The G protein Gα11 is essential for hypertrophic signalling in diabetic myocardium.

AIMS/HYPOTHESIS: Pathological cardiac hypertrophy is an early phenotype in both types 1 and 2 diabetes. The primary stimulus for hypertrophic growth in diabetes is yet unknown and may involve neurohumoral stimulation of Gq-coupled receptors as well as direct glucose-dependent mechanisms. To discriminate between these hypertrophic stimuli we analyzed hypertrophic signalling pathways in wildtype and Gα11-knockout mice. METHODS: Experimental diabetes was induced in wildtype and knockout mice by intraperitoneal injection of streptozotocin. 8 weeks after induction of diabetes myocardial function and structure was assessed by echocardiography before sacrifice. To identify prohypertrophic signalling pathways expression and translocation of protein kinase C isoforms α, ßII, δ, ε and ζ were analyzed by immunohistochemical staining and immunoblot analysis after tissue fractionation. Changes in calcineurin signalling were identified by immunoblot analysis and functional assays. Expression levels of transcription factors GATA4 and NF-κB were quantified by real-time RT-PCR. RESULTS: Diabetic wildtype mice developed myocardial hypertrophy with preserved cardiac function. Calcineurin signalling was not different between the two groups. However, diabetic wildtype mice showed increased protein levels of PKC-α and PKC-ζ, translocation of PKC-α, -δ and -ε to cellular membranes and higher levels of NF-κB expression. In contrast, diabetic Gα11-knockout mice showed no altered phenotype and no changes in NF-κB or PKC expression, although translocation of PKC-ε occurred as in wildtypes. CONCLUSIONS: Gα11 is essential for the development of cardiac hypertrophy in type 1-diabetes. Stimulation of hypertrophic signalling through PKC-α, PKC-δ, PKC-ζ, and NF-κB appears to be receptor-dependent, whereas PKC-ε is activated by hyperglycemia, independent of Gα11.

[Transcatheter aortic valve replacement: when should it be used in aortic stenosis?]. / Kathetergestützter Aortenklappenersatz: eine neue therapeutische Option der Aortenklappenstenose?

30% of patients with significant aortic stenosis are not considered for operative aortic valve replacement because of the high perioperative risk. An alternative catheter based option for these patients is the transcatheter aortic valve replacement (TAVI). In general, there are two approaches for TAVI: transfemoral and transapical. Transfemoral aortic valve replacement is performed by transcatheter replacement of an aortic valve via the femoral arteries. Transapical valve replacement is achieved by transcatheter implantation via the fifth intercostal space. The most common complications are vessel injuries, bleeding complications, new onset of AV-block, development of paravalvular insufficiency, acute kidney injury, stroke and TIA. The first long-term observations suggest positive results. First clinical trials in a high-risk population show a promising outcome. Therefore TAVI offers a reasonable therapy option for patients with high perioperative risk. Further long-term clinical trials are still pending.

Clinical impact of atrial fibrillation in patients with pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is associated with progressive impairment of right ventricular function, reduced exercise capacity and a poor prognosis. Little is known about the prevalence, clinical manifestation and impact of atrial fibrillation (AF) on cardiac function in PH. METHODS: In a four year single-centre retrospective analysis 225 patients with confirmed PH of various origins were enrolled to investigate the prevalence of AF, and to assess the clinical manifestation, 6-minute walk distance, NT-proBNP levels, echocardiographic parameters and hemodynamics obtained by right heart catheterization in PH with AF. RESULTS: AF was prevalent in 31.1%. In patients with PH and AF, parameters of clinical deterioration (NYHA/WHO functional class, 6-minute walk distance, NT-proBNP levels) and renal function were significantly compromised compared to patients with PH and sinus rhythm (SR). In the total PH cohort and in PH not related to left heart disease occurrence of AF was associated with an increase of right atrial pressure (RAP) and right atrial dilatation. While no direct association was found between pulmonary artery pressure (PAP) and AF in these patients, right ventricular function was reduced in AF, indicating more advanced disease. In PH due to left heart failure the prevalence of AF was particularly high (57.7% vs. 23.1% in other forms of PH). In this subgroup, left atrial dilatation, increase of pulmonary capillary wedge pressure, PAP and RAP were more pronounced in AF than in SR, suggesting that more marked backward failure led to AF in this setting. CONCLUSION: PH is associated with increased prevalence of AF. Occurrence of AF in PH indicates clinical deterioration and more advanced disease.

Comparison between ATS/ERS age- and gender-adjusted criteria and GOLD criteria for the detection of irreversible airway obstruction in chronic heart failure.

BACKGROUND: Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (FEV1/FVC <70%) are extensively used for diagnosis of chronic obstructive lung disease in heart failure (HF). The American Thoracic Society (ATS)/European Respiratory Society (ERS) recommends the use of age- and gender-specific lower limit of normal (LLN) for FEV1/FVC. We compared the impact of these definitions on apparent prevalence of airway obstruction in chronic HF. METHODS: Standardized pre- and post-bronchodilator spirometry was performed in HF patients. Airway obstruction was defined by ATS/ERS criteria as diagnostic standard. Additionally, airway obstruction was calculated using the GOLD criteria. RESULTS: Of the 89 participants who fulfilled the ATS criteria for acceptability and reproducibility, 24.7% met ATS/ERS and 43.8% GOLD criteria for airway obstruction (Chi-square p = 0.007, McNemar <0.001). Sensitivity of GOLD criteria was 100%, specificity 74.6%, positive predictive value 56.4% and negative predictive value 100%. Among all individuals with an FEV1/FVC > LLN, 25.4% were falsely identified when using the GOLD criteria. A majority of false positives qualified for airway obstruction GOLD stage I (FEV1% ≥80%), which was significantly less often observed among true positives (76.5 vs. 31.8%; p < 0.001). Only 31.8% of patients with irreversible airway obstruction detected by the ATS/ERS criteria reported a history of COPD. CONCLUSIONS: In all HF patients with persistent dyspnoea despite optimal HF treatment, spirometric testing should be performed. Application of the GOLD criteria leads to overdiagnosis of irreversible airway obstruction in patients with HF, which may result in inappropriate medical therapy and health-care decisions.

Glycogen synthase kinase 3ß transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels.

Despite compelling evidence supporting key roles for glycogen synthase kinase 3ß (GSK3ß), mitochondrial adenosine triphosphate-sensitive K(+) (mitoK(ATP)) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3ß activated mitoK(ATP). PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoK(ATP) channels through GSK3ß, and this GSK3ß signal was mediated via mitochondrial Cx43. Moreover, (i) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3ß-S9A mice; (ii) Cx43 and GSK3ß proteins associated in mitochondria; and (iii) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3ß inhibition results in mitoK(ATP) opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoK(ATP) channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide (43)GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoK(ATP)-mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3ß transfers cytoprotective signaling through mitochondrial Cx43 onto mitoK(ATP) channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.

Local versus general anesthesia for transfemoral aortic valve implantation.

BACKGROUND: Transcatheter aortic valve implantation (TAVI) represents a novel option for elderly with severe aortic valve stenosis who are denied surgical aortic valve replacement due to high perioperative risk. While transfemoral TAVI generally is being performed in general anesthesia (GA), TAVI under local anesthesia plus mild sedation (LAPS) might be an effective and safe alternative. METHODS: In a single-centre analysis, we assessed clinical data, preoperative risk scores (STS-Score), echocardiography, periprocedural data and labor costs in 74 patients undergoing transfemoral TAVI under GA (n = 33) and LAPS (n = 41). RESULTS: Patients who underwent TAVI in LAPS presented significantly more often with pulmonary hypertension and impaired renal function, and tended to have a higher STS score and more severe symptoms (higher NYHA class) versus the GA group. There were no significant differences in procedure-related 30-day mortality or complications between groups. The peak systolic and mean central aortic pressure were significantly higher in the LAPS group, while at the same time these patients required significantly less often periprocedural adrenergic support. Intervention time was shorter in the LAPS group due to avoidance of surgical cut-down of the access site. Moreover, total procedure time was significantly shorter and labor costs were lower in the LAPS group. Patients who underwent TAVI in LAPS could be mobilized significantly earlier. CONCLUSION: Our study indicates that TAVI under LAPS is as effective and safe as TAVI under GA. Furthermore, total procedure time, intervention time and labor costs could be reduced by LAPS. Mobilization of patients could be achieved earlier. We therefore consider LAPS to be favorable in patients undergoing transfemoral TAVI.

Postprocedural atrial fibrillation after transcatheter aortic valve implantation versus surgical aortic valve replacement.

BACKGROUND: Transcatheter aortic valve implantation (TAVI) represents an alternative option for elderly patients with severe aortic valve stenosis who are denied surgical aortic valve replacement (SAVR) because of high perioperative risk. The impact of TAVI on postprocedural atrial fibrillation is undefined. METHODS: In a single-center analysis, we assessed clinical data, preoperative risk scores (Society for Thoracic Surgeons score, logistic European System for Cardiac Operative Risk Evaluation), preprocedural electrocardiograms, and 72-hour postprocedural rhythm monitoring of 170 patients undergoing TAVI (n=84) or SAVR (n=86). In a subanalysis, transapical (n=43) and transfemoral TAVI (n=41) were compared. RESULTS: Expectedly, TAVI patients were significantly older, presented with more severe symptoms, had higher Society for Thoracic Surgeons score, higher logistic European System for Cardiac Operative Risk Evaluation score, and revealed more frequently intermittent atrial fibrillation compared with SAVR patients. Despite this more compromised health state, prevalence of postprocedural atrial fibrillation was significantly lower in the TAVI group (6.0%, versus 33.7% after SAVR, p<0.05). More than two thirds of TAVI patients but no SAVR patient with atrial fibrillation in preprocedural electrocardiograms had stable sinus rhythm during 72-hour postprocedural monitoring. Notably, no atrial fibrillation was observed after transfemoral TAVI. Whereas atrial fibrillation onset in the SAVR group predominantly occurred on postoperative day 3, atrial fibrillation onset after transapical TAVI was obtained within the first 24 hours after the intervention. CONCLUSIONS: Our results indicate that TAVI, compared with SAVR, reduces the risk of periprocedural atrial fibrillation. Furthermore, preprocedural atrial fibrillation may be converted into sinus rhythm particularly after transfemoral TAVI, suggesting an impact of decreased intracardiac pressures in the absence of adverse periprocedural factors that might promote atrial fibrillation.

Conservative management of Candida infection of prosthetic aortic graft by means of caspofungin and fluconazole alone.

Candida albicans infections after prosthetic graft implantation due to acute aortic dissection are rare. A combination of surgical resection and lifelong antifungal drug therapy is the gold standard for treatment of aortic graft infection, yet surgical interventions are associated with high mortality rates. Herein, we present the case of a 57-year-old man who presented with peripheral microembolism due to late-onset C. albicans infection of a prosthetic graft of the thoracic aorta, which was diagnosed by positron emission tomographic imaging. Given the high risk of reoperation, the patient was treated with intravenous caspofungin for 4 weeks, followed by oral administration of fluconazole. During a follow-up of 500 days, he remained asymptomatic, with slightly elevated inflammatory markers. This case suggests that in some instances, particularly in patients with high operative risk, Candida prosthetic graft infection can be managed conservatively with antifungal therapy alone. However, such an approach should be applied with caution and necessitates close follow-up on a long-term basis.

Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes.

Potassium (K+) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K+ (mitoKATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoKATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoKATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide 43GAP27 each substantially reduced diazoxide-mediated stimulation of mitoKATP channels. Suppression of mitochondrial Cx43 inhibited mitoKATP channel activation by PKC. MitoKATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoKATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoKATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury.

Effects of KCNE2 on HCN isoforms: distinct modulation of membrane expression and single channel properties.

Hyperpolarization-activated cation (HCN) channels give rise to an inward current with similar but not identical characteristics compared with the pacemaker current (I(f)), suggesting that HCN channel function is modulated by regulatory beta-subunits in native tissue. KCNE2 has been proposed to serve as a beta-subunit of HCN channels; however, available data remain contradictory. To further clarify this situation, we therefore analyzed the effect of KCNE2 on whole cell currents, single channel properties, and membrane protein expression of all cardiac HCN isoforms in the CHO cell system. On the whole cell level, current densities of all HCN isoforms were significantly increased by KCNE2 without altering voltage dependence or current reversal. While these results correlated well with the KCNE2-mediated 2.2-fold and 1.6-fold increases of membrane protein levels of HCN2 and HCN4, respectively, no effect of KCNE2 on HCN1 expression was obtained. All HCN subtypes displayed faster activation kinetics upon coexpression with KCNE2. Most importantly, for the first time, we demonstrated modulation of single channel function by KCNE2, thus supporting direct functional interaction with HCN subunits. In the presence of KCNE2, the single channel amplitudes and conductance of HCN1, HCN2, and HCN4 were significantly increased versus control recordings. Mean open time was significantly increased in cells coexpressing HCN2 + KCNE2, whereas it was unaffected in HCN1 + KCNE2 cotransfected cells and reduced in HCN4 + KCNE2 cotransfected cells compared with the respective HCN subunits alone. Thus, we demonstrate KCNE2-mediated distinct effects on HCN membrane expression and direct functional modulation of HCN isoforms, further supporting that KCNE2 surves as a regulatory beta-subunit of HCN channels.

Regulation of the human cardiac mitochondrial Ca2+ uptake by 2 different voltage-gated Ca2+ channels.

BACKGROUND: Impairment of intracellular Ca(2+) homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca(2+) uptake is thought to be mediated by the Ca(2+) uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive. METHODS AND RESULTS: By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca(2+)-selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 micromol/L). Both mitochondrial Ca(2+) channels are unaffected by blockers of other possibly Ca(2+)-conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca(2+) uptake and likely Ca(2+)-induced metabolism. CONCLUSIONS: Thus, we conclude that the human mitochondrial Ca(2+) uptake is mediated by these 2 distinct Ca(2+) channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red-insensitive/low-sensitivity non-MCU-type mitochondrial Ca(2+) uptake.

Effect of bacterial endotoxin LPS on expression of INF-gamma and IL-5 in T-lymphocytes from asthmatics.

Epidemiological evidence, in vitro studies and animal models suggest that exposure to the bacterial endotoxin lipopolysaccharide (LPS) can influence the development and severity of asthma. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 and 2 responses, it is unclear whether the LPS ligand TLR 4 is expressed on CD4(+) and CD8(+) T-lymphocytes and if so, whether LPS could modulate the T(H)1 or T(H)2 response in this context. The present authors have, therefore, examined the expression of TLR 4 on peripheral blood CD4(+) and CD8(+) T-lymphocytes using RT-PCR method and FACS analyses. Furthermore, the authors have studied the IL-12-induced expression of the T(H)1-associated cytokine INF-gamma and the IL-4-induced expression of the T(H)2-specific cytokine IL-5 in the presence of LPS using ELISA and compared nine atopic asthmatic subjects and eleven nonatopic normal volunteers. There was an increased anti-CD3/anti-CD28-induced IL-5 expression in T cells of asthmatics compared with normals (p<0.01). In the presence of IL-4 (10 ng/ml), there was an additional increase in IL-5 expression and this additional increase was greater in T cells of normals compared with asthmatics (p<0.05). There was an expression of INF-gamma in anti-CD3/anti-CD28-induced T-lymphocytes without differences between both groups (NS). In the presence of IL-12 (10 ng/ml), there was an increase in INF-gamma release without differences between normals and asthmatics (NS). In the presence of different concentrations of LPS (10 ng/ml, 1 mug/ml), there was a decrease in IL-4-induced IL-5 expression without differences in both groups, indicating an intact T(H)2 response to bacterial endotoxin LPS in asthma. Interestingly, LPS increased the IL-12-induced INF-gamma release in a concentration-dependent manner in T-lymphocytes of normals but this could not be found in T cells of asthmatics, indicating an impaired T(H)1 response to bacterial endotoxin LPS in asthma. In addition, there was a TLR 4 expression on CD4(+) T-lymphocytes of normals and to a lesser extent in asthmatics but this TLR 4 expression could not be found on CD8(+) T cells of both groups. In conclusion, there may be an impaired concentration-dependent LPS-induced T(H)1 rather than a T(H)2 response in allergic adult asthmatics compared with normal volunteers. One reason for this could be a reduced TLR 4 expression on CD4(+) T-lymphocytes of asthmatic subjects.

Functional adenylyl cyclase inhibition in murine cardiomyocytes by 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate.

Beta1-adrenergic receptor activation stimulates cardiac L-type Ca2+ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio-]triphosphate (MANT-GTPgammaS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPgammaS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPgammaS on L-type Ca2+ currents (ICa,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5-/-) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPgammaS attenuated ICa,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77+/-13%; 100 nM MANT-GTPgammaS, +43+/-6%; 1 microM MANT-GTPgammaS, +21+/-9%; p<0.05). The leftward shift of current-voltage curves was abolished by 1 microM but not by 100 nM MANT-GTPgammaS. In myocytes from AC5-/- mice, the residual stimulation of ICa,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute beta-adrenergic stimulation in WT mice. Interestingly, basal ICa,L was lowered by 1 microM but not by 100 nM MANT-GTPgammaS. The decrease was less pronounced in myocytes from AC5-/- mice compared with wild types (-23+/-1 versus -40+/-7%), indicating basal ICa,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of ICa,L by MANT-GTPgammaS, both under basal conditions and following beta-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in ICa,L activation and that MANT-GTPgammaS predominantly acts via AC5 inhibition.