Lung protection in cardio-pulmonary bypass.

Since the invention of the heart-lung machine paediatric cardiac surgery developed rapidly. For correction of complex cardiac malformations the application of a cardio-pulmonary bypass (CPB) has become indispensable but possible negative effects of this technique should not be neglected. Especially, both bypassed organs i.e. heart and lung are not perfused during the procedure and therefore are threatened by ischemia and reperfusion injury. Additionally, CPB was developed with a non-pulsatile flow but there are clinical observations that pulsatile flow might be superior with improved patient outcomes. Thus, the aim of our study was to evaluate the effect of CPB on lung structure and to assess whether different flow modalities (pulsatile vs. non-pulsatile flow) or application of the antibiotic minocycline might be advantageous. Thirty five piglets of four weeks age were examined and divided into five experimental groups: control (no CPB) without or with minocycline, CPB (non-pulsatile flow) without or with minocycline and CPB with pulsatile flow. CPB was performed for 90 min followed by a 120 min reperfusion and recovery phase. Thereafter, adenosine triphosphate-content of lung biopsies and histology was carried out. We found that CPB was associated with a significant thickening of alveolar wall accompanied by an infiltration of neutrophil leucocytes. Moreover, markers for hypoxia, apoptosis, nitrosative stress, inflammation and DNA damage were significantly elevated after CPB. These cellular damages could be partially inhibited by minocycline or pulsatile flow. Both, minocycline and pulsatile flow attenuate lung damage after CPB.

Immunological monitoring of extracorporeal photopheresis after heart transplantation.

Extracorporeal photopheresis (ECP) has been used as a prophylactic and therapeutic option to avoid and treat rejection after heart transplantation (HTx). Tolerance-inducing effects of ECP such as up-regulation of regulatory T cells (T(regs)) are known, but specific effects of ECP on regulatory T cell (T(reg)) subsets and dendritic cells (DCs) are lacking. We analysed different subsets of T(regs) and DCs as well as the immune balance status during ECP treatment after HTx. Blood samples were collected from HTx patients treated with ECP for prophylaxis (n = 9) or from patients with histologically proven acute cellular rejection (ACR) of grade ≥ 1B (n = 9), as well as from control HTx patients without ECP (HTxC; n = 7). Subsets of T(regs) and DCs as well as different cytokine levels were analysed. Almost 80% of the HTx patients showed an effect to ECP treatment with an increase of T(regs) and plasmacytoid DCs (pDCs). The percentage of pDCs before ECP treatment was significantly higher in patients with no ECP effect (26·3% ± 5·6%) compared to patients who showed an effect to ECP (9·8% ± 10·2%; P = 0·011). Analysis of functional subsets of CD4⁺CD25(high)CD127(low) T(regs) showed that CD62L-, CD120b- and CD147-positive T(regs) did not differ between the groups. CD39-positive T(regs) increased during ECP treatment compared to HTxC. ECP-treated patients showed higher levels for T helper type 1 (Th1), Th2 and Th17 cytokines. Cytokine levels were higher in HTx patients with rejection before ECP treatment compared to patients with prophylactic ECP treatment. We recommend a monitoring strategy that includes the quantification and analysis of T(regs), pDCs and the immune balance status before and up to 12 months after starting ECP.

Cardiac fibroblasts inhibit ß-adrenoceptor-dependent connexin43 expression in neonatal rat cardiomyocytes.

Cardiac fibroblasts play an important role in adverse cardiac remodelling. As in many cardiac diseases connexin43 (Cx43) is altered, we wanted to elucidate whether fibroblasts may influence cardiac Cx43 expression. We used four different cell culture systems of neonatal rat cardiomyocytes (CM) and fibroblasts (FB): type 1, pure CM culture; type 2, co-culture of CM/FB; type 3, pure FB culture; type 4, Transwell® system: CM/FB co-cultured but separated by a microporous membrane. Stimulation of types 1-3 cell culture models with isoprenaline significantly enhanced Cx43-protein and Cx43-mRNA expression as well as phosphorylation of ERK and translocation of AP1 and CREB only in the CM cultures; whereas, the CM/FB co-cultures and the FB cultures did not respond to isoprenaline. Similarly, if CM and FB were separated by a microporous membrane (Transwell® system) the isoprenaline-induced increase in CM Cx43 was completely suppressed, suggesting the existence of a soluble factor responsible for the suppressant effect of FB. Angiotensin II determination in types 1 and 2 cell culture supernatants revealed that the CM/FB co-cultures exhibited a significant higher angiotensin II release than the CM cultures. Furthermore, we aimed to inhibit angiotensin II signal transduction pathway: blockade of AT1 receptors or PKC inhibition restored the responsiveness of CM/FB co-cultures to isoprenaline. Moreover, external addition of angiotensin II to CM cultures also resulted in suppression of isoprenaline-stimulated Cx43 expression in an AT1-receptor- and PKC-dependent manner. Thus, our study indicates that cardiac fibroblasts inhibit ß-adrenoceptor-dependent Cx43 signalling in CM involving angiotensin II.

Cardioprotective effects of low-dose cyclosporin A added to histidine-tryptophan-ketoglutarate cardioplegia solution prior to total myocardial ischemia: an in vitro rabbit heart study.

BACKGROUND/AIMS: Mitochondrial permeability transition pore (MPTP) opening appears to play a key role in myocardial cell survival after ischemia-reperfusion injury and can be inhibited by cyclosporin A (CsA). We investigated whether low-dose CsA added to histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution could improve myocardial protection during longer periods of global myocardial ischemia as encountered during cardiac surgery. METHODS: Rabbit hearts perfused on a Langendorff apparatus were arrested with cold HTK solution containing 1 µmol/l CsA. After 90 min of ischemia, the hearts were reperfused and pmax, max dp/dt, min dp/dt, myocardial stiffness, pO(2), coronary flow and heart rate recorded. Tissue ATP and malondialdehyde (MDA) were measured to assess cell energy content and oxidative stress, respectively. RESULTS: CsA-treated hearts recovered pmax (p = 0.026), max dp/dt (p = 0.028) and min dp/dt (p = 0.025) more quickly and to a greater extent than non-treated hearts. They required markedly less oxygen (p = 0.041) in the first 10 min of reperfusion. Hearts treated with CsA produced 44% less MDA (1.09 vs. 1.93, p = 0.008), while ATP levels were unchanged. CONCLUSIONS: HTK cardioplegia solution containing CsA at a dose well below that expected to cause systemic immunosuppressive effects leads to a significant and timelier recovery of myocardial contractility, while consuming less oxygen.

Effects of metoprolol therapy on cardiac gap junction remodelling and conduction in human chronic atrial fibrillation.

BACKGROUND AND PURPOSE: We investigated the influence of metoprolol on gap junction proteins connexin43 (Cx43) and connexin40 (Cx40) in atrial tissue from patients with/without atrial fibrillation (AF). EXPERIMENTAL APPROACH: Left atrial tissue samples from 160 patients with AF or sinus rhythm (SR) with or without metoprolol (mean daily dose: 65.2 ± 9.1 mg·day⁻¹) were analysed for Cx43 and Cx40 by Western blot and immunohistology. Transverse and longitudinal conduction velocities were determined by 64 multi-electrode mapping. KEY RESULTS: Both Cx43 and Cx40 expression were significantly increased in patients with AF versus SR. Cx43-expression in AF was significantly higher in patients receiving metoprolol, while Cx40 expression was unaffected by metoprolol treatment. In AF, the ratio of lateral/polar expression of Cx43 and Cx40 was enhanced due to increased expression at the sides of the cells (lateral) and a loss at the cell poles. This AF-induced increase in lateral/polar expression of Cx43, but not of Cx40, was significantly antagonized by metoprolol treatment. Functionally, in AF patients, transverse conduction velocity in atrial samples was significantly enhanced and this change was also significantly antagonized by metoprolol. CONCLUSIONS AND IMPLICATIONS: AF induced enhanced lateral expression of Cx43 and Cx40 together with enhanced transverse conduction velocity in left atrial tissue. Alterations in localization of Cx43 and conduction changes were both antagonized by metoprolol, showing that pharmacological modulation of gap junction remodelling seems, in principle, possible. This finding may open new approaches to the development of anti-arrythmic drugs.

Activation of cardiomyocytes depending on their proximity to human bone marrow stem cells.

Our study aimed to elucidate whether bone marrow stem cell (BMC) treatment might result in a cellular response in cardiomyocytes IN VITRO. Subconfluent neonatal rat cardiomyocyte cultures were cocultured for three days with Vybrant CM-DiI labeled BMC from human sternal bone marrow and underwent immunohistological staining for the proto-oncogene c-Myc and the cell cycle proteins CDK2, CDK4 and ATF-3. ß-adrenoceptor density was analyzed using [125I]-iodocyanopindolol (ICYP) histoautoradiography. Quantitative analysis of immunohistochemical images revealed significantly increased expression and upregulation of c-Myc, and its downstream targets ATF-3, CDK2 and CDK4 in neighboring cardiomyocytes to BMC, depending on their distance to the BMC compared to cardiomyocytes far from the BMC. Histoautoradiography revealed a significantly higher ß-adrenoceptor density in cardiomyocytes in the immediate vicinity to the BMC. With increasing distance to the BMC, ß-adrenoceptor density in cardiomyocytes declined. Thus, a small number of BMC can affect a larger number of cardiomyocytes by activating an intracellular signaling cascade and enhancing ß-adrenoceptor density.

The aging heart: changes in the pharmacodynamic electrophysiological response to verapamil in aged rabbit hearts.

The aim of this study was to investigate whether the L-type calcium current (I(Ca.L)) may be altered in aged hearts and whether the classical calcium antagonist verapamil may exhibit altered pharmacological profile in aged hearts. We examined male New Zealand rabbits aged either 6 months or 26 months. To examine I(Ca.L) whole-cell patch-clamp technique was performed on isolated cells. Moreover, activation-recovery intervals (ARI) of isolated hearts (Langendorff method) were assessed using an epicardial 256 channel mapping system. We found that the I(Ca.L) density, normalised to the cell volume was significantly reduced (p<0.001). Maximum conductance was also significantly decreased (p=0.01) and steady state inactivation was shifted to more positive potentials in aged hearts (p<0.001). A slightly reduced effect of beta-adrenergic modulation of the I(Ca.L) in aged hearts, and a significantly reduced effect of carbachol on isoprenaline-stimulated I(Ca.L) in aged hearts was observed. L-type alpha 1c subunit, SERCA2-ATPase and the Na(+)/Ca(2+)-exchanger expression were neither significantly different in atrial and ventricular tissues nor between young and old animals. Using the mapping system, isolated hearts were exposed to verapamil (0.005, 0.01, 0.02, 0.05 microM/L). While verapamil did not affect ARI in young hearts, in aged hearts ARI was concentration-dependently reduced and the negative inotropic effect of verapamil was significantly attenuated in aged hearts (p<0.05). From these results we conclude that there are distinct alterations in the electrophysiology of I(Ca.L) (reduced maximum conductance, a shift of the steady state inactivation) in the aged heart which may influence the response to verapamil.

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by beta-adrenoceptors.

BACKGROUND AND PURPOSE: In mammalian heart, connexin43 (Cx43) represents the predominant connexin in the working myocardium. As the beta-adrenoceptor is involved in many cardiac diseases, we wanted to clarify the pathway by which beta-adrenoceptor stimulation may control Cx43 expression. EXPERIMENTAL APPROACH: Cultured neonatal rat cardiomyocytes were stimulated with isoprenaline. Cx43 expression as well as activation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, JUN NH(2)-terminal kinase (JNK) and nuclear translocation of the transcription factors activator protein 1 (AP1) and CRE-binding protein (CREB) were investigated. Additionally, we assessed Cx43 expression and distribution in left ventricular biopsies from patients without any significant heart disease, and from patients with either congestive heart failure [dilated cardiomyopathy (DCM)] or hypertrophic cardiomyopathy (HCM). KEY RESULTS: Isoprenaline exposure caused about twofold up-regulation of Cx43 protein with a pEC(50) of 7.92 +/- 0.11, which was inhibited by propranolol, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole) (p38 inhibitor), PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (MAPK 1 kinase inhibitor) (Alexis Biochemicals, San Diego, CA, USA) or cyclosporin A. Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44 and JNK, and by translocation of AP1 and CREB to the nucleus. Analysis of Cx43 protein and mRNA in ventricular biopsies revealed that in patients with DCM, Cx43 content was significantly lower, and in patients with HCM, Cx43 content was significantly higher, relative to patients without any cardiomyopathy. More importantly, Cx43 distribution also changed with more Cx43 being localized at the lateral border of the cardiomyocytes. CONCLUSION AND IMPLICATION: Beta-adrenoceptor stimulation up-regulated cardiac Cx43 expression via a protein kinase A and MAPK-regulated pathway, possibly involving AP1 and CREB. Cardiomyopathy altered Cx43 expression and distribution.

Postoperative development of aspirin resistance following coronary artery bypass.

BACKGROUND: Aspirin therapy is known to substantially reduce mortality and the rate of ischaemic complications after coronary artery bypass grafting (CABG). Rates of perioperative aspirin resistance cited in the literature are up to 50% and could be influenced by extracorporeal circulation. Thus, aspirin resistance after CABG may have a significant clinical relevance. MATERIALS AND METHODS: In 59 patients undergoing CABG (on-pump, off-pump and combined procedures) aspirin resistance was investigated by arachidonic acid induced platelet aggregometry. Clinical relevance was assessed with 12-month follow up. RESULTS: Two types of resistance were observed: A preoperative resistance (despite oral aspirin or in vitro addition) was present in 29% and a postoperative developing type was seen in 49% resulting in only 22% of patients with a 'normal' reaction to aspirin. If patients were already on oral aspirin at admission, the rate of resistance was significantly reduced. Off-pump surgery or pump-times exceeding 120 min had no significant impact on resistance. During the 12-month follow up (98.3%), there were three deaths (one stroke, one intestinal ischaemia, one mediastinitis after postoperative delirium) in patients with the perioperative resistance and none in other patients (P = 0.345). In none of those patients who presented with perioperative aspirin resistance, could this aspirin resistance be demonstrated when tested again after 12 months? CONCLUSIONS: Aspirin resistance is a transient phenomenon present in the majority of patients undergoing CABG. The three deaths in the resistant group may - although not statistically significant - indicate the possibility of a worse outcome for patients with aspirin resistance.

Safety of the novel atrial-selective K+-channel blocker AVE0118 in experimental heart failure.

Congestive heart failure (CHF) is often associated with atrial fibrillation. The safety of many antiarrhythmic drugs in CHF is limited by proarrhythmic effects. We aimed to assess the safety of a novel atrial-selective K(+)-channel blocker AVE0118 in CHF compared to a selective (dofetilide) and a non-selective IKr blocker (terfenadine). For the induction of CHF, rabbits (n = 12) underwent rapid right ventricular pacing (330-380 bpm for 30 days). AVE0118 (1 mg/kg) dofetilide (0.02 mg/kg) and terfenadine (2 mg/kg) were administered in baseline (BL) and CHF. A six-lead ECG was continuously recorded digitally for 30 min after each drug administration. At BL, dofetilide and terfenadine significantly prolonged QTc interval (218 +/- 30 ms vs 155 +/- 8 ms, p = 0.001 and 178 +/- 23 ms vs. 153 +/- 12 ms, p = 0.01, respectively) while QTc intervals were constant after administration of AVE0118 (p = n.s.). In CHF, dofetilide and terfenadine caused torsades de pointes and symptomatic bradycardia, respectively, and prolonged QTc interval (178 +/- 30 ms vs. 153 +/- 14 ms, p = 0.02 and 157 +/- 7 ms vs. 147 +/- 10 ms, p = 0.02, respectively) even at reduced dosages, whereas no QTc-prolongation or arrhythmia was observed after full-dose administration of AVE0118. In conclusion, atrial-selective K(+)-channel blockade by AVE0118 appears safe in experimental CHF.

Ischemic and inflammatory lung impairment by extracorporeal circulation: effect of PARP-inhibition by INO1001.

OBJECTIVE: Among the complications after cardiac surgery the development of postoperative pulmonary distress is a serious problem. Typically, the patients leave the operating theatre with good blood gas values and O(2)-saturation, but develop their respiratory problems within the next hours/days. We investigated whether extracorporeal circulation may induce biochemical and histological changes in the lungs which may help to explain this development. METHODS: Piglets (6-10 kg) were anaesthetized using isoflurane and underwent extracorporeal circulation (ECC) with hypothermic (25-28 degrees C) cardioplegic arrest for 90 min followed by 3h reperfusion. An additional group received a poly(ADP-ribose) polymerase (PARP)-Inhibitor, INO1001. Cardiopulmonary monitoring was performed during the whole procedure. Finally, lungs were explanted and investigated by histomorphometry and immunohistology for heat shock protein HSP70 (indicator for cellular damage) and TNFalpha in comparison to normal piglets without ECC. RESULTS: Histologically we found significant swelling of the type I alveocytes (thickness increased from 2.4 to 3.2 microm), interstitial oedema, intra-alveolar erythrocyte (4.8 versus 0.4 erythrocytes/alveole) and granulocyte accumulation and fibrinous exudates. There was a significant up-regulation of TNFalpha and of the cellular repair enzyme HSP70, while in control piglets only minimal levels were observed. INO1001 significantly reduced ECC-induced elevation in TNFalpha and in HSP70. Despite the dramatic changes after heart-lung-machine (HLM), blood gases and gas transport were almost not affected at that time. CONCLUSIONS: ECC can lead to early significant histological and histochemical changes which have similarities with a beginning early stage shock lung, although - at 3h reperfusion - gas transport is still sufficient. INO1001 can partially antagonize these changes.

Olea europaea leaf extract exerts L-type Ca(2+) channel antagonistic effects.

ETHNOPHARMACOLOGICAL RELEVANCE: In Southern Europe Olea europaea leafs are known as a folk remedy for hypertension. Cardiovascular diseases are still the leading causes of morbidity and mortality in industrialized countries with hypertension being one of the main risk factors. AIM OF THE STUDY: We investigated effects of a commercial Olea europaea leaf extract (OLE) on isolated hearts and cultured cardiomyocytes. MATERIALS AND METHODS: Isolated rabbit hearts were perfused according to the Langendorff technique and connected to a 256-channel epicardial mapping system. Voltage clamp experiments were performed in cultured neonatal rat cardiomyocytes using a perforated-patch technique. RESULTS: OLE caused a concentration-depended decrease in systolic left ventricular pressure and heart rate as well as an increase in relative coronary flow and a slight, but not significant prolongation of PQ-time. There were no significant changes between the groups in the activation-recovery interval and its dispersion, total activation time, peak-to-peak amplitude, percentage of identical breakthrough-points and similar vectors of local activation. Voltage clamp experiments in cultured neonatal rat cardiomyocytes showed a significant decrease in maximum I(Ca,L) by OLE which was reversible upon wash-out. CONCLUSIONS: OLE suppresses the L-type calcium channel directly and reversibly. Our findings might help to understand the traditional use of OLE in the treatment of cardiovascular disease.

A new role for extracellular Ca2+ in gap-junction remodeling: studies in humans and rats.

We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca(++) for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10-100 nM), cyclosporin A (1 microM),and BMS605401 (100 nM), a specific inhibitor of Ca(2+)-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100-200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was >or=2.2 mM, while Cx43 was independent from extracellular [Ca(++)]. In cultured cells, 4 mM Ca(++)-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca(2+)-induced changes. Moreover, cyclosporin A completely abolished the Ca(2+)-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.

Effects of autologous stem cells on immunohistochemical patterns and gene expression of metalloproteinases and their tissue inhibitors in doxorubicin cardiomyopathy in a rabbit model.

This study aims to investigate the expression of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in chronic doxorubicin cardiomyopathy in a rabbit model and to evaluate the effects of bone marrow-derived mesenchymal stem cell (MSC) transplantation in this disease. Thirty-nine 3-month-old New Zealand rabbits were divided into 4 groups: group 1 (n = 9) was the untreated control. Groups 2-4 were treated with 6 weeks of doxorubicin (3 mg/kg). Group 2 (n = 6) received no further treatment. In group 3 (n = 9), animals were treated with culture medium (CM) alone. In group 4 (n = 15), autologous MSCs (1.5-2.0 x 10(6)/ml) were injected in the left ventricular (LV) wall. Hearts were stained with HE and picrosirius red. MMP-1, -2, -3 and -9 and TIMP-2 and -3 were detected immunohistochemically. The mRNA levels were determined by real-time polymerase chain reaction. The results confirmed that doxorubicin treatment resulted in minimal myocardial fibrosis and showed that expression of MMPs increased and TIMP-3 decreased. The injection procedure resulted in increased myocardial fibrosis in groups 3 and 4. After MSC injection, MMP-1, MMP-2, and TIMP-3 expression was higher than that in group 2. CM injection led to more fibrosis, elevated TIMP-3, but diminished MMP-1 and MMP-2 expression compared with MSC injection. The mRNA levels of MMPs and TIMPs were not significantly different among all groups. In conclusion, chronic doxorubicin cardiomyopathy was characterized by increased MMP and decreased TIMP-3 expression. MSCs injection into the LV resulted in marked differences of collagen content and MMP/TIMP expression in the whole heart, although significant numbers of living MSCs were not detected after 4 weeks.

Ultrastructural findings in porcine hearts after extracorporeal long-term preservation with a modified Langendorff perfusion system.

Preserved ultrastructure is an important precondition for functional regeneration after heart transplantation. We investigated the effectiveness of a newly developed modified Langendorff system in extracorporeal heart perfusion. (Experiment I) Cardioplegia and cold ischaemia were performed in six pigs. Hearts were connected to a modified Langendorff system, and perfused with leucocyte depleted autologous blood. (Experiment II) The untreated hearts of three healthy pigs served as controls. Forty-seven myocardial biopsies at different timepoints (I: n = 29, II: n = 18) were investigated by transmission electronmicroscopy. Cardioplegia/hypothermia (I) induced mild-to-moderate mitochondrial swelling, mild myofibrillar degeneration in cardiomyocytes and moderate endothelial oedema. After 4 h reperfusion cardiomyocytes showed moderate myofibrillar and mild sarcolemmal damage. Moderate endothelial degeneration, mild interstitial oedema and haemorrhages appeared. Untreated hearts (II) showed severely damaged mitochondria and nuclei after 30 min while the myofibrillar structure remained unaffected until 4 h later. This is a promising model for extracorporeal heart perfusion. However, ultrastructural findings indicated that some necessary modifications to prevent cellular damages during reperfusion were needed.

Pharmacodynamics of T-cell function for monitoring immunosuppression.

OBJECTIVES: Recent studies show that measuring pharmacodynamic (PD) effects offers a unique possibility to predict immunosuppression. Thus, in this study we have monitored the PD properties of immunosuppressants on diverse T-cell functions in heart transplant (HTx) recipients. MATERIALS: PDs and blood concentrations (PK) of three different basis-immunosuppressive drugs were studied: cyclosporin A (CsA); tacrolimus (TRL) and sirolimus (SRL). T-cell function was analysed by expression of proliferating cell nuclear antigen (PCNA) labelling, expression of cytokines (IL-2, IFN-gamma) and surface antigen (for example, CD25) by FACS analysis. RESULTS: In group I, at time points C0 and C2, increased CsA-PK significantly inhibited expression of IL-2, IFN-gamma, PCNA and CD25 (P < 0.05). Correlations (r(2)) at C2 between inhibition of T-cell functions (PD) with PK and with drug doses were: CsA-PK: 0.71-0.91 and CsA-dose: 0.73-0.87. In group II, increased TRL-PK over time did not further inhibit expression of CD25, but inhibited PCNA expression more on day 3, and IL-2 and IFN-gamma expression was significantly higher on days 2 and 3 compared to PD effects of CsA (P < 0.05). Blood SRL concentrations in C0 group III, increased on day 1 and remained stable at days 3 and 4. Expression of PCNA was not altered in the SRL-PK category, whereas expression of CD25 was higher and expression of cytokines was lower than PD effects of CsA. CONCLUSIONS: Our results show that PD effects on T-cell function can be used to monitor immunosuppression bringing potential to increase the efficacy and safety of immunosuppressive therapy after HTx.

Segment-dependent expression of muscarinic acetylcholine receptors and G-protein coupling in the equine respiratory tract.

Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6+/-0.7 and 1.9+/-0.3 nmol/L. [3H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720+/-59 nmol/L) than in bronchi (438+/-48 nmol/L) or lung (22 +/- 3 nmol/L). Competitive binding studies using [3H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M1), methoctramine and himbacine (M2), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3), tropicamide (M4) and mamba toxin (MT-3) (M4) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24-28%: M2>M3>M1), whereas in bronchus and trachea M2 subtypes (87-90%) predominated over M3 (14-22%), and M1 subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31-36%) or presence of guanosine 5'-triphosphate (GTP) (approximately 100%). Western blotting for G-protein alpha-subunits showed a much more robust expression of G(alphai1/2) in the trachea (with highest receptor density) than in the lung or bronchi, whereas G(alphas)-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.

Subchronic alpha- and beta-adrenergic regulation of cardiac gap junction protein expression.

Gap junction channels are essential for intercellular electrical communication in the heart. The most important cardiac gap junction proteins are connexin43 (predominantly) (Cx43), connexin40 (Cx40), and in early developmental stages connexin45. Since catecholamines play an important role in cardiac (patho)physiology, we wanted to elucidate whether catecholamines may affect expression of Cx43 and Cx40. Cultured neonatal rat cardiomyocytes were exposed for 24 h to increasing concentrations of noradrenaline (1-10000 nM) (physiological agonist at alpha and beta-adrenoceptors), resulting in significantly increased Cx43-expression, while Cx40 was unaffected. In further experiments cells were incubated with either phenylephrine (alpha-adrenergic agonist) or isoproterenol (beta-adrenergic agonist) (0.1-1000 nM) for 24 h. Both catecholamines lead to a concentration-dependent increase in Cx43 protein and mRNA expression (EC50: 10-20 nM). Inhibition experiments showed that the phenylephrine effect was transduced via PKC, while the isoproterenol effect was mediated by PKA. Dual whole-cell voltage clamp demonstrated that increased Cx43-expression was accompanied by significant increases in gap junction current. In additional in vivo experiments, adult rats were subjected to 24-h infusion of isoproterenol or phenylephrine showing again significant increase in Cx43 but not Cx40. Adrenergic stimulation of cardiomyocytes can enhance Cx43 expression thereby increasing cellular coupling, indicating a possible role for catecholamines in the regulation of cardiac gap junction expression in cardiac disease.

C0h/C2h monitoring of the pharmacodynamics of cyclosporin plus mycophenolate mofetil in human heart transplant recipients.

UNLABELLED: Pharmacokinetic (PK) parameters like C2h have improved efficacy of immunosuppressive therapy. However, drug interactions, toxicities, and individual differences to drug effects still remain challenging. Therefore, this study was designed to assess pharmacodynamic (PD) effects of the combination cyclosporin (CsA) plus mycophenolate mofetil (MMF) on lymphocyte functions in peripheral blood of stable heart transplant recipients (HTx) using our established FACS assays. METHODS: Blood from 25 HTx patients was drawn before (C0h) and 2 hours after dosing (C2h). CsA and mycophenolic acid (MPA) concentrations were measured by EMIT. FACS assessed expression of cytokine production (IL-2, TNF-alpha), lymphocyte proliferation (PCNA), and T-cell activation (CD25, CD95). RESULTS: Evening doses of CsA (25/50/75 or 100 mg) and MMF (250/500 or 1000 mg) produced C0h levels as follows: CsA, 162 +/- 12 ng/mL; MPA, 1.7 +/- 0.2 mg/L. Morning doses of CsA (50/75 or 100 mg) and MMF (250/500/1000 or 1500 mg) produced C2h-levels as follows: CsA, 589 +/- 56 ng/mL and MPA, 7.4 +/- 1.3 mg/L. PD effects at C0h/C2h (% expression +/- SEM, all P < .05) were IL-2, 18 +/- 3/10 +/- 2; TNF-alpha, 12 +/- 2/7 +/- 1; PCNA, 8 +/- 1/5 +/- 1; CD25, 26 +/- 4/13 +/- 2; CD95, 23 +/- 4/11 +/- 2). Correlations (r2) at time point C2h between inhibition of lymphocyte functions (PD) with drug concentrations (PK) and with drug doses were CsA-PK, 0.71 to 0.91; MMF-PK, 0.55 to 0.76; CsA-dose, 0.73 to 0.87; MMF-dose, 0.61 to 0.80. CONCLUSION: For the first time, the immunosuppressive effects of the combination CsA plus MMF were quantified in whole blood of human HTx at different time points. PD assays may offer the opportunity to optimize clinical immunosuppressive drug therapy.

Expression of connexins 40 and 43 in human left atrium in atrial fibrillation of different aetiologies.

OBJECTIVE: To test the hypothesis that atrial fibrillation (AF) is associated with changes in the expression of connexins 40 and 43 in the left atrium with more pronounced changes in mitral valve disease than in lone AF. METHODS: Protein concentrations of connexin 40 and connexin 43 were analysed in left atrial tissue of patients undergoing cardiac surgery. One group of patients had lone AF (n = 41), one group had AF and mitral valve repair (n = 36), and one group in sinus rhythm served as controls (n = 15). RESULTS: Western blot analysis of connexin 40 and connexin 43 expression showed an increase of both gap junctional proteins (connexin 43 > connexin 40) in patients with AF of all forms compared with patients in sinus rhythm (p = 0.01 and p = 0.011, respectively). Subgroup analysis showed increased concentrations of connexin 40 in lone AF and AF with mitral valve disease compared with sinus rhythm (p = 0.06 and p = 0.029, respectively), whereas the same analysis for connexin 43 reached significance only in the mitral valve disease group (p = 0.031). No differences in connexin 40 and connexin 43 expression were detectable between lone AF and AF with mitral valve disease. Within the groups connexin 40 and connexin 43 expression did not differ between patients with paroxysmal AF and patients with chronic AF. CONCLUSION: The present study shows for the first time that AF can induce changes in the left atrium with increased connexin expression. Furthermore, no systematic differences between patients with paroxysmal and chronic AF were detected.