Title Page Sildenafil does not prevent heart hypertrophy and fibrosis induced by cardiomyocyte AT 1 R signaling

List of non-standard abbreviations: cGMP-dependent protein kinase type I (cGKI); protein kinase G (PKG); natriuretic peptides (NPs), cyclic guanosine-3´,5´-monophosphate (cGMP); nitric oxide (NO); cyclic adenosine-3´,5´-monophosphate (cAMP); Angiotensin II (AngII); Angiotensin II receptor type 1 (AT 1 R); sildenafil (SIL); cardiomyocyte (CM), smooth muscle cell (SMC); angiotensin converting enzyme (ACE), cGKIβ rescue mice (βRM), phosphodiesterase (PDE); Förster resonance energy transfer (FRET); 3-isobutyl-1-methylxanthine (IBMX), C-type natriuretic peptide (CNP). Section assignment: Cardiovascular Number of words: 4141 (excluding abstract, material and methods, reference list and figure legends) Number of words (abstract): 250 Number of references: 67 Page count: 37 JPET#226092 3 Abstract Analyses of several mouse models imply that the phosphodiesterase 5 (PDE5) inhibitor sildenafil (SIL), via increasing cyclic guanosine-3',5'-monophosphate (cGMP), affords protection against angiotensin II (AngII) stimulated cardiac remodeling. However, it is unclear which cell types are involved in these beneficial effects, because AngII may exert its adverse effects by modulating multiple reno-vascular and cardiac functions via AngII type 1 receptors (AT 1 R). To test the hypothesis that SIL/cGMP oppose cardiac stress provoked by amplified AngII/AT 1 R directly in cardiomyocytes (CMs), we studied transgenic mice with CM-specific overexpression of the AT 1 R under the control of the α -myosin-heavy chain promoter (αMHC-AT 1 R tg/+ ). The extent of cardiac growth was assessed in absence or presence of SIL and defined by referring changes in heart-weight to body-weight or tibia length. Hypertrophic marker genes, extracellular matrix-regulating factors and expression patterns of fibrosis markers were examined in α MHC-AT 1 R tg/+ ventricles (±SIL) and corroborated by investigating different components of the natriuretic peptide (NP)/PDE5/cGMP pathway as well as cardiac functions. cGMP levels in heart lysates and intact CMs were measured by competitive immunoassays and FRET. We find higher cardiac and CM cGMP levels and up-regulation of the cGMP-dependent protein kinase I (cGKI) with AT 1 R over-expression. However, even a prolonged SIL treatment regimen did not limit the progressive CM growth, fibrosis or decline in cardiac functions in the αMHC-AT 1 R tg/+ model suggesting that SIL does not interfere with the pathogenic actions of amplified AT 1 R signaling in CMs. Hence, the cardiac/non-cardiac cells involved in the cross-talk between SIL-sensitive PDE activity and AngII/AT 1 R need to be identified

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3(-/-)) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythmand for the determination of behavior. Contrary to previous suggestions we find that HCN3(-/-) mice show normal visual, photic, and non-photic circadian function. In addition, HCN3(-/-) mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure

Ultrafast Volumetric Optoacoustic Imaging of Whole Isolated Beating Mouse Heart

The Langendorff-perfused heart technique has become the model of choice for multiparametric optical mapping of cardiac function and electrophysiology. However, photon scattering in tissues represents a significant drawback of the optical imaging approach, fundamentally limiting its mapping capacity to the heart surface. This work presents the first implementation of the optoacoustic approach for 4D imaging of the entire beating isolated mouse heart. The method combines optical excitation and acoustic detection to simultaneously render rich optical contrast and high spatio-temporal resolution at centimeter-scale depths. We demonstrate volumetric imaging of deeply located cardiac features, including the interventricular septum, chordae tendineae, and papillary muscles while further tracking the heart beat cycle and the motion of the pulmonary, mitral, and tricuspid valves in real time. The technique possesses a powerful combination between high imaging depth, fast volumetric imaging speed, functional and molecular imaging capacities not available with other imaging modalities currently used in cardiac research