Artery Myogenic Tone Research Articles

Cryo-EM investigation of ryanodine receptor type 3.

Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in dendritic spines and regulates synaptic plasticity. It controls myogenic tone in arteries and is upregulated in skeletal muscle in early development. RyR3 has a unique functional profile with a very high sensitivity to activating ligands, enabling high gain in Ca2+-induced Ca2+ release. Here we solve high-resolution cryo-EM structures of RyR3 in non-activating and activating conditions, revealing structural transitions that occur during channel opening. Addition of activating ligands yields only open channels, indicating an intrinsically high open probability under these conditions. RyR3 has reduced binding affinity to the auxiliary protein FKBP12.6 due to several sequence variations in the binding interface. We map disease-associated sequence variants and binding sites for known pharmacological agents. The N-terminal region contains ligand binding sites for a putative chloride anion and ATP, both of which are targeted by sequence variants linked to epileptic encephalopathy.

CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries

The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca2+] is a key determinant of constriction, tied to L-type (CaV1.2) Ca2+ channels. While important, other Ca2+ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca2+ channel (CaV3.1) using C57BL/6 wild type and CaV3.1−/− mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1−/− phenotype relative to C57BL/6. CaV3.1−/− mice had absent CaV3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20–60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca2+ wave generation, asynchronous events of Ca2+ release from the sarcoplasmic reticulum, insensitive to L-type Ca2+ channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP3R1/CaV3.1 close physical association. IP3R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a CaV3.1−/− contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.

Fentanyl disrupts autoregulation and leads to exacerbated vasodilation in resistance arteries independent of mu-opioid receptor activation

According to the CDC, 107,375 people in the United States died of drug overdoses and poisonings in the 12-month period ending in January 2022 with 67% involving synthetic opioids like fentanyl. Recent studies have implicated the use of prescription opioids as a risk factor for cardiovascular diseases (CVD), with reported associations including myocardial infarction, coronary artery disease and vascular deterioration. With small doses of fentanyl being particularly dangerous to those who have little to no tolerance, our primary hypothesis was that acute exposure to fentanyl will abolish myogenic activity and induce exacerbated vasodilation. While the mu-opioid receptor (MOR) serves as the primary target for exogenous opioids, it has been shown that the endogenous opioids like spinorphin can induce calcium flux in neutrophils via the N-formyl peptide receptor (FPR-1). With this premise, our second hypothesis was that the vasodilatory effects of fentanyl were in part contributed by inhibition of FPR1. To test these hypotheses, mesenteric resistance arteries were isolated from 12-13-week-old male C57BL/6J mice and vascular function evaluated. Myogenic tone was evaluated via a pressure-response curve (10-140 mmHg) after a 30-minute incubation with fentanyl 10 -6 M. Contractility was evaluated via concentration-response curves to phenylephrine (Phe) in arteries pretreated with: Fentanyl 10 -6 M (Fen), Naloxone 10 -5 M (Nal, MOR antagonist), Naloxone 10 -5 M+Fentanyl 10 -6 M (Nal+Fen), Cyclosporin H 10 -5 M (Cyclo, FPR-1 antagonist), or Cyclosporin H 10 -5 M+Fentanyl 10 -6 M (Cyclo+Fen). Arteries were incubated with antagonists 15 minutes prior to a 30-minute fentanyl incubation. Control (Cont) levels were established prior to any incubations. Pressure data were analyzed using student t test and contractility data using one way anova (p<0.05* vs Cont). Fen abolished myogenic tone in arteries when compared to Cont at 40 mmHg (Cont 7.8±2.2 vs Fen 1.5±1.5*, %, n=3). Further, Fen significantly decreased contraction when compared to Cont [Area under the curve (AUC): Cont 54±5 vs. Fen 15±3 n=4*]. However, the presence of the antagonists for FPR-1 [AUC: Fen 15±3 vs. Cyclo+Fen 12 ±2, n=4, p>0.05) or mu-opioid receptor (AUC: Fen 15±3, vs. Nal+Fen 13±2, n=4, p>0.05) did not change these responses. The presence of the antagonists alone decreased contraction. This suggests a constitutive effect of FPR-1 or mu-opioid receptor on Phe-induced contractility (AUC: Cont 54±5 vs. Nal 32±10* vs. Cyclo 17±2*). Overall, our data suggest that acute treatments of Fentanyl lead to a significant disruption of autoregulation and exacerbated vasodilation in resistance arteries independent of mu and FPR-1 receptors. Therefore, chronic exposure to this opioid may lead to dysfunction and loss of myogenic tone and, subsequently, CVD. R00GM118885, R01HL149762, R00HL151889, and NHLBI (R00HL151889) This is the full abstract presented at the American Physiology Summit 2023 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.

MicroRNA‐210‐mediated mtROS confer hypoxia‐induced suppression of STOCs in ovine uterine arteries

Background and PurposeHypoxia during pregnancy is associated with increased uterine vascular resistance and elevated blood pressure both in women and female sheep. A previous study demonstrated a causal role of microRNA‐210 (miR‐210) in gestational hypoxia‐induced suppression of Ca2+ sparks/spontaneous transient outward currents (STOCs) in ovine uterine arteries, but the underlying mechanisms remain undetermined. We tested the hypothesis that miR‐210 perturbs mitochondrial metabolism and increases mitochondrial reactive oxygen species (mtROS) that confer hypoxia‐induced suppression of STOCs in uterine arteries.Experimental ApproachResistance‐sized uterine arteries were isolated from near‐term pregnant sheep and were treated ex vivo in normoxia and hypoxia (10.5% O2) for 48 h.Key ResultsHypoxia increased mtROS and suppressed mitochondrial respiration in uterine arteries, which were also produced by miR‐210 mimic to normoxic arteries and blocked by antagomir miR‐210‐LNA in hypoxic arteries. Hypoxia or miR‐210 mimic inhibited Ca2+ sparks/STOCs and increased uterine arterial myogenic tone, which were inhibited by the mitochondria‐targeted antioxidant MitoQ. Hypoxia and miR‐210 down‐regulated iron–sulfur cluster scaffold protein (ISCU) in uterine arteries and knockdown of ISCU via siRNAs suppressed mitochondrial respiration, increased mtROS, and inhibited STOCs. In addition, blockade of mitochondrial electron transport chain with antimycin and rotenone inhibited large‐conductance Ca2+‐activated K+ channels, decreased STOCs and increased uterine arterial myogenic tone.Conclusion and ImplicationsThis study demonstrates a novel mechanistic role for the miR‐210‐ISCU‐mtROS axis in inhibiting Ca2+ sparks/STOCs in the maladaptation of uterine arteries and provides new insights into the understanding of mitochondrial perturbations in the pathogenesis of pregnancy complications resulted from hypoxia.

The heat is on! TRPV1 channels and resistance artery myogenic tone.

The heat is on! TRPV1 channels and resistance artery myogenic tone.

Role of TRPC6 in kidney damage after acute ischemic kidney injury

Transient receptor potential channel subfamily C, member 6 (TRPC6), a non-selective cation channel that controls influx of Ca2+ and other monovalent cations into cells, is widely expressed in the kidney. TRPC6 gene variations have been linked to chronic kidney disease but its role in acute kidney injury (AKI) is unknown. Here we aimed to investigate the putative role of TRPC6 channels in AKI. We used Trpc6−/− mice and pharmacological blockade (SH045 and BI-749327), to evaluate short-term AKI outcomes. Here, we demonstrate that neither Trpc6 deficiency nor pharmacological inhibition of TRPC6 influences the short-term outcomes of AKI. Serum markers, renal expression of epithelial damage markers, tubular injury, and renal inflammatory response assessed by the histological analysis were similar in wild-type mice compared to Trpc6−/− mice as well as in vehicle-treated versus SH045- or BI-749327-treated mice. In addition, we also found no effect of TRPC6 modulation on renal arterial myogenic tone by using blockers to perfuse isolated kidneys. Therefore, we conclude that TRPC6 does not play a role in the acute phase of AKI. Our results may have clinical implications for safety and health of humans with TRPC6 gene variations, with respect to mutated TRPC6 channels in the response of the kidney to acute ischemic stimuli.

Modulation of p66Shc impairs cerebrovascular myogenic tone in low renin but not low nitric oxide models of systemic hypertension.

Cerebral blood flow and perfusion are tightly maintained through autoregulation despite changes in transmural pressure. Oxidative stress impairs cerebral blood flow, precipitating cerebrovascular events. Phosphorylation of the adaptor protein p66Shc increases mitochondrial-derived oxidative stress. The effect of p66Shc gain or loss of function in nonhypertensive rats is unclear. We hypothesized that p66Shc gain of function would impair autoregulation of cerebral microcirculation under physiological and pathological conditions. Three previously established transgenic [salt-sensitive (SS) background] p66Shc rats were used, p66-Del/SS (express p66Shc with a nine-amino acid deletion), p66Shc-knockout (KO)/SS (frameshift premature termination codon), and p66Shc signaling and knock-in substitution of Ser36Ala (p66Shc-S36A)/SS (substitution of Ser36Ala). The p66Shc-Del were also bred on Sprague-Dawley (SD) backgrounds (p66-Del/SD), and a subset was exposed to a hypertensive stimulus [NG-nitro-l-arginine methyl ester (l-NAME)] for 4 wk. Active and passive diameters to increasing transmural pressure were measured and myogenic tone was calculated in all groups (SS and SD). Myogenic responses to increasing pressure were impaired in p66Shc-Del/SS rats relative to wild-type (WT)/SS and knock-in substitution of Ser36Ala (S36A; P < 0.05). p66-Del/SD rats did not demonstrate changes in active/passive diameters or myogenic tone relative to WT/SD but did demonstrate attenuated passive diameter responses to higher transmural pressure relative to p66-Del/SS. Four weeks of a hypertensive stimulus (l-NAME) did not alter active or passive diameter responses to increasing transmural pressure (P = 0.86-0.99), but increased myogenic responses relative to p66-Del/SD (P < 0.05). Collectively, we demonstrate the functional impact of p66Shc within the cerebral circulation and demonstrate that the genetic background of p66Shc rats largely drives changes in cerebrovascular function.NEW & NOTEWORTHY We demonstrate that the modulation of p66Shc signaling impairs cerebral artery myogenic tone in a low renin model of hypertension. This impairment is dependent upon the genetic background, as modulated p66Shc signaling in Sprague-Dawley rats does not impair cerebral artery myogenic tone.

Traumatic Brain Injury Alters PAQR7‐induced Vasorelaxation in Pressurized Cerebral Arteries

Traumatic brain injury (TBI) induces acute changes in cerebrovascular function, including alterations to cerebral artery contractility, which ultimately increase the incidence of both ischemic and hemorrhagic events. Cerebrovascular tone is controlled by hormonal, neurogenic, endothelial, and myogenic responses. Progesterone, a steroid hormone, has been evaluated as a potential treatment to improve TBI outcome; however, progesterone has failed to translate to patient benefits in clinical trials, potentially due to its pleiotropic effects on multiple receptors. Progestin &amp; adipoQ receptor 7 (PAQR7) is a plasma membrane progesterone receptor involved in the vasorelaxation of human blood vessels. However, little is known about the smooth muscle‐specific role of PAQR7 in resistance size cerebral arteries following TBI. Here, we examined PAQR7 function in pressurized endothelium‐denuded cerebral resistance size arteries 24 hours following moderately severe TBI (controlled cortical impact). Baseline myogenic tone of middle cerebral arteries (MCA) was attenuated following TBI. PAQR7‐induced vasorelaxation in pressurized MCA from TBI rats was attenuated in a concentration‐dependent manner as compared to sham controls (10−9M‐10−7M). Moreover, pre‐incubation with inhibitors of adenylyl cyclase (AC), PKA, or PKG attenuated PAQR7‐induced vasorelaxation in both groups. In conclusion, smooth muscle‐specific PAQR7 vasodilation involves an AC‐PKA/PKG pathway in cerebral resistance size arteries, and moderately severe TBI attenuates cerebral artery myogenic tone, which reduces overall arterial vasodilatory capacity.Support or Funding InformationSupported by The Florida State University Graduate School Dissertation Award Grant.

Enhanced Vascular Smooth Muscle Calcium Sensitivity and Loss of Endothelial Vasodilator Influence Contribute to Myogenic Tone Development in Rat Radial Uterine Arteries during Gestation

Uterine artery myogenic tone (MT) develops during pregnancy in hemochorial placentates such as rats and humans. The physiological reason for its appearance is not clear, and we reasoned that it may be a late pregnancy (LP) event in preparation for controlling hemorrhage during parturition. We also hypothesized that gestational increases in RhoA-induced vascular smooth muscle (VSM) calcium sensitivity are contributory and occur under the tonic influence of nitric oxide (NO). Second-order pre-placental radial arteries from early-pregnant (day 12, n = 5), mid-pregnant (day 16, n = 5) and LP (day 20, n = 20) rats were used in combination with arteriography, VSM calcium measurements, pharmacological RHO/Rho-associated protein kinase (ROCK) and nitric oxide synthase (NOS) inhibition, and Western blotting. A subgroup of LP animals (LP + LN; n = 5) treated with L-NAME from gestational days 10 to 20 were used to determine the effects of NOS inhibition on MT and RhoA expression. MT was evident throughout pregnancy, but its expression in pressurized vessels was masked by endothelial NO-induced vasodilation during early gestation. RhoA protein expression was upregulated in LP and attenuated by in vivo NOS inhibition (as was MT). In vitro RHO/ROCK inhibition decreased MT in a concentration-dependent manner without reducing VSM calcium. In summary, pressure-dependent uterine artery tone increases with gestational age due to a combination of RhoA-mediated increases in VSM calcium sensitivity and a loss of endothelial NO influence.

Increased TRPM4 Activity in Cerebral Artery Myocytes Contributes to Cerebral Blood Flow Reduction After Subarachnoid Hemorrhage in Rats.

Cerebral blood flow (CBF) reduction underlies unfavorable outcomes after subarachnoid hemorrhage (SAH). Transient receptor potential melastatin-4 (TRPM4) has a pivotal role in cerebral artery myogenic tone maintenance and CBF regulation under physiological conditions. However, the role of TRPM4 in CBF reduction after SAH is unclear. In this study, we aimed at testing whether TRPM4 would contribute to CBF reduction after SAH in vivo and determining underlying mechanisms. Rat SAH model was established by stereotaxic injection of autologous nonheparinized arterial blood at the suprasellar cistern. A TRPM4 blocker, 9-phenanthrol (9-Phe), was infused through an intraventricular catheter connected to a programmed subcutaneous pump to evaluate the contribution of TRPM4 to SAH outcomes. TRPM4 expression and translocation in cerebral artery myocytes were detected by immunoblotting. Macroscopic currents in cerebral artery myocytes were determined by whole-cell patch clamp. Myogenic tone of cerebral arteries was studied by pressurized myography. Cortical and global CBFs were measured via laser Doppler flowmetry and fluorescent microspheres, respectively. After SAH, TRPM4 translocation and macroscopic current density increased significantly. Furthermore, TRPM4 accounted for a greater proportion of myogenic tone after SAH, suggesting an upregulation of TRPM4 activity in response to SAH. Cortical and global CBFs were reduced after SAH, but were restored significantly by 9-Phe, implying that TRPM4 contributed to CBF reduction after SAH. Collectively, these discoveries show that increased TRPM4 activity has a pivotal role in CBF reduction after SAH, and provide a novel target for the management of cerebral perfusion dysfunction following SAH.

Traumatic Brain Injury Alters BK Channel Subunit Expression, Localization, and Function in Cerebral Arterial Myocytes

Traumatic brain injury (TBI) induces acute changes in cerebrovascular function, which include alterations to cerebral artery myogenic tone and autoregulation. In arterial smooth muscle cells (myocytes), large‐conductance calcium‐activated potassium (BK) channels play a vital role in modulating plasma membrane potential and myogenic tone. Here, we examined BK channel expression and function in cerebral resistance size arteries one week following moderately‐severe TBI induced by controlled cortical impact with craniotomy. Total BKα (pore‐forming) and β1 (regulatory) subunit expression were reduced one week post‐TBI. Biotinylation of plasma membrane‐bound proteins revealed a compensatory increase of β1 subunits to the plasma membrane following TBI, which normalized single BK channel activation as assessed using patch‐clamp electrophysiology. However, the loss of total BKα subunits resulted in diminished whole cell, transient BK currents. Pressurized cerebral arteries from TBI rats exhibited greater myogenic tone at 60 mmHg and a diminished response to iberiotoxin, a BK channel pore blocker. In conclusion, moderately‐severe TBI enhances cerebral artery contractility, in part, through reductions in arterial myocyte BK channel subunit expression and function. Moreover, these TBI‐induced alterations in cerebral artery function can persist well beyond the initial 24–48 hour period post‐injury.Support or Funding InformationFlorida State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pregnancy Increases Ca2+ Sparks/Spontaneous Transient Outward Currents and Reduces Uterine Arterial Myogenic Tone.

Spontaneous transient outward currents (STOCs) at physiological membrane potentials of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ. We hypothesize that heightened STOCs play a key role in uterine vascular adaptation to pregnancy. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Ca2+ sparks were measured by confocal microscopy, and STOCs were determined by electrophysiological recording in smooth muscle cells. Percentage of Ca2+ spark firing myocytes increased dramatically at the resting condition in uterine arterial smooth muscle of pregnant animals, as compared with nonpregnant animals. Pregnancy upregulated the expression of RyRs (ryanodine receptors) and significantly boosted Ca2+ spark frequency. Ex vivo treatment of uterine arteries of nonpregnant sheep with estrogen and progesterone imitated pregnancy-induced RyR upregulation. STOCs occurred at much more negative membrane potentials in uterine arterial myocytes of pregnant animals. STOCs in uterine arterial myocytes were diminished by inhibiting large-conductance Ca2+-activated K+ (BKCa) channels and RyRs, thus functionally linking Ca2+ sparks and BKCa channel activity to STOCs. Pregnancy and steroid hormone treatment significantly increased STOCs frequency and amplitude in uterine arteries. Of importance, inhibition of STOCs with RyR inhibitor ryanodine eliminated pregnancy- and steroid hormone-induced attenuation of uterine arterial myogenic tone. Thus, the present study demonstrates a novel role of Ca2+ sparks and STOCs in the regulation of uterine vascular tone and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Relaxin Deficiency Leads to Uterine Artery Dysfunction During Pregnancy in Mice.

The uterine vasculature undergoes profound adaptations in response to pregnancy. Augmentation of endothelial vasodilator function and reduced smooth muscle reactivity are factors contributing to uterine artery adaptation and are critical for adequate placental perfusion. The peptide hormone relaxin has an important role in mediating the normal maternal renal vascular adaptations during pregnancy through a reduction in myogenic tone and an increase in flow-mediated vasodilation. Little is known however about the influence of endogenous relaxin on the uterine artery during pregnancy. We tested the hypothesis that relaxin deficiency increases myogenic tone and impairs endothelial vasodilator function in uterine arteries of late pregnant relaxin deficient (Rln−/−) mice. Reactivity of main uterine arteries from non-pregnant and late pregnant wild-type (Rln+/+) and Rln−/− mice was studied using pressure and wire myography and changes in gene expression explored using PCR. Myogenic tone was indistinguishable in arteries from non-pregnant mice. In late pregnancy uterine artery myogenic tone was halved in Rln+/+ mice (P < 0.0001), an adaptation that failed to occur in arteries from pregnant Rln−/− mice. The role of vasodilator prostanoids in the regulation of myogenic tone was significantly reduced in arteries of pregnant Rln−/− mice (P = 0.02). Agonist-mediated endothelium-dependent vasodilation was significantly impaired in non-pregnant Rln−/− mice. With pregnancy, differences in total endothelial vasodilator function were resolved, although there remained an underlying deficiency in the role of vasodilator prostanoids and alterations to the contributions of calcium-activated K+ channels. Fetuses of late pregnant Rln−/− mice were ~10% lighter (P < 0.001) than those of Rln+/+ mice. In conclusion, relaxin deficiency is associated with failed suppression of uterine artery myogenic tone in pregnancy, which likely contributes to reduced uteroplacental perfusion and fetal growth restriction.

Short-term (48hours) intravenous serelaxin infusion has no effect on myogenic tone or vascular remodeling in rat mesenteric arteries.

Short-term IV sRLX (recombinant human relaxin-2) infusion enhances endothelium-dependent relaxation in mesenteric arteries. This is initially underpinned by increased NO followed by a transition to prostacyclin. The effects of short-term IV sRLX treatment on pressure-induced myogenic tone and vascular remodeling in these arteries are unknown. Therefore, we investigated the effects of sRLX infusion on pressure-induced myogenic tone and passive mechanical wall properties in mesenteric arteries. Mesenteric artery myogenic tone and passive mechanics were examined after 48-hours and 10-days infusion of sRLX. Potential mechanisms of action were assessed by pressure myography, qPCR, and Western blot analysis. Neither 48-hours nor 10-days sRLX treatment had significant effects on myogenic tone, passive arterial wall stiffness, volume compliance, or axial lengthening. However, in 48-hours sRLX -treated rats, incubation with the NO synthase blocker L-NAME significantly increased myogenic tone (P<.05 vs placebo), demonstrating an increased contribution of NO to the regulation of myogenic tone. eNOS dimerization, but not phosphorylation, was significantly upregulated in the arteries of sRLX -treated rats. In mesenteric arteries, 48-hours sRLX treatment upregulates the role of NO in the regulation of myogenic tone by enhancing eNOS dimerization, without altering overall myogenic tone or vascular remodeling.

Pulse pressure-dependent cerebrovascular eNOS regulation in mice

Arterial blood pressure is oscillatory; whether pulse pressure (PP) regulates cerebral artery myogenic tone (MT) and endothelial function is currently unknown. To test the impact of PP on MT and dilation to flow (FMD) or to acetylcholine (Ach), isolated pressurized mouse posterior cerebral arteries were subjected to either static pressure (SP) or a physiological PP (amplitude: 30 mm Hg; frequency: 550 bpm). Under PP, MT was significantly higher than in SP conditions (p < 0.05) and was not affected by eNOS inhibition. In contrast, under SP, eNOS inhibition increased (p < 0.05) MT to levels observed under PP, suggesting that PP may inhibit eNOS. At a shear stress of 20 dyn/cm2, FMD was lower (p < 0.05) under SP than PP. Under SP, eNOS-dependent production contributed to FMD, while under PP, eNOS-dependent NO was responsible for FMD, indicating that PP favours eNOS coupling. Differences in FMD between pressure conditions were abolished after NOX2 inhibition. In contrast to FMD, Ach-induced dilations were higher (p < 0.05) under SP than PP. Reactive oxygen species scavenging reduced (p < 0.05) Ach-dependent dilations under SP, but increased (p < 0.05) them under PP; hence, under PP, Ach promotes ROS production and limits eNOS-derived NO activity. In conclusion, PP finely regulates eNOS, controlling cerebral artery reactivity.

Tumor Necrosis Factor/Sphingosine-1-Phosphate Signaling Augments Resistance Artery Myogenic Tone in Diabetes

Diabetes strongly associates with microvascular complications that ultimately promote multiorgan failure. Altered myogenic responsiveness compromises tissue perfusion, aggravates hypertension, and sets the stage for later permanent structural changes to the microcirculation. We demonstrate that skeletal muscle resistance arteries isolated from patients with diabetes have augmented myogenic tone, despite reasonable blood glucose control. To understand the mechanisms, we titrated a standard diabetes mouse model (high-fat diet plus streptozotocin [HFD/STZ]) to induce a mild increase in blood glucose levels. HFD/STZ treatment induced a progressive myogenic tone augmentation in mesenteric and olfactory cerebral arteries; neither HFD nor STZ alone had an effect on blood glucose or resistance artery myogenic tone. Using gene deletion models that eliminate tumor necrosis factor (TNF) or sphingosine kinase 1, we demonstrate that vascular smooth muscle cell TNF drives the elevation of myogenic tone via enhanced sphingosine-1-phosphate (S1P) signaling. Therapeutically antagonizing TNF (etanercept) or S1P (JTE013) signaling corrects this defect. Our investigation concludes that vascular smooth muscle cell TNF augments resistance artery myogenic vasoconstriction in a diabetes model that induces a small elevation of blood glucose. Our data demonstrate that microvascular reactivity is an early disease marker and advocate establishing therapies that strategically target the microcirculation.

RGS2 squelches vascular Gi/o and Gq signaling to modulate myogenic tone and promote uterine blood flow.

Uterine artery blood flow (UABF) is critical to maintaining uterine perfusion in nonpregnant states and for uteroplacental delivery of nutrients and oxygen to the fetus during pregnancy. Impaired UABF is implicated in infertility and several pregnancy complications including fetal growth restriction, small for gestational age, and preeclampsia. The etiology of abnormal UABF is not known. Here, we determined whether deficiency or loss of RGS2, a GTPase-activating protein for Gq/11 and Gi/o class G proteins, affects UABF in nonpregnant mice. We used Doppler ultrasonography to assess UABF in wild type (WT), Rgs2 heterozygous (Rgs2+/-), and homozygous knockout (Rgs2-/-) mice. Video microscopy was used for exvivo examination of uterine artery myogenic tone and fura-2 imaging for invitro assessment of internal stores Ca(2+) release. We found that baseline UABF velocity was markedly decreased while impedance measured as resistive index (WT=0.58±0.04 vs. Rgs2-/-=0.71±0.03, P<0.01) and pulsatile index (WT=0.90±0.06 vs. Rgs2-/-=1.25±0.11, P<0.01) was increased in Rgs2-/- mice. Uterine artery tone was augmented in Rgs2+/- and Rgs2-/- mice, which was normalized to WT levels following Gi/o and Gq inactivation. Conversely, blockade of ryanodine receptors increased WT myogenic tone to RGS2 mutant levels. The data together indicate that RGS2 deficiency decreases UABF by increasing myogenic tone at least partly through prolonged G protein activation. Mutations that decrease vascular RGS2 expression may be a predisposition to decreased uterine blood flow. Targeting G protein signaling therefore might improve uterine and uteroplacental underperfusion disorders.

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

Piezo1 in Smooth Muscle Cells Is Involved in Hypertension-Dependent Arterial Remodeling.

The mechanically activated non-selective cation channel Piezo1 is a determinant of vascular architecture during early development. Piezo1-deficient embryos die at midgestation with disorganized blood vessels. However, the role of stretch-activated ion channels (SACs) in arterial smooth muscle cells in the adult remains unknown. Here, we show that Piezo1 is highly expressed in myocytes of small-diameter arteries and that smooth-muscle-specific Piezo1 deletion fully impairs SAC activity. While Piezo1 is dispensable for the arterial myogenic tone, it is involved in the structural remodeling of small arteries. Increased Piezo1 opening has a trophic effect on resistance arteries, influencing both diameter and wall thickness in hypertension. Piezo1 mediates a rise in cytosolic calcium and stimulates activity of transglutaminases, cross-linking enzymes required for the remodeling of small arteries. In conclusion, we have established the connection between an early mechanosensitive process, involving Piezo1 in smooth muscle cells, and a clinically relevant arterial remodeling.

Intermittent hypoxia in rats reduces activation of Ca2+ sparks in mesenteric arteries.

Ca(+) sparks are vascular smooth muscle cell (VSMC) Ca(2+)-release events that are mediated by ryanodine receptors (RyR) and promote vasodilation by activating large-conductance Ca(2+)-activated potassium channels and inhibiting myogenic tone. We have previously reported that exposing rats to intermittent hypoxia (IH) to simulate sleep apnea augments myogenic tone in mesenteric arteries through loss of hydrogen sulfide (H2S)-induced dilation. Because we also observed that H2S can increase Ca(2+) spark activity, we hypothesized that loss of H2S after IH exposure reduces Ca(2+) spark activity and that blocking Ca(2+) spark generation reduces H2S-induced dilation. Ca(2+) spark activity was lower in VSMC of arteries from IH compared with sham-exposed rats. Furthermore, depolarizing VSMC by increasing luminal pressure (from 20 to 100 mmHg) or by elevating extracellular [K(+)] increased spark activity in VSMC of arteries from sham rats but had no effect in arteries from IH rats. Inhibiting endogenous H2S production in sham arteries prevented these increases. NaHS or phosphodiesterase inhibition increased spark activity to the same extent in sham and IH arteries. Depolarization-induced increases in Ca(2+) spark activity were due to increased sparks per site, whereas H2S increases in spark activity were due to increased spark sites per cell. Finally, inhibiting Ca(2+) spark activity with ryanodine (10 μM) enhanced myogenic tone in arteries from sham but not IH rats and blocked dilation to exogenous H2S in arteries from both sham and IH rats. Our results suggest that H2S regulates RyR activation and that H2S-induced dilation requires Ca(2+) spark activation. IH exposure decreases endogenous H2S-dependent Ca(2+) spark activation to cause membrane depolarization and enhance myogenic tone in mesenteric arteries.