Principal role of adenylyl cyclase 6 in K+ channel regulation and vasodilator signalling in vascular smooth muscle cells

AIMS: Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (K(ATP))] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential. METHODS AND RESULTS: AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of K(ATP) current. K(ATP) current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation. CONCLUSION: AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure

Conserved expression and functions of PDE4 in rodent and human heart

PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation–contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has ‘global’ effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts

Applying Ultrasound Elastography To Detecting Skeletal Muscle Injury

Skeletal muscle injuries are frequent in sports and exercise, accounting for between 2055% of all sports injuries, with the vast majority of these injuries being muscle strains or contusions. Although muscle injuries are primarily diagnosed through symptomatology and examination of the injury mechanism, medical imaging techniques such as magnetic resonance imaging (MRI) and ultrasound imaging (US) are gaining popularity for assistance with both diagnoses and prognoses. These imaging techniques provide valuable structural and physiological information that is provides more insight into the nature and extent of the damage to guide image treatment. Currently, MRI is the gold standard for use with musculoskeletal applications, but US is becoming more widespread due to its affordability, portability, and real-time imaging capabilities. Ultrasound elastography (USE) is a family of techniques that are used to allow the visual analysis and quantification of the mechanical properties of soft tissue. Injury induced changes in muscle structure affect the mechanical properties of the tissue, and can be detected using USE. Since the echogenicity of a tissue is not directly related to its mechanical properties, ultrasound elastography can extract important information about tissue stiffness and deformability that might not otherwise be attainable. Such mechanical information is especially useful in distinguishing between fibrotic tissue and fatty infiltration. The goal of this thesis is to develop USE as an affordable, fast, and readily available alternative to MRI for early stage diagnosis of acute muscle injury. In pursuit of this goal, we first developed a technique to obtain repeatable and reproducible strain images using USE. We found that a simple averaging procedure performed on 4-8 repeated USE compression cycles significantly improved both the reproducibility and repeatability of the resulting strain images compared to strain images generated using an automated USE system. We then showed that USE combined with principal component analysis can be used to quantify and locate muscle injury in a finite element model, and can feasibly be applied to strain images generated using USE. Finally, we attempted to use the procedures developed in the first two studies to detect and monitor contusion injury in a rat model. However, due to equipment limitations, the results of this study were inconclusive. This thesis is concluded by a summary of the overall findings and discussion of future work

Exploring the Tactor Configurations of Vibrotactile Feedback Systems for Use in Lower-Limb Prostheses

Vibrotactile feedback may be able to compensate for the loss of sensory input in lower-limb prosthesis users to improve the mobility function. Designing an effective vibrotactile feedback system requires that users are able to perceive and respond to vibrotactile stimuli correctly and in a timely manner. Our study explored four key tactor configuration variables (i.e., tactors’ prosthetic layer, vibration intensity, prosthetic pressure, and spacing between adjacent tactors) through two experiments. The vibration propagation experiment investigated the effects of tactor configurations on vibration amplitude at the prosthesis–limb interface. Results revealed a positive relationship between vibration amplitude and intensity and a weak relationship between vibration amplitude and prosthetic pressure. Highest vibration amplitudes were observed when the tactor was located on the inner socket layer. The second experiment involving a sample of ten able-bodied and three amputee subjects investigated the effects of tactor configurations on user perception measured by response time, accuracy identifying tactors’ stimulation patterns, and spatial error in locating the tactors. Results showed that placing the tactors on the inner socket layer, greater spacing between adjacent tactors, and higher vibration intensity resulted in better user perception. The above findings can be directly applied to the design of vibrotactile feedback systems to increase the user response accuracy and decrease the response time required for dynamic tasks such as gait. They can also help to inform future clinical trials informing the optimization of tactor configuration variables

Averaging improves strain images of the biceps brachii using quasi-static ultrasound elastography

OBJECTIVE:Quasi-static ultrasound elastography is a technique for measuring tissue deformation (strain) under externally applied loading and can be used to identify the presence of abnormalities. The objective of this study was to demonstrate the efficacy of averaging strain images from repeated compression cycles in mitigating user-induced error using quasi-static ultrasound elastography.METHODS:Freehand compressions were performed with an ultrasound transducer on the biceps brachii of nine participants (five males and four females), as well as with a custom automated compression system. Sets of strain images from the freehand techniques were averaged to create single representative images and compared against strain images from the automated compressions using both qualitative and quantitative metrics.RESULTS:Significant improvements in intra-operator repeatability and interoperator reproducibility can be achieved by averaging strain images from four to eight repeated compressions. The resulting strain images did not lose significant image data compared with strain images from single automated compressions.CONCLUSION:Averaging is introduced as a feasible and appropriate technique to improve strain image quality without sacrificing important image data

The Effect of Platelet-Rich Plasma (PRP) on Muscle Contusion Healing in a Rat Model.

Background: Current therapy for muscle contusions is usually limited to nonsteroidal anti-inflammatory drugs and/or use of the RICE principle (rest, ice, compression, elevation); thus, other forms of treatment that can potentially accelerate the rate of healing are desirable. Hypotheses: A local injection of platelet-rich plasma (PRP) would lead to accelerated healing rates compared with controls; also, delayed administration of PRP would lead to a blunted response compared with immediate treatment. Study Design: Controlled laboratory study. Methods: Forty-six male Lewis rats each underwent a single blunt, nonpenetrating impact to the gastrocnemius muscle via a drop-mass technique and subsequently received either a single injection of saline into the area of injury immediately after injury (controls, n = 11) or rat PRP (either immediately after injury [PRP day 0, n = 12], the first day after injury [PRP day 1, n = 12], or the third day after injury [PRP day 3, n = 11]). The primary outcome was maximal isometric torque strength of the injured muscle, which was assessed before injury as well as on postinjury days 1, 4, 7, 10, and 14. All animals were sacrificed on postinjury day 15. Histological and immunohistochemical analyses were performed on 6 specimens from each group after sacrifice. Results: The mean platelet concentration in the PRP was 2.19 × 106 (±2.69 × 105)/μL. The mean white blood cell count in the PRP was 22.54 × 103/μL. Each group demonstrated statistically significant decreases in maximal isometric torque strength after injury when compared with preinjury levels, followed by significant increases back toward baseline values by postinjury day 14 (controls, 90.6% ± 7.90%; PRP day 0, 105.0% ± 7.60%; PRP day 1, 92.4% ± 7.60%; PRP day 3, 77.8% ± 7.90%) (P = .121). There were no statistically significant differences between the treatment and control groups at any of the time points. There were also no statistically significant differences between any of the groups in the percentage of centronucleated fibers (controls, 3.31% ± 5.10%; PRP day 0, 0.62% ± 1.59%; PRP day 1, 3.24% ± 5.77%; PRP day 3, 2.13% ± 3.26%) (P = .211) or the presence of inflammatory cells and macrophages. Conclusion: In this rat contusion model, a local injection of PRP into the injured gastrocnemius muscle resulted in no significant differences in functional or histological outcomes, indicating no likely benefit to healing. Additionally, there was no significant difference between immediate or delayed administration of PRP. Clinical Relevance: Before PRP can be recommended for the treatment of muscle contusion injuries, further translational and clinical investigations need to be performed

Thoracic aortic geometry correlates with endograft bird-beaking severity

Objective: Aortic geometry has been shown to influence the development of endograft malapposition (bird-beaking) in thoracic endovascular aortic repair (TEVAR), but the extent of this relationship lacks clarity. The aim of this study was to develop a reproducible method of measuring bird-beak severity and to investigate preoperative geometry associated with bird-beaking. Methods: The study retrospectively analyzed 20 patients with thoracic aortic aneurysms or type B dissections treated with TEVAR. Computed tomography scans were used to construct three-dimensional geometric models of the preoperative and postoperative aorta and endograft. Postoperative bird-beaking was quantified with length, height, and angle; categorized into a bird-beak group (BBG; n = 10) and no bird-beak group (NBBG; n = 10) using bird-beak height ≥5 mm as a threshold; and correlated to preoperative metrics including aortic cross-sectional area, inner curvature, diameter, and inner curvature 7 diameter as well as graft diameter and oversizing at the proximal landing zone. Results: Aortic area (1002 \ub1 118 mm2 vs 834 \ub1 248 mm2), inner curvature (0.040 \ub1 0.014 mm−1 vs 0.031 \ub1 0.012 mm−1), and diameter (35.7 \ub1 2.1 mm vs 32.2 \ub1 4.9 mm) were not significantly different between BBG and NBBG; however, inner curvature 7 diameter was significantly higher in BBG (1.4 \ub1 0.5 vs 1.0 \ub1 0.3; P =.030). Inner curvature and curvature 7 diameter were significantly correlated with bird-beak height (R = 0.462, P =.041; R = 0.592, P =.006) and bird-beak angle (R = 0.680, P <.001; R = 0.712, P <.001). Conclusions: TEVAR bird-beak severity can be quantified and predicted with geometric modeling techniques, and the combination of high preoperative aortic inner curvature and diameter increases the risk for development of TEVAR bird-beaking