Individual Smooth Muscle Cells Research Articles

Pressure regulation of L-type Ca2+ channels and its role in myogenic response

L-type calcium (Ca2+) channels hold a pivotal position in governing intracellular Ca2+ concentration within smooth muscle and the establishment of myogenic tone. Their primary regulatory mechanism has historically been linked to voltage. Nevertheless, it remains a subject of inquiry whether voltage represents the exclusive modulator or whether these channels also exhibit mechanosensitivity. This study seeks to elucidate the influence of pressure stimuli on L-type Ca2+ channels in the resistance arteries of rodents (rats and mice). The investigative approach spans from the scrutiny of individual smooth muscle cells employing methodologies such as whole-cell and cell-attached patch-clamp electrophysiology, immunohistochemistry, proximity ligation assay, and super-resolution microscopy to the evaluation of intact arteries utilizing pressure myography. The findings obtained through whole-cell patch-clamp electrophysiology unveil a notable augmentation in L-type Ca2+ current in response to pressure stimuli. Subsequent scrutiny at the level of single-channel activity discloses that this augmentation is ascribed to an enhancement in functional coupling. Further inquiry reveals that the intensified functional coupling in response to pressure is contingent upon two principal factors: 1) the facilitation of cooperative gating mediated by protein kinase C (PKC), and 2) the traffcking of subunits to caveolae. Notably, functional experiments, involving depolarization elicited through the application of 30 mM KCl, underscore that arterial tone and cytosolic Ca2+ concentration responses are markedly more pronounced when the arteries are subjected to a pressure of 80 mmHg, as opposed to 20 mmHg. The culmination of these observations from both patch-clamp experiments and functional investigations supports the assertion that L-type Ca2+ channels are indeed amenable to mechanosensory influences, in addition to their well-documented voltage-dependent behavior. In summation, this investigation underscores that the regulatory landscape of L-type Ca2+ channels transcends the confines of voltage control, emphasizing the significance of considering pressure sensitivity in both physiological and pathological contexts. Ongoing research endeavors aim to delineate with precision the signaling complexes within vascular smooth muscle that respond to mechanical forces. Supported by the Canadian Institute for Health Research, National Institute of Health and the Rorabeck Chair in Vascular Biology and Neuroscience. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Apatite nanosheets inhibit initial smooth muscle cell proliferation by damaging cell membrane.

Understanding how nanostructured coatings interact with cells is related to how they manipulate cell behaviors and is therefore critical for designing better biomaterials. The apatite nanosheets were deposited on metallic substrates via biomimetic precipitation. Cell viability of apatite nanosheets towards to smooth muscle cells (SMCs) were investigated, and the underlying mechanism was proposed. Apatite nanosheets presented inhibitory activity on SMC growth, and caused rupture of cell membranes. On the basis of measuring changes in intracellular calcium ([Ca2+]i), observing cell contraction and apatite nanosheets - SMC interaction, it was found that calcium ions released from apatite led to rises in [Ca2+]i, which induced vigorous SMC contraction on apatite nanosheets. Consequently, the cell membrane of individual SMCs was cut/penetrated by the sharp edges of apatite nanosheets, resulting in cell inactivation. This damage of cell membranes suggests a novel mechanism to manipulate cell viability, and may offer insights for the better design of calcium-based nanostructured coatings or other biomedical applications.

Tissue-specific smooth muscle cell subtypes identified by transcriptional profiling.

Smooth muscle cells (SMCs) are specialized cells present in many organs where they serve diverse tissue-specific functions. Using the Tabula Muris compendium of single-cell RNA sequencing data, we extracted individual SMC transcriptomes from eight mouse tissues to investigate the transcriptomic landscape of tissue-specific SMCs. We identified marker genes, signaling pathways, and biological processes enriched in tissue-specific SMCs, and inferred potential ligand-receptor interaction between SMC and other cell types. Our analysis also identified sex differences in SMC gene expression in different tissues. Lastly, we used unsupervised clustering to identify novel SMC subtypes based on their downstream targets of transcription factors. Our results highlight the variable SMC phenotypes and underscore this cell's remarkable adaptability to contribute to diverse tissue function.

Fructose Consumption Increases Blood Pressure and Induces Changes in Renal Microvascular Function

A drastic increase in fructose consumption over recent decades has been paralleled by a growing prevalence and incidence of hypertension, along with comorbidities, such as diabetes, renal failure, and cardiovascular disease. High fructose corn syrup is a commonly found ingredient in the Western diet. However, the use of fructose as a sweetener has been shown to promote salt‐sensitive hypertension. The mechanisms by which fructose induces an increase in blood pressure and its effect on renal microvascular function are not completely understood. The goal of this study was to understand the mechanisms that lead to fructose‐induced impairment in renal microvascular function and its relation to the development of hypertension. Salt‐resistant Sprague Dawley rats (8 weeks old) were kept on a low salt (LS; 0.4% NaCl) with or without addition of 20% fructose (FT) to the drinking water provided ad libitum. After two weeks control and FT‐treated groups were switched to a high salt (HS; 8% NaCl) diet. Blood pressure was monitored with surgically implanted telemetry devices. Electrolyte balance and changes in renal function were analyzed among experimental groups using urinary (24 hrs collections) and plasma samples. After two weeks on HS, animals were euthanized, and renal microvessels were isolated to assess renal smooth muscle cell (SMC) excitability. We found that FT‐treated animals experienced elevated systolic blood pressure at LS to compare with control, and this difference were significantly higher in HS‐treated groups (107 ± 3 vs 113 ± 3, and 114 ± 4 vs 131 ± 5, mmHg; LS vs LS+FT, and HS vs HS +FT, correspondingly). Furthermore, these changes inversely correlated with the shift in diuresis and electrolyte balance in HS‐treated animals (70 ± 15 vs 39 ± 15 ml/day, 284 ± 91 vs 123 ± 70 Na/Cre ratio, 36 ± 12 vs 14 ± 10 K/Cre ratio, 0.7 ± 0.4 vs 1.4 ± 0.3 Ca/Cre ratio, HS vs HS+FT correspondingly). No changes in albuminuria, compared with controls, were noted in any of the groups. Two‐photon microscopy analysis of the renal microvascular vasomotion revealed a decrease in amplitude of individual SMC Ca2+ activity at the resting, spontaneous state in both LS and HS FT‐treated groups. However, both amplitude and period of Ca2+ fluctuations in SMC was significantly increased when vessels were exposed to ET‐1 in HS+FT group. Moreover, the amplitude of ET‐1‐induced Ca2+ transient in FT‐treated animals were split into two distinct groups with either higher or lower amplitudes relative to control. Overall, renal microvessels exposed to FT revealed a significant decrease in spontaneous vasomotion, which is accompanying by high responsiveness to FT at the HS with significant decrease in frequency of spontaneous events. These effects further promote high sensitivity (above normal) to ET‐1 stimulation, cell damage and finally low responsiveness (below normal) in FT‐treated renal microvessels. In conclusion, these results indicate the detrimental effects of FT on intracellular Ca2+ homeostasis in SMC, renal microvascular function and blood pressure regulation under high dietary salt.Support or Funding InformationNational Institute of Health R35 HL135749 (to A.S.), Michael Keelan, Jr., MD, CVC Research Foundation Grant (to O.P.), and Advancing a Healthier Wisconsin Endowment (to J.D.I.)

A Novel in situ Approach to Studying Detrusor Smooth Muscle Cells in Mice

The aim of our study was to develop a novel approach to investigating mouse detrusor smooth muscle cell (SMC) physiological activity, utilizing an acute tissue dissection technique and confocal calcium imaging. The bladder of a sacrificed adult female NMRI mouse was dissected. We used light and transmission electron microscopy to assess morphology of SMCs within the tissue. Calcium imaging in individual SMCs was performed using confocal microscopy during stimulation with increasing concentrations of carbamylcholine (CCh). SMCs were identified according to their morphology and calcium activity. We determined several parameters describing the SMC responses: delays to response, recruitment, relative activity, and contraction of the tissue. CCh stimulation revealed three different SMC phenotypes: spontaneously active SMCs with and without CCh-enhanced activity and SMCs with CCh-induced activity only. SMCs were recruited into an active state in response to CCh-stimulation within a narrow range (1–25 µM); causing activation of virtually all SMCs. Maximum calcium activity of SMCs was at about 25 µM, which coincided with a visible tissue contraction. Finally, we observed shorter time lags before response onsets with higher CCh concentrations. In conclusion, our novel in situ approach proved to be a robust and reproducible method to study detrusor SMC morphology and physiology.

The organisation of vascular smooth muscle cells; a quantitative Fast Fourier Transform (FFT) based assessment

BackgroundThe existence of a helical arrangement of vascular smooth muscle cells in the wall of relatively large blood vessels has been known of for at least 80 years. In that time various workers have proposed mathematical models of vascular function that have incorporated one or more helix. However, most of this work has focussed on large conduit vessels such as aorta and carotid arteries. The smaller resistance arteries are more difficult to study and do not have such an apparent helical arrangement, although there are suggestions of its existence in the patterning of smooth muscle cells in the tunica media. MethodWe have employed confocal laser scanning microscopy (CLSM) and a relatively simple Fast Fourier Transform (FFT) based image analysis method to examine a collection of extended focus (z-projection) images of the tunica media of mouse superior mesenteric artery. A fluorescent nuclear stain (Syto 61) was used to identify smooth muscle cell orientation and arrangement. Thirty-six CLSM datasets representing the tunica media of mesenteric arteries taken from 12 C57/black mice were collected for analysis. ResultsMeasurements of Z-projection (extended focus) images, of the tunica media, were compared with measurements made on inverse FFT images where the high frequency information was removed. The results, of both measurement types, indicated the existence of an underlying basic helical arrangement of cells that has a pitch angle of 49°. 3D electron microscopy-based models of individual smooth muscle cells (from the tail artery) were generated to highlight their heterogeneity and orientational position within the tunica media. ConclusionInverse FFT images provide a more objective means of measuring cell organisation and therefore could be used in automated processes. Whilst we propose the existence of an underlying helical arrangement of smooth muscle cells, we do not discount the existence of other helices each with different angles, but these may be more difficult to detect and may extend radially (deep within the tunica media) as well as longitudinally.

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 µN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 µN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue.

Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels

Vascular smooth muscle cells (VSMCs) show pronounced heterogeneity across and within vascular beds, with direct implications for their function in injury response and atherosclerosis. Here we combine single-cell transcriptomics with lineage tracing to examine VSMC heterogeneity in healthy mouse vessels. The transcriptional profiles of single VSMCs consistently reflect their region-specific developmental history and show heterogeneous expression of vascular disease-associated genes involved in inflammation, adhesion and migration. We detect a rare population of VSMC-lineage cells that express the multipotent progenitor marker Sca1, progressively downregulate contractile VSMC genes and upregulate genes associated with VSMC response to inflammation and growth factors. We find that Sca1 upregulation is a hallmark of VSMCs undergoing phenotypic switching in vitro and in vivo, and reveal an equivalent population of Sca1-positive VSMC-lineage cells in atherosclerotic plaques. Together, our analyses identify disease-relevant transcriptional signatures in VSMC-lineage cells in healthy blood vessels, with implications for disease susceptibility, diagnosis and prevention.

The transition of smooth muscle cells from a contractile to a migratory, phagocytic phenotype: direct demonstration of phenotypic modulation.

Key points Smooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned.Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist.This study used a novel, prolonged time‐lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs.In response to serum, highly‐elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity.This study provides a direct demonstration of the transition of fully contractile SMCs to a non‐contractile, migratory phenotype with phagocytic capacity that may act as a macrophage‐like cell. Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme into a ‘synthetic’ migratory phenotype, so‐called phenotypic modulation, whilst plaque macrophages are thought to derive from blood‐borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long‐term, high‐resolution, time‐lapse microscopy to track the fate of unambiguously identified, fully‐differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly elongated, contractile SMCs initially rounded up, for 1–3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell‐cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage‐like behaviour was confirmed by their internalisation of 1 μm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low‐density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling.

MARKERS OF CONNECTIVE TISSUE REMODELING IN GENITAL PROLAPSE

Purpose: to expand the conception of molecular and biochemical changes in genital prolapse (GP) based on the study of morphological and immunohistochemical features in connective tissue structures of the ligamentous apparatus of the pelvic floor and their dependence on genetic polymorphisms MMP / TIMP. Materials and Methods: the study involved 178 women aged 35 to 65, 134 of them with GP relapses (after hysterectomy by vaginal access because of a total and partial uterus and vaginal walls prolapse). Patients were randomized into the following groups: I – with manifestations of undifferentiated connective tissue dysplasia (CTD) (11.7 points on average) (n = 86); II – with no CTD signs (n = 48). Control group lll consisted of healthy women without any GP signs (among 15 patients abdominal hysterectomy was performed in connection with uterus hyperplastic processes) (n = 44). Used: morphological method of studies, immunohistochemical (to assess tissue biopsies of sacrum-uterine and round uterine ligaments), the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), genotyping by polymerase chain reaction of MMP / TIMP polymorphisms. Results: the morphological study of women’s ligamentous apparatus in cases with GP revealed significant fibrosis, coarser collagen septa among bundles of smooth muscle fibers and degenerative changes in individual smooth muscle cells. The group with GP and CTD features showed diffuse atrophy, hyaline or mucinous degeneration of smooth muscle tissue and evident edema of extracellular matrix in 65% of samples. Pathobiochemical disorders in cases of pelvic descencia were determined by an imbalance in collagen type I and III content, with the predominance of the latter, less durable; a decrease in elastin levels and its considerable fragmentation. The greatest expression of tissue degradation was observed among women with GP and CTD manifestations on account of increased MMP-1 and -2 levels; TIMP-1 content was lowest in the group. Associations with GP development have been established among women with CTD signs for genetic polymorphisms: rs3918242 СT gene MMP9 (0,54) (p = 0,007; OR = 3,2; 95% CI 1,3-7,6), rs17576 AG gene MMP9 (0.62 vs. 0,32, p = 0,01; OR = 2,9; 95% CI 1,2-7,0); rs3025058 5A6A gene MMP3 (0.52 vs. 0.45, p = 0.009; OR = 3.7; 95% CI 1,3-10,1); rs2285053 (rs2285052) CT gene MMP2 (0.44 vs. 0.27, p = 0,007; OR = 3,2; 95% CI 1,3-7,5). Statistical significance for the groups was preserved after the correction for multiple comparisons. Summary: the data obtained reveal pathogenetic aspects of genital prolapse – the prevalence of extracellular matrix degradation in a dysplastic morphogenesis. Genetic predictors of pelvic floor remodeling including the formation of its insolvency were established, allowing to extend the range of diagnostic possibilities of the disease progression at early stages or detection the risk of recurrence after surgical treatment. Personification of high-risk groups conducting provides for the exclusion or modification of all the factors predisposing to the development of the disease and performing timely treatment and preventive measures.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype.

Background/AimsSmooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized.MethodsWe generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence.ResultsCongenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRαlow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRαlow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression.ConclusionsSRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRαlow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

Two‐Photon Imaging of Intracellular Ca 2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of Isolated Rat Vessels

In order to understand how intracellular calcium ([Ca2+]i) and nitric oxide (NO) are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies two photon microscopy to measure calcium handling (or NO production) in individual cells of freshly isolated blood vessels. To validate the procedure, endothelial cell [Ca2+]i and NO activity were measured in response to application of acetylcholine (ACh) in a freshly isolated aorta of a Dahl salt-sensitive rat loaded with the intracellular [Ca2+]i ionophore, Fluo-4 AM, or with DAF-FM to indicate NO production. Application of ACh produced a detectable transient increase of the selected ionophore within individual endothelial cells of the aorta. To determine whether the protocol was sufficient to measure [Ca2+]i within small resistance arteries, renal microvessels were isolated, incubated in Fluo-4 AM dye, and imaged. Following addition of Endothelin-1, the smooth muscle cells (SMC) responded by increasing [Ca2+]i and emitting a strong fluorescent signal, which allowed us to calculate calcium transients within individual SMCs of the microvessels. This approach was further used to assess Ca2+ handling and NO production in vessels isolated from Plekha7 and p66 Shc knockout rats. Using this experimental procedure along with gene-editing resources, we have now begun testing the contribution of endothelial and SMC to vascular dysfunction and other pathological states within isolated vessels.

Advanced Age Increases the Amplitude of ATP‐sensitive K + Channel Currents in Murine Resistance Artery Smooth Muscle Cells

Advanced age impairs skeletal muscle blood flow regulation. We tested the hypothesis that advanced age decreases ATP-sensitive K+ channel (KATP) function of smooth muscle cells (SMCs) in skeletal muscle resistance arteries. Superior epigastric arteries (SEAs) were dissected from Young (Y; 蠅3 mo) or Old (O; 蠅24 mo) male C57BL/6 mice and either dissociated to yield individual SMCs for perforated patch clamp recording (holding potential, -30 mV) or cannulated and pressurized to 80 cm H20 for pressure myography. The KATP antagonist, glibenclamide (Glib, 10 µM) blocked small outward currents that were similar in Y and O (0.25±0.08 vs. 0.12±0.09 pA/pF, n = 6, p > 0.05). The KATP agonist levcromakalim (Lev, 10 µM) activated Glib-sensitive outward currents that were smaller in Y than O (0.14±0.05 vs. 1.11±0.37 pA/pF, n = 6, p < 0.05). However, Glib (10 µM) had no effect on resting diameter of SEAs from Y (control: 161±5; Glib: 166±5 μm, n=11, p>0.05) or O (control: 155±12; Glib: 155±11 μm, n=4, p>0.05). Lev induced similar maximal dilation in SEAs from Y (80±4%, n = 11) and O (74±12%, n = 7) with no difference in sensitivity (LogEC50: -6.98±0.12 vs. -6.83±0.23, p > 0.05). Thus, advanced age increases the functional expression of KATP in SEA SMCs. We suggest that this effect may not translate into a change in vasomotor function if it compensates for other age-related alterations in the regulation of SEA myogenic tone. (NIH R01-HL086483).

The effect of temperature changes on in vitro slow wave activity in the equine ileum.

Slow waves are rhythmic pacemaker currents generated by the gastrointestinal pacemaker cells, the interstitial cells of Cajal, and represent the rate-limiting step for small intestinal smooth muscle contractions. Therefore, factors that affect slow wave activity may also influence contractile activity. It is not known how temperature changes may influence slow wave activity in the horse. This could be of relevance during colic surgery if cooling of exposed intestine resulted in reduced slow wave activity potentially exacerbating post operative ileus. To evaluate the effect of temperature changes on in vitro slow wave activity of normal equine ileum using intracellular recording techniques. In vitro experimental study. A segment of ileum was collected immediately following euthanasia from 9 horses for reasons unrelated to the gastrointestinal tract. Intracellular recordings of membrane potentials were made from individual smooth muscle cells. The temperature of the tissue bath was altered during the course of each experiment across a range of 27-41°C. All data were recorded and stored using a computer-interfaced acquisition system. A software package was used to analyse slow wave frequency, duration, amplitude and resting membrane potential. In all 9 horses, slow wave frequency was highly temperature sensitive and approximately linearly related to the temperature over the range studied, increasing by 0.5 cycles/min for each 1°C increase in temperature (P<0.001). The initial slow wave frequency resumed when the temperature was returned to 37°C. The recovery time appeared to be directly related to the duration for which the temperature had been changed. Slow wave frequency in the equine ileum is highly temperature sensitive. As post operative ileus is a major cause of morbidity and mortality in the horse, the negative effect of lower temperatures on slow waves, and therefore contractile activity, should be considered.

Gap Junctions Suppress Electrical but Not [Ca2+] Heterogeneity in Resistance Arteries

Despite stochastic variation in the molecular composition and morphology of individual smooth muscle and endothelial cells, the membrane potential along intact microvessels is remarkably uniform. This is crucial for coordinated vasomotor responses. To investigate how this electrical homogeneity arises, a virtual arteriole was developed that introduces variation in the activities of ion-transport proteins between cells. By varying the level of heterogeneity and subpopulations of gap junctions (GJs), the resulting simulations shows that GJs suppress electrical variation but can only reduce cytosolic [Ca2+] variation. The process of electrical smoothing, however, introduces an energetic cost due to permanent currents, one which is proportional to the level of heterogeneity. This cost is particularly large when electrochemically different endothelial-cell and smooth-muscle-cell layers are coupled. Collectively, we show that homocellular GJs in a passively open state are crucial for electrical uniformity within the given cell layer, but homogenization may be limited by biophysical or energetic constraints. Owing to the ubiquitous presence of ion transport-proteins and cell-cell heterogeneity in biological tissues, these findings generalize across most biological fields.

The influence of gap junction network complexity on pulmonary artery smooth muscle reactivity in normoxic and chronically hypoxic conditions

Experiments on intrapulmonary arteries (IPAs) isolated from rats maintained in normoxia and chronic hypobaric hypoxia showed that in normoxia, the IPA contractile sensitivity to KCl was not modified by gap junction inhibition. In contrast, chronic hypoxia induced an endothelium-independent hypersensitivity, which was suppressed by gap junction inhibition. For the theoretical analysis of these results, we developed a model of interconnected myocytes. Given that smooth muscle cells in IPAs are known to communicate via gap junctions, we regard the cytoarchitecture of the IPA as a spatial network, in which nodes represent individual smooth muscle cells and the links signify intercellular communication. A single-cell model that drives the dynamics of individual nodes includes the major elements of voltage-dependent Ca(2+) signalling. In addition, interindividual variability of SMCs is introduced by distributing the reversal potentials for K(+). Cell-to-cell connection consists of passive Ca(2+) diffusion and electrical coupling, and connection between cells is determined by the topology of the intercellular network. Model predictions indicate that the experimental results can be explained by topological modifications and not by changes in the number of gap junctions. According to the model, in normoxia the myocytes are connected in a complex network, whereas chronic hypoxia is related to loss of complexity, leading to hypersensitivity. Our results thus indicate that chronic hypoxia entails gap junction network rearrangements, leading to disturbances in the intercellular communication pathways.

Aging increases the amplitude of spontaneous transient outward currents in murine resistance artery smooth muscle cells

Large conductance Ca2+‐activated K+ channels (BKCa) contribute to negative feedback regulation of microvascular tone. However, the effects of aging on BKCa function are unclear. The purpose of this study was to determine: 1) if enzymatically‐isolated smooth muscle cells (SMCs) from skeletal muscle resistance arteries display BKCa‐ and ryanodine receptor (RyR)‐dependent spontaneous transient outward currents (STOCs); and 2) whether aging affects STOC properties. Using perforated patch clamp recording, individual SMCs from Young (≥ 3 months) and Old (≥ 24 months) male C57BL/6 mice displayed STOCs at membrane potentials ≥ −30 mV, with the amplitude and frequency of STOCs increasing with membrane depolarization (n = 19–24; p &lt; 0.05). STOCs were inhibited by BKCa channel blockers (1 μM paxilline, 100 nM iberiotoxin) and by a RyR antagonist (100 μM tetracaine) (n ≥ 5; p &lt; 0.05 each). At +30 mV holding potential, STOC amplitude = 3.2 ± 0.32 pA/pF and frequency = 3.88 ± 0.41 Hz (n = 67) in SMCs from Young. STOC amplitude increased to 4.97 ± 0.5 pA/pF (n = 61; p &lt; 0.05) in SMCs from Old, with no significant difference in frequency (3.15 ± 0.31 Hz; p &gt;; 0.05). SMCs from murine resistance arteries display BKCa‐ and RyR‐dependent STOCs and aging increases STOC amplitude. Age‐related change in BKCa activity may impact the regulation of SMC function and contribute to age‐induced alterations in muscle blood flow regulation. (NIH R01HL086483).

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo-4, Propidium iodide, and di-8-Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin-eosin, Masson's tri-chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure.

Selective estrogen receptor modulators (SERMs) for uterine leiomyomas

Uterine fibroids are benign tumours that arise from individual smooth muscle cells of the uterus. Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that act as estrogens in some tissues while blocking estrogen activity in others. There have been many clinical studies of various SERMs for uterine fibroids. However, their effectiveness is controversial. To evaluate the effectiveness and safety of selective estrogen receptor modulators in women with uterine fibroids. We searched the Cochrane Menstrual Disorders and Subfertility Group Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, PubMed, EMBASE, the Register of Chinese trials developed by the Chinese Cochrane Centre, and the Chinese Med Database (Chinese Biomedical Disc), VIP, and China National Knowledge Infrastructure. We handsearched a number of journals and searched reference lists, and searched databases of ongoing trials and the Internet. The searches were conducted in March and April 2012. We included randomised controlled studies of selective estrogen receptor modulators versus other forms of medical therapy, placebo or no treatment in women of reproductive age (18 to 45 years old) with confirmed uterine fibroids. Two review authors independently extracted data and assessed trial quality. As the studies identified were not sufficiently similar, we did not do a meta-analysis but summarised the data in a narrative format. Three studies involving 215 participants were included, the trial size varied from 25 to 100 women. The SERM in all cases was raloxifene. In one study women in both arms received gonadotrophin releasing hormone (GnRH) analogue. Comparison interventions included no treatment and placebo. Two of the three included studies found a significant benefit from raloxifene, but the third study found no benefit at three or six-month follow-up. The overall quality of the evidence was low or very low. All three studies mentioned adverse reactions but data were limited. There is no consistent evidence from the limited number of studies that SERMs reduce the size of fibroids or improve clinical outcomes. Further studies are required to establish evidence of benefit of SERMs in treating women with uterine fibroids. This updated review did not find any new study for inclusion.

Multiple Factors Influence Calcium Synchronization in Arterial Vasomotion

The intercellular synchronization of spontaneous calcium (Ca2+) oscillations in individual smooth muscle cells is a prerequisite for vasomotion. A detailed mathematical model of Ca2+ dynamics in rat mesenteric arteries shows that a number of synchronizing and desynchronizing pathways may be involved. In particular, Ca2+-dependent phospholipase C, the intercellular diffusion of inositol trisphosphate (IP3, and to a lesser extent Ca2+), IP3 receptors, diacylglycerol-activated nonselective cation channels, and Ca2+-activated chloride channels can contribute to synchronization, whereas large-conductance Ca2+-activated potassium channels have a desynchronizing effect. Depending on the contractile state and agonist concentrations, different pathways become predominant, and can be revealed by carefully inhibiting the oscillatory component of their total activity. The phase shift between the Ca2+ and membrane potential oscillations can change, and thus electrical coupling through gap junctions can mediate either synchronization or desynchronization. The effect of the endothelium is highly variable because it can simultaneously enhance the intercellular coupling and affect multiple smooth muscle cell components. Here, we outline a system of increased complexity and propose potential synchronization mechanisms that need to be experimentally tested.