CaMKII Splice Variants in Vascular Smooth Muscle Cells: The Next Step or Redundancy?

Vascular smooth muscle cells (VSMCs) help to maintain the normal physiological contractility of arterial vessels to control blood pressure; they can also contribute to vascular disease such as atherosclerosis. Ca2+/calmodulin-dependent kinase II (CaMKII), a multifunctional enzyme with four isoforms and multiple alternative splice variants, contributes to numerous functions within VSMCs. The role of these isoforms has been widely studied across numerous tissue types; however, their functions are still largely unknown within the vasculature. Even more understudied is the role of the different splice variants of each isoform in such signaling pathways. This review evaluates the role of the different CaMKII splice variants in vascular pathological and physiological mechanisms, aiming to show the need for more research to highlight both the deleterious and protective functions of the various splice variants.

Nontargeted detection of designer androgens: Underestimated role of in vitro bioassays.

Androgens, both steroidal and nonsteroidal in nature, are among the most commonly misused substances in competitive sports. Their recognized anabolic and performance enhancing effects through short- and long-term physiological adaptations make them popular. Androgens exist as natural steroids, or are chemically synthesized as anabolic androgenic steroids (AAS) or selective androgen receptor modulators (SARMs). In order to effectively detect misuse of androgens, targeted strategies are used. These targeted strategies rely heavily on mass spectrometry, and detection requires prior knowledge of the targeted structure and its metabolites. Although exquisitely sensitive, such approaches may fail to detect novel structures that are developed and marketed. A nontargeted approach to androgen detection involves the use of cell-based in vitro bioassays. Both yeast and mammalian cell androgen bioassays demonstrate a clear ability to detect AAS and SARMS, and if paired with high resolution mass spectrometry can putatively identify novel structures. In vitro cell bioassays are successfully used to characterize designer molecules and to detect exogenous androgens in biological samples. It is important to continue to develop new and effective detection approaches to prevent misuse of designer androgens, and in vitro bioassays represent a potential solution to nontargeted detection strategies.

A cell-free bioassay for the detection of androgens.

Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for nontargeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/androgen response element (ARE) regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/ml (3.7 × 10-7 to 5 × 10-11 M), with an EC50 of 6.63 ng/ml (23 nM). To reduce complexity, Stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labelling the RNA molecule with fluorophore-labelled nucleotide triphosphates, Stage 3 involved reverse transcription-polymerase chain reaction (PCR) of the RNA molecule, and Stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The Stage 4 product detected testosterone across the range of 106.7-0.0001 ng/ml (3.7 × 10-7 to 5 × 10-13 M), with an EC50 of 0.04 ng/ml (0.155 nM). Further to this, we show that the Stage 4 product can detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, nontargeted androgen screen.

Unravelling androgens in sport: Altrenogest shows strong activation of the androgen receptor in a mammalian cell bioassay.

Altrenogest is a commonly used progestogen for the suppression of oestrus and associated distracting behaviours that interfere with training and performance of female racehorses. The steroid is derived from 19-nor testosterone and is structurally similar to the anabolic androgenic steroid, trenbolone. In this study, the relative androgen potency of altrenogest was determined by a kidney (HEK293) cell androgen bioassay. The HEK293 bioassay shows that in its pure form, altrenogest has a high relative potency compared with testosterone but is not as strong as ß-trenbolone. Our results also show that altrenogest is able to activate the androgen receptor at the concentrations relevant to the administration regime of racehorses and retains its activity ex vivo. Thus, we show unequivocally that altrenogest, a progestogen used widely in female racehorses, acts as a strong androgen in a mammalian cell bioassay.

A Timing Effect of 17-ß Estradiol on Atherosclerotic Lesion Development in Female ApoE-/- Mice.

Differences in size or composition of existing plaques at the initiation of estrogen (E2) therapy may underpin evidence of increased risk of atherosclerosis-associated clinical sequelae. We investigated whether E2 had divergent effects on actively-growing versus established-advanced atherosclerotic lesions. Eight weeks of subcutaneous bi-weekly injections of 3 µg/g 17ß-estradiol (n = 18) or vehicle control (n = 22) were administered to female Apolipoprotein null-mice aged 25- or 45 weeks old. Histological assessment of lesion size within the brachiocephalic artery was conducted. Lesion composition was also assessed with acellular, calcification and fibrosis areas measured and other cellular features (intimal thickening, foam cells, lipid pools and cholesterol) scored (0-3) for severity. The comparison showed increased lesion size and calcified area with advancing age but no effect of E2. However, subtle changes in composition were observed following E2. Within the younger group, E2 increased intima thickening and acceleration of calcification. In the older group, E2 increased the thickness of the lesion cap. Therefore, this study shows different effects of E2 depending on the underlying stage of lesion development at the time of initiation of treatment. These divergent changes help explain the controversy of the adverse effects of E2 treatment in cardiovascular disease.

Shear force sensing of epithelial Na+ channel (ENaC) relies on N-glycosylated asparagines in the palm and knuckle domains of αENaC.

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.

Epithelial Na+ channel differentially contributes to shear stress-mediated vascular responsiveness in carotid and mesenteric arteries from mice.

A potential "new player" in arteries for mediating shear stress responses is the epithelial Na+ channel (ENaC). The contribution of ENaC as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance), is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (third-order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no flow) and in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and after inhibition of endothelial nitric oxide synthase (eNOS) by Nω-nitro-l-arginine methyl ester (l-NAME; 100 µM). Under no-flow conditions, amiloride increased internal and external diameters of carotid (13 ± 2%, P < 0.05) but not mesenteric (0.5 ± 0.9%, P > 0.05) arteries. In response to increased intraluminal pressure, amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress-strain curves of mesenteric arteries. With increased shear stress, ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2 ± 5.3%) compared with control (no amiloride, 6.2 ± 3.3%, P < 0.05). In mesenteric arteries, amiloride induced a flow-mediated constriction (-11.5 ± 6.6%) compared with control (-2.2 ± 4.5%, P < 0.05). l-NAME mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. ENaC-mediated effects were associated with NO production but may involve different (artery-dependent) downstream signaling pathways. NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) contributes to shear sensing in conduit and resistance arteries. In conduit arteries ENaC has a role as a vasoconstrictor, whereas in resistance arteries ENaC contributes to vasodilation. Interaction of ENaC with endothelial nitric oxide synthase/nitric oxide signaling to mediate the effects is supported; however, cross talk with other shear stress-dependent signaling pathways cannot be excluded.

Practical and effective stomal sphincter creation: evaluation in pigs.

PURPOSE: Stoma creation frequently presents complications for which there is no satisfactory surgical solution. We reexamined the feasibility of managing stoma continence with an artificial sphincter, addressing the outstanding issues of geometry, electrode disposition, and fatigue resistance. METHODS: In 6 pigs, 1 rectus abdominis muscle was preconditioned with electric stimulation for 4 weeks by an implanted stimulator. A sphincter was then constructed and tested for its ability to provide continence against saline at a typical intestinal pressure. The result was compared with a sphincter fashioned from the unconditioned contralateral (control) muscle. In each case, stimulation was applied alternately to longitudinal segments. RESULTS: A 2-layered wrap was required to achieve continence. Sphincters created from the preconditioned muscles could sustain continence continuously for at least 90 minutes. CONCLUSION: This study establishes a practical approach to the creation of a sphincter from the rectus abdominis muscle in stoma patients. Continence can be achieved only with a double-layered wrap. Fatigue during long-term operation can be avoided by a combination of preconditioning and segmental stimulation of intramuscular nerve branches.

Therapeutic stimulation of denervated muscles: the influence of pattern.

Muscular atrophy due to denervation can be substantially reversed by direct electrical stimulation. Some muscle properties are, however, resistant to change. Using a rabbit model of established denervation atrophy, we investigated whether the extent of restoration would vary with the stimulation protocol. Five patterns, delivering 24,000-480,000 impulses/day, were applied for 6 or 10 weeks. The wet weight, cross-sectional area, tetanic tension, shortening velocity, and power of denervated muscles subjected to stimulation all increased significantly. The fibers were larger and more closely packed and there was no evidence of necrosis. There was a small increase in excitability. Isometric twitch kinetics remained slow and fatigue resistance did not improve. The actual pattern of stimulation had no influence on any of these findings. The results, interpreted in the context of ultrastructural changes and an ongoing clinical study, reaffirm the clinical value of introducing stimulation during the initial non-degenerative phase. They indicate that there would be little therapeutic benefit in adopting regimes more energetically demanding than those in current use, and that the focus should now shift to protocols that represent the least intrusion into activities of daily living.

Effects of chronic electrical stimulation on long-term denervated muscles of the rabbit hind limb.

We investigated the extent to which activity induced by chronic electrical stimulation could restore the mass and contractile function of rabbit tibialis anterior (TA) muscles that had undergone atrophy as a result of prolonged denervation. Denervation was carried out by selectively interrupting the motor nerve branches to the ankle dorsiflexors in one hind limb. Stimulators were implanted, with electrodes on the superficial and deep surfaces of the denervated TA muscle. Ten weeks later, the mass and mid-belly cross-sectional area (CSA) of TA muscles subjected to denervation alone had fallen to approximately 40% of normal. At this stage, stimulators in the other rabbits were activated for 1 h/day to deliver 20-ms rectangular bipolar constant-current pulses of 4 mA amplitude at 20 Hz with a duty cycle of 1s ON/2s OFF, a total of 24,000 impulses/day. The animals were examined after a further 2, 6 or 10 weeks. Stimulation restored the wet weight of the denervated muscles to values not significantly different to those of normal, innervated controls. It increased CSA from 39% to 66% of normal, and there was a commensurate increase in maximum isometric tetanic force from 27% to 50% of normal. Light and electron microscopic examination revealed a marked improvement in the size, packing, and internal organization of the stimulated-denervated muscle fibres, suggestive of an ongoing process of restoration. Excitability, contractile speed, power, and fatigue resistance had not, however, been restored to normal levels after 10 weeks of stimulation. Similar results were found for muscles that had been denervated for 39 weeks and then stimulated for 12 weeks. The study demonstrates worthwhile benefits of long-term electrical stimulation in the treatment of established denervation atrophy.

Evaluation of barrier creams against sulphur mustard: (II) In vivo and in vitro studies using the domestic white pig.

Previous studies in our laboratory have demonstrated that barrier creams, comprising perfluorinated polymers, are effective against the chemical warfare agent sulphur mustard (SM) when evaluated using human skin in vitro. The purpose of this follow-up study was to further evaluate three candidate (perfluorinated) barrier creams against SM (vapour) using the domestic white pig. The severity and progression of the resulting skin lesions were quantified daily for three weeks post-exposure using biophysical measurements of transepidermal water loss (TEWL) and skin reflectance spectroscopy (SRS). Skin biopsies obtained post-mortem were evaluated by light microscopy and additional skin samples were obtained from adjacent (unexposed) skin sites for a comparative in vitro skin absorption study. Samples of SM vapour within the dosing chambers were measured ex vivo to ascertain the exposure dose (Ct). The three creams were highly effective against SM in vivo (Ct approximately 5000 mg.min.m(-3)): After 3 weeks, barrier cream pre-treated sites were not significantly different from control (unexposed) skin when evaluated by TEWL, SRS or histology. In contrast, skin exposed to SM without pre-treatment showed evidence of persistent damage that was consistent with the slow healing time observed in humans. The amount of SM absorbed in vitro in untreated pig skin was similar to that required to cause comparable lesions in human skin (8-20 and 4-10 microg.cm(-2), respectively), further validating the use of pigs as a toxicologically-relevant dermal model for SM exposure.

Counterpulsation from the skeletal muscle ventricle and the intraaortic balloon pump in the normal and failing circulations.

BACKGROUND: The intra-aortic balloon pump (IABP) is the device that is in most common use to provide cardiovascular support. A skeletal muscle ventricle (SMV) was configured to produce counterpulsation in the thoracic aorta similar to that obtained with an IABP. The hemodynamic effects of an IABP and a SMV in the same animal and in both normal and failing circulations were assessed. METHODS AND RESULTS: SMVs were connected to and IABPs were placed in the thoracic aorta of 12 anesthetized pigs. Hemodynamic parameters during the IABP- or the SMV-assisted beat were compared with those during the preassist beat. Acute heart failure was induced in 6 of the pigs by snaring the left anterior descending coronary artery (LAD). The hemodynamic effects of the IABP and the SMV were then reassessed. In the assisted cycles, SMV activation increased the mean aortic diastolic pressure (MADP) by 26.5+/-3.5%, the mean diastolic LAD flow by 48.4+/-7.2%, and endocardial viability ratio (EVR) by 31.6+/-3.8% (P<0.0001). In the same animals, IABP assist increased MADP by 19.8+/-2.3%, mean diastolic LAD flow by 37.2+/-3.9%, and EVR by 21.4+/-3.0% (P<0.0001). Under acute heart failure conditions, both SMV and IABP assist significantly increased MADP, mean diastolic LAD flow, and EVR. CONCLUSIONS: In both the normal and failing circulations, the SMV was an effective counterpulsator, providing cardiac assist that was at least equal to that available from an IABP. The SMV may therefore provide the proven benefits of an IABP in ambulant patients.

Functional electrical stimulation of denervated muscles: basic issues.

Denervating injuries result in flaccid paralysis and severe atrophy of the affected muscles. This work reviews the potential for functional restoration of such muscles by electrical stimulation, focusing on the basic scientific issues.

Determination of the chronaxie and rheobase of denervated limb muscles in conscious rabbits.

Measurements of the rheobase and chronaxie can be used to define the excitability of nerves and muscles. The aim of this study was to obtain a record over many weeks of changes in the rheobase and chronaxie of denervated rabbit tibialis anterior muscle (TA). A custom-built electronic stimulator was implanted into the peritoneal cavity of New Zealand White rabbits. Large stainless steel electrodes were placed on the denervated TA muscle. Rheobase and chronaxie were measured noninvasively at weekly intervals by means of a laptop PC, which communicated with the stimulator via a radio-frequency link. At each setting the denervated TA was palpated manually to detect the response of the muscle. During the first few days after denervation the rheobase increased transiently to 0.8 +/- 0.13 mA, approximately twice the value for normal innervated muscle, then decreased to normal for the remainder of the experimental period. Chronaxie underwent a significant 3-fold increase from 4.5 +/- 1.1 ms to 14.1 +/- 1.1 ms during the first two weeks of denervation and remained elevated throughout. The custom-built implantable electronic stimulator allowed changes in muscle excitability to be studied over a long period of denervation within individual animals, providing an accurate assessment of the time course of denervation-induced changes in muscle excitability.