Tillman Dahme

PINCH Proteins Regulate Cardiac Contractility by Modulating Integrin-Linked Kinase-Protein Kinase B Signaling

ABSTRACT Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

Paxillin and Focal Adhesion Kinase (FAK) Regulate Cardiac Contractility in the Zebrafish Heart

An orchestrated interplay of adaptor and signaling proteins at mechano-sensitive sites is essential to maintain cardiac contractility and when defective leads to heart failure. We recently showed that Integrin-linked Kinase (ILK), ß-Parvin and PINCH form the IPP-complex to grant tuned Protein Kinase B (PKB) signaling in the heart. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure. Two components of IPP, ILK and ß-Parvin directly bind to Paxillin; however, the impact of this direct interaction on the maintenance of heart function is not known yet. Here, we show that targeted gene inactivation of Paxillin results in progressive decrease of cardiac contractility and heart failure in zebrafish without affecting IPP-complex stability and PKB phosphorylation. However, we found that Paxillin deficiency leads to the destabilization of its known binding partner Focal Adhesion Kinase (FAK) and vice versa resulting in degradation of Vinculin and thereby heart failure. Our findings highlight an essential role of Paxillin and FAK in controlling cardiac contractility via the recruitment of Vinculin to mechano-sensitive sites in cardiomyocytes.

Clinical outcome of 2nd generation cryoballoon pulmonary vein isolation in patients over 75 years of age

BACKGROUND Pulmonary vein isolation is an established therapy for symptomatic atrial fibrillation. Despite the fact that incidence and prevalence of atrial fibrillation increases with age, patients over 75 years of age have been excluded in all major atrial fibrillation ablation trials. Pulmonary vein isolation with the cryoballoon has been shown to be equally effective compared to irrigated radiofrequency catheter ablation, but patients over 75 years have also been excluded. The 2nd generation cryoballoon has shown superior efficacy compared to the 1st generation cryoballoon. The aim of the study was to assess the efficacy of pulmonary vein isolation with the 2nd generation cryoballoon for symptomatic atrial fibrillation in elderly patients over 75 years. METHODS Patients over 75 years of age presenting with symptomatic paroxysmal or persistent atrial fibrillation refractory or intolerant to at least one class I or class III antiarrhythmic drug who underwent pulmonary vein isolation with the 2nd generation cryoballoon were included in this single-center observational study. RESULTS A total of 40 patients with a mean age of 78.3±2.7 years with paroxysmal (n=31; 77.5%) or persistent (n=9; 22.5%) atrial fibrillation were identified. All patients had a successful pulmonary vein isolation procedure with 100% of veins isolated. After a 3-month blanking period during a mean follow-up of 15.1±8.2 months there were 9 (22.5%) arrhythmia recurrences, while 31 patients (77.5%) maintained stable sinus rhythm. Freedom from arrhythmia recurrence was 86.4% at 12 months and 80.2% at 24 months. CONCLUSIONS Pulmonary vein isolation with the 2nd generation cryoballoon appears to be an effective treatment for symptomatic atrial fibrillation also in patients over 75 years of age.

Time-to-isolation guided titration of freeze duration in 3rd generation short-tip cryoballoon pulmonary vein isolation – Comparable clinical outcome and shorter procedure duration

BACKGROUND The optimal freeze duration in cryoballoon pulmonary vein isolation (PVI) is unknown. The 3rd generation cryoballoon facilitates observation of the time-to-isolation (TTI) and thereby enables individualized cryoenergy titration. To evaluate the efficacy of an individualized freeze duration we compared the clinical outcome of patients treated with a TTI-guided ablation protocol to the outcome of patients treated with a fixed ablation protocol. METHODS We compared 100 patients treated with the 3rd generation cryoballoon applying a TTI-based protocol (TTI group) to 100 patients treated by a fixed freeze protocol (fixed group). In the fixed group a 240s freeze cycle was followed by a 240s bonus freeze after acute PV isolation. In the TTI group freeze duration was 180s if TTI was ≥30s and reduced to only 120s, if TTI was <30s. In case of a TTI >60s a 180s bonus freeze was applied. RESULTS Freedom from atrial arrhythmia recurrence off class I/III antiarrhythmic drugs after one year was not different between the TTI group (73.6%) and the fixed group (75.7%; p=0.75). Mean procedure duration was 85.8±27.3min in the TTI group compared to 115.7±27.1min in the fixed group (p<0.001). Mean fluoroscopy time was 17.5±6.6min in the TTI group and 22.5±9.8min in the fixed group (p<0.001). CONCLUSIONS TTI-guided cryoenergy titration leads to reduced procedure duration and fluoroscopy time and appears to be as effective as a fixed ablation strategy. A single 2-minute freeze seems to be sufficient in case of short TTI.

Mutation of the Na + /K + -ATPase Atp1a1a.1 causes QT interval prolongation and bradycardia in zebrafish

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na+/K+-ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na+/K+-ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na+/K+-ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na+/K+-ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na+/K+-ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization.