No abstract

CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner.

Prolonged electrograms localized to epicardial RVOT with variable low voltage were identified in all patients with BrS. J-point and ST-segment elevation correlated with greater transmural dispersion of late activation and was independent of total low-voltage area. Despite normalization of spontaneous type 1 pattern in all patients after ablation, recurrence was still observed, suggesting the implantable cardioverter-defibrillator as the cornerstone therapy for BrS.

CREB-binding protein (CBP) is an important transcriptional cofactor for various intracellular signaling pathways, including Ca(2+)- and cAMP-mediated gene activation. The loss of one CBP allele causes the human Rubinstein-Taybi syndrome, which is characterized by mental retardation and other severe developmental defects. Deletion of both CBP alleles in the mouse leads to early embryonic lethality. To address the function of CBP in late embryogenesis and in adult physiology, a floxed CBP allele (CBP(fl)) was generated. Using the Cre/loxP recombination system, CBP function was disrupted in principal forebrain neurons by breeding with a transgenic CaMKIIalpha-Cre mouse line to obtain CBP(fl/fl;CaMKIIalphaCre) mice. These mice contain CBP(stop523) alleles specifically in principal forebrain neurons, presumably resulting in the production of a truncated CBP protein unable to interact with a number of transcription factors, including phosphorylated CREB.

No abstract

p22phox-dependent NADPH oxidases contribute to the pathophysiology of PH induced by hypoxia.

Aims The optimal procedural endpoint to achieve permanent pulmonary vein isolation (PVI) during ablation of atrial fibrillation (AF) remains unknown. We aimed to compare the impact of prolonged waiting periods and adenosine triphosphate (ATP) testing after PVI on long-term freedom from AF. Methods and results In total, 538 patients (median age 61 years, 62% male) undergoing first-time radiofrequency ablation for paroxysmal AF were randomized into four groups: Group 1 [PVI (no testing), n = 121], Group 2 (PVI + 30min waiting phase, n = 151), Group 3 (PVI+ATP, n = 131), and Group 4 (PVI + 30min+ATP, n = 135). The primary endpoint was freedom from AF. Repeat mapping to assess for late pulmonary vein (PV) reconnection was performed in patients who remained AF-free for >3 years (n = 46) and in those who had repeat ablation for AF recurrence (n = 82). During initial procedure, acute PV reconnection was observed in 33%, 26%, and 42% of patients in Groups 2, 3, and 4, respectively. At 36 months, no significant differences in freedom from AF recurrence were observed among all four groups (55%, 61%, 50%, and 62% for Groups 1, 2, 3, and 4, respectively; P = 0.258). Late PV reconnection was commonly observed, with a similar incidence between patients with and without AF recurrence (74% vs. 83%; P = 0.224). Conclusion Although PVI remains the cornerstone for AF ablation, intraprocedural techniques to assess for PV reconnection did not improve long-term success. Patients without AF recurrence after 3 years exhibited similarly high rates of PV reconnection as those that underwent repeat ablation for AF recurrence. The therapeutic mechanisms of AF ablation may not be solely predicated upon durable PVI.

exposure to chronic hypoxia results in pulmonary hypertension characterized by increased vascular resistance and pulmonary vascular remodeling, changes in functional parameters of the pulmonary vasculature, and right ventricular hypertrophy, which can eventually lead to right heart failure. The underlying mechanisms of hypoxia-induced pulmonary hypertension have still not been fully elucidated while no curative treatment is currently available. Commonly employed pre-clinical analytic methods are largely limited to invasive studies interfering with cardiac tissue or otherwise ex vivo functional studies and histopathology. In this work, we suggest volumetric optoacoustic tomography (VOT) for non-invasive assessment of heart function in response to chronic hypoxia. Mice exposed for 3 consecutive weeks to normoxia or chronic hypoxia were imaged in vivo with heart perfusion tracked by VOT using indocyanide green contrast agent at high temporal (100 Hz) and spatial (200 µm) resolutions in 3D. Unequivocal difference in the pulmonary transit time was revealed between the hypoxic and normoxic conditions concomitant with the presence of pulmonary vascular remodeling within hypoxic models. Furthermore, a beat-to-beat analysis of the volumetric image data enabled identifying and characterizing arrhythmic events in mice exposed to chronic hypoxia. The newly introduced noninvasive methodology for analysis of impaired pulmonary vasculature and heart function under chronic hypoxic exposure provides important inputs into development of early diagnosis and treatment strategies in pulmonary hypertension. Pulmonary hypertension (PH) is a disorder characterized by pulmonary vascular remodeling, right ventricular hypertrophy and increased pulmonary arterial pressure. PH has been associated with various disorders while, according to the recent WHO classification, it is regarded as a separate entity when associated with hypoxia or chronic diseases of the respiratory system 1. The latter include chronic obstructive pulmonary disease (COPD), interstitial lung diseases, sleep disordered breathing, but also chronic exposure to high altitude and some rare neonatal diseases 2,3. Murine models have been widely used to gain deeper insight into lung-heart interactions under chronic hypoxic conditions 4. The hypoxia-inducible factor (HIF) has been highly implicated in the development of PH 5 where several preclinical studies focus on the molecular mechanisms of HIF and its role in PH 6-9. However, there is a lack of direct functional studies of the heart in response to chronic hypoxia. Methods for assessing pulmonary and cardiac structural alterations in PH are usually limited to histopathological ex vivo analyses looking at right ventricular (RV) hypertrophy and pulmonary vasculature changes. RV catheterization is an invasive in vivo procedure to measure pressure differences as a surrogate parameter of increased pulmonary arterial pressure 10-13 .