The angiotensin II receptor type 1b (AT R ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT R activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT R or AT R with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT R mRNA is ∼30-fold higher than AT R in whole cerebral arteries and ∼45-fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT R for pressure-induced vasoconstriction ABSTRACT: Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT R - AT R and AT R - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT R increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT R knockout animals compared with controls. In patch-clamp experiments using native cerebral arterial myocytes, membrane stretch-induced cation currents were blocked by the TRPM4 inhibitor 9-phenanthrol in both groups. Further, the AT R blocker losartan (1 μm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT R mRNA was ∼30-fold greater than AT R in cerebral arteries, and knockdown of AT R selectively diminished myogenic constriction. We conclude that AT R , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

ObjectivesSmartphone-enabled ECG devices have the potential to improve patient care by enabling remote ECG assessment of patients with potential and diagnosed arrhythmias. This prospective study aimed to assess the usefulness of pediatric ECG tracings generated by the AliveCor device (Oklahoma City, OK) and to assess user satisfaction.Study DesignEnrolled pediatric patients with documented paroxysmal arrhythmia used the AliveCor device over a yearlong study period. Pediatric electrophysiologists reviewed all transmitted ECG tracings. Patient completed surveys were analyzed to assess user satisfaction.Results35 patients were enrolled with the following diagnoses: supraventricular tachycardia (SVT, 57%), atrial fibrillation (AF, 11%), ectopic atrial tachycardia (EAT, 6%), atrial tachycardia (AT, 3%), and ventricular tachycardia (VT, 23%). A total of 238 tracings were received from 20 patients, 96% of which were of diagnostic quality for sinus rhythm, sinus tachycardia, SVT, and AF. 126 patient satisfaction surveys (64% from parents) were completed. 98% of the survey responses indicated that it was easy to obtain tracings, 93% found it easy to transmit the tracings, 98% showed added comfort in managing arrhythmia by having the device, and 93% showed interest in continued use of the device after the study period ended.ConclusionsSmartphone-enabled ECG devices can generate tracings of diagnostic quality in children. User satisfaction was extremely positive. Use of the device to manage certain patients with AF and SVT showcases the future role of remote ECGs in the successful outpatient management of arrhythmias in children by potentially reducing Emergency Department visits and healthcare costs.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

Background and Purpose: In cardiac myocytes, cyclic AMP (cAMP) produced by both β 1-and β 2-adrenoceptors increases L-type Ca 2+ channel activity and myocyte contraction. However, only cAMP produced by β 1-adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca 2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β 2-adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. Experimental Approach: The cAMP responses produced by β 1-and β 2-adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex.

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