bluuebunny/crossref_metadata_2025 huggingface dataset:
Atrap interacts with the cardiac Ca2+‐ATPase SERCA2A and facilitates ventricular relaxation (1150.7)
Authors: Mederle Katharina , Gess Bernhard , Castrop Hayo
Published: June 2021

The angiotensin II type 1 receptor‐associated protein (Atrap) is highly expressed in the heart. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. To identify novel interacting partners of Atrap, we performed pull‐down assays. In 3 independent experiments, sequencing of the complexes by MALDI‐MS showed that Atrap interacts with the cardiac Ca2+‐ATPase SERCA2A. The physical interaction of Atrap and SERCA2A was confirmed by co‐immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy (Biacore). The binding of SERCA2A to Atrap resulted in a dose‐dependent increase in the SPR signal with a KD of 7.1*10‐8 M. To evaluate the functional relevance of this protein interaction in vivo, the left ventricular function was assessed in WT and Atrap‐/‐ mice. The heart rates (608±8 vs. 585±12 bpm; p=.20) and ejection fractions (65±2% vs. 64±1%; p=.60) were similar in WT and Atrap‐/‐ mice (n=8). The maximum ventricular relaxation (dV/dt max) was significantly decreased in Atrap‐/‐ (652±42 µl/s) compared to WT mice (1139±184 µl/s; p=.01; n=8), whereas peak contraction velocity (dV/dt min) was inconspicuous (‐778±56 µl/s in Atrap‐/‐ vs. ‐782±102 μl/s in WT mice; p=.97). In summary, we identified Atrap as a novel regulatory protein of the cardiac SERCA2A. We suggest that Atrap enhances the activity of SERCA2A and, consequently, facilitates ventricular relaxation.
Increased type VI collagen gene expression in the post‐MI human myocardium (1152.11)
Authors: Bryant Jennifer
Published: June 2021

Cardiac fibroblasts and myofibroblasts are responsible for post‐myocardial infarction (MI) remodeling via regulation of extracellular matrix (ECM) formation. Accelerated post‐MI remodeling leads to excessive ECM deposition and fibrosis which can contribute to impaired contractile function, arrhythmias, and heart failure. Type VI collagen has been shown to induce myofibroblast differentiation in cultured cardiac fibroblasts and could contribute to accelerated post‐MI remodeling. Type VI collagen is elevated in the myocardium 7 days, 14 days, and 20 weeks post‐MI in the rat. The absence of type VI collagen improved cardiac function post‐MI in a knock out mouse model. The purpose of this study was to determine if collagen VI gene expression was elevated in human myocardium post‐MI. Autopsy samples were obtained from subjects who had died within seven days of an MI (acute MI group) and subjects who had died of a non‐heart related issue with no documented prior MI (control group). Quantitative real time PCR reactions were performed with RNaseP as a reference gene for normalization. Type VI collagen gene expression in the acute MI group was 414% greater than the control group expression, but did not reach statistical significance in this small study. However, this preliminary data indicate that type VI collagen gene expression may be significantly elevated in the post‐MI human myocardium and warrants further study.

Grant Funding Source: Supported by Shenandoah University School of Pharmacy Professional Practice Plan
Inhibiting interferon regulatory factor 5 attenuates endothelial mesenchymal transition (1152.5)
Authors: Weihrauch Dorothee , Bamkole Omoshalewa , Krolikowski John , Pritchard Kirkwood
Published: June 2021

Introduction: Systemic Sclerosis (SSc) is an autoimmune connective tissue disease characterized by oxidative stress, fibrosis and inflammation of skin and internal organs. Tight skin (Tsk/+) mice are an established model of SSc that displays inflammatory and fibrotic features that are consistent with SSc. When endothelial cells (ECs) are cultured on extracellular matrix (ECM) isolated from the skin of Tsk/+ mice they undergo endothelial mesenchymal transition (EndoMT). 4F, an apoA‐I mimetic that inhibits atherosclerosis by many mechanisms, not only decreases myocardial fibrosis in Tsk/+ mice but also decreases interferon regulator factor 5 (IRF5), a transcription factor considered a molecular switch for autoimmunity. The goal of the present study was to determine if directly targeting IRF5 reduces inflammation and oxidative stress in ECs cultured on cardiac ECM of Tsk/+ mice. Methods: KEP20 was designed and developed to specifically inhibit IRF5.The oxidative state of cardiac ECM was determined using the advanced oxidized protein product (AOPP) assay and western blot analysis for 3‐Nitrotyrosine and advanced glycation end products (AGE). ECs were cultured on Tsk/+ cardiac ECM and effects on expression of TGF β, Twist, collagen‐I in ECs ± KEP20 were determined by western blot analysis. Results: Tsk/+ ECM increase the levels of AOPP (13%), 3‐Nitrotyrosine (21%), and AGE (86%) in EC cultures as compared to EC maintained on cardiac ECM C57BL/6J. KEP20 treatments (50µg/mL) of EC cultured on Tsk/+ ECM reduces expression of TGFβ (221%), Twist (35 ± 14.51%), Collagen‐I (137%) compared ECs on Tsk/+ ECM. KEP20 also reduces Twist by 73%, TGFβ by 34% and collagen‐I by 56% compared to the levels in ECs on Tsk/+ ECM. Conclusion: These data indicate inhibiting IRF5 prevents Tsk/+ cardiac matrix from inducing EndoMT as well as increasing EC inflammation and fibrosis and demonstrate that IRF5 is a novel therapeutic target for SSc.
Extracellular ROS formation in contracting soleus muscle in mice with lung TNF‐α overexpression (1153.1)
Authors: Zuo Li , Hallman Allison , Roberts William , Wagner Peter , Hogan Michael
Published: June 2021

Chronic obstructive pulmonary disease (COPD) commonly results in elevated levels of tumor necrosis factor‐alpha (TNF‐α). However, the mechanism of extracellular reactive oxygen species (ROS) formation in skeletal muscle isolated from pulmonary TNF‐α overexpression mice, has been overlooked. We hypothesized that ROS can be released from contracting skeletal muscle when there is increased TNF‐α formation in the lung and that this causes muscle early fatigue. Using wild type (WT) and pulmonary TNF‐α overexpression mice (Tg+), we performed a cytochrome c (cyt c) assay to measure extracellular superoxide (O2•–, one of the major types of ROS) production in isolated contracting soleus muscle. Our results showed that Tg+ soleus released higher levels of O2•– than WT during the contraction period (n = 5; P < 0.05). Tg+ soleus muscle also presented a reduced fatigue resistance compared to WT muscle. Perfusion of the contracting soleus with membrane‐impermeable superoxide dismutase, an extracellular O2•– scavenger, resulted in less cyt c reduction confirming that the type of ROS released from the Tg+ muscles was O2•–. We conclude that TNF‐α overexpression in the lung induces more O2•– release from Tg+ than WT muscle, and that the extracellular formation of O2•– markedly reduces the fatigue resistance in the Tg+ muscle. Thus, this study highlights O2•– as a potential mechanistic mediator in COPD induced muscle impairment.

Grant Funding Source: Supported by NHLBI Grant PPG‐HL‐091830, NIAMS Grant AR040155, OSU‐HRS 013000
Sex influences systemic and peripheral blood mononuclear cell production of superoxide in older adults (1153.11)
Authors: Schanzle Jennifer , Deo Shekhar , Fadel Paul
Published: June 2021

Previous work suggests that elevations in oxidative stress are associated with cardiovascular disease. Although it is well known that oxidative stress increases with age, whether this occurs differently in men and women remains unclear. Likewise, recent work indicating that mRNA expression for NADPH oxidase is greater in peripheral blood mononuclear cells (PBMCs) of older subjects did not differentiate sex effects. Therefore, we measured plasma and intracellular levels of superoxide (O2●‐), along with PBMC expression of NADPH oxidase subunits in 12 healthy older post‐menopausal women and 10 age‐matched older men. Older men demonstrated greater plasma O2●‐ levels (lucigenin chemiluminescence; men 202.73 ± 9.28 vs. women 171.67 ± 7.72, Relative Light Units; P<0.05) as well as intracellular O2●‐ in PBMCs (dihydroethidium fluorescence; men 1.94 ± 0.20 vs. women 0.96 ± 0.14, Relative Fluorescence Units; P<0.05) compared to older women. Similarly, western blot analysis demonstrated that older men exhibit greater NADPH oxidase subunit protein expression for gp91phox, p22phox, and p47phox in PBMCs (e.g., gp91phox; men 1.48 ± 0.38 vs. women 0.31 ± 0.07; P<0.05) and p67phox tended to be higher (P=0.14). These preliminary findings suggest that systemic O2●‐ levels are greater in older men compared to older women and PBMC‐derived O2●‐ production via NADPH oxidase is a likely contributing source.

Grant Funding Source: Supported by NIH R01 HL093167
Interplay among superoxide, nitric oxide and hydrogen peroxide in hyperglycemia‐induced endothelial dysfunction (1153.7)
Authors: Patel Hemang , Chen Juan , Kavdia Mahendra
Published: June 2021

Free radicals have critical involvement in both physiological and pathological processes in cardiovascular system. Their role is not clear in the regulation of hyperglycemia‐induced endothelial dysfunction. In this study, we investigated the interplay amongst the superoxide (O₂‾), nitric oxide (NO), and hydrogen peroxide (H₂O₂) levels in endothelial cells following 24 hours of hyperglycemia. Furthermore, hyperglycemic‐endothelial cells were treated with superoxide dismutase (SOD) and catalase. Hyperglycemia significantly increased O₂‾ and H₂O₂ levels and decreased NO levels in endothelial cells. Following the exposure to exogenous SOD and catalase, O₂‾ levels decreased to control levels and NO levels significantly increased in hyperglycemic endothelial cells. Regulation of O₂‾, NO and H₂O₂ following SOD and catalase exposure, indicated a tight interplay resulting in increased NO levels upon neutralization of either O₂‾ or H₂O₂. Our findings provide a direct evidence for H₂O₂ led NO level regulation through O₂‾ depletion in hyperglycemia‐induced endothelial dysfunction.

Grant Funding Source: NIH R01 HL084337
Elevated levels of reactive oxygen species in heart failure patients: role for increased mononuclear NADPH oxidase expression (1153.9)
Authors: Deo Shekhar , Zimmerman Matthew , Zucker Irving , Fadel Paul
Published: June 2021

Although heart failure (HF) patients exhibit elevated levels of ROS, the sources and mechanisms contributing to the increase in ROS remain unclear. Recent work has shown that peripheral blood mononuclear cells (PBMCs) are a major source of NADPH oxidase‐derived superoxide (O2●‐) production. Therefore, we measured systemic ROS, O2●‐, and expression of NADPH oxidase and its potential activators, angiotensin II type 1 receptors (AT1R) and Rho Kinase (ROCK) 1, in PBMCs from 6 HF (ejection fraction 22±4%) and 6 age‐matched healthy controls. Total ROS and O2●‐ levels were measured in blood using electron paramagnetic resonance spectroscopy. Protein expression of NADPH oxidase subunits (gp91phox, p22phox, p47phox and p67phox), AT1R and ROCK1 were assessed from PBMCs using western blot. Total ROS and O2●‐ levels were elevated in blood from HF patients compared to controls (P<0.05). HF patients showed augmented protein expression of all NADPH oxidase subunits (e.g. 4±1 fold increase in gp91phox; P<0.05 vs. control). ROCK1 protein expression was also greater in HF patients (3±1 fold; P<0.05 vs. control), whereas AT1R tended to be higher (2±1 fold; P=0.13 vs. control). These preliminary findings suggest that PBMC‐derived NADPH oxidase expression is up‐regulated in HF patients and may contribute to the elevated levels of ROS, including O2●‐, through a ROCK1 and/or AT1R dependent mechanism.

Grant Funding Source: Supported by NIH RO1 HL 038690‐22
Left ventricular dysfunction following short‐term high fat feeding in a canine model: influence of dietary fat composition (1155.2)
Authors: Nelson Michael , Broussard Josiane , Kolka Cathryn , Asare Bediako Isaac , Szczepaniak Lidia , Bergman Richard
Published: June 2021

Short‐term high fat feeding causes left ventricular dysfunction in a canine model of dietary obesity; however, the mechanism remains unknown. It is hypothesized that dietary fat composition plays an important role. To address this hypothesis, mongrel dogs were fed a normal diet (40/30/30, carb/fat/protein) supplemented with 6 g/kg of fat composed of either lard(n = 8, high in saturated fatty acids) or salmon oil (n = 8, high in polyunsaturated fatty acids); resulting in identical caloric composition (30/50/20, carb/fat/protein). Cardiac magnetic resonance imaging was performed to evaluate left ventricular (LV) volumes, mass, and myocardial deformation (by tissue tagging) at week 0 and week 6. Lard‐feeding dramatically worsened (all p<0.05) circumferential strain (mean+SE: from ‐12.0 + 0.9 to ‐8.99 + 0.8%) and LV torsion (from 6.7+0.7° to 4.4+0.6°) in all 8 dogs, with no changes in LV mass, arterial blood pressure or heart rate. In contrast, no changes in LV function were observed with salmon oil‐feeding, despite comparable increases in body mass between groups (+8% vs +10%, lard vs. salmon oil). Together, these data advance the hypothesis that saturated fatty acid uptake is cardiotoxic, and may have important implications for cardiovascular disease in obesity and diabetes.
Increased α‐adrenoceptor sensitivity in Zucker diabetic fatty rats during anesthesia (1155.6)
Authors: Bussey Carol , de Leeuw Anne , Lamberts Regis
Published: June 2021

Diabetics increasingly require surgical intervention for medical complications, and they experience more perioperative cardiovascular complications than their non‐diabetic counterparts. The sympathetic nervous system, acting via adrenoreceptors (ARs), is crucial for cardiovascular regulation, and is modulated by anesthesia. Therefore, we aimed to determine how diabetes alters cardiovascular AR responsiveness in vivo, and how this is affected by anesthesia.

Male Zucker Diabetic Fatty (ZDF) rats and lean littermates (n=7) underwent surgery at 16 weeks. A radiotelemeter positioned in the abdominal aorta provided hemodynamic data, and a vascular access port in the femoral vein provided intravenous access. α‐ or β‐ARs were stimulated with phenylephrine (1‐100µg/kg) or dobutamine (2‐120 µg/kg) respectively, under conscious and anesthetized (2% isoflurane) conditions.

Conscious diabetic rats had reduced β‐AR sensitivity (58±7 vs. 27±8 bpm; p<0.05 at 15µg/kg), which was lost during anesthesia (49±8 vs. 63±8 bpm; NS at 15µg/kg). Diabetic animals exhibited a slightly higher top α‐AR response while conscious (80±3 vs. 95±4 mmHg; p<0.05 at 100µg/kg). However, a marked increase in α‐AR sensitivity was evident in anesthetized diabetic animals (34±1 vs. 54±6 mmHg; p<0.001 at 8µg/kg). In addition, diabetes prolonged α‐AR responses, which was exacerbated during anesthesia in diabetic rats.

These data indicate the importance of α‐AR mediated cardiovascular dysfunction in diabetes, particularly in the surgical setting. Inability of diabetic individuals to properly control blood pressure during anesthesia may cause undue pressures on the heart and contribute to poor cardiac outcomes.
Impact of natural cocoa ingestion on microvascular endothelial function in healthy older adults (1156.10)
Authors: Harrison Michelle , Christmas Kevin , Preston Amy , Crozier Stephen , Brothers R.
Published: June 2021

Impact of natural cocoa ingestion on microvascular endothelial function in healthy older adults Michelle L. Harrison1, Kevin M. Christmas1, Amy G. Preston2, Stephen J. Crozier2, R. Matthew Brothers1 1Environmental and Autonomic Physiology Laboratory, Department of Kinesiology and Health Education, University of Texas at Austin; 2The Hershey Company, Hershey, Pennsylvania Aging is associated with a progressive decline in endothelial function that is significantly correlated with increased cardiovascular disease risk. The objective of this study is to investigate the role of natural cocoa (NC) on cutaneous microvascular endothelial function in older individuals. The skin represents an easily accessible vascular bed and evidence is suggesting that changes in the microcirculation may precede overt clinical signs of disease. This study is testing the hypothesis that older individuals will exhibit impaired thermal reactivity in response to a local heating stimulus and that this impairment will be restored following acute NC ingestion (13g NC, The Hershey Company). 11 individuals (68±5 years) were tested on two separate days; they were provided either a NC‐free placebo or a NC‐containing beverage (randomized order, double‐blinded). Cutaneous vascular conductance (CVC) was assessed while the skin was clamped at 34°C and 40°C and values were normalized to a maximal value obtained during 43°C heating for 30 min. This was done pre and 2 hours post NC ingestion. There is a trend for post‐CVC to be increased following flavanol ingestion at both 34°C (Pre: 11±3 % max vs. Post: 14±5 % max; P=0.08) and 40°C (Pre: 58±10 % max vs. Post: 64±14 % max; P=0.10). Our preliminary data suggest that microvascular endothelial function may be improved in older individuals following acute NC ingestion.
Comparison of two techniques for measuring pulse wave velocity in children (1156.5)
Authors: Philips Nicole , Faught Brent , Hay John , Cairney John , O'Leary Deborah
Published: June 2021

Pulse wave velocity (PWV) is used as an indicator of arterial stiffness. Carotid‐femoral PWV is the gold standard method. However, femoral pressure is difficult to measure in children and therefore carotid‐toe PWV has been used as an alternate method. Furthermore, measurement of carotid artery pressure requires highly skilled technicians, while participants are required to remain still over a time period, making this measure difficult to obtain in children. As a result, this study was undertaken to determine the effectiveness of using ECG R‐wave‐toe as an alternate method to measure PWV. Carotid‐toe PWV and R‐wave‐toe PWV were calculated for 31 healthy adolescents (16 females). Ten consecutive beats were averaged for each subject to determine the pulse transit time for each PWV method. PWV was calculated by dividing the pulse transit time by the distance travelled between the two recording sites for each method. A paired t‐test and Pearson correlation were conducted and significance was set at p<0.05. PWV estimated by the R‐wave‐toe technique was significantly lower than that estimated by the carotid‐toe technique (3.86 vs. 4.50 m/s, p<0.001). As well, a positive correlation was found between the two methods (r = 0.86, R2 = 0.75, p<0.001). Although based on a small sample, these results suggest that R‐wave‐toe measurement technique may be a simple and useful alternate method to obtain PWV in children.

Grant Funding Source: Supported by Canadian Institutes of Health Research
Intermittent parathyroid hormone administration attenuates age‐related endothelial dysfunction (1156.8)
Authors: Guers John , Prisby Rhonda , Edwards David , Lennon‐Edwards Shannon
Published: June 2021

Aging is an independent risk factor for cardiovascular disease and is characterized by a decline in conduit artery function. Parathyroid hormone (PTH) treatment has been shown to increase endothelial nitric oxide synthase (eNOS) expression. The purpose of this study was to test the hypothesis that 15 days of intermittent PTH administration would improve endothelial function in old rodents. Old (24 mo) and young (4 mo) Fischer‐344 rats were given injections of PTH (43 µg/kg/day; n=8) or phosphate buffered saline (PBS; 100 µl/day; n=9). In vitro endothelium‐dependent relaxation (EDR) and endothelium‐independent relaxation (EIR) were assessed in aortic segments to acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Aortic eNOS protein expression was assessed by western blot. EDR response to cumulative doses of ACh was significantly reduced in the old compared to young rats (p<0.05) as was maximal relaxation (Old = 72.5% ± 6.0 vs. Young = 94% ± 1.4; p<0.05). These decreases in age‐related EDR were improved significantly in the old PTH in response to cumulative doses of ACh (p<0.05) and at maximal dilation (Old = 72.5% ± 6.0 vs. Old PTH = 87% ± 1.8; p<0.05). When compared to young rats expression of eNOS was reduced (p<0.05) in old rats but not young or old PTH treated rats. These data suggest a potential role for PTH in the treatment of age‐related declines in endothelial function.

Grant Funding Source: Research supported in part by NIH Grant: 1R15AR062882‐01(RDP)
Mitochondrial N‐formyl peptides cause hypotension via formyl peptide receptor activation (1157.6)
Authors: Wenceslau Camilla
Published: June 2021

Hypotension is the major pathophysiological characteristic of systemic inflammatory response syndrome (SIRS) and sepsis. Mitochondrial N‐formyl peptides (F‐MIT) are a damage‐associated molecular pattern that activate the innate immune system via the formyl peptide receptor (FPR). We hypothesized that F‐MIT activates FPR and induces hypotension. Male Wistar rats (12‐weeks old) were cannulated (femoral artery and vein) for blood pressure recordings and drug infusion. F‐MIT infusion (0.02 or 0.2 mg/rat) decreased blood pressure (Low dose: Basal: 82±3 vs. F‐MIT infusion: 60±4*mmHg; High Dose: Basal: 79±3 vs. F‐MIT infusion: 41±3*mmHg;*p<0.05). WRW4 (FPR‐2 antagonist: 0.2 mg/rat), L‐NAME (NOS inhibitor: 15 mg/rat) and ODQ (guanylate cyclase (GC) inhibitor: 0.6 mg/rat) all abolished F‐MIT‐induced hypotension and cyclosporine H (FPR‐1 antagonist: CsH, 3 mg/rat) partially reduced this response. Also, isolated mesenteric resistance arteries were used to assess F‐MIT on vascular function. F‐MIT (10‐30 μM) induced relaxation (~30%) but ODQ or WRW4 abolished this response. However, L‐NAME (100 μM) or CsH (1 μM) only partially inhibited the vasodilatation. These data suggest that FPR‐2>FPR‐1 activation induces hypotension via NO. However, the vasodilatation induced by FPR‐2 is mediated by mechanisms that are NO‐independent but GC‐dependent. Thus, F‐MIT may be a link between trauma, sterile SIRS and sepsis.

Grant Funding Source: Supported by NIH, CNPq and AHA
Sarcolipin orchestrates calcium signaling to regulate muscle metabolism (1162.1)
Authors: Pant Meghna , Sopariwala Danesh , Rafael‐Fortney Jill , Periasamy Muthu
Published: June 2021

Sarcolipin(SLN) is 31 amino acid long protein that regulates the Sarcoplasmic reticulum calcium ATPase (SERCA) activity in skeletal and cardiac muscle. Recently, our lab identified that SLN plays an important role in muscle‐based thermogenesis. Mice lacking SLN are cold sensitive and susceptible to diet induced obesity. Interestingly SLN is induced in skeletal muscle during neonatal development, high fat diet, muscle dystrophies, and inflammation. Since these are the states of increased oxidative metabolism in skeletal muscle we hypothesize that SLN is recruited to maintain this oxidative phenotype. Here we show that SLN overexpressing muscle fibers have high resting calcium and increased recruitment of calcium mediated signaling when there is increased demand. To determine the mechanism regulating SLN induction and downstream signaling we made use of C2C12 cells. We show that inhibiting the calcium signaling pathways regulated by calcineurin downregulates the expression of SLN. Based on our data we propose that SLN forms a feedback loop with calcium mediated signaling pathways and plays an important role to support the oxidative metabolism in skeletal muscle.

Grant Funding Source: Supported by NIH, MDA
Sarcolipin overexpression in mice increases their endurance exercise capacity (1162.2)
Authors: Sopariwala Danesh , Shaikh Sana , Pant Meghna , Rowland Leslie , Periasamy Muthu
Published: June 2021

Sarcolipin (SLN) is a transmembrane protein that interacts with Sarco(endo)plasmic Reticulum Calcium ATPase (SERCA) and is expressed in cardiac and skeletal muscle. SLN is known to inhibit Vmax of SERCA, but its role in muscle physiology is yet to be fully understood. We have shed some light on this front, by showing that SLN knock out (SLN KO) mice are highly sensitive to acute cold and are prone to develop obesity when fed a high fat diet. Moreover, we showed that the SLN KO mice can be rescued from hypothermia by over expression of the SLN transgene; suggesting that SLN is essential for heat generation and increasing metabolic rate. The aim of this study was to further explore how SLN affects muscle function and metabolism; by the use of SLN over expression (SLN OE) mouse model, which expresses SLN in all skeletal muscles up to 10 fold higher than in wild type (WT) mice. Here, we show that SLN OE mice have a higher basal metabolic rate (by indirect calorimetry) than WT mice. The SLN OE mice also have a significantly higher endurance capacity (treadmill running) than their WT littermates. Isolated muscle studies using Tissue Oxygen bath system, show that SLN OE muscle has similar force frequency response as WT muscle, but is more resistant to develop fatigue and consumes more oxygen. Analysis of muscle oxidative metabolic markers and oxygraph measurements using isolated mitochondria; suggest an increased oxidative capacity in the SLN OE muscle compared to WT. Although skeletal muscle can increase metabolic rate up to hundred fold, a decline in the rate of ATP production is a major reason for muscle fatigue during prolonged physical activity. Hence, these results indicate that SLN enhances muscle’s ability to hydrolyze substrates and generate ATP in times of increased energy demand, which in turn increases endurance capacity.

Grant Funding Source: Supported by NIH
Does cAMP‐signaling regulate atrogenes expression in skeletal muscle upon motor denervation? (1162.9)
Authors: Lustrino Danilo , Gonçalves Dawit , Zanom Neusa , Kettelhut Ísis , Navegantes Luiz Carlos
Published: June 2021

Recent studies have focusing on the role of cAMP‐signaling in modulating the protein degradation via de ubiquitin‐proteasome pathway during muscle wasting. Decreases in this proteolytic system during chronic muscle disuse are thought to prevent the excessive loss of muscle mass. However, the intracellular pathways that elicit this response are elusive. In turn, this study was performed to shed light on the molecular mechanisms involved in this process. For this, C57/BL6 male mice (~30g) were anesthetized and submitted to motor denervation (DEN) by unilateral sciatic nerve section. After 3, 7 and 14 days, tibialis anterior muscles were harvested to examine: mRNA and protein levels of atrophy‐related genes (i.e atrogenes; MuRF1 and atrogin‐1) and different components of cAMP‐signaling, including muscle cAMP levels, the PKA activity (estimated by phospho‐PKA substrates) and the phospho‐CREB and mRNA levels of Sik1, a CREB‐target gene. Sham‐operated and denervated animals were treated with either saline or clenbuterol (CB, 3mg/kg/day; s.c.) during 14 days. The loss of muscle mass induced by DEN started on 7th day (13%) and reached 34% at 14 days after DEN. Skeletal muscles from 3‐days denervated mice showed an increase in the gene expression of atrogin‐1 (243%) and MuRF1 (553%). However, after the 7th day of DEN, atrogenes expression was normalized. This effect was associated with an increase of muscle cAMP content (25% in relation to innervated muscles), phospho‐PKA substrates (~2.2 fold), Sik1 mRNA expression (~5.2 fold) and phospho‐CREB (~4 fold) content. Furthermore, CB treatment for 3 days increased muscle cAMP content (16%) and blunted the up‐regulation of both atrogin‐1 and MuRF1 mRNAs. These results suggest the existence of an adaptive mechanism responsible to restrain the activation of atrogenes during motor denervation that seems to be mediated by activation of cAMP/PKA/CREB signaling.

Grant Funding Source: FAPESP (2012/05697‐7; 2012/24524‐6)
Reductions in REDD1 expression correspond to enhanced rates of protein synthesis following eccentric contractions (1163.16)
Authors: Gordon Bradley , Steiner Jennifer , Lang Charles , Kimball Scot , Jefferson Leonard
Published: June 2021

Resistance exercise induces skeletal muscle hypertrophy due to enhanced rates of protein synthesis. Signaling through mTORC1 plays a vital role in integrating signals generated by resistance exercise to control skeletal muscle growth. REDD1 is a negative regulator of mTORC1 signaling whose mRNA expression is reduced in human skeletal muscle following resistance exercise. The purpose of the present study was to determine how changes in REDD1 protein expression correspond with changes in mTORC1 signaling and protein synthesis, following electrical stimulation of the sciatic nerve to induce contraction of the tibialis anterior in mice. Thirty min post stimulation, phosphorylation of p70S6K1 (T389) was increased 146% while rates of protein synthesis and REDD1 expression were unchanged compared to control. Four h post stimulation, phosphorylation of p70S6K1 (T389) was further elevated 4.5‐fold and rates of protein synthesis were increased compared to control. At this time point, REDD1 expression was reduced 40%, which was accompanied by a 71% increase in Akt (T308) phosphorylation. These data demonstrate that a reduction in REDD1 expression is not necessary for the initial, weak contraction‐induced stimulation of mTORC1 signaling. However, a subsequent fall in REDD1 expression leads to a more dramatic stimulation of mTORC1 signaling resulting in elevated rates of protein synthesis.

Grant Funding Source: NIH Grants DK‐15658, GM38032
Simvastatin reduces myosteatosis after skeletal muscle injury (1163.19)
Authors: Korn Michael , Davis Max , Gumucio Jonathan , Mendias Christopher
Published: June 2021

Chronic skeletal muscle injuries or tears can lead to the induction of myosteatosis, which is muscle atrophy combined with an accumulation of lipid plaques. Simvastatin is a drug that has demonstrated the ability to prevent lipid accumulation in other injured tissues. We hypothesized that administering simvastatin would reduce myosteatosis following muscle injuries. As the rotator cuff muscle group is especially prone to the development of myosteatosis, to test our hypothesis we performed a full‐thickness supraspinatus tenectomy and administered vehicle or simvastatin for 4 weeks. Compared to controls, simvastatin treatment increased muscle fiber specific force by 19% and decreased visible fat around fibers. Expression of several genes associated with fat accumulation, inflammation, macrophage accumulation, and extracellular matrix fibrosis was significantly decreased. Simvastatin also reduced the percent of pathological type IIB muscle fibers by 39%. These results suggest that simvastatin can protect muscles from the development of myosteatosis, and be useful in the clinical treatment of chronic muscle injuries and tears.
Astaxanthin attenuates lipopolysaccharide‐induced atrogin‐1 expression in C2C12 myotubes (1163.22)
Authors: Takegaki Junya , Kondo Hiroyo , Fujino Hidemi
Published: June 2021

Atrogin‐1, as a muscle specific ubiquitin ligase, is elevated in skeletal muscle during cachectic state, causing muscle atrophy. The elevation of atrogin‐1 expression level is induced by inflammatory cytokines, and they are promoted by oxidative stress. Astaxanthin, a member of carotenoid family, exerts high antioxidative capacity. We investigated preventive effect of astaxanthin on atrogin‐1 expression and muscle atrophy caused by lipopolysaccharide (LPS) treatment. In C2C12 myotubes, LPS treatment markedly increased the level of atrogin‐1 mRNA expression, leading to myotube atrophy. Astaxanthin treatment significantly attenuated the increased atrogin‐1 mRNA expression, and prevented myotube atrophy induced by LPS in C2C12 myotubes. These results suggest the efficacy of astaxanthin could provide a preventive approach against cachexia‐related muscle atrophy.
Growth hormone influences atrophy pathways in skeletal muscle of heart failure rats (1163.3)
Authors: Lima Aline , Damatto Ricardo , Pagan Luana , Bonomo Camila , Martinez Paula , Cezar Marcelo , Damatto Felipe , Zanati Silmeia , Okoshi Katashi , Okoshi Marina
Published: June 2021

Skeletal muscle atrophy is usually observed in heart failure. Growth hormone (GH) increases muscular mass in several clinical settings. Objective: To evaluate GH administration effects on myogenin, MyoD, MRF4, myostatin, follistatin, PI3K, Akt, atrogin‐1 and MuRF‐1 protein expression, in the gastrocnemius muscle of rats with aortic stenosis‐induced heart failure. Methods: Male Wistar rats were subjected to thoracotomy for ascending aortic stenosis induction. After developing tachypnea, rats were evaluated by echocardiogram and subdivided into three groups (n=8): Sham, aortic stenosis (AS) and AS treated with GH (AS‐GH, 2mg/kg/day for 14 days). At the end of treatment, animals were subjected to echocardiogram and euthanized. Protein expression was analized by Western‐Blot. Statistical analysis: Anova and Tukey. Results: Before treatment, cardiac variables did not differ between AS and AS‐GH groups. GH administration preserved body weight (BW, Sham 492±32; AS 420±32; AS‐GH 486±69 g; p<0.05 AS vs Sham and AS‐GH). Left ventricular posterior wall thickness (Sham 3.22±0.20; AS 3.80±0.36; AS‐GH 4.37±0.67 mm) and right ventricle weight/BW ratio (Sham 0.55±0.03; AS 1.15±0.25; AS‐GH 1.18±0.15 mg/g) were higher in AS and AS‐GH than Sham. MyoD expression was higher in AS group than Sham and unchanged in AS‐GH and Sham (Sham 1.00±1.28; AS 1.71±1.37; AS‐GH 1.21±1.21 arbitrary units). MRF4 levels were higher in AS‐GH than Sham and similar in AS and Sham (Sham 1.00±0.73; AS 1.58±0.42; AS‐GH 3.14±1.61 arbitrary units). Atrogin‐1 was lower decreased in AS‐GH than Sham and AS (Sham 1.00±0.58; AS 1.14±0.87; AS‐GH 0.31±0.16 arbitrary units). Protein expression of myostatin, follistatin, myogenin, PI3K, Akt and MuRF‐1 did not differ between groups. Conclusion: Growth hormone administration modulates myogenic regulatory factors and atrogin‐1 expression in the gastrocnemius muscle of heart failure rats.

Grant Funding Source: Supported by Fapesp
The preventive effects of middle frequency electrical stimulation combined with eccentric contraction on disuse atrophy in deep portion of calf muscles (1163.5)
Authors: Tanaka Minoru , Murakami Shinichiro , Fujita Naoto , Fujino Hidemi
Published: June 2021

The purpose of the present study is to evaluate the combined effect of electrical stimulation using middle frequency (ES) and eccentric contraction on disuse atrophy in deep portion of calf muscles induced by hindlimb unloading . Rats were randomly divided into the control, hindlimb unloading for 2 weeks (HU), HU plus ES combined concentric (HU+cES), isometric (HU+iES), and eccentric (HU+eES) contraction groups. The animals in the ES groups were stimulated electrically using middle frequency current twice a day during the unloading period. Animals undergoing HU for 2 weeks exhibited the significant loss of muscle mass, decreased cross‐sectional area of muscle fibers, and overexpression of ubiquitinated protein in the soleus muscles compared with the control animals. HU+eES inhibited the HU‐induced loss of muscle mass, decreased cross‐sectional area of muscle fibers in the soleus muscles. In addition, the HU+eES inhibited the overexpression of ubiquitinated protein in the soleus muscles. Furthermore, the HU+eES inhibited the HU‐induced above factors in the soleus muscles compared with ES combined using other types of contraction groups. These results suggested that the combination of electrical stimulation with eccentric contraction might contribute to higher preventive effects on muscle atrophy in the deep portion of calf muscle.

Grant Funding Source: Supported by The Japanese Society of Physio Therapeutics
Muscle slow to fast fiber plasticity is triggered by low frequency stimulation, membrane depolarization and ATP (1164.11)
Authors: Casas Mariana , Llanos Paola , Jorquera Gonzalo , Jaimovich Enrique
Published: June 2021

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers; we realized that the muscle fiber signals through extracellular ATP to regulate important cellular processes, including regulating fast to slow muscle fiber phenotype changes. In adult muscle fibers, 20 Hz electrical stimulation activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin channels, purinergic receptors activation and IP3‐dependent calcium signals, to finally lead to gene transcription related to fast to slow muscle fiber type transition. ATP release plays a key role in this process, being able to activate, in the absence of electrical stimulation, most of the transcriptional changes observed after 20 Hz stimulation. In fibers depolarized to using external K+, we documented calcium signals and transcriptional changes similar to those observed after 20 Hz stimulation.

Addition of 100‐500 µM external ATP induced muscle fiber depolarization and that transcriptional changes observed after stimulation of fibers with 30 µM external ATP, were abolished by 25 µM nifedipine, a Cav1.1 inhibitor. These two events were completely inhibited by the IP3R blocker Xestospongin B, suggesting that IP3‐dependent events are triggered at these membrane depolarization values.

Grant Funding Source: Supported by Fondecyt 1110467, ACT 1111
Upstream activators of PGC‐1α transcription with acute contractile activity (1164.4)
Authors: Carter Heather , Shuen Michael , Hood David
Published: June 2021

PGC‐1α is a potent transcriptional coactivator of numerous genes involved in contractile activity‐induced mitochondrial biogenesis. We sought to describe the role of transcription factors that regulate the PGC‐1α promoter in the context of acute contractile activity along with established downstream targets. A 1.5kb upstream region of the PGC‐1α promoter fused to a luciferase reporter was injected and electroporated into the rat tibialis anterior (TA) muscle bilaterally. Five days after injection the left TA was subjected to in situ contractile activity for 15 mins (5 mins at 1 Hz, 10 mins at 10Hz) while the right TA served as a resting control. Animals were allowed to recover for 2 hours, and then tissues were extracted. During stimulation, muscle force declined to 40% of initial tension. Transcriptional activity of the PGC‐1α promoter was elevated 3.6‐fold in the stimulated TA, while PGC‐1α mRNA was increased by 1.4‐fold. COX IV and NRF‐1, transcriptional targets of PGC‐1α, were each elevated by 1.2‐fold. NF‐E2 related factor 2 (Nrf2), a transcription factor with a putative binding site on the PGC‐1α promoter was increased by 1.4‐fold at the mRNA level, while GATA4, previously identified as a transcriptional regulator of PGC‐1α, remained unchanged. Thus, contractile activity signals lead to large increases in PGC‐1α transcription along with smaller increments in upstream activators, and downstream targets.

Grant Funding Source: Supported By NSERC.
Ambulatory moderate plus vigorous physical activity is associated with greater bone mineral density in the hip in children with Prader‐Willi syndrome (1166.2)
Authors: Duran Andrea , Castner Diobel , Tucker Jared , Rubin Daniela
Published: June 2021

Individuals with Prader‐Willi Syndrome (PWS) present hypotonia, low lean mass and a fairly low amount of spontaneous ambulatory physical activity (PA), possibly influencing bone mineral density (BMD). This study explored the differences in BMD between more and less active children with PWS. Twenty five children with PWS participated (age: 10.8±2.0 y, height: 141.4±11.7 cm, lean mass [LM]: 28.32±10.31 kg). Moderate plus vigorous PA (MVPA) was assessed via accelerometry. Participants were categorized into more active (n=13, MVPA: 44±3 min/day) and less active (n=12, MVPA: 23±2 min/day) groups based on a MVPA median split of 33 min/day. LM, bone mineral content (BMC), BMD and BMD z‐score (BMDz) of the hip, spine and total body minus the head were measured by dual energy x‐ray absorptiometry. Group differences were assessed using analyses of covariance, controlling for age, height and LM depending on the model. More active children had a significantly greater hip BMD (0.86±0.03 vs. 0.76±0.03 g/cm2), BMDz (0.28±0.28 vs. ‐0.86±0.29) and BMC (19.37±0.72 vs. 17.20±0.75 g) than their less active peers (p<0.05). There were no significant group differences for any spine or total body bone parameters. Children with PWS should incorporate moderate plus vigorous weight‐bearing activities as a critical component of their daily routine.

Grant Funding Source: Supported by USAMRAA Award W81XWH‐08‐1‐0025
Cardiovascular responses to orthostatic stress after 90 minutes head‐down bed rest and artificial gravity exposure in hypovolemic men and women (1169.18)
Authors: Zhang Qingguang , Knapp Charles , Smith Jeffrey , Evans Joyce
Published: June 2021

Intermittent artificial gravity (AG) training was proved to improve orthostatic tolerance limit (OTL) and alter cardiovascular regulations in response to orthostatic stress. The effect of short duration AG was not determined, especially in deconditioned subjects. To test the hypothesis that cardiovascular responses to orthostatic stress would be altered by a short bout of AG compared with head‐down bed rest (HDBR), nine men and seven women were studied during combined 70º head‐up tilt (HUT) and progressive lower body negative pressure (LBNP) until the onset of presyncope after 90‐min HDBR and AG exposure in hypovolemic condition (20 mg intravenous furosemide). To assess associated changes in cardiovascular regulation, blood pressure and heart rate variability were analyzed by spectral analysis and spontaneous baroreflex activity was evaluated by cross‐spectral analysis. Orthostatic tolerance limit was significantly increased after AG compared with HDBR. Women and men had similar responses to orthostasis for most of the cardiovascular parameters following short duration AG compared with HDBR. Following AG, women and men had a greater low frequency (0.04‐0.15 Hz) phase delay between systolic blood pressure and RR intervals at supine and did not change in response to orthostasis. Following HDBR, the supine phase delay increased in response to orthostatic stress. We conclude that a short bout AG alters some cardiovascular responses to orthostatic stress.

Grant Funding Source: Supported by KY NASA EPSCoR Grant #NNX07AT58A, KY State Matching Grants, the NASA Johnson Space Cent
Interactive effects of trigeminal nerve stimulation and muscle metaboreflex activation on muscle sympathetic nerve activity in healthy humans (1170.5)
Authors: Fisher James , Fernandes Igor , Barbosa Thales , Coote John , Nóbrega Antonio , Vianna Lauro
Published: June 2021

We investigated the independent and interactive effects of trigeminal cutaneous receptor stimulation and activation of metabolically sensitive skeletal muscle afferents (muscle metaboreflex) on muscle sympathetic nerve activity (MSNA). Heart rate (HR; ECG), blood pressure (BP; Finometer) and MSNA (microneurography) were continuously recorded in eight healthy men (29±2 yr) during three experimental conditions; 1) facial cooling [0°C, trigeminal stimulation, TGS], 2) muscle metaboreflex activation by post‐exercise ischemia [PEI] following isometric handgrip at 30% maximum voluntary contraction, and 3) simultaneous TGS and PEI. Trials were counterbalanced and respiratory rate and tidal volume controlled at baseline, TGS, PEI and TGS+PEI. TGS produced a significant decrease in HR (Δ‐5±2 bpm; mean±SE), and increase in mean BP (Δ14±3 mmHg) and MSNA total activity (Δ353±118% AU). During PEI, HR remained at resting levels (Δ3±2 bpm; mean±SE), while mean BP (Δ25±3 mmHg) and MSNA total activity (Δ234±83% AU) increased. Finally, during combined TGS and PEI, HR was unchanged (Δ0±2 bpm; mean±SE), while mean BP (Δ30±3 mmHg) and MSNA total activity (Δ331±100% AU) were increased. Thus, excitatory inputs from the trigeminal nerve and metabolically sensitive skeletal muscle afferents do not appear to summate algebraically in determining a MSNA response, but rather exhibit neural occlusion.

Grant Funding Source: Supported by CAPES, CNPq, FAPERJ.
Effect of sub‐occipital release on steady‐state and dynamic oscillatory features of the cardiovascular system (1170.7)
Authors: Wilson Thad , Wagler Josiah , LePorte Andrew , Vijayakumar Amirtha , Kaya Ryan , Metzler‐Wilson Kristen , Walkowski Stevan
Published: June 2021

To determine if sub‐occipital release (SOR) modulates sympathetic control during basal conditions and in conditions with acutely increased activity, 9 healthy subjects (5 male, 4 female) participated in 2 protocols: 1) cold stress (15°C water perfused through a high‐density tube‐lined suit for 20 min) to increase sympathetic tone and 2) normothermia (34°C). In each thermal condition, subjects received in random order, SOR and yaw sham (30 cycles/min) for 2 min. Measurements included: heart rate (HR; ECG); mean arterial blood pressure (MAP; photoplethysmography); cutaneous vascular conductance (CVC; laser‐Doppler flux/MAP); stroke volume (SV; Modelflow); systemic vascular resistance (SVR; Modelflow); and low frequency (LF; 0.04‐0.15 Hz) spectral power (Fast‐Fourier Transform). Cold stress increased MAP (SOR: 90±2 to 98±2; yaw: 87±2 to 99±2 mmHg) and SVR (SOR: 15±2 to 17±2; yaw: 15±2 to 17±2 units), decreased CVC (SOR: 0.52±0.22 to 0.26±0.14; yaw: 0.46±0.15 to 0.24±0.11 units, normothermia to cold stress, respectively), and did not significantly change HR or SV. Blood pressure and laser‐Doppler flux LF spectral power also increased during cold stress. No significant differences were observed between SOR and yaw during normothermia or cold stress. These data indicate that cold stress increases sympathetic noradrenergic tone but SOR does not have the capability to modulate these variables.

Grant Funding Source: Supported by a grant from the American Osteopathic Association
Increased ATP delivery to contracting skeletal muscle blunts sympathetic vasoconstriction in humans (1172.5)
Authors: Hearon Christopher , Racine Matthew , Richards Jennifer , Luckasen Gary , Larson Dennis , Dinenno Frank
Published: June 2021

Sympathetic stimulation of postjunctional α‐adrenergic receptors elicits vasoconstriction in resting skeletal muscle that is blunted during exercise in an intensity dependent manner. This ‘sympatholysis’ is hypothesized to occur due to increased circulating ATP. We tested the hypothesis that infusion of a non‐sympatholytic low dose of ATP during mild exercise will evoke sympatholysis in humans. In young adults (n=8), we measured forearm blood flow (Doppler Ultrasound) and calculated vascular conductance (FVC) to local intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during: 1) control vasodilator infusion (low dose adenosine triphosphate, ATP; sodium nitroprusside, SNP), 2) mild (5% maximum voluntary contraction; MVC) or moderate (15% MVC) intensity handgrip exercise, and 3) combined ATP or SNP + 5% exercise. Vasoconstriction to PE during mild exercise (ΔFVC: ‐30±1%) was similar to that observed during ATP and SNP (ΔFVC: ‐30±3 and ‐35±3%; NS), however this was blunted during moderate exercise (ΔFVC: ‐17±3%; P<0.05). ATP + 5% exercise blunted PE mediated vasoconstriction similar to that seen during moderate intensity exercise (ΔFVC: ‐18±3%). In contrast, SNP + 5% exercise did not blunt PE mediated vasoconstriction (ΔFVC: ‐34±5%). These data suggest that increasing ATP delivery to contracting muscle during exercise can blunt sympathetic vasoconstriction in humans.

Grant Funding Source: Supported by: H HL102720L