bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative stress is required for mechanical ventilation‐induced protease activation in the diaphragm
Authors: Hudson Matthew B , Whidden Melissa A , Smuder Ashley J , Wu Min , Nelson W Bradley , Powers Scott K
Published: June 2021

Prolonged mechanical ventilation (MV) results in diaphragmatic weakness due to both fiber atrophy and contractile dysfunction. Recent work reveals that activation of the proteases calpain and caspase‐3 are required for MV‐induced diaphragmatic atrophy and contractile dysfunction. However, the mechanism to trigger activation of these proteases remains unknown. To address this issue, we tested the hypothesis that oxidative stress is essential for the activation of calpain and caspase‐3 in the diaphragm during MV. We tested this postulate by preventing MV‐induced diaphragmatic oxidative stress using the antioxidant trolox. Treatment of animals with trolox was successful in the prevention of MV‐induced protein oxidation and lipid peroxidation in the diaphragm. Importantly, this avoidance of MV‐induced oxidative stress inhibited the activation of both calpain and caspase‐3 in the diaphragm during MV. Further, the prevention of MV‐induced oxidative stress not only averted the activation of these proteases but also rescued the diaphragm from MV‐induced diaphragmatic atrophy and contractile dysfunction. Collectively, these findings support the hypothesis that oxidative stress is required for MV‐induced activation of calpain and caspase‐3 in the diaphragm and suggest that antioxidant therapy could be beneficial in the prevention of diaphragmatic weakness during prolonged MV.
ATM plays a role in insulin‐stimulated phosphorylation of AS160 independent of Akt in mouse soleus muscle
Authors: Ching James Kain , Armon Jennifer L. , Renth Allyson , Collins Roy L. , Fisher Jonathan S.
Published: June 2021

Recent data have emerged suggesting that ataxia telangiectasia mutated (ATM) is a factor that modulates insulin signaling. However, the manner in which ATM impacts the insulin signaling pathway has not been addressed in skeletal muscle. The aim of this study was to determine how ATM impacts proximal insulin signaling in mouse soleus muscle. Isolated soleus muscles from wild type (ATM+/+) mice, ATM deficient (ATM−/−) mice, and mice lacking one functional ATM allele (ATM+/−), were incubated with or without insulin (100 nM). Western blot analysis showed that insulin‐stimulated phosphorylation of Akt S473 and Akt T308 was normal in ATM−/− and ATM +/− mice. Interestingly, however, insulin‐stimulated phosphorylation of Akt substrate of 160 kDa (AS160) was prevented in muscles from ATM−/− mice. Similarly, soleus muscles from ATM+/− mice showed a significant decrease in insulin‐stimulated phosphorylation of AS160, indicating that even partial loss of ATM is sufficient to impair insulin signaling at AS160. Our data suggest that ATM modulates insulin signaling leading to phosphorylation of AS160, independent of Akt.

This work is supported by NIH grant R15DKO80437
Myosin loss and muscle wasting in an experimental ICU model: Underlying mechanisms
Authors: Larsson Lars , Ochala Julien , Diaz Monica Llano , Gustafson Ann‐Marie , Dworkin Barry R , Nair Sreekumaran K , Gautel Mathias
Published: June 2021

The muscle wasting and impaired muscle function associated with critical llness in ICU patients have negative consequences for recovery from primary disease, but are also the most persistent and debilitating of problems for ICU survivors years after hospital discharge. This study aims at improving our understanding of the mechanisms underlying the muscle wasting in ICU patients by conducting time‐resolved analyses of skeletal muscle structure and function together with measurements of protein synthesis rate, myofibrillar protein and gene expression and intracellular signaling in response to post‐synaptic block of neuromuscular transmission (NMB), sedation, mechanical ventilation and muscle unloading at durations varying from hours to two wks by using a unique experimental rat ICU model. Skeletal muscle structure and function were well maintained during the first four days, followed by a dramatic and progressive decline in muscle size and function. The preferential loss of the molecular motor protein myosin and impaired forcegenerating capacity (specif force) are the major causes underlying the impaired skeletal muscle function in the ICU model. Muscle unloading due to lack of weightbearing (external unloading) and muscle membrane depolarization (internal unloading) are forwarded as very important underlying factors.

Supported by the Sweish Research Council and AFM
Muscle hypertrophy and metabolic signaling after two different resistance exercises in young men
Authors: Hulmi Juha J , Walker Simon , Ahtiainen Juha P , Nyman Kai , Kraemer William J , Häkkinen Keijo
Published: June 2021

Muscle mTOR/MAPK signaling is important in mediating hypertrophy/metabolic adaptations to resistance exercise (RE). However, possible differences in signaling responses to a RE protocol designed primarily for large gains in muscle hypertrophy (HRE) and another associated with neural adaptations and limited hypertrophy (maximal strength, MRE), are not well known. Eight untrained men (28.4±3.7yrs) conducted bilateral leg press HRE and MRE protocols in a randomized order with one week between REs. HRE was five sets of ten repetition maximums (5x10RM) and MRE 15x1RM. Six men (26.5±3.6yrs) served as controls. Vastus lateralis muscle biopsies were taken before and 0.5h after RE or resting in controls. The decrease in isometric force and increase in blood lactate after RE, was larger after HRE than after MRE (P<0.05). Western blotting results revealed that no changes were seen in the controls, but the phosphorylation (p) of p70S6K, p85S6K, rpS6, and MAPKs p38α and p38γ were increased after both REs. However, the increase in p‐p70S6K, p‐rpS6 and p‐p38γ were larger after HRE. p‐Erk1 and p‐Erk2 MAPKs increased only after HRE. In conclusion, mTOR/MAPK signaling increases after both maximal strength and hypertrophic protocols but it is greater in the latter, possibly mediating adaptations specific to these types of training. Supported by: DAVID Sports Ltd, the Finnish Cultural Foundation.
MARK4 is a Novel CaMKKα Substrate and Contraction‐Regulated Kinase in Mouse Skeletal Muscle
Authors: Witczak Carol A , Toyoda Taro , Rockl Katja SC , Middelbeek R. Jan‐Willem , Hirshman Michael F , Goodyear Laurie J
Published: June 2021

Ca2+/calmodulin‐dependent protein kinase kinase α (CaMKKα) regulates contraction‐induced glucose uptake in skeletal muscle, but the signaling pathway underlying this effect is not known. Microtubule affinity regulated kinases 1–4 (MARK1‐4) are homologous to known CaMKKα substrates, yet to date no studies have assessed MARKs as CaMKKα substrates. Our goal was to determine if MARKs are CaMKKα substrates in vitro and in skeletal muscle. Recombinant CaMKKα phosphorylated recombinant MARK4, but not MARK1, 2 or 3 in vitro. Expression of active CaMKKα in mouse tibialis anterior muscle increased MARK4 activity ~2‐fold, but did not affect MARK1 or MARK2/3 activities. In female, ICR mice (a standard outbred strain), 5 and 10 min of in situ contraction increased MARK4 activity 2‐ and 3‐fold, respectively, but did not affect MARK1 or MARK2/3 activities. In female, CaMKKα knockout mice, in situ contraction did not increase MARK4 activity. These results demonstrate that: 1] CaMKKα phosphorylates MARK4, but not MARK1, 2 or 3 in vitro; 2] Expression of active CaMKKα in muscle only stimulates MARK4 activity in skeletal muscle; 3] Contractile activity only increases MARK4 activity in skeletal muscle; and 4] CaMKKα is the main MARK4 kinase during muscle contraction. In conclusion, MARK4 is a newly identified CaMKKα substrate and contraction‐regulated kinase in mouse skeletal muscle.
A physiological role of heat shock factor 1‐related stress response in the regrowth of atrophied soleus muscle in mice
Authors: Goto Katsumasa , Ohno Yoshitaka , Goto Ayumi , Nakamura Ayane , Nakai Akira , Sugiura Takao , Ohira Yoshinobu , Yoshioka Toshitada
Published: June 2021

Hypertrophic stimuli, such as mechanical loading and heat stress, could up‐regulate heat shock proteins (HSPs) in muscle cells. Although it has been proposed that HSPs has several cellular functions, the physiological roles for HSPs induction in skeletal muscles is still unclear. The induction of HSPs expression is regulated by heat shock factor 1 (HSF1), which binds to heat shock elements located on the upstream region of all Hsp genes. In the present study, using the HSF1 knockout (HSF1‐KO) mice, we examined the role of HSPs inthe reloading‐associated regrowth of atrophied soleus muscle. The regrowth was induced by the reloading on soleus following 2‐week‐hindlimb suspension. Increment in the protein content and the up‐regulations of HSPs of soleus muscles in HSF1‐KO mice was smaller than that of wild type animals. Results from this study indicated that the up‐regulation of HSPs may facilitate the protein synthesis in skeletal muscle cells. This study was supported, in part, by Grant‐in‐Aid for Scientific Research (B, 20300218, KG; A, 18200042, TY; S, 19100009, YO) from Japan Society for the Promotion of Science, and Research Grant from KAO Health Science Research (KG).
Mild Heat Shock Promotes Hypertrophy in Cardiac, Skeletal and Smooth Muscle Cells
Authors: Hall Todd , Craig Robin , Touchberry Chad , Silswal Neerupma , Elmore Christopher , Baker Kendra , Andresen Jon , Wacker Michael , Brotto Marco
Published: June 2021

Heat‐Shock Proteins (HSPs) are involved in growth and development of uscles, however, the exact role that hyperthermia and HSPs play in muscle hypertrophy and muscle repair is unknown. Here, for the first time, we present our findings on the effects of heat shock (HS) in representative cell culture models of cardiac (HL‐1), skeletal (C2C12) and smooth (A7R5) muscle cells. We used fully differentiated cells, and exposed them to a mild heat shock protocol (43ºC for 20 min). Cells were returned after HS to their normal culture environment and 24 hrs after HS, we measured cell area. Surprisingly, we discovered that HS induced significant hypertrophy in all three types of muscle cells. To begin deciphering the molecular machinery involved with the hypertrophic response we used a real‐time PCR array that included specific genes involved in hypertrophy, cell signaling and transduction, and excitation‐contraction coupling. In C2C12, 6 hrs after HS, we found an increase in the expression of Hsp genes and downregulation of IP3R‐1, TLR2, MHC6, and Titin. This is the first report to show that HS promotes cardiac, skeletal, and smooth muscle hypertrophy that associates with changes in a small subset of genes.
Plasma level Hsp27 as a Potential Biomarker of Doxorubicin‐induced Heart Failure
Authors: Ilangovan Govindasamy , Karthikeyan Krishnamoorthy
Published: June 2021

Doxorubicin (Dox) is known to cause oxidative stress‐induced heart failure among the cancer patients who were treated with this drug. An early detection of a reliable biomarker of cardiotoxicity will be useful to modify the future regimens of this drug for the patients who are under potential risk of heart failure. We have found that plasma level Hsp25 increases upon treating with Dox in animal models by the time the cardiac dysfunction starts. Mice were treated with Dox (6mg/kg ) for 4 weeks and blood samples were collected once in a week for 4 weeks of post drug treatment. Plasma level Hsp25 remained almost the same for the first three weeks, but from fourth week onwards it increased more than eight fold (8.75±.68, p = < 0.001, n = 12). From fourth week onwards echo showed symptoms of cardiac failure (%FS reduced from 46.7±3.6 (n=12) to 26.8±1.2 (n=12, p < 0.001). We propose that the HSF‐1 responds to Dox induced oxidative stress and increased Hsp25. To confirm this, we found the HSF‐1 knock out mice (HSF‐1−/−) were resistant to Dox induced heart failure. For HSF‐1−/− mice, the 50% survival was found to be beyond 33 ± 2 days (Log‐Rank test). In this case, 40% of the animals survived until the study was completed (100 days), unlike the wild type where the 50% survival was only 23±3 days, and almost all the animals died by end of the study. Thus increased level in Hsp25 may serve as a potential marker of heart failure among Dox treated cancer patients.
Phenol increases twitch tension by increasing Ca2+ transients in intact single Xenopus myofibers
Authors: Nogueira Leonardo , Knapp Amy E. , Hogan Michael C.
Published: June 2021

Phenol is a neurolytic agent used for management of spasticity in patients with neuron injury. The effects of phenol on skeletal muscle function are unknown. We investigated the effects of phenol on force production and Ca2+ release in intact single Xenopus skeletal muscle fibers. To determine the relationship between force production and Ca2+ release in the absence and presence of phenol (0.005% – 0.05% w/v), intact single muscle fibers were dissected and microinjected with FURA‐2 followed by electrically stimulated single‐twitches and tetanic contractions (1–150Hz, 250 ms train, 2 ms stimulus, 8–10V). During single‐twitches at activation frequencies that elicited far less force than maximal, phenol reversibly increased the force and peak Ca2+ ratios compared to the untreated condition in a concentration‐dependent manner (from 10 ± 4% to 57 ± 10% of maximum tetanic force and from 47 ± 11% to 68 ± 10% of the maximum FURA‐2 ratio, both at 0.05% phenol, P<0.01). At higher concentrations (>0.05%), phenol completely blocked force production. These results suggest that phenol increases twitch force by increasing Ca2+ release from the sarcoplasmic reticulum in skeletal muscle fibers. This mechanism remains to be elucidated; however, phenol may prove to be a new agent used to study skeletal muscle function.

Supported by NIH grant 1P01HL091830‐01A18101
Reduced mitochondrial volume contributes but cannot fully explain the increase in matrix free calcium after addition of ADP
Authors: Boelens Age Douwe , Stowe David F , Aldakkak Mohammed , Gadicherla Ashish K , Dash Ranjan K , Haumann Johan , Camara Amadou K S
Published: June 2021

In an earlier study we showed a transient doubling of mitochondrial matrix (m) [Ca2+] in response to added ADP and proposed that greater buffering of Ca2+ by ATP than by ADP is largely responsible for the increase. Previous studies have described mitochondrial volume contraction in response to ADP. Thus we explored if reduced mitochondrial volume with ADP also contributes to the apparent increase in m[Ca2+]. Guinea pig heart mitochondria were isolated by differential centrifugation. Mitochondria were suspended in respiration media, including 1 mM EGTA, 1 mM MgCl2, 140 mM KCl, and 5 mM K2HPO4. Mitochondria were energized with pyruvic acid followed by 250 μM ADP. Mitochondrial volume was measured by light scattering at a wavelength of 520 nm. Alamethicin (5 μg/ml) was added at the end to attain maximal matrix volume. Changes in matrix volume after addition of ADP were calculated as a percentage of maximal volume change. We showed a matrix contraction in response to ADP of approximately 5% of the maximal change in matrix volume. We conclude that although matrix contraction can contribute to the increase in [Ca2+] after addition of ADP, it explains only a small proportion of the rise in [Ca2+] during phosphorylation of ADP, suggesting differential Ca2+ buffering of ADP and ATP remains a major factor.
Prostaglandin E2 (PGE2) Type 1 Receptors (EP1R) in the Subfornical Organ (SFO) Contribute to Slow‐Pressor AngII Sympathoexcitation and Hypertension (HTN) via NADPH Oxidase (Nox)‐Dependent Signaling
Authors: Cao Xian , Wang Gang , Peterson Jeffrey , Burmeister Melissa , Sharma Ram , Iadecola Costantino , Davisson Robin
Published: June 2021

The SFO is a critical forebrain region that mediates slow‐pressor AngII‐induced sympathoexcitation and HTN via reactive oxygen species (ROS) formation. We have shown that the HTN and sympathetic outflow caused by slow‐pressor AngII are abolished in EP1R null mice. Here we tested the hypothesis that EP1R in the SFO contribute to these AngII responses by activating Nox‐dependent ROS signaling. Real‐time PCR revealed that EP1R is the predominant PGE2 receptor in SFO at baseline compared to EP2R, EP3R or EP4R (p<.05; n=3). Slow‐pressor AngII infusion for 2 wks (600ng/kg/min, sc) caused a gradual increase in EP1R mRNA selectively in SFO, which peaked (1.89±.3‐fold day 0, p<.05; n=3) before BP began to rise. ICV infusion of the EP1R antagonist SC‐51089 (6ug/hr) prevented AngII‐induced HTN (ΔMAP: 5±5 vs vehicle 35±9 mmHg, p<.05, n=6–7) and ROS formation in SFO (1.07±.2 vs vehicle 2.3±.4, p<.05, n=6–8) during week 2. In vitro, AngII and PGE2 dose‐dependently elicited increases in ROS production in isolated SFO cells with EC50 = 63 nM and 53.2 nM, respectively. The AT1 antagonist losartan blocked the response to AngII but not PGE2. Both responses were eliminated in SFO cells treated with gp91ds‐tat or derived from Nox2 or EP1R null mice. This suggests that activation of EP1R is a critical step that links AT1 and Nox signaling in the SFO, which may contribute to sympathetic activation in this model of AngII HTN. (HL096571)
Pravastatin normalizes peripheral sympathetic hyperactivity in the Spontaneously Hypertensive Rat by reducing cardiac angiotensin 2 levels
Authors: Herring Neil , Lee Chee Wan , Sunderland Nicholas , Wright Kathryn , Paterson David J
Published: June 2021

Hypertension is associated with heightened sympathetic tone and increased renin‐angiotensin system activity. Recent evidence suggests statins can reduce angiotensin 2 (AT2) signalling independent of cholesterol lowering. We therefore investigated whether 2 weeks treatment with pravastatin (100mg/L in drinking water, n=41 v's n=32 control) could alter the heart rate (HR) response to sympathetic nerve stimulation (SNS) via an AT2 dependent pathway in the spontaneously hypertensive rat (SHR). Pravastatin significantly (p<0.05) lowered resting HR in‐vivo despite unaltered LDL cholesterol levels. In right atrial preparations pravastatin significantly reduced release of 3H labelled norepinephrine (NE) during field stimulation to levels observed in WKY rats. In atria/right stellate ganglion preparations, the HR response to SNS was also significantly reduced by pravastatin whilst the HR response to NE remained similar. Pravastatin significantly reduced left ventricular AT2 levels whilst levels of the AT2 receptor (ATR1) assessed by Western blotting remained similar. The ATR1 antagonist losartan (5μM) equalised release of 3H‐NE to comparable levels in treated and untreated SHRs. This suggests that pravastatin reduces levels of local cardiac AT2 in SHRs and prevents its facilitatory effect on NE release thereby normalizing sympathetic hyper‐responsiveness.

Supported by the British Heart Foundation
Longitudinal Association between Weight Gain and Hypertension: China Health and Nutrition Survey, 1991—2006
Authors: Du Shufa , Wang Huijun , Wang Xiaoshan , Popkin Barry M
Published: June 2021

While studies showed that weight gain may increase the risk of hypertension, there are ethnic differences in associations and few studies among the Chinese population. Using longitudinal data on 46,601 subjects aged 18—69 y followed over six waves of the China Health and Nutrition Survey, we studied the effect of weight gain on blood pressure and hypertension. Trained nutritionists obtained weighed dietary data over 3 days combined with individual 24‐h records. Trained physicians collected 3 measures of blood pressure with a 30‐secondinterval between cuff inflations, weight and height data with standardized methods. Pattern mixture generalized linear mixed model was used to estimate the association. The average follow‐up was 7.5 years (2–15). Prevalence of hypertension increased rapidly from 15% in 1991 to 27.8% in 2006. The cumulative incident hypertension was 23.9%. The odds ratio (OR) of weight gain (1 kg/year) was 1.14 (95%CI: 1.10—1.20, p<0.01), controlled for age and weight at baseline, gender, education, overweight status and interaction between weight gain and overweight and other dummies. Sodium intake did not modify this relationship. Weight gain significantly increased the risk of hypertension in Chinese, particularly in the overweight and obese population.

This research was supported by NIH R01‐HD30880.
Dairy attenuation of oxidative and inflammatory stress in metabolic syndrome
Authors: Zemel Michael B. , Stancliffe Renee
Published: June 2021

We previously demonstrated that dairy components (calcium, leucine, and dairy peptides) suppress adipocyte and macrophage‐mediated oxidative and inflammatory stress. Since oxidative and inflammatory burdens are magnified in metabolic syndrome, we have assessed the effects of low (<0.5 servings) vs. adequate (3.5 servings) dairy (LD vs. AD) diets on oxidative and inflammatory stress in the absence of caloric restriction in 40 metabolic syndrome subjects with habitually low dairy intakes. Diet had no effect on body weight over the 12‐week study, but AD significantly reduced waist circumference and DXA‐measured trunk fat (p<0.01 for both), while LD had no effect. AD attenuated oxidative stress, with a 35% decrease in MDA evident at 7 days (p<0.01) and maintained through 12 weeks and a progressive decrease in oxidized LDL evident at 7 days (11%) and progressing to a 25% decrease at 12 weeks (p<0.01), while LD had no effect. Similarly, inflammatory markers were suppressed by AD, with decreases in TNF‐&α evident at seven days and further reductions through 12 weeks (35%, p<0.05), IL‐6 (21%, p<0.02), MCP‐1 (14% drop at four weeks, 24% drop at 12 weeks, p<0.05), and a corresponding 55% increase in adiponectin (55% at 12 weeks, p<0.01). LD exerted no effect on these markers. Thus, increasing dairy intake results in attenuation of oxidative and inflammatory stress in metabolic syndrome. Supported by the National Dairy Council.
Silencing of brain p44/42 mitogen‐activated protein kinase ameliorates aldosterone‐induced sympathetic excitation in rats
Authors: Yu Yang , Zhang Zhi‐Hua , Wei Shun‐Guang , Felder Robert B
Published: June 2021

Brain mitogen‐activated protein kinase (MAPK) pathways appear to contribute to aldosterone‐induced sympathetic excitation in rats. We tested whether silencing of brain p44/42 MAPK with small interfering RNAs (siRNAs) would prevent aldosterone‐induced sympathetic excitation in rats. Four days after intracerebroventricular (ICV) injection of SMART pool siRNAs targeting p44/42 MAPK, control siRNA or vehicle, we recorded renal sympathetic nerve activity (RSNA), mean blood pressure (MBP) and heart rate (HR) at baseline and for 4 hours during intravenous infusion of aldosterone. Hypothalamic tissues were then collected for molecular analysis. Treatment with ICV p44/42 siRNA, but not with ICV vehicle or control siRNA, reduced total and phosphorylated p44/42 in the hypothalamus and ameliorated the aldosterone‐induced increases (p<0.05) in RSNA, HR and MBP. The p44/42 siRNA treatment had no effects on hypothalamic p38 MAPK (Western blot). These data strengthen our previous studies showing that brain p44/42 MAPK plays a critical role in mediating renin‐angiotensin‐aldosterone system influences on sympathetic drive. Brain p44/42 MAPK may be a new target for therapeutic intervention in the heart failure syndrome. Supported by a VA Merit Review Award and NIH RO1 HL073986.
Brain p44/42 mitogen‐activated protein kinase contributes to the sympathetic response to blood‐borne TNF‐α in rats
Authors: Zhang Zhihua , Yu Yang , Wei Shun‐Guang , Felder Robert B.
Published: June 2021

Blood‐borne pro‐inflammatory cytokines act upon the central nervous system to increase sympathetic drive in normal and pathophysiological conditions including heart failure. The underlying signaling pathways are still poor understood. We tested the hypothesis that brain p44/42 mitogen‐activated protein kinase (MAPK) mediates tissue necrosis factor ‐ alpha (TNF‐α) induced sympatho‐excitatory responses. In urethane‐anesthetized rats, a centrally directed intracarotid artery (ICA) injection of TNF‐α elicited significant (p<0.05) increases in renal sympathetic nerve activity (RSNA), mean blood pressure (MBP) and heart rate (HR), and in hypothalamic phosphorylated p44/42 MAPK expression (Western blot and immunofluorescence). Intracerebroventricular (ICV) pretreatment (4 days before) with small interfering RNA (siRNA) using SMART pool siRNA targeting p44/42 MAPK significantly reduced the increases in RSNA, MBP and HR induced by ICA TNF‐α, but pretreatment with vehicle or control siRNA had no effect. TNF‐α induced increases in RSNA, MBP and HR were similarly diminished by a continuous ICV infusion of the p44/42 MAPK inhibitor PD98059. These data suggest that blood‐borne TNF‐α activates the brain p44/42 MAPK pathway to elicit sympatho‐excitatory responses. Supported by a VA Merit Review Award and NIH RO1 HL073986.
Central organization of sympathetic pathways controlling kidney or spleen revealed by transneuronal retrograde labeling with isogenic strains of pseudorabies virus
Authors: Sved Alan , Card J. Patrick , Cano Georgina
Published: June 2021

Recent evidence suggests that hypertension in rats caused by angiotensin and elevated salt intake is associated with increased celiac sympathetic outflow but reduced renal sympathetic nerve activity (Osborn and Fink, 2009). In this study we used transneuronal tracing with isogenic, phenotypically‐distinct strains of pseudorabies virus injected into left kidney and spleen to identify brain circuits involved in selective control of renal sympathetic nerve activity versus an organ supplied by the celiac ganglion. In spinal cord, sympathetic preganglionic neurons were selectively infected from a single target, whereas spinal interneurons were single‐ and dual‐infected. In brain, all presympathetic areas contained intermixed populations of dual‐ and single‐infected neurons; proportions differed across regions, with A5 and RVLM having particularly high numbers of dual‐infected neurons. Studies are being expanded to examine three targets simultaneously, providing greater insight into the extent to which presympathetic neurons control unique versus multiple targets. Organ‐specific (single‐infected) and common (multiple‐infected) neurons provide the neural substrates for the two modalities (discrete or global) of sympathetic control. Specific pathways likely underlie the unique sympathetic signatures associated with different physiological or disease states. (NIH HL076312, NSF IOS0918867)
Lateralized changes in renal sympathetic activity evoked by unilateral stimulation of lateral/dorsolateral periaqueductal gray
Authors: Xavier Carlos Henrique , Ianzer Danielle , Nalivaiko Eugene , Fontes Marco Peliky
Published: June 2021

Periaqueductal gray (PAG) is considered an important synaptic relay for behavioral, thermal and cardiovascular responses to stress. Stimulation of lateral/dorsolateral PAG (l/dl PAG) causes fight/flight reaction and sympathetically mediated cardiovascular changes. In adult male anesthetized rats (n=5), we compared changes in heart rate (HR), mean arterial pressure (MAP) and renal sympathetic activity (RSNA) produced by chemical activation (NMDA 100pmol/100nl) of right (R) and left (L) sides of l/dl PAG. RSNA was recorded only on the L side. We observed greater increases in left RSNA following ipsilateral l/dl PAG stimulation (Δ: L 92±14 vs. R 17±4%; P=0.0012). These responses were accompanied by increases in MAP and HR (average Δ: 17mmHg and 35bpm, respectively), but no differences in the magnitude of these responses were found between left or right l/dl PAG stimulation. Maximal changes in MAP and RSNA occurred at the same time, about 30s after l/dl PAG stimulation. Intriguingly, however, the maximal response for HR was delayed (≈90s), following a different time course. We conclude that the functional descending pathways from l/dl PAG to renal sympathetic nerves are prominently lateralized. The mechanisms involved in the differential time course responses between MAP/RSNA and HR require further investigation. Support: FAPEMIG‐PPM and CNPq.
Chronic central nervous system MC3/4R activation exacerbates the cardiovascular responses to peripheral nitric oxide synthase inhibition
Authors: do Carmo Jussara M , Bassi Mirian , da Silva Alexandre A , Hall John E
Published: June 2021

We examined if nitric oxide synthase inhibition exacerbates the cardiovascular responses of central nervous system (CNS) melanocortin‐3/4 receptor (MC3/4R) activation. Sprague‐Dawley rats implanted with telemetry device, venous catheter and intracerebroventricular (ICV) cannula were divided in 2 groups: After 5 days of control measurements, L‐NAME was infused (14 mg/kg/day, i.v.) for 17 days. Starting on day 7 of L‐NAME infusion, the MC3/4R agonist MTII (240 ng/day, n=7 – group 1), or vehicle (n=6 – group 2) was infused ICV for 10 days. L‐NAME + MTII caused a larger increase in blood pressure (BP: 110±3 to 183±3 mmHg) and heart rate (HR: 383±9 to 437±10 bpm) compared to L‐NAME + vehicle treatment (103±2 to 161±6 mmHg and 371±7 to 364±17 bpm). The increase in BP and HR in the L‐NAME + MTII group occurred despite a 60% reduction in food intake during the first 6 days of MTII infusion. MC3/4R activation also reduced glucose and insulin levels by ~30% and 35%. We then tested if the exacerbated pressor responses to combined L‐NAME + MTII treatment was due to a reduction in nitric oxide (NO) levels in the brain by infusing L‐NAME alone, or in combination with MTII, into the CNS. Central infusion of L‐NAME + MTII caused only ~10 mmHg increase in BP with no change in HR, whereas L‐NAME alone had no effect on BP. These results suggest that peripheral, but not central, reduction in NO production augments the sensitivity to CNS MC3/4R activation
Electrical vagal afferent stimulation inhibits sympathetic nervous activity irrespective of baroreflex activation without lowering blood pressure
Authors: Hosokawa Kazuya , Kawada Toru , Ando Makoto , Tanaka Atsushi , Sakamoto Takafumi , Onitsuka Ken , Murayama Yoshinori , Ide Tomomi , Sunagawa Kenji
Published: June 2021

Background

The blunted arterial baroreflex control and the imbalance of autonomic nervous system are a salient feature of heart failure. Although electrical vagal stimulation has been shown to improve survival, its effect on arterial baroreflex function remains unknown.

Methods and Results

We vascularily isolated the bilateral carotid sinus baroreceptor regions in rats after myocardial infract (MI, n = 5) and in rats without MI (Control, n = 6). We ligated the vagal nerves and attached a pair of electrode to the central end for afferent stimulation (aVNS). We recorded sympathetic nerve activity (SNA) at the celiac region. We changed baroreceptor pressure (CSP) stepwise with and without aVNS (1 Hz, 3.0‐5.0 V). Increases in CSP sigmoidally suppressed SNA, and baroreflex induced suppression in SNA quasilinearily decreased arterial pressure (AP). aVNS induced a parallel downward shift of sigmoidal CSP‐SNA relation both in Control (−16 ± 17%, p < 0.05) and in MI (−17 ± 18%, p < 0.05), whereas it upward shifted the quasilinear SNA–AP relation both in Control (+16.2 ± 12.0 mmHg, p < 0.05) and MI (+27.2 ± 10.6 mmHg, p < 0.05). As a result of these opposite modulations, aVNS did not significantly impact the CSP‐AP relation.

Conclusion

aVNS inhibits SNA irrespective of baroreflex activation and thereby might have beneficial effects in heart failure. This study was supported in part by research grants from the Ministries of Japan.
Angiotensin‐(1‐7) inhibits angiotensin II intra‐neuronal signaling
Authors: Yang Rui‐Fang , Yin Jing‐Xiang , Li Yu‐Long , Schultz Harold D. , Zimmerman Matthew C.
Published: June 2021

Elevated levels of superoxide (O2•−) induced by angiotensin II (AngII) play a vital role in the sympathoexcitation associated with many neuro‐cardiovascular diseases, such as hypertension and heart failure. Recent studies report that angiotensin‐(1‐7) [Ang‐(1‐7)] increases neuronal levels of nitric oxide (NO•), which can rapidly react with O2•− to limit its potential signaling. We hypothesized that Ang‐(1‐7) inhibits AngII intra‐neuronal signaling by modulating O2•− levels. Using differentiated CATH.a neurons, we observed that the AngII (100 nM)‐induced increase in O2•−, as measured by EPR spectroscopy and DHE fluorescence, was markedly attenuated by pretreatment with 100 nM Ang‐(1‐7) (EPR arbitrary units/103 neurons: vehicle = 938 ± 67; AngII = 1310 ± 92*; Ang‐(1‐7) + AngII = 1004 ± 95; *P<0.05 vs. vehicle and Ang‐(1‐7) + AngII). This inhibition was blocked by the Ang‐(1‐7) receptor antagonist A‐779 (1261 ± 107; P<0.05 vs. Ang‐(1‐7) + AngII). Further, AngII‐induced inhibition of neuronal K+ current (IKv; 49 ± 9% inhibition) was attenuated by Ang‐(1‐7) (22 ± 4% inhibition; P<0.05 vs. AngII alone). These findings indicate that Ang‐(1‐7) attenuates the AngII‐induced increase in O2•− and inhibition of IKv. Ang‐(1‐7) signaling in neurons may play an important protective role counterbalancing AngII sympathoexcitation in the pathogenesis of cardiovascular diseases. NIH P20RR017675 and P01HL062222
Are Aging and Heart Failure Similar Syndromes of Sympathetic Dysregulation?
Authors: Kenney Michael J. , Margiocco Marco L. , Musch Timothy I. , Hageman K. Sue , Fels Richard J.
Published: June 2021

Both heart failure (HF) and aging alter the basal regulation of sympathetic nerve discharge (SND) and the majority of HF patients are aged, however, the effect of combined aging and HF on SND regulation to acute stress is not well established. We tested the hypothesis that aging and HF are not similar syndromes of sympathetic nervous system dysregulation, as evidenced by differences in SND (renal and splenic) responses to heat stress in young HF compared with aged HF rats and in aged HF compared with aged non‐HF rats. Anesthetized young (3–6 months) and aged (24–25 months) F344 rats received either a surgically‐induced myocardial infarction or a sham operation. Heating experiments (internal body temperature, Tc, increased to 41.5°C) were completed in chloralose‐urethane anesthetized rats 4–6 weeks after the myocardial (or sham) infarction procedure. Left ventricular (LV) function was determined by measuring left ventricular end diastolic pressure (LVEDP) and comparing dimensions of the left atrium (LA) and the aorta (Ao) (LA/Ao ratio) using echocardiographic techniques. Preliminary results indicate that LVEDP and LA/Ao ratio and SND responses to acute thermal stress may differ in young HF compared with aged HF rats and in aged HF compared with aged non‐HF rats. These findings suggest that both age and HF should be considered when determining mechanisms regulating SND responses to acute environmental stress. Funding provided by NIH HL091342.
Spinal cord stimulation (SCS) modifies cardiac‐related dorsal root ganglia (DRG) afferent neurons in response to myocardial ischemia
Authors: Gibbons David D , Southerland E. Marie , Armour J. Andrew , Ardell Jeffrey L.
Published: June 2021

Objective

To determine if SCS modulates the capacity of cardiac sympathetic afferent neurons to transduce regional myocardial ischemia.

Methods

Using extracellular recording techniques in anesthetized canines, cardiac‐related DRG neurons (T1‐T3 spinal levels) were identified by multimodal stimulation of the left anterior descending (LAD) region. Then neuronal responses to transient (1 min) LAD coronary artery occlusion (CAO) were evaluated prior to and following SCS (T1‐T3 spinal level; 50Hz, 90% motor threshold; 20 min).

Results

LAD CAO caused a ~4 fold increase in cardiac afferent neuronal activity that persisted for at least 30 minutes following reperfusion. SCS doubled basal cardiac afferent neuronal activity, doing so in a stochastic manner. Following SCS, LAD CAO evoked no further increase in such activity.

Conclusion

Transient myocardial ischemia activates primary afferent inputs to the spinal cord well beyond stressor application. Pre‐emptive SCS obtunds sensory transduction of the ischemic myocardium to spinal cord neurons. (Supported by HL71830.)
TRPV1‐MEDIATED MODULATION OF SYNAPTIC INPUTS IN IDENTIFIED KIDNEY‐RELATED PRE‐SYMPATHETIC IML NEURONS
Authors: Derbenev Andrei V , Duale Hanad
Published: June 2021

Synaptic balance in the neurons of the intermediolateral cell column (IML) is a critical component in the central pathways mediating sympathetic innervation of the kidney. Transient receptor potential vanilloid type 1 (TRPV1) have potent effects on autonomic motor neurons and are expressed in the IML, but effects of TRPV1 activation in the IML is not known. Whole‐cell patch‐clamp recordings in longitudinal spinal cord slices were used to characterize synaptic activity of kidney‐related pre‐sympathetic IML neurons identified with the transynaptic retrograde viral vector PRV‐152, to test the hypothesis that activation of TRPV1 enhances synaptic activity to these neurons. The resting membrane potential was −51±4 mV and input resistance was 448±39 MΩ. At a holding potential of −60 mV, we observed spontaneous EPSCs at a frequency of 3.1±1 Hz with an average amplitude of 11.5±2 pA. At a holding potential of −10 mV, we observed spontaneous IPSCs with a frequency of 1.4±0.3 Hz and an amplitude of 19±9 pA. Immunohistochemical studies showed that TRPV1 were distributed throughout the IML and co‐localized with kidney‐related IML neurons. Application of the TRPV1 agonist capsaicin increased the frequency of spontaneous IPSCs and EPSCs in kidney‐related pre‐sympathetic IML neurons by 150%. This mechanism can modulate blood pressure and, by inference, hypertension. Supported by NIH HL091293‐01A1; 3R21HL091293‐01A1S1.
Particulate matter exposure decreased spiking response to depolarization and increased potassium current of cardiac vagal neurons in mice
Authors: Chen Chao
Published: June 2021

Epidemiologic studies show that exposure to fine particulate matter (PM2.5) increases the total daily cardiovascular mortality. W previously showed that 3 d of PM2.5 exposure (6 hr/d) in the form of iron‐soot reduced heart rate variability in mice. We further showed that PM2.5 exposure reduced neuronal responsiveness to excitation in cardiac vagal neurons in the nucleus ambiguus. Here, we tested the hypothesis that PM2.5 exposure‐induced increases in potassium currents contribute to the decreased neuronal excitability. Using patch‐clamp techniques, we determined the spiking response to depolarization and potassium currents in anatomically identified cardiac vagal neurons, retrogradely labeled with a fluorescent dye applied to the SA node. PM2.5 exposure decreased spiking responses of identified cardiac vagal neurons to depolarization in the presence of glutamatergic and GABAergic receptor antagonists (p < 0.05). PM exposure also increased the peak (by 24%) and stead state (by 17%) potassium currents. The data suggest that PM2.5 exposure‐induced increase in potassium channel conductance maybe one mechanism contributing to the decreased heart rate variability. (Support R01 ES012957)
Nucleus Ambiguus Processing of Electroacupuncture Cardiovascular Inhibitory Reflex Responses: Role of Opioids and GABA
Authors: Tjen‐A‐Looi Stephanie C , Li Peng , Longhurst John C
Published: June 2021

Stimulation of cardiopulmonary receptors with phenylbiguanide (PBG) elicits cardiovascular reflex responses, including decreases in blood pressure and heart rate mediated by the brainstem parasympathetic cardiac neurons in the nucleus ambiguus (NA). The present study examined the NA mechanisms underlying the influence of electroacupuncture (EA) on the PBG‐induced hypotension and bradycardia. We hypothesized that somatic stimulation during EA modulates PBG responses through opioid and γ‐aminobutyric acid (GABA) mechanisms in the NA. Anesthetized and ventilated cats were studied during repeated stimulation with PBG or cervical vagus while low frequency EA (2 Hz) was applied at P5‐6 overlying the median nerve for 30 min and NA neuronal activity, heart rate and blood pressure were recorded. Microinjection of kainic acid into the NA attenuated the PBG‐induced bradycardia from 67±17 to 46±14 bpm. EA likewise reduced the PBG‐induced depressor and bradycardia reflex by 39 and 68%, respectively. Cervical vagus evoked activity in the NA was reduced rapidly by EA for at least 40 min. Blockade of opioid or GABAA receptors using naloxone and gabazine reversed the EA‐related modulation of the evoked vagal activity by 73 and 53%, respectively. Thus, EA at P5‐6 decreases PBG evoked hypotension and bradycardia as well as the vagus evoked NA activity through opioid and GABA systems. Supported by HL‐63313.
Age‐related changes in brain monoamines in a murine model of Parkinson's disease
Authors: Nayyar Tultul , Bubser Michael , Ferguson Marcus C , Deutch Ariel Y , Ansah Twum A
Published: June 2021

Aging is a well established risk factor for Parkinson's disease (PD). In the present studies, we determined age‐related effects of 1‐methyl‐4‐phenyl‐1,2.3,6‐tetraphydropyridine (MPTP) treatment on DA, norepinephrine (NE) and serotonin (5‐HT) in the striatum and prefrontal cortex (PFC). Male C57BL/6J mice were subjected to i.p. administration of (1) chronic MPTP treatment (20 mg/kg MPTP for 5 days) at 9 weeks of age or at 18 months of age and sacrificed at 3 weeks after MPTP treatment, and (2) acute MPTP treatment (4 doses of 20 mg/kg at 2 hrs interval) at 9 weeks of age and were sacrificed either at 12 weeks of age or at 18 months of age. Saline was used as vehicle control. Monoamines concentrations in the striatum and PFC were analysed by HPLC. Chronic MPTP treatment decreased striatal DA concentration by 25% in the 9‐ week old mice and 67% in the 18‐month old mice; decreased significantly striatal NE concentrations in the 18‐month old mice. However, striatal 5‐HT and monoamines concentrations at PFC were not altered following chronic MPTP treatment. After acute MPTP treatment, the striatal DA concentrations were significantly decreased at both survival periods and significant decreases in 5‐HT and NE concentrations in the PFC at both survival periods. The data suggest that aging may increase the vulnerability of the dopaminergic, adrenergic and serotonergic systems to MPTP.
Ketone pretreatment reduces superoxide production in human U87 glioblastoma cells exposed to normoxia and hyperoxia
Authors: D'Agostino Dominic , Dean Jay B
Published: June 2021

Unlike normal brain cells, cancer cells lack the metabolic flexibility to use ketones as fuel. Brain cancer cells have defective mitochondria and derive energy primarily through aerobic glycolysis (the Warburg effect). Cancer growth is intimately linked to glucose‐stimulated reactive oxygen species (ROS) production. We hypothesized that ketones would reduce mitochondrial ROS production in cultured human U87 glioblastoma cells. Fluorescence microscopy and confocal spectral imaging were used to detect real‐time intracellular superoxide production with dihydroethidium (DHE). Cultured U87 cells were maintained in media containing 1mM glucose in the presence or absence of 2‐10mM D‐beta‐hydroxybutyrate (BHB) for 4‐6 days. Results demonstrate that superoxide production (measured as DHE fluorescence intensity units; FIU) was approximately 300% higher in untreated cells versus BHB‐treated cells (2 mM). Measurement of superoxide production during normoxia (60 minutes) increased FIUs 49 ± 3, 14 ± 2 and 38 ± 2 in untreated, 2mM and 10mM BHB, respectively. Hyperoxia‐induced mitochondrial superoxide production was abolished in U87 cells pretreated with 2mM BHB. We conclude that supplemental ketones significantly reduce superoxide production in U87 cells. These results suggest that supplemental ketones may confer protection against malignant brain cancer.
Enzymatic analysis of the American locust jumping muscle during development
Authors: Kirkton Scott D , Nyberg Elizabeth T. , Fox Kristin M.
Published: June 2021

Insects have the highest mass‐specific metabolic rates among animals due in part to their tracheal respiratory system. However, it has been hypothesized that larger/older insects may have greater oxygen delivery problems than smaller/younger insects because longer tracheae may be less efficient. This effect of size on oxygen delivery may be most dramatic during development or locomotion when oxygen demands are greatest. We tested this hypothesis by comparing the metabolic profile of the jumping muscle during development in the American locust (Schistocerca americana). Developing grasshoppers increase their body mass over 200 times from 1st instar juveniles to adults. To determine whether there is a developmental shift in the metabolic profile of the jumping muscle, we compared how enzymatic markers for aerobic (citrate synthase) and anaerobic metabolism (lactate dehydrogenase) varied in 2nd, 4th, and 6th instar as well as adult grasshoppers. Lactate dehydrogenase increased 2.8 fold with age from the 2nd instar to the adult. In contrast, citrate synthase decreased 1.9 fold with age from the 2nd instar to the adult. These findings support the hypothesis that the metabolic profile of S. americana jumping muscle gradually increases its reliance on anaerobic metabolism with development. Our research supports previous findings that larger/older grasshoppers produce more powerful jumps that result in greater lactic acid production than smaller/younger grasshoppers.
The evolution of diabetes in non human primates: comparative physiology implications for human type 2 diabetes mellitus (T2DM)
Authors: Hansen Barbara Caleen
Published: June 2021

T2DM develops naturally and spontaneously in humans and non human primates. We have studied prospectively a colony of >220 adult rhesus monkeys that were continuously maintained with ad libitum feeding of a high fiber low fat “healthy” diet throughout life. 87 individuals were identified that developed frank T2DM. Longitudinal analysis of the emerging dysglycemia and dyslipidemia has allowed the determination of the glycemic threshold for diagnosis (dx) of prediabetes: 80 mg/dl (4.4 mmol/L) for monkeys, shown by modeling to be comparable to 100 mg/dl (5.5 mmol/L) in humans; and overt DM dx occurs at 100 mg/kg (5.5 mmol/L) in monkeys compared to the current ≥126 mg/dl (6.9 mmol/L) defined for humans. All monkeys reaching the prediabetes diagnosis, if not intervened on to prevent progression, did in fact proceed to overt diabetes (2/3 in < 3 yrs; ~<10 yrs in humans). Sequence analysis to model the diabetes trajectory showed that acute beta cell response to glucose peaked 2 to 6 years prior to DM dx; fasting plasma insulin 1 to 4 yrs pre DM dx; and nearly all were hypertriglyceridemic 5 to 12 yrs pre DM dx. These carefully controlled studies show that in humans fasting insulin, β cell functional responses and dyslipidemia should be used to identify those progressing toward diabetes in order to implement true preventive strategies prior to β cell failure.

Supported by: supported by NIA N01AG31012 and NIA HHSN2532008002C.