Vascular Smooth Muscle Contractility Research Articles

TRPML1 channels initiate Ca2+ sparks in vascular smooth muscle cells.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

Biophysical and Pharmacological Characterization of Atp-Sensitive Potassium Channels in Mice Kir6.1WT/V65M Mirroring the Human Cantu′ Syndrome

Cantu syndrome(CS) arises from GOF mutations in KCNJ8 or ABCC9, the genes encoding for Kir6.1 and SUR2 subunits of ATP-sensitive potassium(KATP) channels. Kir6.1 is prominent in vascular and skeletal muscle, but absent from cardiac muscle. Heterozygous Kir6.1[V65M] mice, in which the CS mutation was knocked in to the endogenous locus by CRISPR/Cas9 gene editing, exhibit KATP over-activity and reduced contractility in vascular smooth muscle, but not cardiomyocite. No information is available for other tissues. We measured the forelimb strength and ultrasonography of diaphragm and skeletal muscle, and investigated the KATP channel properties in native flexor digitorum brevis (FDB) and soleus (SOL) fibers, skin fibroblasts and long bones/calvaria-derived osteoblasts from WT and Kir6.1wt/V65M mice, by patch clamp technique. Forelimb forces were lower, and ultrasonography evaluation of the hind-limb showed significant enhancement of echodensity, in Kir6.1wt/V65M mice with respect to WT. Increased organ/tissue weights were observed in Kir6.1wt/V65M mice with respect to WT. In patch clamp experiments, higher KATP amplitude, reflecting the GOF associated with the mutation was observed in FDB fibers, skin fibroblasts and long bones/calvaria- derived osteoblasts from Kir6.1wt/V65M mice with respect to WT. No differences in KATP amplitude were detected between WT vs Kir6.1wt/V65M SOL fibers. A marked loss of channel sensitivity to Glib, also characteristic of the mutation was observed in all the mutant cells vs controls. Finally, a lower FDB fiber survival was observed in Kir6.1wt/V65M mice respect to the WT mice within 24 hours of isolation. The CS mutation Kir6.1wt/V65M has significant effects on KATP currents in multiple skeletal muscle and other tissues, reducing skeletal muscle contractility and fiber viability. This suggests that skeletal muscle insufficiency could be a significant issue in CS patients suffering from Kir6.1 GOF mutations.

Loss of smooth muscle α-actin effects on mechanosensing and cell-matrix adhesions.

Mutations in ACTA2, encoding smooth muscle α-actin, are a frequent cause of heritable thoracic aortic aneurysm and dissections. These mutations are associated with impaired vascular smooth muscle cell function, which leads to decreased ability of the cell to sense matrix-mediated mechanical stimuli. This study investigates how loss of smooth muscle α-actin affects cytoskeletal tension development and cell adhesion using smooth muscle cells explanted from aorta of mice lacking smooth muscle α-actin. We tested the hypothesis that reduced vascular smooth muscle contractility due to a loss of smooth muscle α-actin decreases cellular mechanosensing by dysregulating cell adhesion to the matrix. Assessment of functional mechanical properties of the aorta by stress relaxation measurements in thoracic aortic rings suggested two functional regimes for Acta2−/−mice. Lower stress relaxation was recorded in aortic rings from Acta2−/−mice at tensions below 10 mN compared with wild type, likely driven by cytoskeletal-dependent contractility. However, no differences were recorded between the two groups above the 10 mN threshold, since at higher tension the matrix-dependent contractility may be predominant. In addition, our results showed that at any given level of stretch, transmural pressure is lower in aortic rings from Acta2−/−mice than wild type mice. In addition, a three-dimensional collagen matrix contractility assay showed that collagen pellets containing Acta2−/−smooth muscle cells contracted less than the pellets containing the wild type cells. Moreover, second harmonic generation non-linear microscopy revealed that Acta2−/−cells locally remodeled the collagen matrix fibers to a lesser extent than wild type cells. Quantification of protein fluorescence measurements in cells also showed that in absence of smooth muscle α-actin, there is a compensatory increase in smooth muscle γ-actin. Moreover, specific integrin recruitment at cell–matrix adhesions was reduced in Acta2−/−cells. Thus, our findings suggest that Acta2−/−cells are unable to generate external forces to remodel the matrix due to reduced contractility and interaction with the matrix.Impact statementThoracic aneurysm formation is characterized by progressive enlargement of the ascending aorta, which predisposes the aorta to acute aortic dissection that can lead to sudden death. SMCs in the aorta play an integral role in regulating vessel wall contractility and matrix deposition in the medial layer. Recent studies show that mutations in genes associated with actomyosin apparatus reduce SMC contractility, increasing susceptibility to TAAD. Single-cell experiments enable discrete measurements of transient microscopic events that may be masked by a macroscopic average tissue behavior. Biophysical methods combined with microscopy techniques aid in understanding the specific roles of adhesion and cytoskeletal proteins in regulating SMC mechanosensing when SMα-actin is disrupted. Our findings suggest that Acta2−/−cells have increased SMγ-actin and decreased integrin recruitment at cell–matrix adhesion, hence a synthetic phenotype with reduced cellular mechanosensing.

STIM1 Maintains Stable Peripheral Coupling in Fully Differentiated Contractile Vascular Smooth Muscle Cells Independently of Ca2+ Store Depletion

STIM1 Maintains Stable Peripheral Coupling in Fully Differentiated Contractile Vascular Smooth Muscle Cells Independently of Ca2+ Store Depletion

P1936Selective inhibition of the Mek1/2-Erk1/2 signalling pathway induces the differentiation of human cardiac pericyte-like cells into contractile vascular smooth muscle cells

Abstract Background Arteriogenesis is key for tissue repair but whether myocardial stromal cells contribute to this phenomenon remains unknown. Purpose Investigate if cardiac pericytes are a druggable target for therapeutic arteriogenesis. Methods and results The localization of pericyte-like cells (PCs) was assessed in the human and murine heart by immunohistochemistry of typical antigenic markers. CD34+ PCs co-expressing NG2 and PDGFRβ but not endothelial cell (EC) or vascular smooth muscle cell (VSMC) antigens were identified in peri-arterial position in normal hearts. Interestingly, we also found rare PCs co-expressing αSMA in the peri-infarct myocardium, suggesting these cells could represent a transitory phenotype between PCs and VSMCs. Next, we isolated human cardiac PCs by immunosorting for CD31 and CD34 and established the purity of the isolated CD34+CD31- fraction by flow cytometry. Following culture expansion, cardiac PCs maintain the typical antigenic profile except for CD34. Moreover, we confirmed the PCs' ability to promote angiogenesis in-vitro. The withdrawal of EGF and bFGF from the culture media for 10 days induced the differentiation of PCs into mature VSMCs, as documented by a massive upregulation of contractile genes MYH11, CNN1 and ACTA2 (200-, 35- and 15-folds increase versus naïve PCs, p<0.01), which was followed by the induction of SM-MHC, Smoothelin B, αSMA, Calponin and SM22α proteins (p<0.05 versus PCs). In addition, PC-derived cells lost migratory capacity, secreted elastin, and responded to endothelin-1 in a contraction assay, thus phenocopying the behaviour of control coronary artery-derived VSMCs. We excluded contamination of the PC preparation by verifying similar phenomena occur in PCs expanded from single cell clonogeneic assays. Moreover, the process is partially reversible, with PC-derived VSMCs being able to reacquire some intermediate markers following EGF/bFGF re-challenge. ECs secrete EGF and bFGF, with this GF signalling being enhanced by hypoxia, suggesting ECs may control the PC phenotype in a paracrine fashion. Mechanistic studies revealed the Mek1/2-Erk1/2-Elk1 signalling is accountable for the transcriptional repression of VSMC genes in PCs. Accordingly, a selective Mek1/2 inhibitor (PD0325901) was able to switch the definitive VSMC phenotype of PCs maintained in full media. The drug prevented the phosphorylation of Erk1/2 and its downstream target Elk1. This likely relieves the complex SRF/MyocD and abolishes the transcriptional repression at the gene promoter. Conclusions Cardiac PCs have a VSMC potential which is under the inhibitory control of the Mek1/2-Erk1/2-Elk1 signalling. Mek1/2 inhibitors showed promises for the treatment of melanoma and solid tumours. A novel application of this class of compounds to improve arteriogenesis in myocardial ischemia is fascinating. The caveat about their potential cardiotoxicity could be less relevant with short duration treatments of myocardial ischemia. Acknowledgement/Funding British Heart Foundation Centre for Regenerative Medicine Award (II) - “Centre for Vascular Regeneration” (RM/17/3/33381)

Brain Endothelial Cells Release Apical and Basolateral Microparticles in Response to Inflammatory Cytokine Stimulation: Relevance to Neuroinflammatory Stress?

Microparticles (MP) are regarded both as biomarkers and mediators of many forms of pathology, including neurovascular inflammation. Here, we characterized vectorial release of apical and basolateral MPs (AMPs and BMPs) from control and TNF-α/IFN-γ treated human brain endothelial monolayers, studied molecular composition of AMPs and BMPs and characterized molecular pathways regulating AMP and BMP release. The effects of AMPs and BMPs on blood-brain barrier properties and human brain microvascular smooth muscle tonic contractility in vitro were also evaluated. We report that human brain microvascular endothelial cells release MPs both apically and basolaterally with both AMP and BMP release significantly increased following inflammatory cytokine challenge (3.5-fold and 3.9-fold vs. control, respectively). AMPs and BMPs both carry proteins derived from parent cells including those in BBB junctions (Claudin−1, −3, −5, occludin, VE-cadherin). AMPs and BMPs represent distinct populations whose release appears to be regulated by distinctly separate molecular pathways, which depend on signaling from Rho-associated, coiled-coil containing protein kinase (ROCK), calpain as well as cholesterol depletion. AMPs and BMPs modulate functions of neighboring cells including BBB endothelial solute permeability and brain vascular smooth muscle contractility. While control AMPs enhanced brain endothelial barrier, cytokine-induced AMPs impaired BBB. Cytokine-induced but not control BMPs significantly impaired human brain smooth muscle contractility as early as day 1. Taken together these results indicate that AMPs and BMPs may contribute to neurovascular inflammatory disease progression both within the circulation (AMP) and in the brain parenchyma (BMP).

Sleep disorders, nocturnal blood pressure, and cardiovascular risk: A translational perspective.

Cardiovascular disease (CVD) represents the first cause of death globally. The nighttime is generally a period of relative protection from CVD events such as myocardial infarction, sudden cardiac death, and stroke, at least compared to the early morning period. The nighttime also generally entails lower values of arterial blood pressure (ABP) and heart rate (HR) and higher cardiac parasympathetic modulation. These day-night cardiovascular rhythms are ultimately driven by circadian molecular oscillators in the hypothalamic suprachiasmatic nucleus and in peripheral cells, including those in the heart, blood vessels, and kidneys. The wake-sleep states are intermediate mechanisms of circadian cardiovascular regulation, with non-REM sleep decreasing ABP and HR and increasing cardiac parasympathetic modulation at the beginning of the night. Obstructive sleep apnea, insomnia, and the restless legs syndrome have high prevalence in the general population and may increase nighttime cardiovascular activity and CVD risk. CVD risk is better predicted by ABP values during nighttime sleep than during daytime wakefulness. Higher nighttime values of ABP and HR increase cardiac work and vessel wall stress. During the night, circadian rhythms may enhance cardiac responses to hypertrophic stimuli, increase vascular smooth muscle Rho kinase activity and contractility, decrease endothelial nitric oxide production and vascular responses to vasodilators, and increase circulating monocytes with the potential to infiltrate atherosclerotic plaques. Together, these factors configure a "perfect storm" scenario that may make increased cardiovascular activity during the night a final common mechanism linking sleep disorders to CVD risk.

Inhibitory effects of rubratoxin A, a potent inhibitor of protein phosphatase 2, on the Ca2+-dependent contraction of skinned carotid artery from guinea pig.

Rubratoxin A, a potent inhibitor of PP2A, is known to suppress smooth muscle contraction. The inhibitory role of PP2A in smooth muscle contraction is still unclear. In order to clarify the regulatory mechanisms of PP2A on vascular smooth muscle contractility, we examined the effects of rubratoxin A on the Ca2+-induced contraction of β-escin skinned carotid artery preparations from guinea pigs. Rubratoxin A at 1 µM and 10 µM significantly inhibited skinned carotid artery contraction at any Ca2+ concentration. The data fitting to the Hill equation in [Ca2+]-contraction relationship indicated that rubratoxin A decreased Fmax-Ca2+ and increased [Ca2+]50, indices of Ca2+ sensitivity for the force and myosin-actin interaction, respectively. These results suggest that PP2A inhibition causes downregulation of the myosin light chain phosphorylation and direct interference with myosin-actin interaction.

Biomechanical properties of veins cultured in vitro under elevated internal pressure.

The venous response to elevated blood pressure (BP) is of major importance because it is closely related to the etiology of venous diseases and the competency of vein grafts. In vitro culture experiments may provide useful information on the function of vein grafts because it is easier to separate mechanical and hemodynamic effects from other systemic influences compared to in vivo experiments. To study the effects of BP elevation on wall dimensions and mechanical properties of in vitro cultured veins. Rabbit femoral veins were cultured in vitro under internal pressures of 1 to 50 mmHg for 1 week, and their wall dimensions, biomechanical properties, and histology were determined. No significant differences were observed in internal vein diameter and wall thickness among vessels cultured at 10-50 mmHg compared to non-cultured control vessels. For an internal pressure of 10 mmHg applied to vessels during culture (equivalent to in vivo working BP), wall circumferential stress was maintained within control levels. There were no significant effects of pressure on basal tone and contractility of vascular smooth muscle and vascular compliance. The in vitro results were essentially similar to those obtained from previous in vivo animal experiments, indicating that in vitro tissue culture techniques are applicable to studies of venous remodeling.

Enhancement of Vascular Smooth Muscle Contractility by Alterations of Membranous Architecture.

Plasma membrane (PM) of smooth muscle cells hosts channel molecules regulating the flow of various ions. An intact architecture of PM is essential to orchestrate proper channel functions in order to complete agonist-mediated contraction, which includes Ca2+ release from the sarcoplasmic reticulum (SR) to initiate contraction, and subsequent Ca2+ refilling into SR through PM to sustain muscle contraction. The Junctional Complex (JC), comprising of junctional SR, and its apposing PM and neighboring caveolae, provides a quasi-enclosed microdomain housing receptors as well as ion channels and also restricting ion diffusions into the cytosol so the cell achieves optimal performance. The spatial arrangement of the JC is believed to ensure an uninterrupted Ca2+ cycling route. Full understanding of the functional role of the JC is the key to elucidating the contractile mechanisms of vascular smooth muscle and the physiological function of vessel contraction. The JC can be further divided into two sub-divisions, namely the PM-SR and caveolar regions. Previously, we demonstrated the role of the PM-SR region in the initiation of muscle contraction using pharmacological tools on the inferior vena cava (IVC) of rabbit. In the current study, we further dissected the caveolar region using a cholesterol-disrupting agent to investigate the role of the caveolar region. We conclude that disruption of the caveolar region in rabbit IVC smooth muscle results in augmented muscle contraction in response to adrenergic stimulation and the altered Ca2+ signaling may underlie the augmented contractility. Anat Rec, 302:186-192, 2019. © 2018 Wiley Periodicals, Inc.

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

Serum electrolyte levels may be associated with prefrontal hemodynamic responses in near infrared spectroscopy

Neuronal activity is tightly coordinated with blood flow to ensure proper brain function. This is achieved by neurovascular coupling. Although this mechanism is maintained by numerous mediators such as nitric oxide, ion channels, and astrocytes, its signaling process is much more complex. Frontal-temporal brain activity as measured by near infrared (NIR) spectroscopy has been suggested to be decreased or disturbed in many psychiatric disorders. To investigate the relationship between serum electrolyte levels and prefrontal hemodynamic responses, we evaluated psychiatric patients who underwent NIR spectroscopy with a 22-channel topography system and measurement of serum electrolyte levels. Changes in the concentration of oxygenated hemoglobin were evaluated during a verbal fluency test. The data were analyzed to see any significant correlation between NIR spectroscopy indices (integral value: size of an area during activation, centroid value: centroid time of an area throughout the task, and initial value: axis assessed by an initial change) and serum electrolyte levels (sodium, potassium, and chloride) by gender and cortex region. The study population consisted of 102 males (mean age, 42.4 years) and 133 females (mean age, 47.2 years). Sodium levels were significantly positively correlated with integral values at temporal regions in female subjects, while potassium levels were significantly negatively correlated with initial values at frontal regions in male subjects. However, chloride levels showed no correlations. In conclusion, higher serum sodium levels increase and higher serum potassium levels decrease regional cerebral blood flow with gender variances in psychiatry. The effect of extracellular electrolyte concentrations on hemodynamic responses remains unverified. However, our findings suggest that serum electrolytes levels may be involved in vascular smooth muscle contractility via Na+-K+-ATPase pumps that mediate neurovascular coupling.

Age-related focal loss of contractile vascular smooth muscle cells in retinal arterioles is accelerated by caveolin-1 deficiency.

Cerebral microcirculation is critical for the preservation of brain health, and vascular impairment is associated with age-related neurodegenerative diseases. Because the retina is a component of the central nervous system, cellular changes that occur in the aging retina are likely relevant to the aging brain, and the retina provides the advantage that the entire vascular bed is visible, en face. In this study, we tested the hypothesis that normal, healthy aging alters the contractile vascular smooth muscle cell (VSMC) coverage of retinal arterioles. We found that aging results in a significant reduction of contractile VSMCs in focal patches along arterioles. Focal loss of contractile VSMCs occurs at a younger age in mice deficient in the senescence-associated protein, caveolin-1. Age-related contractile VSMC loss is not exacerbated by genetic depletion of insulin-like growth factor-1. The patchy loss of contractile VSMCs provides a cellular explanation for previous clinical studies showing focal microirregularities in retinal arteriolar responsiveness in healthy aged human subjects and is likely to contribute to age-related retinal vascular complications.

Biomimetic soft fibrous hydrogels for contractile and pharmacologically responsive smooth muscle

The ability to assess changes in smooth muscle contractility and pharmacological responsiveness in normal or pathological-relevant vascular tissue environments is critical to enable vascular drug discovery. However, major challenges remain in both capturing the complexity of in vivo vascular remodeling and evaluating cell contractility in complex, tissue-like environments. Herein, we developed a biomimetic fibrous hydrogel with tunable structure, stiffness, and composition to resemble the native vascular tissue environment. This hydrogel platform was further combined with the combinatory protein array technology as well as advanced approaches to measure cell mechanics and contractility, thus permitting evaluation of smooth muscle functions in a variety of tissue-like microenvironments. Our results demonstrated that biomimetic fibrous structure played a dominant role in smooth muscle function, while the presentation of adhesion proteins co-regulated it to various degrees. Specifically, fibre networks enabled cell infiltration and upregulated expression of actomyosin proteins in contrast to flat hydrogels. Remarkably, fibrous structure and physiologically relevant stiffness of hydrogels cooperatively enhanced smooth muscle contractility and pharmacological responses to vasoactive drugs at both the single cell and intact tissue levels. Together, this study is the first to demonstrate alterations of human vascular smooth muscle contractility and pharmacological responsiveness in biomimetic soft, fibrous environments with a cellular array platform. The integrated platform produced here could enable investigations for pathobiology and pharmacological interventions by developing a broad range of patho-physiologically relevant in vitro tissue models. Statement of SignificanceEngineering functional smooth muscle in vitro holds the great potential for diseased tissue replacement and drug testing. A central challenge is recapitulating the smooth muscle contractility and pharmacological responses given its significant phenotypic plasticity in response to changes in environment. We present a biomimetic fibrous hydrogel with tunable structure, stiffness, and composition that enables the creation of functional smooth muscle tissues in the native-like vascular tissue microenvironment. Such fibrous hydrogel is further combined with the combinatory protein array technology to construct a cellular array for evaluation of smooth muscle phenotype, contraction, and cell mechanics. The integrated platform produced here could be promising for developing a broad range of normal or diseased in vitro tissue models.

Effect of aging on Rho‐kinase activity and vascular smooth muscle contractility in skeletal muscle resistance arteries

IntroductionVasoconstrictor responses decline with age in resistance arteries. The mechanism for the decrement in constrictor function is not fully understood but has been proposed to involve signaling through the RhoA pathway.ObjectiveWe tested whether aging affects Rho kinase (ROCK) activity in aged resistance arteries.MethodsSoleus feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. Smooth muscle cells isolated from one group of SFA were lysed and ELISA assay was used for assessment of both ROCK isoforms, total ROCK 1 and 2, and their activity, phospho ROCK 1 and 2. A separate group of young and old SFA was cannulated with glass micropipettes for assessment of vasoconstrictor function. To isolate the role of smooth muscle in constrictor responses, endothelial cells were removed from cannulated arteries (denuded) by passing 5 ml of air through the lumen of the artery. Vasoconstrictor responses were assessed using increasing whole log doses of norepinephrine (NE; 10−9–10−4 M), angiotensin II (Ang II; 10−11–10−7 M), and phenylephrine (PE; 10−9–10−4 M).ResultsTotal ROCK 1 and ROCK 2 was similar in cells isolated from young and old SFA, whereas phospho‐ROCK 1 and phospho‐ROCK 2 were higher in cells isolated from old SFA relative to young arteries. Constrictor responses of denuded SFA to PE and Ang II were significantly impaired in old SFA relative to young SFA whereas constrictor responses to NE were preserved. In addition, presence of a ROCK inhibitor, Y 27632, reduced further the constrictor response to agonists.ConclusionThe results of this study indicate that smooth muscle contractile function declines with age in SFA. Contractility regulation involves the ROCK pathway, but upregulation of ROCK activity in old arteries is not able to rescue the reduced contractility state in aging.Support or Funding InformationAHA grant 0765043Y, AHA grant 4150031, and Sydney and J.L. Huffines Institute of Sports Medicine and Human Performance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein kinase Cα deletion causes hypotension and decreased vascular contractility

Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.

Regulation of voltage-gated potassium channels in vascular smooth muscle during hypertension and metabolic disorders.

Voltage-gated potassium (KV ) channels are key regulators of vascular smooth muscle contractility and vascular tone, and thus have major influence on the microcirculation. KV channels are important determinants of vascular smooth muscle membrane potential (Em ). A number of KV subunits are expressed in the plasma membrane of smooth muscle cells. Each subunit confers distinct kinetics and regulatory properties that allow for fine control of Em to orchestrate vascular tone. Modifications in KV subunit expression and/or channel activity can contribute to changes in vascular smooth muscle contractility in response to different stimuli and in diverse pathological conditions. Consistent with this, a number of studies suggest alterations in KV subunit expression and/or function as underlying contributing mechanisms for small resistance artery dysfunction in pathologies such as hypertension and metabolic disorders, including diabetes. Here, we review our current knowledge on the effects of these pathologies on KV channel expression and function in vascular smooth muscle cells, and the repercussions on (micro)vascular function.

Differential effects of ageing on porcine arterial vascular smooth muscle contractility and the role of the Rho-kinase pathway

Differential effects of ageing on porcine arterial vascular smooth muscle contractility and the role of the Rho-kinase pathway

Abstract P499: Protein Kinase Cα Deletion Causes Hypotension Due to Decreased Vascular Contractility

Protein kinase Cα (PKCα) regulates multiple cell signaling pathways, including those that impact blood pressure. PKCα activation increases vascular smooth muscle contractility, yet reduces cardiac contractility. PKCα has also been shown to modulate nephron ion transport. We have shown that PKCα deletion leads to hypotension, with compensatory increases in sodium retention. Here, we hypothesized that PKCα deficiency reduces vascular contractility, leading to decreased mean arterial pressure (MAP). MAP, measured by telemetry, was decreased in PKC KO (≈12 mmHg) compared to PKC control (PKC CTL) mice. Aorta and mesenteric arteries were isolated, and concentration response curves (CRCs) to phenylephrine (Phe), acetylcholine (ACh) or sodium nitroprusside (SNP) were performed in the presence of vehicle or the following inhibitors: L-NAME or indomethacin (NOS, COX inhibitor, resp. ). CRCs to KCL were performed to assess receptor-independent vascular responses. In aorta, we observed a striking reduction in KCl-mediated contraction (5.8±0.3mN vs. 10.4±1.1mN control, **p<0.01). PKC KO aorta and mesenteric arteries had decreased contractile responses to Phe, as compared to control (aorta, 12.7±0.5mN R max vs. 16.3±0.5mN R max , and mesenteric 9.9±0.3mN R max vs. 11.8±0.6mN R max ; n=4, **p<0.01), revealing a role for reduced vascular contractility. Endothelium-mediated relaxation responses to ACh were also increased in PKC KO mice, as compared to control (59.3±6.8% R max vs. 45.4±3.2% R max , n=4, *p<0.05). Interestingly, NOS inhibition increased contractility in mesenteric arteries from PKC KO mice (8.55±2.65mN R max vs. 6.95±0.39mN R max control, n=4, ***p<0.001). However, PKC KO aorta had an enhanced response to COX inhibition (12.2±0.7mN R max vs. 10.1±0.6mN R max control, n=4, *p<0.05) suggesting that PKCα may be negatively regulating NOS in mesenteric arteries, and COX-mediated prostaglandin production in the aorta. No differences were observed in the relaxation responses to SNP. These data suggest that global deletion of PKCα results in hypotension due to decreased vascular contractility, and loss of PKCα-mediated inhibition of endothelial relaxing factors. Thus, systemic targeting of PKCα may be beneficial for the reduction of MAP.

Regulation of Pulmonary Vascular Smooth Muscle Contractility in Pulmonary Arterial Hypertension: Implications for Therapy.

There are two primary components that produce pulmonary arterial hypertension (PAH); aberrant structural changes (smooth muscle cell proliferation, smooth muscle cell hypertrophy, and the deposition of matrix proteins within the media of pulmonary arterial vessels), and excess vasoconstriction. However, in PAH, the target and aim of all current therapeutic agents is to reduce the contractility of the pulmonary vasculature; prostaglandins, phosphodiesterase inhibitors, guanylate cyclase stimulators, endothelin antagonists, NO inhalation and Rho kinase inhibitors all influence signaling pathways in the pulmonary vascular smooth muscle to decrease vasoconstriction, and hence, pulmonary vascular resistance (PVR). This review will therefore primarily focus on discussing the signaling pathways regulating contractility in pulmonary vascular smooth muscle, the mechanism for current treatments, as well as highlighting potential targets for the development of novel therapies.