−/0 mice, as shown by birefringence analysis of Sirius red staining and electron microscopy of collagen fibrils. At 21 days after MI, LV hemodynamic parameters were assessed by pressure-volume measurements in vivo to obtain LV end-diastolic pressure, end-diastolic volume, and end-systolic volume. bgn−/0 mice were characterized by aggravated LV dilation evidenced by increased LV end-diastolic volume (bgn−/0, 111±4.2 &mgr;L versus WT, 96±4.4 &mgr;L; P<0.05) and LV end-diastolic pressure (bgn−/0, 24±2.7 versus WT, 18±1.8 mm Hg; P<0.05) and severely impaired LV function (EF, bgn−/0, 12±2% versus WT, 21±4%; P<0.05) 21 days after MI. Conclusion— Biglycan is required for stable collagen matrix formation of infarct scars and for preservation of cardiac hemodynamic function.

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo

BackgroundBacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo.MethodsWild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured.ResultsIn WT mice, CpG-ODN induced a strong activation of pulmonary NFκB as well as a significant increase in pulmonary TNF-α and IL-1β mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice.ConclusionThis study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.

The fibrin-derived peptide Bβ15- 42 is cardioprotective in a pig model of myocardial ischemia-reperfusion injury

Objective:The fibrin-derived peptide B&bgr;15–42 has been shown to reduce infarct size in rodent models of ischemia-reperfusion injury. To increase its potential for translation into the clinic, we studied the effects of B&bgr;15–42 in pigs, whose coronary anatomy is similar to that of humans. In addition, we evaluated the pharmacokinetics and safety of B&bgr;15–42 in several species, including humans. Design:Animal study and phase I trial. Setting:University hospital and contract research laboratories. Subjects:Pigs/healthy volunteers. Interventions:Male farm-bred Landrace pigs were subjected to 1 hr of left anterior descending coronary artery occlusion followed by 3 hrs of reperfusion. At the time of reperfusion, B&bgr;15–42 (2.4 mg/kg, n = 6) or random peptide (control; 2.4 mg/kg, n = 6) was administered as an intravenous bolus. As a positive control, pigs were subjected to ischemic preconditioning (n = 6). Cardiac damage and hemodynamics were recorded. Biodistribution and pharmacokinetics of B&bgr;15–42 were determined in rats and dogs. In a phase I trial involving 30 male healthy volunteers, pharmacokinetics and safety were tested in a randomized, double-blinded, placebo-controlled, parallel-group, single ascending dose study. Measurements and Main Results:B&bgr;15–42 and ischemic preconditioning significantly reduced myocardial infarct size and troponin I levels. B&bgr;15–42 also reduces interleukin-6 levels, underlining its anti-inflammatory properties. Furthermore, in humans, the pharmacokinetics of the peptide B&bgr;15–42 were comparable to those of animals, and no serious adverse effects were observed. Conclusions:B&bgr;15–42 elicits cardioprotection in pigs and is clinically safe in phase I testing of humans. This study confirms the new concept of a pathogenic role of fibrin derivatives in myocardial reperfusion injury, which can be inhibited by peptide B&bgr;15–42.

Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury

Objective:To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. Design:C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01–1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. Setting:University hospital research laboratory. Subjects:Anesthetized male mice and murine cardiomyocytes. Measurements and Main Results:Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. Conclusions:Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.

Priming with Synthetic Oligonucleotides Attenuates Pressure Overload-Induced Inflammation and Cardiac Hypertrophy in Mice

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.

Rosiglitazone is cardioprotective in a murine model of myocardial I/R.

The peroxisome proliferator-activated receptor &ggr; (PPAR-&ggr;) is a regulator of anti-inflammatory genes. One of its agonists, rosiglitazone-widely used in the treatment of type 2 diabetes mellitus-has recently been reported to increase the risk for myocardial infarction. In contrast, various studies provide evidence for a rosiglitazone-induced cardioprotection in different models of acute myocardial I/R. Here, we report that this protection can still be observed after 28 days of reperfusion in a murine model even when treatment commenced after the period of ischemia (reperfusion therapy). In vitro, cells from the rat cardiomyoblast cell line H9c2(2-1) are protected against oxidative stress by incubation with rosiglitazone, which can be abrogated by dexamethasone or cycloheximide. The antioxidant enzyme heme oxygenase 1 is up-regulated in these cells after rosiglitazone treatment. Our data provide further evidence that rosiglitazone exerts protective effects during myocardial I/R and might contribute to the reevaluation of the approved drug rosiglitazone.

In vivo electrophysiological characterization of TASK-1 deficient mice

Background/Aims: TASK-1 is a potassium channel predominantly expressed in heart and brain. We have previously shown that anesthetized TASK-1-/-mice have prolonged QT intervals in surface electrocardiograms (ECGs). In addition, heart rate variability quantified by time and frequency domain parameters was significantly altered in TASK-1-/-mice with a sympathetic preponderance. Aims of the present study were the analysis of QT intervals by telemetric ECGs, to determine potential influences of anesthesia and β-adrenergic stimulation on repolarization in surface ECGs, to investigate in vivo electrophysiological parameters by intracardiac electrical stimulation and to quantify heart rate turbulence after ischemia/reperfusion or ventricular pacing in TASK-1+/+ and TASK-1-/-mice. Methods: Rate corrected QT intervals (QTc) were recorded in conscious mice by telemetry and in surface ECGs following administration of various anesthetics (tribromoethanol (Avertin®), pentobarbital and isoflurane). TASK-1+/+ and TASK-1-/mice were characterized by programmed electrical stimulation using an intracardiac octapolar catheter. The baroreceptor reflex was analyzed by heart rate turbulence (turbulence onset and slope) after ischemia/reperfusion and by stimulated premature ventricular contractions. Results: Telemetric and surface ECGs in mice sedated with Avertin®and pentobarbital, showed a significantly lengthened rate corrected QT interval in TASK-1-/-mice (telemetry: TASK-1+/+ 43±3ms vs. TASK-1-/-49±5ms, n=6, p<0.05; Avertin®: TASK-1+/+ 36±8ms vs. TASK-1-/-48±4ms, n=13/16, p<0.0001). The prolongation of the QT interval was most pronounced at lower heart rates. Isoflurane, known for its stimulatory effects on the TASK channel family, attenuated the rate corrected QT interval prolongation in TASK-1-/-mice. Intracardiac electrical stimulation revealed normal values for electrical conduction and refractoriness. No significant arrhythmias after atrial and ventricular burst stimulation were induced before and after adrenergic challenge in both genotypes. Turbulence onset after premature ventricular contraction was significantly altered in TASK-1-/-mice. Conclusion: TASK-1-/-mice exhibit a phenotype of QT prolongation, which distinct relation to heart rate. TASK-1 deficiency does neither alter key electrophysiological parameters nor increases atrial/ventricular vulnerability after electrical stimulation. The heart rate response after premature ventricular contractions is significantly abolished indicating a diminished baroreceptor reflex in TASK-1-/-mice.

Toll-like receptor 2 signaling triggers fatal arrhythmias upon myocardial ischemia-reperfusion

Objective:Restoration of myocardial blood flow after ischemia triggers an inflammatory response involving toll-like receptors. Toll-like receptor 2 deficiency is associated with a reduced infarct size after myocardial ischemia and reperfusion. Because a marked mortality was observed in C3HeN wild-type mice, which was absent in TLR2−/− mice, we tested whether cardiac arrhythmias are the underlying pathology and aimed to elucidate how toll-like receptor 2 ligation might prevent lethal arrhythmias. Design:Experimental animal model. Setting:University hospital research laboratory. Subjects:Male C3HeN mice. Interventions:Myocardial ischemia and reperfusion was surgically induced by ligation of the left anterior descending coronary artery for 20 mins followed by 24 hrs of reperfusion. Electrocardiography was continuously recorded during the observation period through an implantable telemetry transmitter to detect cardiac arrhythmias during reperfusion. Measurements and Main Results:Toll-like receptor 2 expression was associated with a 51% mortality rate (23 of 45 mice died) after myocardial ischemia and reperfusion. Absence of toll-like receptor 2 improved survival toward 100% (17 of 17 mice survived). Electrocardiography diagnostics in conscious animals and histologic analysis revealed that absence of toll-like receptor 2 signaling prevented the formation of pathologic heart rate turbulence after myocardial ischemia and reperfusion and modulated the density of connexin 43-positive gap junctions in the ischemic area compared with wild-type hearts, indicating arrhythmia as the cause underlying the observed mortality. Conclusions:The results presented here indicate toll-like receptor 2 as a novel target for the prevention of lethal arrhythmic complications after myocardial ischemia and reperfusion.

Therapeutic injection of parp inhibitor ino-1001 preserves cardiac function in porcine myocardial ischemia and reperfusion without reducing infarct size

Pharmacological protection from myocardial reperfusion injury, despite plenty of approaches, has still not been realized in humans. We studied the putative infarct size (IS)-sparing capacity of poly(ADP-ribose)polymerase inhibitor, INO-1001, and focused on cardiac functional recovery during reperfusion. Male farm-bred Landrace pigs were subjected to 1-h left anterior descending coronary artery occlusion followed by 3 h of reperfusion (control). Infarct size was determined by triphenyltetrazolium chloride/Evans blue staining. Plasma markers of myocardial injury (troponin T, creatine kinase, lactate dehydrogenase) were determined upon protocol completion. Cardiac function was continuously assessed via pulmonary and femoral artery catheters. INO-1001 (1 mg/kg) was administered upon reperfusion in the treatment group. As a positive control, untreated pigs were subjected to ischemic preconditioning (10-min left anterior descending coronary artery occlusion followed by 15-min reperfusion before the intervention). Ischemic preconditioning reduced myocardial damage reflected by a smaller IS and lower plasma markers of myocardial injury. INO-1001 did not reduce IS but significantly improved functional recovery (increased stroke volume, cardiac index, and mixed venous oxygen saturation) during reperfusion compared with vehicle-treated control and ischemic preconditioning. Although we could not confirm the IS-sparing capacities of poly(ADP-ribose)polymerase inhibitor, INO-1001, the drug holds the potential of hemodynamic improvement during reperfusion.