bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of vascular smooth muscle contraction through Calcium‐dependent Calcium sensitization
Authors: Alvarez Silvina Monica , Miner Amy S , Ratz Paul H
Published: June 2021

We used Bay‐K‐8644 (Bay‐K), a calcium channel agonist, to examine basal and calcium‐stimulated levels of [Ca2+]i (Ca), force, myosin light chain phosphorylation (MLCp) and myosin phosphatase targeting subunit phosphorylation (MYPT1p) in the absence (control) and presence of inhibitors of ROCK (H‐1152) and MLCK (wortmannin). Also used was staurosporine (St), which inhibits ROCK, MLC kinase (MLCK) and integrin‐linked kinase (ILK) at 1μM and ZIP kinase (ZIPK) at 10µM. Bay‐K induced increases in Ca, MYPT1p‐T853, MLCp and force and not MYPT1p‐T696. A Ca‐free solution inhibited the increases in MYPT1‐pT853, MLCp and force. Wortmannin inhibited the increases in MLCp and force and not MYPT1p‐T853. H‐1152 inhibited the increases in MYPT1p‐T853, MLCp and force but not Ca. These data suggest that Bay‐K caused Ca sensitization by activating ROCK. H‐1152 (1 µM) reduced the basal level of MYPT1‐pT853 but not MLCp. Wortmannin and TFP did not reduce basal MYPT1p‐T853 nor basal MLCp. St (1µM) reduced basal MYPT1‐pT696, MYPT1‐pT853 and MLCp. These data support a model in which constitutive ILK provides basal MLCp through MYPT1p‐T696 induced MLCP inhibition, and increases in Ca can further increase MLCp by activating MLCK and inhibiting MLCP via activation of ROCK and increases in MYPT1‐T853. Constitutive ROCK activity and MYPT1‐pT853 do not appear to play a role in basal MLCp. Support: R01HL061320
Regulation of VSMC migration through association of SLP3 and ASIC2
Authors: McKey Elizabeth , Burford Matthew , Gannon Kimberly , Grifoni Samira , Drummond Heather
Published: June 2021

Previous studies from our laboratory indicate Acid‐Sensing Ion Channel 2 (ASIC2) inhibits chemotactic migration in vascular smooth muscle cells (VSMC). Function of certain ASIC proteins is regulated by stomatin like proteins (SLP), a family of channel‐to‐membrane linking proteins. The goal of the current study was to determine if ASIC2 associates with a specific SLP protein, SLP3, to regulate VSMC migration. In freshly dissociated VSMCs, SLP3 localizes with ASIC2 at the cell surface in infrequent clusters. To determine if SLP3 and ASIC2 biochemically associate, we transfected cultured VSMCs with a myc tagged SLP3 (mycSLP3) expression vector. Using immunoprecipitation, we found ASIC2 coimmunoprecipitates with SLP3 and vice versa. To determine if SLP3 regulates migration, we evaluated migration to platelet derived growth factor bb (PDGF‐bb) in mycSLP3 transfected cells and found SLP3 inhibits migration. Expression of SLP3 did not alter cytoplasmic or surface‐biotinylated ASIC2 expression in the VSMCs, a finding which suggests SLP3 does not inhibit migration by increasing ASIC2 protein levels. However, whether SLP3 increases activity of ASIC2 channels has yet to be determined. These data suggest SLP3 associates with ASIC2 to regulate VSMC migration, a function important in a range of responses including growth and development, tumor metastasis and vascular injury.
Coronary artery vascular smooth muscle‐specific contractile protein expression in Syndrome X
Authors: Smith Erika , Jadhav Rashmi , Rocic Petra
Published: June 2021

A key aspect of coronary collateral growth (CCG) is outward vascular remodeling. We have shown that CCG was impaired in a model of Syndrome X (JCR rat). Outward remodeling is associated with differentiated vascular smooth muscle (VSM), characterized by high VSM‐specific contractile protein expression, vs. inward remodeling, associated with de‐differentiated VSM, characterized by loss of VSM‐specific proteins. We hypothesized that expression of VSM‐specific contractile proteins was significantly lower in Syndrome X. SM‐alpha‐actin, SM‐myosin, smoothelin and caldesmon expression was evaluated by immunohistochemistry and Western blot in coronary arteries (CA) of JCR vs. healthy (WKY) rats. Results show decreased SM‐myosin (25%), SM‐alpha‐actin (50%), smoothelin (30%) and caldesmon (48%) expression in JCR vs. WKY CA. The pattern of expression differed between large (lCA) vs. small (sCA) arteries. Caldesmon was consistently lower in lCA and sCA of JCR rats, but SM‐alpha‐actin and smoothelin exhibited significantly larger decreases in sCA vs. lCA, while SM‐myosin was significantly decreased in lCA but not in sCA. These results demonstrate that coronary artery VSM is de‐differentiated in Syndrome X, but the extent of specific contractile protein downregulation differs between large vs. small coronary arteries, and may provide a partial explanation for impaired CCG in Syndrome X. NIH HL093052
Regulation of the cardiac phenylethanolamine N‐methyltransferase gene in the spontaneously hypertensive rat
Authors: Peltsch Heather , Bizier C. , Nguyen P. , de Wit J. , Crispo J. , Eibl J. , Tai T.C.
Published: June 2021

Phenylethanolamine N‐methyltransferase (PNMT) is the terminal enzyme in the catecholamine biosynthetic pathway involved in the synthesis of epinephrine from norepinephrine. Recent studies have identified cardiac PNMT and endogenous epinephrine in the heart. Genetic mapping studies have linked the PNMT gene to hypertension, and elevated PNMT levels are associated with higher blood pressure in hypertensive rats. The current study examined the expression of the cardiac PNMT gene in the genetic rodent model of hypertension, and associated changes in transcriptional regulators of the gene. Results show upregulation of PNMT mRNA in all four chambers of the heart in spontaneously hypertensive rats (SHR) compared to Wistar‐Kyoto (WKY) rats, with the greatest fold change (2.18) in the right atrium (RA) (p<0.01). The transcription factors Egr‐1, Sp1 and GR have been previously identified as regulators of the PNMT promoter. RT‐PCR analyses show significant increases in Sp1 mRNA in all chambers of the heart of SHR. Further, Egr‐1 mRNA was upregulated in the right and left atria (1.90, 1.45 fold) in SHR. Western blots showed significant fold increases in Sp1 in SHR for the RA (1.74), and the left (2.72) and right (1.46) ventricles. Egr‐1 protein paralleled mRNA expression in SHR. These results show that PNMT gene expression is elevated in SHR, and is likely mediated by altered transcriptional regulation of PNMT in the heart.
Reduction in mononuclear cell mRNA expression of pro‐inflammatory and pro‐oxidant genes with habitual aerobic exercise in older humans
Authors: Gano Lindsey Brooke , Seals Douglas R. , Roeca Cassandra , Hamza Muhammad , Pierce Gary L. , Donato Anthony J.
Published: June 2021

The biological properties of circulating mononuclear cells (MC) interact with and influence the vascular endothelium. Recently we reported that impaired vascular endothelial function (endothelium‐dependent dilation, EDD) with age in healthy adults is associated with a pro‐inflammatory/pro‐oxidant gene expression profile in circulating mononuclear cells (MC). To determine if habitual aerobic exercise can reverse this profile, 11 sedentary older adults (62±1yr) walked 6 days/week for ~45 min/day at a moderate intensity (70‐75% of max heart rate) for 8 weeks. Walking increased measures of maximal aerobic exercise capacity (P<0.05) and improved EDD (brachial artery flow‐mediated dilation) by 28% (P<0.05). Walking reduced MC mRNA expression (real time RT‐PCR) of nuclear factor κB (‐0.8±0.4 fold change (FC), P=0.05), monocyte chemoattractant protein‐1 (‐2.7±0.9 FC, P=0.01), tumor necrosis factor‐α (‐0.9±0.6 FC, P=0.07), NADPH oxidase p47 (‐0.5±0.2 FC, P<0.05) and receptor for advanced glycation end products (‐1.7±0.6 FC, P<0.01), but did not affect interleukin‐6 (P=0.49) or inducible nitric oxide synthase (P=0.21). These preliminary results indicate that regular aerobic exercise may exert anti‐inflammatory and antioxidant influences on gene expression in circulating MC of older healthy adults. Such effects may represent a novel mechanism by which habitual exercise preserves vascular endothelial function in settings of aging and cardiovascular disease. NIH AG013038, AG022241, AG006537, AG029337, AG000279, RR00051
The in vivo role of arginase in cutaneous microvascular dysfunction in hypercholesterolemic humans before and after statin therapy
Authors: Holowatz Lacy A. , Lang James A. , Sorkin Jeremy J. , Kenney W. Larry
Published: June 2021

Elevated low‐density lipoproteins (LDL) are associated with systemic vascular dysfunction evident in the cutaneous microvasculature. Our aim was to determine the role of arginase and freely exchangeable L‐arginine (L‐arg) in vivo in reduced NO‐dependent vasodilation (VD) in hypercholesteroleΔic (HC) skin before and after statin therapy. Three intradermal microdialysis (MD) fibers were placed in 5 normocholesterolemic (NC) (LDL=92 ±8 mg/dl) and 6 HC subjects before (LDL=179±6 mg/dl) and after 3 months of atorvastatin (10 mg) (LDL=100±7 mg/dl): control, arginase‐inhibited (A‐I: 5 mM BEC), and L‐arg (10 mM). Skin blood flow (SkBF) was measured while local skin heating (42°C) induced NO‐dependent VD [quantified (ΔNO) by infusing 10 mM L‐NAME after the plateau (P) in SkBF]. Cutaneous vascular conductance (CVC= flux/MAP) was normalized to max (50 mM SNP). VD was reduced in HC (HC: 77±5 vs. NC: 94±5 % max, p<0.05) as was ΔNO (HC: 37±5 vs. NC: 61±6 % max, p<0.05). P and ΔNO were augmented in HC with A‐I (P: 92±3, ΔNO: 65±3% max, p<0.05) and L‐arg (P: 95±3, ΔNO: 75±4% max, p<0.05) but not in NC. After statin therapy there was no difference between control (P: 92±2, ΔNO: 51±4% max) and A‐I (P: 97±1, ΔNO: 62±6% max, p>0.05) or L‐arg (P: 94±3, ΔNO: 56±5% max, p>0.05) sites. Reduced NOS substrate availability through upregulated arginase contributes to cutaneous microvascular dysfunction with HC, which is corrected with statin therapy.
Chronic aerobic exercise opposes age‐ and high fat diet‐associated vascular endothelial dysfunction: relation to IKKβ and AMPK
Authors: Durrant Jessica R , Folian Brian J , Connell Melanie L , Russell Molly J , Seals Douglas R , Lesniewski Lisa A
Published: June 2021

Age and high fat, ie. Western, diet (WD) are associated with inflammation and impaired endothelium‐dependent dilation (EDD), whereas aerobic exercise has opposite effects. Activation of the pro‐inflammatory signaling molecule inhibitor of kappa B kinase beta (IKKβ), as modulated by adenosine monophosphate activated protein kinase (AMPK), may contribute to these differential effects. Old (O: 29‐31 mo) B6D2F1 mice fed either normal chow (NC) or WD (40% fat, 8 weeks) either were sedentary (SED) or performed voluntary wheel running (VR). In SED, carotid artery EDD to acetylcholine was reduced after WD (max dilation: 62 ± 8 vs. NC: 76 ± 2%, P < 0.05) and this was associated with increased aortic IKKβ activation (phosphorylated (pSer177/181):total IKKβ: 2.1 ± 0.8 vs. 0.5 ± 0.1 AUs, P = 0.01) and reduced AMPK activation (pThr172:total AMPKα: 0.7 ± 0.1 vs. 1.0 ± 0.1 AUs, P = 0.05). VR improved EDD in both NC (96 ± 2%, P < 0.01) and WD (91 ± 7%, P < 0.05) fed mice, while reducing IKKβ activation by ~60% (P < 0.01 in NC, P = 0.1 in WD) and increasing AMPK activity by 30‐40% (P = 0.1 in NC, P < 0.05 in WD). Here we demonstrate that habitual voluntary aerobic exercise may reverse age‐ and WD‐associated vascular endothelial dysfunction by increasing AMPK activity and inhibiting IKKβ‐linked pro‐inflammatory signaling.

NIH AG013038, AG006537, AG015897, AG022241, AG000279
Cold‐induced cutaneous vasoconstriction: effects of age and gender
Authors: Ware Stuart K. , Ware Jeffrey
Published: June 2021

Cold stress elicits cutaneous vasoconstriction through neural and local mechanisms to conserve body heat. In this study, the cutaneous sympathetic vasoconstrictor (VC) response to a contralateral arm cold challenge was evaluated in the middle finger (glabrous skin with abundant arteriovenous anastomoses) and posterior forearm (nonglabrous skin with fewer AVAs) in relation to age and gender. Cutaneous blood flow was measured using laser Doppler flowmetry (LDF) in the non‐dominant arm. Cutaneous vascular conductance was calculated as LDF/mean arterial blood pressure and used to assess VC. Seven men (mean age 64.2 yrs) and 8 women (mean 59.6 yrs) composed the older group. The younger group was composed of 9 men (mean age 23.1 yrs) and 9 women (mean 22.3 yrs). All subjects were healthy and not taking medications. Although the variability of response was greater in the older group, the VC responses at both the finger and forearm were attenuated compared to the younger group, but only at the finger was this statistically significant (P<0.05). VC due to deep inspiratory gasp was also evaluated and found to be more rapid and significant in the younger subjects at both the finger and forearm (P<0.05). With regards to gender, cold‐induced finger VC was greater in the young females compared to the men (P<0.05); this response was also greater in the older females compared to the men, but nonsignificantly. It thus appears that age attenuates the cold‐induced vasoconstriction, especially in glabrous skin, and that females show a greater cold‐induced VC effect compared to age‐matched men.
Glomerular podocytes in developing quail kidney
Authors: Karasawa Tamaki , Nameta Masaaki , Yaoita Eishin , Yang Yimu , Kawachi Hiroshi , Yamamoto Tadashi , Uchiyama Makoto , Nishimura Hiroko
Published: June 2021

In metanephric kidneys of Japanese quail, the number of glomeruli increases exponentially during the later embryonic stage and early postnatal days. In the surface area of developing metanephric kidneys, condensed areas that contain undifferentiated cells are seen. In this mesenchymal region, no mature tubules are present. In contrast, in the inward area, mature glomeruli, proximal tubules consisting of columnar epithelial cells, and cuboidal distal tubules are seen. The condensate zone decreases in size with maturation, but is still detectable at 3‐postnatal week. We examined podocytes (glomerular epithelial cells) and slit diaphragms in developing quail kidneys. Podocyte foot processes with irregular size and intervals are recognized at embryonic day 15 and postnatal day 5 (D5) of metanephros. Foot processes are fused in some location. Slit membranes connecting adjacent foot processes are more clearly seen after D14. Podocin and nephrin, which are important in maintaining integrity and permeability of the slit membrane, were recognized histochemically (immunoreactive against anti‐rat or anti‐mouse antibody) at D4 and D14, respectively, and thereafter. These results suggest that in quail kidneys that 1) nephrogenesis continues after birth and 2) molecular constituents of glomeruli also develop with structural maturation (NSF IOB0615359, NIH HL52881, JSPS17390247).
H2S induces lysosomal rupture in erythrocytes from an H2S‐tolerant marine polychaete
Authors: Ortega Jessica A , Moorthy Shhyam , Julian David
Published: June 2021

H2S and H2O2 can each react with divalent metals to produce free radicals through Fenton‐type reactions. Lysosomes, which accumulate iron, are ruptured by H2O2 exposure in cultured mammalian cells, leading to cell death, and this effect is minimized by antioxidants. Many marine invertebrates are exposed to potentially toxic concentrations of H2S in their environment, but it is unknown whether this can result in loss of lysosomes. We tested whether H2S can cause lysosomal rupture in cells from a H2S‐tolerant marine invertebrate in vitro, and whether this effect can be minimized by antioxidants. We exposed erythrocytes from the marine mudflat polychaete Glycera dibranchiata to 0.01‐1 mM H2O2 for 1 h or 0.03‐1% H2S for 2 h in vitro, after which we used LysoTracker Red (LTR) and acridine orange to quantify lysosome number and staining intensity. Both H2O2 and H2S exposure decreased the integrated intensity of LTR by up to 63% (p=0.0051) and 76% (p<0.0001), respectively, and lysosomal area by 61% (p=0.0150) and 81% (p<0.0001), respectively. However, the effects of H2S were not reduced by the antioxidants glutathione ethyl ester, N‐acetylcysteine or hypotaurine. Therefore, H2S exposure causes loss of lysosomes similar to that of H2O2 exposure, even in an animal adapted to H2S, but it remains unclear whether this effect is via oxidative damage.
Comparison of two peroxidase knockout mouse models: effect of oxygen exposure
Authors: Liu Geng , Fisher Donald , Dodia Chandra , Hong Nan Kang , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Peroxiredoxin 6 (Prdx6), a recently described enzyme with glutathione peroxidase (GPX) activity, plays an important role in lung anti‐oxidant defense. Since Prdx6, unlike GPX1, can reduce phospholipid hydroperoxides, we compared the effects of knock‐out of the two enzymes on the response of mice to hyperoxia. The time (h) to 50% lethality (LT50) on exposure to 85% O2 was 148±12 for wild type (C57 Bl/6), 123±10 for GPx1‐/‐, and 98.5±8.6 for Prdx6‐/‐. After 100% O2 exposure for 60 h, protein content and nucleated cells in the bronchoalveolar lavage fluid and lung wet/dry increased significantly in wild type lungs; changes with GPx1 ‐/‐ lungs were similar to wild type, but Prdx6‐/‐ lungs showed markedly increased injury. Lung thiobarbituric acid reactive substances (TBARS) were 114 ± 8.5 in wild type, 148 ±16.3 in Gpx1‐/‐, and 256 ±17.2 in Prdx6‐/‐ (pmol/mg). Exposure of isolated mouse pulmonary microvascular endothelial cells to 50 μM t‐butyl hydroperoxide (t‐BOOH) for 2 hrs resulted in 34 % cell death for wild type, 65.2% for GPX1‐/‐, and 96.6% for Prdx6‐/‐. Thus, Prdx6 null mice are significantly more sensitive to hyperoxic stress than the GPX1 null indicating that repair of lipid peroxidation (i.e. membrane damage) plays a major role in antioxidant defense.
Intestinal permeability during hyperthermia in the mouse: no protection of superoxide scavenging
Authors: Oliver Scott Ryan , Clanton Thomas L
Published: June 2021

Severe heat stroke is associated with loss of intestinal barrier function resulting in release of endotoxin into the blood. In rat, intestinal permeability is elevated at or above 41.5 °C. The purpose of this study was to determine the sensitivity of mouse intestine to hyperthermia and to evaluate if oxidant production contributes to increases in permeability. Mouse small intestines were excised, cleaned, everted over a glass rod and filled with pre‐oxygenated Ringers solution. Gut sacs (1‐2 cm) were then placed into cuvettes containing oxygenated buffer, with 4kD fluorescein isothiocyanate (FITC) dextran (0.3mM) for 90 min. Leakage of FITC‐dextran into the gut sac was found by fluorometery to be 2.8 ±0.6, 2.2 ± 0.2 and 7.8 ±0.4 μmoles /cm2 at 37, 40.5 and 41.5 °C, respectively. Tiron (1mM), a superoxide scavenger, showed no protective effect on intestinal permeability at 41.5 °C (7.9 μmoles /cm2 ±0.47) and no effect at 37 °C (3.1 μmoles/cm2 ±0.8). The results demonstrate that a threshold temperature of 41.5ºC is necessary for significant loss of intestinal barrier function in mouse (P< 0.01) and that superoxide scavenging does not significantly alter permeability. NHLBI 53333
Pancreatic Beta and Alpha Cell Expression Patterns Across Avian Species
Authors: Hostetter Kimberly , Sweazea Karen L , Braun Eldon J
Published: June 2021

The mammalian pancreas has a remarkable ability to maintain glucose homeostasis. This is accomplished through the production of the blood glucose lowering hormone insulin as well as its counter‐regulatory hormone glucagon. The concept of avian diabetes arose in early studies conducted by Minkowski in the late 1800's which demonstrated that removal of the pancreas in domestic fowl and geese did not result in the development of diabetes. Further studies by others showed that this may be a common characteristic of birds. A species that deviates from this is horned owls which develop hyperglycemia and eventually die following pancreas removal. Prior studies have demonstrated that the chicken pancreas contains significantly more glucagon‐producing alpha cells compared to insulin‐producing beta cells. This is reflected in the observed low blood levels of insulin in chickens. We hypothesize that species differences may exist in the expression patterns of pancreatic islet cells in birds allowing some to persist after pancreatectomy, whereas other birds perish as a result of hyperglycemia. In the present study, we employed immunohistochemistry to compare the density of alpha and beta cells in pancreatic tissue from birds consuming different diets. Pancreatic tissue from Wistar rats was used for comparison to avian pancreatic tissue.
CDK‐dependent assembly of replication proteins at the initiation step of chromosomal DNA replication
Authors: Araki Hiroyuki , Hirai Kazuyuki , Li Yan , Tanaka Tamon , Muramatsu Sachiko , Tanaka Seiji
Published: June 2021

Replication proteins assemble onto origins in two successive steps; formation of the pre‐RC in late M and G1 phases and assembly of other replication proteins including DNA polymerases to initiate DNA synthesis when CDK is activated from G1/S boundary. Sld2 and Sld3 are phosphorylated by CDK and bind to N‐terminal and C‐terminal tandem BRCT domains of Dpb11, respectively. Their interactions are essential and sufficient for the CDK‐dependent initiation of chromosomal DNA replication. However, how these interactions promote initiation of chromosomal DNA replication has not been elucidated.

We have argued that the CDK‐dependent interaction between Sld2 and Dpb11 facilitates formation of the pre‐LC containing Dpb11, Sld2, Polε and GINS. The pre‐LC is a fragile complex that is detected only with the aid of a cross‐linking agent in vivo and is formed in a CDK‐dependent and pre‐RC‐independent manner. We can now reconstitute the pre‐LC from purified proteins. To recruit replication proteins, such as GINS and Polε, to origins, the pre‐LC may bind to phosphorylated Sld3, which associates with origins in a Cdc45‐dependent manner. Moreover, Sld3 seems to facilitate formation of an active replicative helicase, the CMG complex consisting of Cdc45, MCM and GINS. We, thus, propose that the interactions between Sld2, Sld3 and Dpb11 recruit replication proteins to origins and remodel them to form an active helicase at origins.
Discovery and functional characterization of the novel muscle‐specific inositol phosphatase MIP (MTMR14)
Authors: Qu Cheng‐Kui , Shen Jinjua , Yu Wen‐Mei , Guo Caiying , Stoddard Christopher , Brotto Marco , Nosek Thomas M.
Published: June 2021

We discovered a novel phosphatase by hidden Markov database mining and named it muscle‐specific inositol phosphatase (MIP). Both human and mouse homologies shared 90% amino acid sequences identity. MIP was predominantly expressed in skeletal and heart muscle and localized in the cytoplasm and enriched in the SR. MIP dephosphorylated a variety of phosphoinositides, especially PI(3,5)P2, but not PI(4)P, similar to PTEN and myotubularin and myopathy related (MTMR) phosphatases. The Mustalin group (Cell 117, 699‐711; 2004) also identified this phosphatase and listed it as the 14th member of the MTMR family. To characterize the physiological function of MIP, we generated MIP deficient mice through gene targeting. MIP KO mice were born at the Mendelian ratio and showed no obvious deficiencies during the first 12 months. We found that PI(3,5)P2 in MIP KO SR doubled compared to WT controls. Ultrastructure of KO cells appeared normal except that SR terminal cisternae were slightly swollen. Behavior tests (rotarod, inclined screen, and running on a treadmill until exhaustion) revealed decreased muscle function in MIP KO mice. Recently, inactivating mutations in MIP/MTMR14 were identified in two cases of human autosomal centronuclear myopathy. Whether these mutations play a causal role in this disease has not yet been determined. (Support: TREC NCI Grant U54‐CA116867 to TMN, MB, and CKQ and NIH HL068212 and HL082670 to CKQ)
Loss of muscle‐specific inositol phosphatase (MIP) and increased phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2) inhibit muscle contractility and produce a Brody's disease phenotype
Authors: Nosek Thomas M. , Brotto Leticia , Qu Cheng‐Kui , Brotto Marco
Published: June 2021

MIP knockout mice (KO) in which cellular PI(3,5)P2 is increased display behavioral characteristics that suggest a loss of muscle function and increased fatigability. To test muscle function, fast‐twitch extensor digitorum longus and slow‐twitch soleus muscles were isolated from the control and KO animals used in treadmill experiments 1‐2 weeks after the treadmill tests. Maximum isometric tetanic force was significantly reduced in both muscle types and the force vs. frequency relationship was shifted to the right in KO soleus muscles. Resting force was increased and the relaxation of KO muscles was prolonged. Muscles from KO animals fatigued more quickly, to a greater extent, and recovered to a lesser extent than muscles from the WT controls. In primary myotubes and adult muscle fibers, resting Ca2+ was elevated in the KO and the SR Ca2+ content was decreased. Store‐operated Ca2+ entry (SOCE) was also inhibited in KO myotubes. Perfusion of PI(3,5)P2 in WT myotubes recapitulated the defects in Ca2+ signaling of KO cells. Thus, ablation of MIP increases PI(3,5)P2 and causes muscle weakness and increased fatigability via a reduction of SR Ca2+ content by Ca2+ leak from the SR and inhibition of SOCE. These overall phenotypes recapitulate the symptomatology of Brody disease and certain forms of muscular dystrophies. (Support: TREC NCI Grant U54‐CA116867 to TMN, MB, and CKQ; AHA 0535555N to MB; NIH HL068212 and HL082670 to CKQ)
The effects of phosphatidylinositol 3,5‐bisphosphate on cardiac muscle
Authors: Wacker Michael J. , Touchberry Chad D. , Rohrberg Travis J. , Qu Cheng‐Kui , Brotto Marco P.
Published: June 2021

The downstream product of phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2), inositol 1,4,5‐trisphosphate (IP3), is known to induce calcium (Ca) release from the sarcoplasmic reticulum and elicit alterations in contraction of the heart and arrhythmias. What has not been studied is the effects of other phosphoinositides (PIs) such as phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2) and phosphatidylinositol 3‐phosphate (PI(3)P) on the heart. A striated muscle specific phosphatase (MIP/MTMR14) dephosphorylates PI(3,5)P2, and is present in skeletal and cardiac muscle. MIP knockout animals display reduced exercise performance, providing a strong rationale that PI(3,5)P2 may be involved in heart function. We tested 0.5 µM PI(3,5)P2, PI(4,5)P2, and PI(3)P on left ventricular muscle strips from adult mice paced at 1 Hz. All PIs tested induced an increase in the magnitude of the twitch‐force of the left ventricle. PI(3,5)P2 was also applied to single isolated cardiac myocytes and induced an increase in intracellular Ca levels and altered spontaneous Ca waves. While the mechanism of these actions remains to be determined, these data suggest that PI(3,5)P2, PI(4,5)P2, and PI(3)P may play a role in altering cardiac function. It will be essential to determine the potential role of this novel signaling mechanism in heart‐related diseases where Ca homeostasis is known to be modified.
Inhibition of ataxia telangiectasia mutated (ATM) prevents the prolonged increase in phosphorylation of Akt substrate of 160 kDa (AS160) subsequent to activation of the AMP‐activated protein kinase (AMPK)
Authors: Lewis LaQuinta R. , Davis Jennifer , Akinola Ayotola , Fisher Jonathan S.
Published: June 2021

We hypothesized that activation of AMPK would result in a lingering increase in AS160 phosphorylation (P‐AS) and that inhibition of ATM, a reported regulator of AMPK and Akt, would prevent this effect. L6 myotubes were incubated for 30 min with DMSO (vehicle) or the ATM inhibitor KU55933 (KU) at a concentration (1 μM) that would inhibit ATM but would be unlikely to interfere with the related kinases PI3K or mTOR. Then, myotubes were incubated 60 min ± 2 mM AICAR (an AMPK activator) and ± KU, after which cells were incubated 1 h ± KU, then 1 h without additives, and finally 20 min ± 100 nM insulin. There was a ~40% increase in AMPK phosphorylation (P‐AMPK) immediately after 1 h of AICAR treatment, but this had worn off by 140 min later. In myotubes not exposed to KU, P‐AS tended to be elevated by ~70% in myotubes previously exposed to AICAR. Insulin also tended to increase P‐AS by about 2‐fold, and effects of insulin and prior exposure to AICAR tended to be more than additive. KU caused an overall elevation in P‐AS, but prevented any further effects of insulin or prior exposure to AICAR on P‐AS. KU increased Akt phosphorylation (P‐Akt, S473 and T308) in the absence of insulin but did not interfere with insulin‐stimulated increases in P‐Akt. Previous exposure to KU depressed P‐AMPK by about 40%. It appears that exposure to an AMPK activator leads to long‐term elevation of P‐AS, and ATM may play some role in this effect.

NIH DK08437 supported this work.
Ataxia telangiectasia mutated (ATM) is required in insulin‐like growth factor‐1 (IGF‐1) signaling through the PI3K/Akt pathway
Authors: Ching James Kain , Luebbert Stephen H. , Zhang Zhihong , Marupudi Nandhini , Banerjee Sankha , Hurd Robin , Collins Roy L. , Ralston Lyle , Fisher Jonathan S.
Published: June 2021

Reports that ATM is required for full activation of Akt raise the hypothesis that ATM plays a role in IGF‐1 signaling through the PI3K/Akt pathway. Differentiated C2C12 cells harboring either ATM‐targeting shRNA or non‐targeting shRNA and myotubes from a C2C12 lineage previously exposed to empty vector lentivirus were incubated in the presence or absence of 10 nM IGF‐1. Western blot analysis showed that IGF‐1 stimulated phosphorylation of Akt S473, Akt T308, and p70 S6 kinase (S6K) in cells expressing non‐targeting shRNA and in empty vector controls, but the IGF‐1 effects were significantly reduced in cells with shRNA‐mediated ATM knockdown. Consistent with these findings, the ATM inhibitor KU55933 (10 μM) prevented stimulation of S6K phosphorylation in C2C12 myotubes exposed to IGF‐1. Furthermore, IGF‐1‐stimulated phosphorylation of Akt S473, Akt T308, and S6K was lower in isolated soleus muscles from mice expressing only one functional ATM allele (ATM +/‐) compared to muscles from wild‐type (ATM +/+) mice. Our data suggest that ATM is a modulator of IGF‐1 signaling through the Akt pathway.

This work is supported by NICHD/NCMRR through a Pilot Project award from the National Skeletal Muscle Research Center.
Enhanced three‐dimensional visualization of rat phrenic motoneurons.
Authors: Ermilov Leonid G. , Zhan Wen‐Zhi , Issa Amine N. , Sieck Gary C. , Mantilla Carlos B.
Published: June 2021

Retrograde transport of fluorescent dyes permits imaging of nearly the entire pool of motoneurons when administered in the target muscle or axon shaft, but the extent of dendritic labeling is usually limited. We used an intracellular injection of Lucifer Yellow CH (LY) into retrogradely labeled phrenic motoneurons (PMNs) to enhance dendritic structure. PMNs were specifically labeled by Alexa Fluor 488‐conjugated cholera toxin fragment B (CTB) injected into the pleural space of 18‐days old rats. After a 72‐hr survival period, animals were transcardially perfused with 4% paraformaldehyde and 4% sucrose in 0.1 M phosphate buffer. Cervical spinal cords were sectioned in coronal plane. Individual CTB‐labeled PMNs were intracellularly injected with LY under direct visual control and subsequently imaged with an Olympus FluoView 200 laser‐scanning confocal system. Image processing and analyses were performed using Metamorph 7.4 software. PMNs were extensively labeled and reconstructed in 3D. The range of visible primary, secondary and tertiary processes was 3 ‐ 7, 2 ‐ 4 and 0 ‐ 2 per PMN, respectively. Average length of a single dendritic tree was 218.0±20.1 μm and surface area was 1295±199 μm2. The described technique provides 3D quantitative morphometry of a large sample of retrogradely‐labeled PMNs in great detail. Supported by NIH grants HL37680 and AR51173.
Ventilatory impairment in a rat model of central dysmyelination
Authors: Johnson Rebecca Ann , Duncan Ian D. , Mitchell Gordon S.
Published: June 2021

Although respiratory complications are the major cause of morbidity/mortality in advanced myelin disorders (i.e. MS), little is known concerning mechanisms whereby lack of myelin impairs ventilation, or how patients compensate for such changes (i.e. plasticity). To establish a model for future studies concerning mechanisms of ventilatory impairment/compensation, we tested the hypothesis that respiratory function progressively declines with advancing disease in a genetic model of CNS dysmyelination, the Long Evans shaker rat (les). Ventilatory measurements were made via whole‐body plethysmography in les and wild‐type rats at baseline and during hypercapnia (7%CO2). In young (P59) rats, there were no differences in breathing between les and wild‐type groups in either condition (p>0.05). However, by P150, significant decreases between groups were observed in mean inspiratory flow (Vt/Ti), breathing frequency and minute ventilation during baseline (40%, 47% and 37%, respectively) and hypercapnia (40%, 44% and 50%, respectively) (all p<0.05). Thus, ventilatory capacity is maintained in young, dysmyelinated rats, but ventilatory impairment develops as les rats age. This study provides the foundation for mechanistic studies of respiratory insufficiency and therapeutic interventions in myelin disorders. Support: NIH NS057778, UW ICTR via NIH CTSA 1UL1RR025011, NMSS TR3761
Modulating GABAA receptors that contain alpha 2/3 subunits corrects respiratory disturbances in a mouse model of Rett syndrome
Authors: Bissonnette John M , Knopp Sharon J
Published: June 2021

We have recently shown that augmenting γ‐aminobutyric acid (GABA) A inhibition in a mouse model of Rett syndrome corrects periodic apneas and restores cycle regularity to that of wild type (2008 EB abstracts [on CD‐ROM], Abstract#1232.12). These mice are deficient in α2 subunits of GABAA receptors (J Neurophysiol 99:112, 2008). Here we show that allosteric modulation of GABAA receptors with a compound that is relatively specific for receptors that contain α2/3 subunits (L‐838,417, Nature Neurosci 3:587, 2000) is equally efficient as a broad‐spectrum benzodiazepine in correcting the respiratory disorder. Studies were performed in heterozygous methyl‐CpG‐binding protein 2 (Mecp2) deficient female mice with continuous monitoring of pleural pressure by telemetry. Following ip injection of L‐838,417 (20 mg/Kg) or vehicle respiratory pattern was examined for 3 hrs.

Adjusting for differences in wake/sleep pattern from animal activity (Physiol Genomics 28:232, 2006) did not alter the effect of L‐838,417.

Support: MOD #6‐FY‐314 and IRSF #0802
Attenuation of Heart Rate Control and Neural Degeneration in Nucleus Ambiguus (NA) of OVE26 Transgenic Mice of Type 1 Diabetes
Authors: yan binbin , li Lihua , Harden Scott W , Cheng Zixi (Jack)
Published: June 2021

Baroreflex control of heart rate (HR) is impaired by diabetes mellitus. Previously, we found a deficit of central mediation of baroreflex‐bradycardia. In this study, we aimed to assess whether diabetes‐induced neural degeneration in NA and reduced HR response to microinjection of L‐Glutamate (L‐Glu) into the NA. FVB control and OVE26 diabetic mice (6mo) were anesthetized with sodium pentobarbital. HR and mean arterial blood pressure (MAP) responses to L‐Glu microinjections (0.1 0.2, 0.3, 0.4, 0.5, 1, 5 mMol/L, 20nl) into the left NA were measured. Brainstem slices ‐600, ‐300, 0, +300, and +600 μm relative to the obex were processed using Nissl staining. The NA region was identified by vagal motoneurons retrogradely‐labeled by TMR‐D injection into the ipsilateral nodose ganglion. Compared to FVB control, we found that: 1) diabetes attenuated the HR response (ΔHR: % of maximum) to L‐Glu injection into the NA at low doses (ANOVA, p<0.05, n=7/group), but MAP responses (ΔMAP: % of maximum) were unchanged. 2) After i.v. injection of β1‐bloker atenolol, ΔHR were reduced (to about 10% of the maximum HR response); subsequential injection of M‐blocker methylatropine, ΔHR were reduced additionally (to about 6.5% of the maximum HR response) in both FVB and OVE26 mice. 3) Diabetes reduced the number of vagal motoneurons in the NA region [the total number of NA motoneurons (left and right): 299.3 ± 17.8 (FVB) vs. 182.2 ± 13.6 (OVE26); p <0.05; n=6/group]. We suggest that degeneration of NA motoneurons may contribute to impairment of baroreflex sensitivity in OVE26 mice. Supported by NIH HL79636.
Forearm blood flow during mental stress before and after sympathetic denervation in patients with hyperhidrosis
Authors: Elvebak Rachel Leigh , Wehrwein Erica A , Atkinson John L , Fealey Robert D , Eisenach John H
Published: June 2021

The complex forearm vasodilator response during mental stress (MS) is mediated in part by unchanged or decreased forearm muscle sympathetic nerve activity. However, immediately after MS, nerve activity is increased. To determine the effect of chronic sympathetic denervation on forearm blood flow (FBF, [ml/100ml forearm volume/min]) response to MS, we studied patients pre and post sympathotomy (ETS) for palmar hyperhidrosis. 15 healthy subjects (11 F; 19‐32 yr) with a mean BMI of 23 ± 5 kg/m2 were studied 1 day‐6 mo before ETS and 1‐6 mo after ETS. FBF (plethysmography) was recorded during baseline, 3 min Stroop test, and 2 min post‐MS recovery. ETS did not affect resting HR, BP or FBF. However, resting forearm vascular resistance (FVR, [MAP/FBF]) was reduced after ETS (50 ± 20 vs 33 ± 14, p < 0.01) and forearm vascular conductance (FVC, [(FBF/MAP)*100]) was increased (3 ± 2 vs 4 ± 2, p < 0.05). MS caused significant increases in FBF and FVC, and a decrease in FVR, and these responses were unaffected by ETS. However during post‐MS recovery, ETS caused a significant increase in FBF (2 ± 1 vs 3 ± 1), FVC (3 ± 1 vs 4 ± 2), and a decrease in FVR (52 ± 22 vs 32 ± 16) (p < 0.01). Chronic sympathetic denervation influences post‐MS forearm vasoconstriction, which likely has minor hemodynamic consequences but confirms a greater role for sympathetic control of FBF in the post‐MS period as opposed to during MS. NIH HL‐089331, NS‐32352
Mental stress during acute loading of the baroreceptors
Authors: Durocher John , Schwartz Christopher E. , Carter Jason R.
Published: June 2021

Neural and cardiovascular responses to mental stress (MS) and acute 6° head‐down tilt (HDT) were examined separately and combined. We hypothesized sympathoexcitation during MS, sympathoinhibition during HDT, and an additive neural interaction during combined MS and HDT. Muscle sympathetic nerve activity (MSNA), mean arterial pressure (MAP), and heart rate (HR) were recorded in 16 healthy subjects in the supine position during three randomized trials: 1) MS (via mental arithmetic), 2) HDT, and 3) combined MS and HDT. MS significantly (P<0.05) increased MSNA (7±1 to 12±2 bursts/min), MAP (91±2 to 103±2 mmHg), and HR (70±3 to 82±3 beats/min). HDT did not change MSNA or HR, but MAP was reduced (91±2 to 89±3 mmHg). Combined MS and HDT significantly increased MSNA (7±1 to 11±1 bursts/min), MAP (88±3 to 99±3 mmHg), and HR (70±3 to 82±3 beats/min). Increases in MSNA and HR during the combination trial were not different from the sum of the individual trials. However, the increase in MAP during the combination trial was significantly greater than the sum of the individual trials (Δ11±1 vs. Δ9±1 mmHg; P<0.05). We conclude that the interaction for MSNA and HR are additive during combined MS and HDT, but that MAP responses are augmented during the combined trial. These findings suggest that increases in MAP may be exaggerated when stress is experienced during a cephalic fluid shift similar to that which occurs in microgravity. Supported by a graduate fellowship from the Michigan Space Grant Consortium.
Adrenergic Regulation of Vasomotor Responses in Skeletal Muscle Composed of Different Fiber Types
Authors: Behnke Brad J , Armstrong Robert B. , Delp Michael D
Published: June 2021

The sympathetic nervous system (SNS) has a profound ability to control blood flow and vascular resistance in skeletal muscle at rest and during exercise. The purpose of this study was to further elucidate the influence of the SNS on vasoconstrictor tone in muscles composed of different fiber types. Blood flow to the red (RGast) and white (WGast) portions of the gastrocnemius and the soleus muscles were measured pre‐ and post‐denervation, and dose‐response relations to alpha1 (phenylephrine) or alpha2 (clonidine) adrenoreceptor agonists were determined in isolated arterioles from these muscles. Denervation increased blood flow 8.7‐fold in the fast low‐oxidative muscle WGast while only eliciting an ∼2.7‐fold increase in the slow oxidative (soleus) and fast oxidative (RGast ) muscles. In arterioles, sensitivity to phenylephrine was greater in WGast and RGast versus the soleus muscle, whereas vasoconstriction to clonidine was greater in WGast (~50%) relative to that in RGast (~31%) and soleus (~17%). These data indicate 1) an intermediate control of vascular resistance by the SNS in fast oxidative muscle versus the other muscle types, and 2) the SNS influence on blood flow could fully account for the magnitude of the hyperemia observed during exercise in fast low‐oxidative muscle.
Modulation of cardiac output alters the mechanisms of the muscle metaboreflex during dynamic exercise
Authors: Ichinose Masashi , Sala‐Mercado Javier A , Coutsos Matthew , Li ZhenHua , Ichinose Tomoko K , O'Leary Donal S
Published: June 2021

Muscle metaboreflex activation (MMA) in conscious dogs during mild to moderate exercise elicits a pressor response primarily due to an increased cardiac output (CO). However, when the ability to increase CO is limited, such as in heart failure or during maximal exercise, the MMA‐induced increases in arterial pressure (AP) occur solely via peripheral vasoconstriction. However, how the mechanisms of this pressor response are altered is unknown. We tested the hypothesis that this change in metaboreflex function from increased CO to increased vasoconstriction is dependent on the level of CO. The muscle metaboreflex was activated during mild dynamic exercise (3.2 km/h) via partial reduction of hindlimb blood flow. MMA increased CO and AP while vascular conductance of all non‐ischemic areas (NIVC) did not change. CO was then reduced to the same level observed during exercise prior to MMA via partial occlusion of the inferior and superior vena cavae. AP dropped rapidly with the reduction in CO but subsequently nearly completely recovered. With the removal of the MMA‐induced rise in CO, NIVC decreased sharply. We conclude that whether vasoconstriction occurs with MMA depends on whether CO rises. NIH HL 55473
Muscle Metaboreflex Control of Coronary Blood Flow and Ventricular Contractility During Dynamic Exercise in Heart Failure
Authors: Coutsos Matthew , Sala‐Mercado Javier , Ichinose Masashi , Li Zhen Hua , O'Leary Donal
Published: June 2021

Muscle metaboreflex activation (MMA) during dynamic exercise increases cardiac work and O2 demand via increases in heart rate, ventricular contractility and afterload. This increase in cardiac work should lead to metabolic coronary vasodilation. However, no change in coronary vascular conductance is seen, indicating that the increased sympathetic activity which increased contractility also caused vasoconstriction. In heart failure, cardiac output does not increase with MMA presumably due to impaired left ventricular contractility, and large decreases in coronary vascular conductance are observed. We tested whether this coronary vasoconstriction could explain in part, the reduced ability to increase cardiac performance during MMA. In conscious, chronically instrumented dogs after pacing induced heart failure, MMA responses during mild exercise were observed before and after α1 ‐ adrenergic blockade (prazosin 50‐100 μg/kg). During MMA, the increases in coronary blood flow, coronary vascular conductance, cardiac output, and +dP/dtmax were significantly greater after α1 ‐ adrenergic blockade. We conclude that during heart failure the coronary vasoconstriction limits the ability of muscle metaboreflex to increase left ventricular contractility. NIH HL 55473
Interaction of the muscle mechanoreflex and graded metaboreflex activation in reducing carotid‐cardiac baroreflex gain in man.
Authors: Walsh Jason , Vianna Lauro C , White Michael J
Published: June 2021

We have shown that muscle mechanoreflex stimulation during concurrent metaboreflex activation reduces maximal gain (Gmax) of the carotid‐cardiac baroreflex in man. The effect of graded metabolite accumulation and metaboreflex activation on mechanoreflex modulation of carotid‐cardiac Gmax is unknown. Mechanoreflex stimulation by a standard passive calf stretch was performed during local circulatory occlusion in the resting limb, and after isometric calf contraction for 90s at 10, 30, 50 and 70% of maximum voluntary contraction (MVC). Heart rate and blood pressure were measured continuously. Throughout the protocol, pulsatile neck pressure trains (+40 to ‐80 Torr) were applied to derive carotid cardiac and vasomotor baroreflex function curves. As expected, there was a graded increase in blood pressure with graded metaboreflex activation. Gmax of the carotid‐cardiac baroreflex function curve was similarly reduced from rest during stretch in the 30, 50 and 70 % trials, by ‐50±7, ‐44±2 and ‐51±16 % respectively (P<0.05). No significant reduction from rest was detected during the 0 and 10% trials. Gmax of the carotid‐vasomotor baroreflex function curves was unaffected by stretch. The results suggest that afferent feedback due to passive stretch in combination with metabolites accumulated during exercise at 30% MVC and above is sufficient to alter carotid‐cardiac baroreflex gain.
A new method to quantify core temperature instability in rodents.
Authors: Gordon Christopher J
Published: June 2021

Methods to quantify instability of autonomic systems such as temperature regulation should be important in toxicant and drug safety studies. Stability of core temperature (Tc) in laboratory rodents is susceptible to a variety of stimuli. Calculating the temperature differential of Tc monitored by radiotelemetry is proposed as a method to quantify the stability of the rodent thermoregulatory system. Mice and rats were implanted with radiotelemetric implants (Data Sciences Int) to monitor Tc at 1‐10 min intervals under a variety of environmental conditions. The absolute value of the Tc differential (ATD; °C/hr) was calculated for periods of > 24 hr. Mean Tc was stable while ATD could vary depending on ambient temperature (Ta), genetic strain, cage bedding, and body size. For example, the ATD was significantly higher in mice than rats. Mean Tc was stable over a wide range of ambient temperatures (Ta) while the ATD changed inversely with Ta. Overall, the ATD is simple to calculate from telemetry data and may be an important parameter to assess the effects of toxicological and pharmacological agents on thermoregulation. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.