bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of a model equation to predict sweating and water requirements
Authors: Gonzalez Richard R. , Cheuvront Samuel N. , Goodman Daniel A. , Blanchard Laurie A. , Berglund Larry G. , Sawka Michael N.
Published: June 2021

We evaluated the original equation (OSE, Shapiro et al. (EJAP 48: 83, 1982)) predicting sweating rate, msw (g•m−2•h−1) = 27.9 • Ereq • (Emax)−0.455 over wide heat stress conditions (HS), clothing systems including chemical protective (CPS), and metabolic activities. Ereq is required sweat loss calculated from heat balance and (Emax) is the maximum evaporative power of a thermal environment. OSE in a Heat Strain Decision Aid (HSDA) over‐predicts actual msw over wide HS, work rates, and times potentially resulting in over‐hydration problems. Data from 101 volunteers (80 men and 21 women) were analyzed including experiments at various activity levels over wide HS (Ta = 15°C – 46°C, Pw= 2 – 33 Torr and (V) = 0.4 – 2.5 m•s−1) and various CPS with body armor. Studies included 4 separate chamber studies, 1 USARIEM field study & 1 study outside the U.S. Data were analyzed using fuzzy piecewise regression. OSE was reliable over a limited HS up to 2‐h and work rates < 450W. OSE predicted high values (p < 0.003) compared to observed msw for 8‐h work periods. A correction to OSE is: msw = 147•exp (0.0012•OSE). We recommend using the sweat prediction algorithm: msw = 147+1.527• (Ereq) − 0.87• (Emax), which accounts for effects of heavy work, CPS, body armor, 8‐h work, and is not gender specific.
Exercise‐induced modulation of renal vs. systemic endothelium
Authors: Moningka Natasha , Ahlgren Joslyn , Hayward Linda , Baylis Chris
Published: June 2021

Endothelial nitric oxide (NO) synthase (eNOS) and extracellular (EC) superoxide dismutase (SOD) are increased in exercise (EX) due to increased shear stress, but in kidney, blood flow is reduced with EX. We investigated the impact of EX on renal and aortic eNOS and SOD. Rats were subjected to 6wks voluntary running wheel EX (EX, n=7; sedentary (SED), n=3). At wk 6, average distance run=4.3±1.6 km/day. Decreases in HR and MAP (by telemetry) were seen in EX rats. Western blots of renal cortex (RC) revealed falls in eNOS and EC SOD protein abundance, while aortic eNOS was unchanged and EC SOD increased with EX (Table). In contrast, there were no significant changes in Mn SOD with EX while Cu/Zn SOD increased with EX in RC. Protein abundance measurements expressed in units of IOD/Ponceau red/Int Std; *p<0.05 vs. SED. The marked reduction in eNOS abundance and tendency for EC SOD to fall with EX in the kidney is predictable based on the reduction in renal blood flow. The increased RC Cu/Zn SOD may help to buffer exercise‐induced oxidative stress but the decreased eNOS will likely compromise endothelium‐dependent renal vasodilation. The long term impact of EX on kidney function and structure in health and in renal disease requires further study.
Increased mitochondrial content in response to mitochondrial dysfunction in skeletal muscle of Cu, Zn superoxide dismutase knockout mice
Authors: Marcinek David Joseph , Smith Steven R , Van Remmen Holly
Published: June 2021

Mitochondrial biogenesis is an important mechanism allowing cells to adapt to energetic stress. Here we test whether skeletal muscle compensates for mitochondrial dysfunction induced by oxidative stress by increasing mitochondrial content in vivo. We used 31P NMR and optical spectroscopies to measure ATP and O2 fluxes in vivo in wild‐type and Cu, Zn‐superoxide dismutase knockout mice (SOD1). Adult SOD1 mice demonstrated significant mitochondrial dysfunction as indicated by a 36% lower energy coupling ratio (P/O) (P=0.03) and a 13% lower resting ATP/PCr (P=0.01). Mitochondrial dysfunction in the SOD1 mice was associated with an increased capacity for mitochondrial ATP production (1.08 ± 0.04 vs. 0.72 ± 0.10 mM ATP s−1, P=0.01) and O2 uptake (O2max) (0.30 ± 0.04 vs. 0.13 ± 0.02 mM O2 s−1, P<0.01). Cytochrome oxidase protein and gene transcript and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PGC1a) transcript levels were positively correlated with O2max and negatively correlated with P/O across treatment groups (P<0.05), supporting a relationship between reduced energy coupling and increased mitochondrial content. These results indicate that skeletal muscle compensates for mitochondrial dysfunction in vivo by activating mitochondrial biogenesis and increasing mitochondrial content. Supported by NIH grants AG028455, AG022385, and the Ellison Medical Foundation.
Metformin Attenuates a Complex I Mediated Increase in Mitochondrial Oxidant Emitting Potential in Skeletal Muscle of Obese Zucker Rats
Authors: Kane Daniel A. , Anderson Ethan J. , Woodlief Tracey L. , Price Jesse W. , Bickman Benjamin T. , Cortright Ronald N. , Neufer P. Darrell
Published: June 2021

Metformin (MET) is currently the most popular antidiabetic drug, although its insulin sensitizing mechanism of action is unknown. To test the effects of metformin (MET) on skeletal muscle mitochondrial function, we measured both mitochondrial respiration and H2O2 emission in saponin‐permeabilized red gastrocnemius muscle fiber bundles from lean and obese Zucker (fa/fa) rats that had been treated with MET for 4 wks. MET treatment significantly (p=.03) improved insulin sensitivity (HOMA) in obese, but not lean rats. In permeabilized fibers, ADP‐stimulated state 3, but not basal state 4, respiration rates were greater in obese vs. lean rats. However, MET treatment did not significantly affect permeabilized fibers respiring on palmitoyl‐carnitine (PC), complex I‐ or complex II‐linked substrates. Surprisingly, H2O2 emission induced by reverse electron flow (titration of complex II substrate succinate) was more than 2‐fold higher in obese vs lean rats. MET treatment significantly (p<.05) attenuated succinate‐ and PC‐induced H2O2 emission in both lean and obese rats. These results suggest that 1) obesity is associated with a marked increase in mitochondrial ROS emitting potential in skeletal muscle, and 2) metformin may exert its antidiabetic effects by inhibiting complex I mediated ROS production, thereby decreasing the susceptibility to conditions favoring ROS emission. Support: NIH DK061314 and DK073488
Mechanisms by which lipoic acid prevents high fat diet‐induced insulin resistance in skeletal muscle.
Authors: Gupte Anisha A , Bomhoff Gregory L. , Morris Jill K. , Gorres Brittany K. , Geiger Paige C.
Published: June 2021

High fat diet‐induced insulin resistance is associated with impaired insulin signal transduction and reduced glucose transport in skeletal muscle. The anti‐oxidant alpha‐lipoic acid (LA) has been shown to improve insulin action in high fat fed animal models, yet little is known about the underlying mechanism. We hypothesize that LA acts by directly inhibiting stress kinases, such as c‐Jun N‐terminal kinase (JNK), known to interfere with insulin signaling intermediates. Male Wistar rats were fed a high fat diet (HFD, 60% calories from fat) for six weeks, while controls received a low fat diet (20% calories from fat). Half of the rats in each group received daily LA injections (30 mg/kg body wt). LA increased whole body glucose disposal in both diet groups as measured by oral glucose tolerance tests. LA treatment effectively restored insulin responsiveness in soleus muscles from rats fed a HFD, as seen by insulin‐stimulated pAkt/Akt and 2‐deoxyglucose uptake. LA treatment reduced the elevation of JNK (pJNK/JNK) seen with the HFD and increased activation of p38 MAPK and AMPK (pAMPK/AMPK), a mediator of insulin independent glucose transport. In addition, LA increased expression of heat shock protein 70 (HSP70), a protein previously shown to directly inhibit JNK activation, in rats fed a HFD. In conclusion, LA reversed HFD induced insulin resistance by reducing JNK, and increasing p38 MAPK, AMPK and HSP70 activation.
Chloroquine stimulates phosphorylation of the AMP‐activated protein kinase (AMPK) and Akt
Authors: Spears Larry D. , Tran Andrew V. , Fisher Jonathan S.
Published: June 2021

Prior research has shown that AMPK may be a target of ataxia telangiectasia mutated (ATM) and that activation of Akt by insulin may require ATM. Accordingly, we hypothesized that ATM activators would increase phosphorylation of AMPK, acetyl coenzyme A carboxylase (ACC, an AMPK substrate), and Akt in cultured muscle cells. C2C12 cells were differentiated into myotubes in DMEM containing 2% horse serum (differentiation medium). Myotubes were incubated in differentiation medium with 0 μM, 250 μM, or 500 μM chloroquine, an ATM activator, for 1 hour. Myotubes incubated in 250 μM and 500 μM chloroquine had two‐ and three‐fold more phosphorylated AMPK (P‐AMPK), respectively, than control cells (P<0.05 and P<0.001, respectively). Consistent with its effect to increase P‐AMPK, 500 μM chloroquine also caused better than a two fold increase in P‐ACC (P<0.01). Additionally, myotubes incubated with 500 μM chloroquine had a two‐fold increase in P‐Akt at serine 473 (P<0.05). Another putative ATM activator, indole‐3‐carbinol, had no affect on AMPK phosphorylation. The chloroquine‐stimulated increase in P‐AMPK, P‐ACC, and P‐Akt may be relevant to previously reported hypoglycemic effects of chloroquine.
Caloric restriction attenuates developmental myosin heavy chain expression and myostatin signaling in aged muscles
Authors: Hepple Russell , Qin Maggie , Huba Elaine
Published: June 2021

Aging is associated with an accumulation of muscle fibers expressing developmental myosin heavy chain (dMHC), suggesting they are in the process of regenerating. The cleaved form of myostatin increases in aged muscles and may be involved in impairing regeneration. Since caloric restriction (CR) slows age‐related muscle atrophy, we sought to determine the effect of CR on dMHC expression and to examine how this related to the myostatin signaling pathway. The gastrocnemius muscle from young adult (YA), late middle aged (LMA) and senescent (SEN) ad libitum fed and CR rats was harvested for either dMHC expression in situ or myostatin and ACVR2 receptor expression by Western immunoblot. CR attenuated dMHC expression with aging and although it did not affect the increase in cleaved myostatin, it slowed the increase in one of its receptors, ACVR2. Since the degree of muscle atrophy with aging correlated (r2=0.72) with the proportion of dMHC positive fibers, our results suggest that CR attenuates age‐related muscle atrophy by limiting the accumulation of incompletely regenerated muscle fibers secondary to attenuated myostatin signaling in aged muscles.

Supported by AHFMR and CIHR.
Modulation of Insulin Signaling via Resistance Exercise
Authors: Hamilton David Lee , Mackenzie Matthew Graeme , Baar Keith Richard
Published: June 2021

Resistance exercise in rats has been shown to substantially elevate S6K1 phosphorylation for up to 36 hr post exercise. Genetically increasing the activity of S6K1 can alter insulin sensitivity by inhibition of IRS‐1 expression and function. We hypothesized that lengthening contractions would increase S6K1 phosphorylation and down regulate the expression and function of IRS‐1. Wistar rats underwent unilateral lengthening contractions of the right Tibialis Anterior muscles and were collected at 0 mins, 30 mins, 3 hrs, 18 hrs and 48 hrs. S6K1389 phosphorylation was elevated by 751 ± 208 %, 3197 ± 1144 %, 10041 ± 2209 % (p < 0.05), at 0 min, 30 min, 3 hr, respectively. IRS‐1636/639 phosphorylation was significantly increased at 30 mins and 18 hrs by 715 ± 160 % and 170 ± 38 %, respectively. IRS‐1 associated P85 was significantly reduced at 30 min and 3 hrs by 57 ± 7 % and 55 ± 7 %, respectively. Due to the time course of the S6K1389 and IRS‐1636/639 phosphorylation we concluded that S6K1 was the most likely kinase mediating the response. To test this further we pretreated female Wistar rats with rapamycin and collected tissues 30 mins post stimulation. We found that rapamycin reduced the increase in IRS‐1636/639 phosphorylation and partially rescued the reduction in IRS‐1 associated P85. We conclude that increased S6K1 activity after resistance exercise enhances IRS‐1 serine phosphorylation, decreasing IRS‐1 associated P85.
Reactive oxygen species and nitric oxide, but not AMPK, mediate stretch‐induced glucose uptake by muscle.
Authors: Smith Melissa A , Goodyear Laurie J , Reid Michael B
Published: June 2021

Diabetes is characterized by a reduction in insulin‐stimulated signaling leading to impaired glucose uptake. An alternative pathway for glucose uptake can be stimulated by exercise and exogenous reactive oxygen species (ROS). We proposed a model wherein mechanical loading, i.e. stretch, stimulates production of ROS to activate AMP‐activated kinase (AMPK) to increase glucose uptake. Previously, we demonstrated stretch increases ROS, AMPK phosphorylation, and glucose transport in murine extensor digitorum longus (EDL) muscle. The current studies demonstrate that the ROS‐selective antioxidants ebselen (catalase mimetic) or combined superoxide dismutase (SOD)‐plus‐catalase suppress stretch‐induced oxidant activity (−20% vs. stretch p<0.05). In contrast, stretch‐induced glucose uptake persists in transgenic mice expressing an inactive form of the AMPKalpha2 catalytic subunit in skeletal muscle (+73% of basal p<0.05). MnTBAP, an SOD mimetic, N‐acteyl cysteine (NAC), a nonspecific antioxidant, and L‐NAME abolish stretch‐induced glucose uptake (2.26, 3.08, 2.88 vs. 4.57 μmol/ml/hr respectively p<0.05). Ebselen or combined SOD‐plus‐catalase decrease stretch‐induced glucose uptake (3.45, 3.68 vs. 4.57 μmol/ml/hr p<0.05, =0.07). These data indicate that stretch‐induced glucose uptake is mediated by a ROS‐dependent but AMPK‐independent mechanism. Supported by AHA 0615263B & DK 066232.
Cycling Training effects on resting p38 MAPK activation in previously untrained young subjects
Authors: Schrager Matthew Alan , Fry Andrew C , Bloomer Richard J , Moore Christopher A , Thomason Donald B
Published: June 2021

p38 is a stress‐activated protein kinase related to apoptosis, but it is not clear what effect an endurance training program has on p38 activation.

OBJECTIVE:

To quantify p38 activation after an 8‐week endurance training program in sedentary subjects.

METHODS:

Three men (X±SE; 26.0 ± 1.2 yr) and 6 women (22.0 ± 1.5 yr) participated in an 8‐week cycling training program 3 dwk−1. Pre‐/post‐biopsies were performed, and Western immunoblot analysis with infrared markers was used to identify resting values of total p38 and p‐p38/total p38. In addition, a p38 ‘Activation Index’ (AI) was calculated (p38 AI = total p38 x [0.01 x [p‐p38/total p38]]).

RESULTS:

VO2 max increased 8.9%, while %body fat decreased 2.1%. Resting total p38 decreased 39.8 ± 15.7% (P < 0.05), but p‐p38/total p38 did not significantly decrease (−8.3 ± 14.9%). However, p38 AI was considerably decreased (pre = 100.0, post = 55.2; P < 0.05).

CONCLUSIONS:

Our results suggest that this effective endurance‐exercise program does not induce basal stress at the level of skeletal muscle in young previously untrained subjects, as indicated by activation levels of p38. Further study is needed to determine whether these findings apply to more vulnerable populations (e.g. older persons, patients with rehabilitating injuries).

Supported by Sigma‐tau HealthSciences, Inc.
Intermittent, normobaric hypoxia evokes adaptive modifications of nitric oxide synthase in canine myocardium
Authors: Ryou Myoung Gwi , Sun Jie , Manukhina Eugenia B , Downey H Fred , Mallet Robert T
Published: June 2021

Background: Physiologically modulated concentrations of nitric oxide (NO) are generally beneficial, but excessive NO can injure myocardium by producing cytotoxic peroxynitrite. Recently we reported that intermittent, normobaric hypoxia conditioning (IHC) produced robust cardioprotection against infarction and lethal arrhythmias in a canine model of coronary occlusion‐reperfusion, and that IHC dampens explosive, cytotoxic NO formation upon reperfusion of occluded coronary arteries. Hypothesis: IHC suppresses myocardial nitric oxide synthase (NOS) activity and contents of NOS isoforms. Methods: Mongrel dogs were conditioned by a 20 d IHC program (FIO2 9.5–10%; 5–10 min hypoxia/cycle, 5–8 cycles/d with intervening 4 min normoxia). One day later, ventricular myocardium was sampled for NOS activity assays, and immunoblot detection of endothelial and inducible NOS isoforms (eNOS, iNOS) and phosphorylated eNOS. Values in IHC dogs were compared with respective values in non‐conditioned, control dogs. Results: IHC lowered left and right ventricular NOS activities by 60%, from 100–115 to 40–45 mU/g protein (P < 0.01), and decreased eNOS and iNOS contents by 30 and 50%, respectively (P<0.05). IHC increased fractional phosphorylation of eNOS S1177 by < 2.5‐fold without altering T495 phosphorylation, a pattern that activates eNOS. Conclusions: IHC suppressed myocardial NOS activity, and eNOS and iNOS contents, but enhanced S1177 phosphorylation of the remaining eNOS. Thus, potentially cardioprotective attenuation of NOS activity may be due primarily to IHC suppression of iNOS. Support: NIH AT‐003598
MnSOD deficiency increases endothelial dysfunction produced by intermittent hypoxia
Authors: Miller Jordan D. , Chu Yi , Brooks Robert M , Heistad Donald D
Published: June 2021

Sleep apnea in humans is characterized by intermittent severe hypoxia, and is associated with endothelial dysfunction. We tested the hypothesis that mitochondrial superoxide scavenging is an important protective mechanism against endothelial dysfunction induced by CIH. Effects of severe, chronic intermittent hypoxia (CIH) on endothelial function in wild‐type mice (MnSOD+/+) or mice deficient in one copy of the MnSOD gene (MnSOD+/−) mice. CIH (90 second cycles, duty cycle = 0.33, nadir fraction of inspired oxygen = 0.05) or comparable intermittent bursts of room air (CTRL) was administered in a plexiglass chamber for 12 hours per day for 14 days. In wild‐type mice, maximal relaxation in response to acetylcholine in aorta was significantly reduced from 81 ± 3% (mean ± SE) in CTRL mice to 72 ± 2% in CIH mice. Endothelial function in MnSOD+/− mice was modestly impaired during CTRL conditions (69 ± 2%), and 14 days of CIH produced marked impairment of endothelial function (47 ± 5%; p < 0.05). Incubation of aortic rings with the superoxide dismutase mimetic tempol normalized vasorelaxation in response to acetylcholine in MnSOD+/+ and MnSOD+/− mice exposed to CIH (p = n.s. versus CTRL). Collectively, these data suggest that dismutation of mitochondrial superoxide is an important protective mechanism against endothelial dysfunction produced by intermittent hypoxia.
Mechanism of hypoxia‐induced α1H (CaV3.2) gene expression: examining the transcriptional regulation
Authors: Sellak Hassan , Liu Bainan , Zhou Chun , Chen Hairu , Wu Songwei
Published: June 2021

T‐type Ca2+ channels have been proposed to be a major effector that mediates cellular responses to hypoxia in a number of tissues. Among three cloned genes encoding the pore‐forming subunits of the T‐type Ca2+ channels, α1G, α1H, and α1I, only α1H has been reported to be upregulated under hypoxic conditions. Despite this observation, mechanism(s) underlying such hypoxia‐dependent α1H‐gene expression remains incompletely understood. The present study sought to examine the involvement of hypoxia‐response elements (HREs) within the promoter region of the α1H‐gene in the regulation of its expression under chronic oxygen deficiency. A ∼3.1‐kb rat α1H 5′‐flanking region upstream of the transcriptional start site was cloned in pGL3 vector harboring luciferase gene reporter. Sequence analysis of this region revealed the presence of 11 potential HRE sites. Transient transfection of PC‐12 cells with this construct showed a slight, but not significant, increase in luciferase activity under chronic hypoxia (3% O2, 48 h) compared to normoxia (21% O2). Serial deletion analysis of the ∼3.1‐kb fragment revealed that none of the potential HRE sites was responsible for hypoxia‐induced upregulation of α1H. In addition, cotransfection of different constructs with hypoxia‐inducible factor (HIF‐1) expressing vector did not show any increase in luciferase activity. Taken together, these data suggest that hypoxic regulation of α1H‐gene expression does not involve HRE sites located within the promoter region. Rather, the regulation might be at the post‐transcriptional level as evidenced by mRNA studies. Supported by HL‐66299.
Striated muscle fatigue is caused by e‐c coupling and metabolic alterations
Authors: Santos Bianca Maria Alves , Chang Ruchan , Garcia Karla Paola , Gonzalez‐Serratos Hugo
Published: June 2021

The mechanism of fatigue is not well understood. There are two propositions: a failure of e‐c(e‐c) as the tubular action potential (TAP); and an increase of ATP by products. We propose that both mechanisms may take place during fatigue development. First e‐c like the TAP and then metabolic alterations. Metabolic alterations may take place later after prolonged repetitive stimulation. To distinguish between these possibilities we used two paradigms, in one we stimulated the muscles at high frequencies continuously, in the other we stimulated them repetitively at high frequency with rest periods for the TAP to recover. When muscles were stimulated continuously with 60 Hz stimulations with out resting periods, the force dropped by 75% within a few seconds. The tension drop time course followed a liner function varying from10 sec at 60 Hz to 45 sec at 20 Hz of stimulation. With 60 Hz the recovery time was within 2 sec and was dependent on the frequency of stimulation. When muscles were stimulated repetitively with resting periods between stimulations (60 Hz 2 sec stimulations every 3 sec, 1 sec resting period) the force dropped by 75% in approximately 480 sec. The recovery time was then around 50 sec. We concluded that the two types of fatigue (metabolic and e‐c coupling) take place. The earlier drop in tensions may be due to failures of TAPs and the later be due to metabolic alterations.
Single fiber physiology differs between three human leg muscles
Authors: Luden Nicholas , Hayes Erik , Minchev Kiril , Louis Emily , Trappe Scott
Published: June 2021

While single fiber contractile function within a given fiber type population is largely similar between muscles, fiber type distribution can vary considerably. Because single fiber contractile function and fiber type distribution both contribute to whole muscle function, we generated composite profiles to compare three leg muscles of interest. Eight recreationally active males (5) and females (3) (23 ± 1 yr, 64 ± 3 kg, 51.8 ± 2.6 ml/kg/min) were biopsied from the vastus lateralis (VL) and soleus (S). To make further comparisons, seven recreationally active males (4) and females (3) (22 ± 1 yr, 68 ± 2 kg, 49.5 ± 2.6 ml/kg/min) were biopsied from the gastrocnemius (G). Single fibers were isolated and investigated for size and function, and subsequently processed for myosin heavy chain (MHC) composition (SDS‐PAGE). A composite value, which is a theoretical representation of the entire muscle, was then calculated for each contractile parameter based on the MHC composition of the studied fibers. The VL had higher specific tension (VL 66% > G and 28% > S), shortening velocity (40% > G; 48% > S), peak power (266% > G; 152% > S), and normalized power (276% > G; 221% > S), when compared to the gastrocnemius and soleus. Additionally, the gastrocnemius had lower peak tension (G 37% < VL; G 31% < S) and lower specific tension (40% < VL; 22% < S) when compared to the vastus lateralis and soleus. These findings highlight the unique intrinsic profile of these select muscles, which should be considered when interpreting how they adapt to exercise training.
Phenotype‐Specific Permeability Transition Pore Regulation in Rat Skeletal Muscle
Authors: Picard Martin , Mattas Jimmy , Ascah Alexis , Csukly Kristina , Godin Richard , Burelle Yan
Published: June 2021

The aim of this study was to determine whether the susceptibility of mitochondria to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the soleus (SOL: 95% type I fibers) and superficial white gastrocnemius (WG: 92% type IIb fibers). Sensitivity to Ca2+‐induced PTP opening was determined with a novel approach using permeabilized myofibers devoid of their contractile apparatus (phantom fibers). Ca2+ uptake in this preparation is specific to mitochondria and calcium retention capacity (CRC) is a good indicator of PTP sensitivity. Mitochondrial volume density was ~ 2 fold greater in SOL vs. WG, as judged by respiratory capacity per mg fiber weight and activity of the marker enzyme citrate synthase (CS). CRC values were 2.2 fold lower in phantom fibers from SOL compared to WG when expressed per unit of CS (CRC: 36.6±9.9 vs. 81.0±26.2 pmol Ca2+/mIU CS; p<0,001; n=4), indicating a pronounced vulnerability to PTP opening in type I vs. IIb fibers. A similar phenomenon was also observed in isolated mitochondria (CRC: <83 and 121 ± 60 nmol Ca2+/mg prot. in SOL and WG). This phenomenon was partly due to a 2‐fold increase in endogenous matrix Ca2+ levels in mitochondria from SOL compared to WG, but not to enhanced ROS production. Expression of the PTP regulatory protein cyclophilin‐D was similar in mitochondria from both muscles. However, expression of ANT, a protein reported to be involved in pore formation and which contributes to Ca2+‐sensitivity of the PTP, was significantly 2.3 fold higher in SOL compared to WG. The present study provides evidence that regulation of the PTP varies according to the phenotype of muscle fibers, which may impact on muscle cell death under conditions of stress.

Funded by NSERC.
Upregulation of In Situ Myosin Light Chain Kinase Activity In Fetal Compared to Adult Ovine Carotid Arteries
Authors: Injeti Elisha R , Graupner Gerhart , Pearce William J
Published: June 2021

Vascular reactivity changes dramatically during postnatal maturation due in large part to developmetal changes in myofilament calcium sensitivity. In turn, our recent findings suggest that reactivity of the thick filament component of calcium sensitivity, which is measured as the relation between cytosolic calcium concentration and the extent of myosin light chain (MLC) phosphorylation, is upregulated in fetal compared to adult arteries. The present study tests the hypothesis that upregulation of fetal thick filament reactivity is due to upregulation of myosin light chain kinase (MLCK) activity. Western blot analysis revealed that MLCK abundance was 6.5±0.4 fold greater in adult than in fetal arteries. Total MLCK activity, estimated as the rate of phosphorylation of the substrate myosin light chain in intact arteries, was greater in fetal (7.9 ± 0.3 %/sec) than in adult (6.6 ± 0.3 %/sec) preparations. When total MLCK activity was normalized relative to MLCK abundance to estimate the specific activity of MLCK (%phosphorylation/sec/μg MLCK), these estimates were dramatically greater in fetal (45.5 ± 2.3) than in adult (5.8 ± 0.5) arteries. Together, these results indicate that upregulation of MLCK specific activity contributes significantly to upregulation of thick‐filament reactivity in fetal compared to adult ovine carotid arteries. Supported by NIH HD31226, HL54120, and HL64867.
NCS‐1 modulates expression of calcium handling proteins
Authors: Telemaque Sabine , Wang Zhen , Liu Meei‐Yueh , Buzdugan Larisa , Liu S. Jesse
Published: June 2021

We have previously shown that neuronal‐calcium sensor‐1 (NCS‐1) was present in rat heart and that overexpression of this small binding protein in isolated ventricular myocytes lead to an increase in contractile function. To further elucidate the physiological role of NCS‐1 in the heart, we examine cellular mechanisms by which overexpression of NCS‐1 could augment contractile function. Isolated adult rat ventricular myocytes were infected with adenovirus constructs (MOI: 100) expressing either NCS‐1 or β‐gal (control) for 48 hr. Cells were then washed and collected. Total cardiac lysate proteins were separated on SDS‐PAGE gels and expression levels of major E‐C coupling‐associated proteins were determined. Preliminary results suggest that expression of SERCA‐2a was decreased in the NCS‐1‐infected cells when compared to control cells. Expression of phospholamban (PLB), a negative regulator of SERCA‐2a activity, was unchanged while levels of phosphorylated PLB were significantly increased in NCS‐1‐infected cells, which is consistent with an increase in cardiac contractility. Finally, expression of NCX‐1 was not altered in the NCS‐1‐infected cells. Taken together, our data suggest that in addition to directly interacting with cardiac myofilament proteins, NCS‐1 may also regulate contractile function by modulating expression of key calcium handling proteins. Supported by UAMS Department of Medicine Seed Money Award (ST).
Endothelial Progenitor Cells Are Decreased in the Circulation of Patients with Sepsis
Authors: Burnham Ellen Lyda , Sutcliffe Diane , Edwards Michael , Taylor W. Robert , Moss Marc
Published: June 2021

Background: Acute lung injury (ALI) and sepsis are associated with widespread endothelial injury. Elaboration of endothelial precursor cells from bone marrow that could participate in repair represents one mechanism to attenuate injury.

Methods: Blood was obtained from 24 ALI and 12 septic patients within 72 hours of diagnosis. Peripheral blood mononuclear cells were isolated and cultured on fibronectin‐coated wells in endothelial‐specific medium. Numbers of endothelial progenitor colony‐forming units (EPC CFUs) were assessed after 7 days.

Results: ALI and sepsis patients were similar for mortality, age, and severity of illness. 81% (9/11) of sepsis patients had septic shock;17% (2/12) had a pulmonary source for sepsis. 54% (13/24) of ALI patients had septic shock. ALI patients had higher EPC CFU numbers than did septic patients [median 56 (19–90) vs 19 (3–50), p<0.04)]. Comparing just patients with septic shock (ALI, n=13 vs. sepsis, n=9) revealed a trend for higher EPC CFUs in ALI patients [median 50 (17–93) vs 19 (11–46), p =0.09). No significant difference in EPC CFU numbers between those with and without septic shock were observed among those with ALI (p=0.58).

Conclusions: More numerous EPC CFUs in those with ALI provides evidence for the role of lung injury in their mobilization. Alternatively, septic shock might hinder their release from the marrow.

AHA Beginning Grant‐in‐Aid
Age‐Related Vascular Endothelial Dysfunction is Associated with Altered Regulation of Nuclear Factor k B and Increased Pro‐Inflammatory Cytokines in Humans
Authors: Donato Anthony J , Lawson Brooke R , Jablonski Kristen L , Gano Lindsey B , Seals Douglas R
Published: June 2021

Inflammation may contribute to vascular endothelial dysfunction with aging. Compared with young adults (Y), endothelial cell protein expression (ECPE) of nuclear factor κ B (NFκB), a pro‐inflammatory nuclear transcription factor, is increased in healthy older adults (O), but the underlying mechanism and potential pro‐ inflammatory effects are unknown. Endothelium‐dependent dilation (EDD: Δforearm blood flow [FBF] with acetylcholine) was 30% lower (peak FBF 12.8±1.3 vs 17.9±2.2 mL tissue−1 min−1, P<0.05), whereas endothelium‐independent dilation (ΔFBF with sodium nitroprusside) was similar (P=0.30) in O (n=13, 62±1yr) vs Y (n=10, 24±1yr). ECPE (immunofluorescence) of NFκB was higher (0.69±0.06 vs 0.39±0.03 NFκB/HUVEC intensity, P<0.05), whereas ECPE of inhibitor of NFκB (IKBα) was lower (0.17±0.02 vs 0.24±0.03 IKBα/HUVEC intensity, P < 0.05) in O; IKBα activator kinase was not different (P>0.05). ECPE of tumor necrosis factor α (0.52±0.06 vs 0.33±0.06 TNF‐α/HUVEC intensity, P < 0.05), monocyte chemoattractant protein 1 (0.59±0.06 vs 0.39±0.02 MCP‐1/HUVEC intensity, P < 0.05) and interleukin 6 (0.43±0.08 vs 0.29±0.02 IL‐6/HUVEC intensity, P = 0.07) were ~50–60% greater in the O vs Y. Older adults with endothelial dysfunction have increased ECPE of NFκB, possibly as a result of lower IKBα. This may contribute to increased ECPE of pro‐inflammatory cytokines. NIH AG013038, AG022241, AG006537, AG000279
The Immunosuppressive drugs rapamycin and FK506 decrease vasodilation by increasing PKCbetai‐mediated phosphorylation of eNOS Thr495
Authors: Chiasson Valorie L. , Mitchell Brett M.
Published: June 2021

PKC is known to phosphorylate eNOS at Thr495, which decreases NO production and endothelial function. We have shown previously that the conventional PKC family (isoforms alpha, betaI, betaII, gamma) mediates the increased eNOS Thr495 phosphorylation and endothelial dysfunction caused by rapamycin and FK506. Here we sought to determine which of these PKC isoforms was responsible for phosphorylating eNOS at this inhibitory site. Endothelium‐intact mouse aortas were treated acutely with rapamycin or FK506 (1 uM, 30 min) with or without the PKCalpha inhibitor Ro‐32‐0432 (0.01 uM), the PKCbetaII inhibitor CG53353 (1 uM), or a PKCbeta inhibitor (0.1 uM) and NO‐mediated relaxation responses and eNOS Thr495 phosphorylation were measured. Inhibition of PKCalpha and PKCbetaII had little effect on NO‐mediated relaxation whereas PKCbeta inhibition completely restored relaxation responses to or beyond control levels in rapamycin‐ and FK506‐treated aortas. Similar effects were seen on eNOS Thr495 phosphorylation in rapamycin‐treated aortas. These preliminary data suggest that PKCbetaI is the isozyme that phosphorylates eNOS Thr495 in response to rapamycin and FK506. We are currently measuring PKC isoform‐specific activation and performing PKC isoform‐specific knockdown studies in rapamycin‐ and FK506‐treated endothelial cells to confirm these findings. (Supported by HL084299 to BMM)
Hyperglycemia‐induced reactive oxygen species are differentially regulated by estrogen in human endothelial cells
Authors: Chakrabarti Subhadeep , Davidge Sandra T
Published: June 2021

Diabetes is a predisposing factor for atherosclerosis. High glucose generates reactive oxygen species such as superoxide and peroxynitrite which lead to pro‐atherogenic changes in the endothelium. Estrogen has a beneficial anti‐oxidant and anti‐inflammatory effect on the vascular endothelium. However, the beneficial effects of estrogen are absent in patients with diabetes. We investigated the effects of estrogen on endothelial cells in the context of pre‐existing hyperglycemia. Human umbilical vein endothelial cells (HUVEC) were used for the studies. Superoxide and peroxynitrite were detected by staining for Dihydroethidium and nitrotyrosine respectively. Enzymes were detected by western blotting. We found that high glucose increased levels of both superoxide and peroxynitrite in the endothelial cells. Estrogen administration had no effect on superoxide levels. Surprisingly, estrogen significantly reduced the peroxynitrite generated under high glucose conditions. On examining the expression of nitric oxide synthase isoforms, we found that estrogen increased eNOS levels under normal glucose, but not under high glucose. The iNOS levels were unchanged under all conditions. Our results show a novel selective anti‐oxidant effect of estrogen on hyperglycemia‐induced peroxynitrite in endothelial cells.

This work has been supported by CIHR, HSF Canada and AHFMR.
BH4 protects endothelial progenitor cell number and function via suppressing thrombospondin‐1 expression in salt‐sensitive hypertension
Authors: Xie He‐Hui , Du Yan‐Hua , Channon Keith M. , Chen Alex F.
Published: June 2021

Background: Endothelial progenitor cells (EPC) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. eNOS regulates EPC mobilization and function, but is uncoupled in DOCA‐salt hypertension due to reduced cofactor BH4. We tested the hypothesis that GTP cyclohydrolase I (GTPCH), the rate‐limiting enzyme of BH4 synthesis, protects EPC and its function in DOCA‐salt mice.

Methods and Results: EPC were isolated from peripheral blood and bone marrow of wild‐type (WT), GTPCH transgenic (Tg‐GCH) and BH4 deficient hph‐1 mice. In WT DOCA‐salt and hph‐1 mice, EPC number was significantly reduced (flow cytometry and fluorescence microscopy, n=7–12, p<0.01), which was rescued in DOCA‐salt Tg‐GCH mice (n=5–8). Compared with WT Sham mice, both EPC adhesion and angiogenesis functions were impaired in WT DOCA‐salt and hph‐1 mice, which were reversed in DOCA‐salt Tg‐GCH mice. Superoxide level (DHE flow cytometry) in EPC was augmented in WT DOCA‐salt and hph‐1 mice, which was blunted in DOCA‐salt Tg‐GCH mice. Western blot showed that the secreted thrombospondin‐1 (TSP‐1, a key inhibitor of EPC function) from EPC was elevated in WT DOCA‐salt and hph‐1 but not in DOCA‐salt Tg‐GCH mice, which was prevented by NOS inhibitor L‐NNA. Conclusion: GTPCH/BH4 pathway protects EPC and its functions in DOCA‐salt hypertension b suppressing TSP‐1 expression.
Diverse TRPC heteromultimers form store‐operated channels in native vascular smooth muscle preparations
Authors: Albert Anthony , Saleh Sohag , Peppiatt‐Wildman Claire , Large William
Published: June 2021

Store‐operated channels (SOCs) are activated following depletion of intracellular Ca2+ stores and the canonical transient receptor potential (TRPC) family of channels are possible molecular candidates of SOCs. Here we describe diverse properties and putative molecular identities of SOCs in different vascular preparations.

In rabbit mesenteric (MA) and coronary artery (CA) myocytes SOCs were activated by the store depleting reagents BAPTA‐AM and cyclopiazonic acid and in 1.5mM [Ca2+]o these SOCs had conductances of about 2pS (Saleh et al. 2006) and 3 pS and reversal potentials (EREV) of about +20mV and −10mV respectively. In 0mM [Ca2+]o the conductances were about 7pS in MA and 3pS in CA whilst EREV were both about −10mV respectively, highlighting different biophysical properties of SOCs in these two preparations.

MA SOCs were selectively inhibited by anti‐TRPC1/C5 whilst SOCs from CA were specifically inhibited by anti‐TRPC1/C5/C6. SOCs recorded from portal vein myocytes had similar biophysical properties to MA SOCs (Albert & Large, 2002) but were blocked by anti‐TRPC1/C5/C7.

These data suggest that TRPC channels can form alternative heteromultimers with a similar SOCE function in different vascular smooth muscle preparations. Moreover, these data show that anti‐TRPC antibodies can be used as ‘immunopharmacological’ reagents with a remarkable degree of selectivity to determine the composition of heteromultimeric ion channel proteins in native tissue.

Funded by British Heart Foundation and The Wellcome Trust.
TRPC1 and STIM1 Mediate Capacitative Calcium Entry in Mouse Pulmonary Arterial Smooth Muscle Cells
Authors: Ng Lih Chyuan , McCormack Mary D. , Shen Xiao‐Ming , Hume Joseph R.
Published: June 2021

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 channel mediates capacitative Ca2+ entry (CCE) but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediate CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured PASMCs loaded with fura‐2, 10μM CPA caused a rise in [Ca2+]i but it was abolished when extracellular Ca2+ was removed. Subsequent addition of 2mM Ca2+ elicited a transient rise in [Ca2+]i that was partially inhibited by 10μM nifedipine, leaving a nifedipine‐insensitive transient and nifedipine‐insensitive sustained rise in [Ca2+]i. The nifedipine‐insensitive sustained, but not transient rise in [Ca2+]i was inhibited in cells pretreated with antibodies raised against extracellular epitope of TRPC1 and STIM1. RT‐PCR revealed TRPC1 and STIM1 mRNAs, whereas Western blot analysis identified TRPC1 and STIM1 proteins in cultured mouse PASMCs. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry, CCE and another as yet unidentified voltage‐independent Ca2+ entry pathway. These data suggest that CCE is mediated by TRPC1 channel possibly through activation of STIM1 protein in mouse PASMCs. (Supported by HL49254 and NCRR P20RR15581)
Sulfonylurea receptor‐dependent and ‐independent pathways for KATP channel opener‐mediated vasodilation
Authors: Adebiyi Adebowale , McNally Elizabeth M , Jaggar Jonathan H
Published: June 2021

KATP channel openers, including diazoxide and pinacidil, activate both plasma membrane and mitochondrial KATP channels. Here, we investigated functional molecular targets for diazoxide and pinacidil in myocytes of murine resistance‐size mesenteric arteries. In pressurized (60 mmHg) arteries, ryanodine, a Ca2+ spark inhibitor, iberiotoxin, a KCa channel blocker, 4‐AP, a Kv channel blocker, MnTMPyP, an antioxidant, and ryanodine+4‐AP reduced diazoxide‐induced dilations by between 45 and 77%, but did not alter pinacidil‐induced dilations. RT‐PCR indicated that isolated mesenteric artery myocytes expressed sulfonylurea receptors (SUR) 2A and 2B, but did not express SUR1. Pinacidil‐induced dilation in arteries of SUR2‐deficient (SUR2−/−) mice was ∼10% of that in wild‐type control arteries, whereas diazoxide‐induced dilation was similar in SUR2−/− and wild‐type. Atpenin, a specific mitochondrial ETC complex II (ETCII) inhibitor, dilated arteries similarly to diazoxide and this effect was attenuated by MnTMPyP and ryanodine+4‐AP. Atpenin also reduced diazoxide‐induced dilation, but did not alter pinacidil‐induced dilation. Data indicate that pinacidil‐induced vasodilation requires SUR2. In contrast, diazoxide‐induced vasodilation does not require SURs and is mediated by ETCII inhibition, a ROS elevation, and Ca2+ spark, KCa channel, and Kv channel activation. NIH/AHA
Gene‐by‐Gene Interactions Between Phenylethanolamine N‐Methyltransferase and the Beta‐2 Adrenergic Receptor and the Ventricular Response to Exercise
Authors: Snyder Eric Michael , Ji Yuan , Joyner Michael J , Turner Stephen T , Weinshilboum Richard M , Johnson Bruce D
Published: June 2021

Epinephrine is a sympathomimetic which is derived from phenylalanine and tyrosine. The final step of epinephrine synthesis, the conversion from norepinephrine, relies on phenylethanolamine N‐methyltransferase (PNMT). The beta‐2 adrenergic receptor (ADRB2) binds to epinephrine and has several common functional polymorphisms including a Glycine (Gly) for Arginine (Arg) substitution at amino acid 16. We assessed the effect of the gene‐to‐gene interaction of these proteins on the ventricular response to exercise in healthy humans (n=69, age=29±6yrs, ht=174±10cm, wt=71±13kg). We found that within the 2*1 haplotype of the 5′FR, which has previously demonstrated greater epinephrine synthesis compared to 2*7 haplotype, the Arg16 genotype demonstrates lower stroke volume (SV) than the Gly16 genotype despite similar epinephrine levels and synthesis rates (Arg16= 75±5, Gly16=89±6ml/beat). Similarly, within the 2*7 haplotype of the 5′FR, which has demonstrated attenuated epinephrine synthesis compared to 2*1, the Arg16 genotype demonstrates a smaller SV than the Gly16 genotype (Arg16= 59±3, Gly16=78±6ml/beat). Additionally, the Arg16 and Gly16 subjects within the 2*1 haplotype of PNMT have greater stroke volume than their ADRB2 counterparts of the 2*7 haplotype of PNMT. These data demonstrate an interaction between the genes that encode PNMT and ADRB2 on the ventricular response to exercise.
Vascular Endothelial Dysfunction with Aging in Healthy Adults is Related to Total White Blood Cell Count and Selective Immune Cell Populations
Authors: Seibert Sara Marian , Donato Anthony J. , Lawson Brooke R. , Seals Douglas R. , Walker Ashley E.
Published: June 2021

Vascular endothelial dysfunction (VED) develops with aging and contributes to increased risk of cardiovascular (CV) diseases. White blood cell count (WBC) is an independent predictor of CV events in middle‐aged and older adults (MA/O), but its relation to age‐associated VED is unknown. In 27 healthy adults free of infections (18–72 y), WBC was inversely related (r=−0.47, P<0.05) to endothelium‐dependent dilation (EDD), but was not related (P>0.05) to endothelium‐independent dilation (maximal forearm blood flow responses to acetylcholine and sodium nitroprusside, respectively). EDD was 41% lower (9.3±1.4 vs. 15.8±2.4 mL tissue−1 min−1, mean±SE, P<0.05) in MA/O with higher WBC (6.0±0.2 109/L, range 5.0–7.1, 65±1y, n=9/5m) than MA/O with lower WBC (4.0±0.2 109/L, 3.4–4.8, 63±1y, n=9/6m) despite no differences in CV risk factors. EDD was similar in the MA/O with lower WBC vs. young controls with higher mean WBC (16.2±2.5 mL tissue−1 min−1; WBC 4.9±0.6 109/L, range 3.0–8.9, 22±2y, n=9/7m,). EDD also was inversely related to WBC differential counts for neutrophils (r=−0.56, P<0.01), monocytes (r=− 0.34, P<0.05), eosinophils (r=−0.46, P<0.01), and basophils (r=−0.36, P<0.05), but was not related to lymphocytes (P=0.38). These results indicate that VED with aging is related to greater total WBC and elevations in selective populations of circulating immune cells. NIH AG006537, AG013038, AG022241, AG000279
Flow mediated dilation response to oscillatory vs. steady shear: evidence for the transduction of the mean shear stimulus
Authors: Pyke Kyra E. , Poitras Veronica , Tschakovsky Michael E.
Published: June 2021

OBJECTIVE: 1) To compare the flow mediated dilation (FMD) response to an increase in brachial artery (BA) shear stress (SS) via forearm heating (FH) vs. forearm exercise (FE). 2) To isolate the effects of the oscillatory pattern of exercise SS. BA diameter (BAD) and mean blood velocity (MBV) were measured with ultrasound in 16 healthy subjects. BA MBV was elevated to the same mean level for 10min via 3 protocols. 1) FHstdy ‐ A steady MBV was created by controlled release of BA compression upstream of heat‐induced forearm vasodilation. 2) FHosc: rhythmic cuff inflation simulated exercise induced oscillations in MBV. 3) FE ‐ Rhythmic isometric handgrip exercise.

RESULTS: The mean increase in shear rate (MBV/BAD) (±SD) was the same in all conditions (FHstdy: 52.2±14.1s−1; FHosc: 51.7±15.7s−1; FE: 50.1±13.0s−1 P=0.13). Neither the percent change in BAD (End‐trial: FHstdy: 7.0±3.0%; FHosc: 7.8±3.4%; FE: 6.7±3.0% P=0.20) nor the initial response speed (tau‐time to 63% of max response) (FHstdy:27.8±14.4s; FHosc: 22.6±16.7s; FE:25.9±23.3s P=0.69) were different between conditions.

CONCLUSION: The FMD was determined by the mean shear independent of stimulus pattern (oscillatory vs. steady) and mode of increase (heating vs. exercise). These data suggest that the endothelium transduces the mean shear stimulus and indicate that exercise may provide a viable technique to investigate human FMD.

Funded by NSERC, ACSM and HSFC
Investigating a Triple Congenic Rat Model to Determine Genes Contributing to Renal Failure
Authors: O'Meara Caitlin Claire , Lazar Josef , Moreno Carol , Provoost Abraham P. , Jacob Howard J.
Published: June 2021

To find genes associated with kidney disease, the fawn‐hooded hypertensive (FHH) rat was crossed with the normotensive, renal resistant August × Copenhagen Irish (ACI) strain. We used a genetic mapping strategy to locate five quantitative trait loci (QTLs) on the rat genome that show linkage to measures of renal impairment (Rf1–5). Following this linkage analysis, single, double and triple congenic animals were made to demonstrate the synergistic relationship between the genes within the various Rf QTLs. Double and triple congenic animals containing Rf‐1 showed more marked measures of renal impairment, which indicates that genes within these QTLs are all interacting with one another to contribute to this complex disease. We have developed Rf‐1+3+4 triple congenic animals to investigate the interaction between these three QTLs. Our current focus is to analyze candidate genes in the Rf‐3 region.

National Institute of Health and the Medical College of Wisconsin