bluuebunny/crossref_metadata_2025 huggingface dataset:
Cigarette smoke attenuates collagen production and migration of cardiac fibroblasts through inhibition of the HIF‐1α pathway
Authors: Bradley Jessica Marie , Doggett Travis M , El Hajj Milad , Pyakurel Kusma , Breslin Jerome W , Gardner Jason D
Published: June 2021

Cigarette smoke (CS) is the major cause of preventable death with 5 million deaths annually worldwide, many due to cardiovascular disease. Previously, we found that 6 wks of CS exposure exacerbates volume overload cardiac remodeling and dysfunction. Our findings indicated that CS induces ventricular dilation by disrupting compensatory collagen production. Key regulators of extracellular matrix remodeling are the cardiac fibroblasts. Our hypothesis is that CS exposure inhibits the HIF‐1α pathway preventing collagen production and impairing migration of cardiac fibroblasts. We examined the effects of CS using isolated adult rat cardiac fibroblasts. After 24 hrs exposure to 0, 5 and 10% CS extract (CSE), Western blot analysis on whole cell lysates demonstrated that 10% CSE reduced expression of HIF‐1α (13% vs. control) and VEGF (6%), a downstream product of HIF‐1α activation. Collagen secretion assessed by media hydroxyproline was reduced after 5 and 10% CSE by 64 and 72%, respectively. Using electric cell‐substrate impedance sensing (ECIS) to evaluate cardiac fibroblast migration after wounding, we found that 5% CSE had little effect; however, 10% CSE impaired migration by 36%. These data suggest that CS may attenuate collagen production and migration of cardiac fibroblasts through inhibition of the HIF‐1α pathway. Louisiana Board of Regents LEQSF2009‐12‐RDA10, NIH P20RR018766 and R01HL098215.
Educational Uses of Molecular Visualization
Authors: Craig Paul A , Michel Lea V. , Bateman Robert C
Published: June 2021

As biochemists, one of our most captivating teaching tools is the use of molecular visualization. It is a compelling medium that can be used to communicate structural information much more effectively in 3D than in 2D. While some of us have tried using true 3D visualization (with the help of stereo glasses), many of us have attempted to incorporate some aspect of molecular visualization in our classrooms, labs or online course materials. A survey and a series of interviews were conducted with the goal of compiling information on the educational uses of molecular visualization. We used a series of questions, but the two overarching issues were

How does molecular visualization impact student learning?

How can we assess this learning effectively?

Results of the survey (n > 100) and the interviews (n ≥ 10) will be presented, as well as a perspective on developing an assessment rubric.
Simulation of Physiologic Strain to Aligned Cells Anchored in 3D Affects Proliferation, Differentiation, and Organization of the Actin Cytoskeleton
Authors: Doroudian Golnar , Gang Anjulie , Curtis Matthew , Russell Brenda
Published: June 2021

Human bone marrow‐derived mesenchymal stem cells (hMSCs) are an attractive cell source for regeneration of damaged tissue. The objective is to understand how mechanical cues affect the morphology, proliferation and differentiation of hMSCs into different cell lineages. The approach used is to deliver 10% strain at 1 Hz frequency to hMSCs grown in culture on flat surfaces, or on substrata with microposts measuring 15 micrometer high and spaced 75 micrometer apart for 48 h (n=5). The actin cytoskeleton with oriented cyclic flexing and topography became elongated spanning between adjacent microposts. The length/width ratio of the nuclei was highest with both strain and microtopography, and the nuclear area was smaller by addition of either topography or strain. Also all strained cells exhibited approximately two‐fold greater proliferation than relaxed cells. Microarray analysis showed more than 1000 genes differing between the flat‐strain and topography‐strain conditions, including altered gene expression related to muscle function and differentiation, actin assembly, adhesion, and extracellular matrix remodeling (P<0.05). However, cardiac‐specific markers were not seen. Therefore, attention to mechanical stimuli in the creation an artificial stem cell niches is essential for advanced applications for regenerative medicine. (Funded by NIH HL0905230).
Real‐time observation of cytoskeletal stresses and subsequent focal adhesion remodeling in live cells
Authors: Hua Susan , Ye Nannan , Verma Deepika , Meng Fanjie , Sachs Frederick
Published: June 2021

Cytoskeletal stresses are important regulators of cell adhesion, migration and growth. While cytoskeleton tension has been shown to promote the development of adhesions, the force transduction mechanism remains unclear, because it requires a measurement of cytoskeletal stresses in live cells. We have developed a group of stress sensitive FRET probes that can be inserted into cross‐linking proteins of the cytoskeleton to measure the stresses in live cells. By co‐expressing FRET probes in actinin and fluorescence labeled paxillin, we simultaneously observed the time‐dependent changes in cytoskeletal stresses and the dynamics of its linking focal adhesions. We show that an increase in cytoskeletal stress at the focal adhesions results in an elongation of the adhesions in the direction of force, and a decrease of local force causes disassembly of the adhesions. The orientation of the adhesion sites maintains parallel with the stress fibers at the cell‐cell contacts. At the open edge, a highly active protrusion is followed by the formation of periphery actin bundles and the rotation of associated adhesions. Interestingly, the change of orientation of adhesions appears to be directed by the force in the stress fibers, involving a separation of actin fibers from adhesion sites and a stress directed lateral displacement of adhesions. These results provide the direct observation of the force transduction at focal adhesions.
Mechanisms Protecting Chronic Pressure Overload by Apoptosis Inhibition
Authors: Park Misun , Gao Shumin , Lee Grace Jung Ah , Yoon Seonghun , Vatner Stephen F , Vatner Dorothy E
Published: June 2021

It is generally believed that chronic pressure overload increases myocyte apoptosis, and that the resultant loss of myocytes is responsible for the cardiac dysfunction. If this reasoning is correct, then protecting myocyte apoptosis with chronic pressure overload should increase numbers of myocytes. We examined the effects of protecting apoptosis by caspase inhibition (CI) with chronic pressure overload induced by thoracic aortic constriction (TAC). Left ventricular (LV) ejection fraction was reduced by TAC (55% vs 70%), but was protected with CI (66%). However, CI reduced myocyte apoptosis by only 0.06%, compared with non‐myocyte apoptosis (0.21%), and did not result in increased total myocyte number, which was similar in sham operated controls to TAC with and without CI. However, other mechanisms were potentially responsible for the protection of cardiac function: 1) CI reduced interstitial fibrosis (2.9%) compared to vehicle (6.8%) after TAC. 2) Ki67 staining of endothelial cells was increased with CI and TAC (3.4±0.7%) compared to vehicle group (1.4±0.6%) indicative of angiogenesis. 3) Ki67‐positive myocytes, indicative of myogenesis, were also higher with CI than vehicle (0.9±0.2% vs 0.3±0.1%). Thus, CI improved LV function after TAC, not by protecting myocyte apoptosis, but rather through angiogenesis and myogenesis, reducing myocardial fibrosis.
Phase Change in Membranes Observed by Variations in Electrical Capacitance
Authors: Doermann Alex , Goggins Landon , Larsen David , Woodbury Dixon J
Published: June 2021

Lipid membranes made of DPPC (Dipalmitoyl‐phosphatidyl‐choline) are well‐documented to undergo phase changes across specific critical temperatures. Here, it is shown that the electrical capacitance of a planar lipid membrane changes dramatically as the membrane changes phase. By cooling a DPPC membrane below the transition temperature, Tm (41.5°C), the capacitance increases >20%. Since capacitance is directly related to surface area and inversely related to membrane thickness, these data provide insight to the fundamental structures of the different phases. Although these phase changes have been measured before using X‐ray, DSC (Differential Scanning Calorimetry), and AFM (Atomic Force Microscopy), these previous studies mostly observed stacked or supported membranes; the present study demonstrates the phase change in a single, fully hydrated membrane. Hence, this method provides the opportunity to measure directly, in a biologically relevant system, the effects that such a change in membrane properties have on membrane proteins and the process of exocytosis.
Ubiquitylation of the plasma membrane KCa3.1 is not required for channel endocytosis but for its lysosomal targeting
Authors: Balut Corina Mihaela , Devor Daniel C.
Published: June 2021

We recently demonstrated that plasma membrane KCa3.1 is ubiquitylated subsequent to endocytosis and targeted for lysosomal degradation. To further elucidate the role of ubiquitylation in KCa3.1 endocytosis and trafficking, we constructed a mutant KCa3.1 in which all 18 predicted cytoplasmic lysine residues were replaced with arginines (K‐less). This K‐less KCa3.1 trafficked normally to the plasma membrane and was functional. The K‐less KCa3.1 internalized normally, indicating that channel ubiquitylation is not required for endocytosis. However, the treatment with the ubiquitin‐activating enzyme (E1) inhibitor UBEI‐41 resulted in a reduced mutant endocytosis, suggesting that ubiquitylation of other adaptor proteins is required for KCa3.1 internalization. Next, we showed that the mutant degradation was dramatically reduced. To determine which lysine(s) are being ubiquitylated and target KCa3.1 for lysosomal degradation, we generated mutants where individual lysines were introduced back into the K‐less KCa3.1. Using tandem ubiquitin‐binding entities to isolate ubiquitylated proteins we could demonstrate that lysines in the N‐terminus and intracellular loops of KCa3.1 are not important in this process. The role of the other lysines remains to be determined. These results provide novel insights into the molecular mechanisms of endocytosis and downstream trafficking of KCa3.1 channels.
Loss of renal Nedd4‐2 in adult mice leads to PHAII compensated by ENaC down‐regulation and ROMK up‐regulation
Authors: Staub Olivier , Ronzaud Caroline , Loffing Dominique , Debonneville Anne , Yang Baoli , Stokes John , Koesters Robert , Hummler Edith , Loffing Johannes
Published: June 2021

Constitutive Nedd4‐2 knockout (KO) mice show Na+ retention and salt‐sensitive hypertension. To determine the role of renal Nedd4‐2 in adult mice, we generated inducible renal tubule‐specific Nedd4‐2 KO mice (Nedd4‐2Pax8/LC1) using a combination of the Tet‐On and Cre‐loxP systems. Under standard and high‐Na+ diets, KO displayed decreased plasma aldosterone but normal Na+/K+ balance. Under high Na+ diet, KO mice showed hypercalciuria and hypertension, both treated by thiazides. NCC expression and phosphorylation were increased in the KO. Interestingly, KO showed increased intracellular β‐ and γENaC abundance, but decreased α‐ and γENaC proteolytic cleavage and decreased αENaC mRNA and protein expression, suggesting down‐regulated ENaC. In contrast, ROMK protein expression and cell surface localization were increased in the distal convoluted tubules and connecting tubules of KO, in keeping with the absence of hyperkalemia. Thus, in adult mice, loss of renal Nedd4‐2 causes a pseudohypoaldosteronism type II‐like syndrome with NCC up‐regulation leading to salt‐sensitive hypertension and hypercalciuria, suggesting that Nedd4‐2 primarily targets NCC. The accompanying hypoaldosteronism with down‐regulation of ENaC‐mediated Na+ reabsorption together with an up‐regulation of K+ secretion via ROMK likely contributes to the compensated phenotype of the KO mice and explains the maintained Na+/K+ balance.
Long QT syndrome type 3 caused by a PY‐motif mutation leading to altered ubiquitylation and increased expression of Nav1.5 in knock‐in mice
Authors: ROUGIER Jean-Sébastien , Albesa Maxime , Remme Carol Ann , Ogrodnik Jakob , Petitprez Séverine , Bankston John , Kass Robert S. , Bezzina Connie R. , Chung Wendy , Abriel Hugues
Published: June 2021

Background

Congenital long QT syndrome type 3 (LQT3) is a cardiac arrhythmias caused by gain‐of‐function mutations in SCN5A, the gene encoding the sodium channel Nav1.5. This channel can be regulated via the ubiquitin ligase Nedd4‐2 which binds to the PY‐motif of Nav1.5.

Aim

We have investigated the mechanisms underlying the phenotype of a LQT3 family harboring a SCN5A mutation located in the PY‐motif (p.Y1981N) of Nav1.5.

Results

In HEK cell, co‐immunoprecipitation experiments revealed that the interaction of Nav1.5 with Nedd4‐2 was abolished by the mutation. While the WT protein was ubiquitylated and the sodium current (INa) down‐regulated upon Nedd4‐2 co‐expression, no such regulation was observed with the mutant channel. In vivo relevance of this mechanism was assessed by generating a knock‐in (KI) mouse line bearing the p.Y1981N mutation. Ubiquitylation of the mutant channel expressed in KI hearts mice was reduced, and the total level of Nav1.5 protein was increased compared to WT littermates. INa and action potential duration were increased in KI cardiomyocytes compare to WT.

Conclusions

These results demonstrate a central role for Nav1.5 ubiquitylation in the regulation of sodium channel density in cardiomyocytes.

Funding from the European Community's Seventh Framework Programme, EUTrigTreat, the Swiss National Science Foundation, and the Swiss Heart Foundation.
Acute changes in dietary sodium lead to sodium retention in the collecting duct NOS1 knockout mouse
Authors: Hyndman Kelly Anne , Brands Michael , Pollock David M , Pollock Jennifer S
Published: June 2021

We previously determined that collecting duct (CD) specific nitric oxide synthase‐1 (NOS1, nNOS) knockout mice (CDNOS1KO) have elevated systolic blood pressure (129±4 vs 113±3 mmHg, p< 0.01) that is further increased after 7 days (d) of a 4.0% NaCl (high salt, HS) diet (142±2, p<0.001). In this current study, we hypothesize that CDNOS1KO mice will have an attenuated increase in Na+, water and NOx (NO2/NO3) excretion following an acute increase in dietary Na+. CDNOS1KO (n=6) and flox (n=6) were acclimated to metabolic cages for 3 d while on a normal salt (NS, 0.4% NaCl) gel diet (switched from a NS pellet diet), then a 2 d of Na+ deficient diet (LS, trace Na+) followed by a 2 d HS. On day 3 of NS gel diet, CDNOS1KO had a lower water intake (1.75±0.6 vs 5.6±1.1 ml/d, p=0.010), urine volume (1.4±0.5 vs 2.7±0.3 ml/d, p = 0.058), Na+ excretion (0.66±0.08 vs 0.38±0.07 mmol/d, p=0.017) and NOx excretion (0.56 ±0.17 vs 1.03±.11 μmol/mg creatinine, p=0.047) compared to flox mice. After 2 d LS diet, CDNOS1KO and flox mice had similar water intake (1.65±0.2 vs 2.5±0.9 ml/d) and Na+ excretion (0.18±0.04 vs 0.15±0.01 ml/d, p>0.05). After 2 d HS diet, CDNOS1KO excreted less Na+ than flox mice (2.75±0.55 vs 4.21mmol/d, p=0.03). On all diets, the mice ate equal amounts of food. These data suggest that CDNOS1KO mice are sensitive to dietary changes (NS pellet vs gel diet) and that deletion of NOS1 from the CD leads to Na+ retention during consumption of a HS diet.
Osmotic Water Absorption Across Toad Skin: C.B. Jorgensen Revisited
Authors: Hillyard Stanley D. , Larsen Erik Hviid
Published: June 2021

Water absorption (Jv) was measured across isolated skin from the pectoral and pelvic regions of toads (Bufo bufo) that were maintained with ad lib water availability. Absorption from 50 mM NaCl was lower than from deionized water (DI) as predicted from the vant'Hoff equation. These results differ from whole animal studies that have shown Jv to be greater from dilute NaCl solutions than from DI in dehydrated toads (Ferriera, H.G. and Jesus, C.H. J. Physiol. 228:583–600. 1973). Absorption from 50 mM Nagluconate was lower than from 50 mM NaCl, suggesting an anion effect on water absorption that might be related to differences between isolated skin and intact toads. The Jv values observed from DI were similar to those reported for AVT‐treated skin with the vasculature perfused (Christensen, C.U. J. Comp. Physiol. 93:93–104, 1974). These results suggest aquaporins may be expressed in the apical membrane of the skin of hydrated toads that utilize ad lib water sources for “anticipatory drinking” (Jorgensen, C.B. Comp.Biochem. Physiol. 109A:311–324. 1994).
Bicarbonate‐sensing soluble adenylyl cyclase in elasmobranch and teleost fishes
Authors: Roa Jinaé Nicole Bartlett , Barron Megan Elizabeth , Tresguerres Martin
Published: June 2021

Soluble adenylyl cyclase (sAC) is an evolutionarily conserved CO2/pH/bicarbonate sensor recently shown to be essential for blood acid/base regulation in the dogfish shark. To elucidate if sAC is present in other elasmobranchs, we performed Western blotting with round ray (Urotrygon reticulata) and leopard shark (Triakis semifasciata) samples, and found immunoractive bands of ~100 kDa in eye, rectal gland, gill, intestine and white muscle. BLAST searches revealed rainbow trout Oncorhynchus mykiss, a teleost fish, also has a sAC ortholog. By RT‐PCR, we detected sAC mRNA in trout testes, but not in other tissues. However, Western blots using anti‐trout sAC antibodies detected prominent 100 and/or 50kDa immunoreactive bands in testes, red blood cells, gill, kidney and muscle, as well as in cultured cell lines derived from gill and intestine. These results suggest sAC is present in multiple tissues from both elasmobranch and teleost fishes and that, similarly to mammals, the trout sAC gene undergoes extensive alternative splicing. This research was supported by a San Diego Fellowship to JNBR.
Lung ventilation is an effector of the baroreflex in the cane toad (Rhinella marina)
Authors: Hedrick Michael S. , Hillman Stanley S. , Drewes Robert C. , Withers Philip C.
Published: June 2021

Lung ventilation plays an important role for regulating lymph flux in anurans by changing the compliance of lymph sacs that surround the lungs during changes in lung volume. Because lymph mobilization in anurans is dependent upon blood volume/pressure status, we hypothesized that lung ventilation, like cardiac and lymph heart effectors, is also an effector of the baroreflex. We tested this hypothesis by measuring lung ventilation frequency (fR) and heart rate (HR) while increasing mean arterial blood pressure (MAP) with phenylephrine (Phe; 0.05–1.0 mg/kg, i.v.) and decreasing MAP with sodium nitroprusside (SNP; 0.05–1.0 mg/kg, i.v.). Decreasing MAP with SNP resulted in a significant increase in fR by an average of 12 breaths min−1 while increasing MAP with Phe decreased fR by an average of 4 breaths min−1. The slope of the baroreflex gain (ΔHR/ΔMAP; beats min−1 kPa−1) for combined Phe and SNP injections was −3.9 ± 0.9 (r2 =0.54; P<0.001) and was similar to the slope of the relationship between ΔfR and ΔMAP (−5.2 ± 0.8; r2 = 0.69; P<0.001). These results suggest that lung ventilation is regulated by MAP, in addition to blood gases and pH, and is an effector of the baroreflex. Supported by NSF‐IOS 0843082.
β‐Adrenoreceptors in the ventricle of the rainbow trout
Authors: Shiels Holly A , Fenna Andrew , Pellowe Sarita , Robinson Ryan , Taylor Rebecca
Published: June 2021

Stimulation of cardiac β adrenergic receptors (βARs) results in increased cardiac function. Despite significant research into mammalian βAR signalling, little is known of βAR signalling in the teleost heart. We applied pharmacological agents known to stimulate βARs in mammalian ventricular myocytes, to myocytes isolated from the rainbow trout heart and assessed changes in the Ca current and in cell shortening. In agreement with previous work, isoprenaline (50 nM and 1 uM) increased % cell shortening and the amplitude of Ca current. Salbutamol (1 uM and 50 uM) a β2‐AR partial agonist also increased these parameters above control levels but not to the same extent as isoprenaline. The β2‐AR antagonist (ICI‐188,551, 1 uM) significantly decreased the contractile and ionic responses to isoprenaline and almost completely abolished the responses to salbutamol. These experiments were repeated in myocytes pre‐incubated with Pertussis toxin (PTX) to inhibit Gi‐signalling and while there was a trend for PTX‐ treated cells to show greater responses than control cells, this was not statistically resolvable. Real time qPCR was used to investigate the βAR subtypes responsible for these functional observations in trout ventricle. mRNA was expressed for β1, β2, β3a, β3b¬‐ARs, with the β2AD the most prominent consistent with the pharmacology.
Variable oxygen affinity among terrestrial and diving vertebrates
Authors: Wright Traver J , Davis Randall W
Published: June 2021

From being the first protein to have its three‐dimensional structure determined to being sequenced for hundreds of species, myoglobin (Mb) has had a distinguished history as a research subject. Although Mb is one of the most studied proteins, surprisingly little is known of its oxygen affinity at physiologically significant temperature or the variability of this oxygen affinity between species. In this study we compared the oxygen affinity (P50) of skeletal muscle Mb from six terrestrial and ten diving vertebrates. After extracting Mb from muscle samples and removing hemoglobin contamination, oxygen affinity was determined using a TCS Hemox Blood Analyzer. We found Mb P50 ranging from 2.5 to 5.3 mmHg at 37°C. Modeling of oxygen transport in muscle tissue will be necessary to determine if this twofold difference in oxygen affinity has significant implications in oxygen transport under various physiological conditions. Variants in Mb structure and function could be adaptive for species that regularly experience muscle hypoxia such as diving vertebrates.
Alterations in Ca2+ regulatory proteins and Ca2+ ‐dependent gene expression in skeletal muscle from ALS mice
Authors: Chin Eva R. , Mazala Davi A.G. , Chen Dapeng
Published: June 2021

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by skeletal muscle atrophy, weakness and paralysis leading to death. The purpose of this study was to examine changes in Ca2+ handling proteins and in expression of Ca2+ ‐regulated skeletal muscle genes in a mouse model of ALS. Gastrocnemius (GAS) and tibialis anterior (TA) muscles were obtained from control (CON) or G93A superoxide dismutase 1 Transgenic (Tg) mice for muscle protein and transcript analysis. Fast and slow isoforms of the Sarcoplasmic Reticulum (SR)/Endoplasmic Reticulum Ca2+ ATPase (SERCA) protein were significantly reduced at the symptomatic stage (120–140d) in GAS muscle: SERCA1 to 44% and SERCA2 to 11% of CON; p<0.05. The Ca2+ buffering protein parvalbumin was reduced in GAS to 60, 75 and 40% of CON levels at 70d, 90d and 120–140d, respectively. This coincided with an up‐regulation of the slow troponin I and myoglobin transcripts by 16.0 and 1.6 –fold and down‐regulation of fast fibre‐type genes (fast troponin I, muscle creatine kinase and GAPDH) in TA muscle of Tg mice. Overall, these data indicate adaptive changes in intracellular Ca2+ regulatory proteins and activation of the slow/oxidative gene expression program beginning in the pre‐symptomatic stage of ALS disease which would help maintain force producing capacity during ongoing muscle atrophy.
Blocking of myostatin and activins increase muscle protein synthesis and mTORC1 signaling but decreases capillary density
Authors: Hulmi Juha J , Oliveira Bernardo M , Silvennoinen Mika , Ma Hongqiang , Pierre Philippe , Hoogaars Willem M.H. , Pasternack Arja , Kainulainen Heikki , Ritvos Olli
Published: June 2021

Blocking of myostatin and activins increases muscle mass but the underlying mechanisms are unknown and whether during muscle growth capillary density is maintained. Soluble activin receptor IIb fused to Fc (ActRIIB/Fc) was produced in house inhibiting myostatin, activin A and activin B signaling in a biological assay. Healthy mice were injected with ActRIIB/Fc either 1x or 2x/wk for 2 weeks. The 2x/wk dosage seemed to increase muscle mass slightly more. In gastrocnemius muscle fiber size increased ~15–20 % whereas the capillary density decreased ~20 %. In muscle homogenate, PGC‐1α and cytochrome C protein decreased 40–50 %. To elucidate mechanisms for these, we next sacrificed mice 1 or 2 days after ActRIIB/Fc or PBS injection. Muscle protein synthesis increased in gastrocnemius ~30 % 2 days after ActRIIB/Fc correlating with increased mTORC1 signaling (increased phosphorylation (p) of rpS6, r=0.8, p<0.01). In contrast, muscle protein ubiquitination and p‐FoxO remained unchanged. Both p‐p38 and p‐Erk 1/2 MAPKs decreased 2 days after ActRIIB/Fc. Of novel proteins possibly associated with TGFβ blocking, REDD1 increased 1–2 days after ActRIIB/Fc. Importance of this finding needs to be studied in the future. In conclusion, blocking of ActRIIB pathway increases muscle protein synthesis and mTORC1 signaling but decreases MAPK signaling after 2 days and decreases muscle capillary density.
The effects of cAMP on force of contraction and recovery from fatigue of fast twitch muscle is dependent on stimulation parameters
Authors: MacDonald Tara L , Murrant Coral L
Published: June 2021

Cyclic adenosine monophosphate (cAMP) is a potent second messenger in signal transduction and a positive inotropic regulator of cardiac muscle contractility, however effects on skeletal muscle mechanics are unclear. To determine the effect of 10−5M dibutyryl cAMP on post‐fatigue recovery in soleus (SOL) and extensor digitorum longus (EDL) in vitro, we stimulated SOL and EDL using varied contraction frequencies (contractions per minute; CPM) and total stimulation duration to elicit diverse fatigue profiles. SOL fatigued for 7 and 9min at 30 and 15CPM and recovered to 91.5±0.7% and 80.4±3.04% force. Long duration stimulation for 90min at 30 and 15CPM elicited 8.5±4.2% and 7.3±3.5% force restitution. SOL exposed to cAMP did not differ significantly from controls. EDL fatigued for 5 and 14 min at 10 and 2 CPM and recovered 84.1±4.1% and 81.2±1.20% force. Stimulation for 90min at 10 and 2CPM produced 7.7±1.5% and 1.2±2.9% recovery of force production. cAMP significantly improved recovery following high‐frequency stimulation (10CPM), but inhibited recovery after low‐frequency stimulation (2CPM). These results suggest that the effects of cAMP on contractility are a) fiber‐type dependent, such that only fast‐twitch fibers (EDL) respond to cAMP as an inotropic factor and b) the observed effect is dependent on stimulation parameters, namely frequency of contraction.
Impaired regeneration in ERRalpha deficient skeletal muscle
Authors: LaBarge Samuel , McDonald Marisa , Huss Janice
Published: June 2021

The nuclear receptor transcription factor, Estrogen‐Related Receptor α (ERRα), regulates mitochondrial glucose and fatty acid oxidation and mitochondrial biogenesis in highly oxidative tissues. However, it is unknown whether ERRα is involved in adaptive skeletal muscle processes requiring metabolic remodeling like muscle regeneration. We employed skeletal muscle‐specific knockout mice (m‐ERRα−/−) to assess ERRα‐dependence of mitochondrial biogenesis and myofiber regeneration in tibialis anterior following cardiotoxin (CTX)‐induced injury. Expression of ERRα and its coactivator, PGC‐1α, were increased in regenerating WT myofibers, supporting their involvement in metabolic and contractile gene regulation during regeneration. Satellite cell (Pax7+) activation and macrophage (CD11b+) infiltration were the same in WT and m‐ERRα−/− injured muscles. In contrast, delayed regeneration in m‐ERRα−/− muscle was revealed by increased centralized nuclei at d15 and reduced myofiber area at d7 and d15 compared to WT mice. Mitochondrial enzyme activities (succinate dehydrogenase, cytochrome oxidase, citrate synthase) were not distinct early (d7) but were significantly lower in d15 m‐ERRα−/− regenerating fibers compared to WT. These results suggest that delayed early regeneration in m‐ERRα−/− muscles is not due to mitochondrial defects but may involve perturbed signaling of MAPK or calcineurin/CaMK pathways, that are modulated by ERRα and involved in muscle regeneration.

(Support by NIH R01DK74700 (J.M.H.) and Morgan and Helen Chu graduate fellowship (S.L.)
Macrophage AMPKα1 is necessary for the resolution of inflammation during skeletal muscle regeneration
Authors: Mounier Rémi , Theret Marine , Cuvellier Sylvain , Sakamoto Kei , Zibrova Darya , Foretz Marc , Viollet Benoit , Chazaud Bénedicte
Published: June 2021

Adult skeletal muscle has the unique capacity to regenerate. Muscle regeneration is always associated with inflammation and notably macrophages (MPs), which play dual role. Soon after injury, inflammatory monocyte‐derived macrophages (M1 phenotype) stimulate myogenic cell proliferation. After phagocytosis of muscle debris, MPs switch their phenotype to acquire an anti‐inflammatory profile (M2) and stimulate myogenic cell differentiation and myofibre growth. Here, we explored the role of AMPK in the resolution of inflammation during muscle repair.

AMPKα1 KO muscle shows both a delay and an impairment of post‐injury regeneration. These deficiencies are also observed in LysM‐CRE;AMPKfl/fl muscle, confirming the MP specificity of AMPK requirement. In vitro, AMPKα1 KO MPs hardly acquire a M2 profile upon cytokine stimulation. Their phagocytic activity is also altered. In vivo analysis of MP subpopulations (using the AMPKα1−/−;CX3CR1GFP/+ mouse) during muscle repair shows that the number of intramuscular MPs exhibiting the M2 phenotype is reduced in the AMPKα1 KO compared to the WT mouse. Accordingly, leukocytes from AMPKα1 KO muscle do not increase their expression of markers associated with the resolution of inflammation during muscle regeneration.

These results strongly support that AMPKα1 is one key regulator of MP switch at time of resolution of inflammation and is essential for a proper muscle repair.
eNOS and nNOS contribution to reflex cutaneous vasodilation during dynamic exercise in humans
Authors: McNamara Tanner C , Keen Jeremy , Simmons Grant H , Holowatz Lacy A , Wong Brett J
Published: June 2021

Recent data suggests nNOS mediates NO‐component of reflex cutaneous vasodilation with passive heat stress. Our hypothesis was nNOS but not eNOS inhibition would attenuate reflex cutaneous vasodilation during dynamic exercise. Protocol 1: subjects performed a VO2 peak test on a supine cycle ergometer. Protocol 2: with experimental arm at heart level subjects cycled in supine posture at 60% VO2 peak to raise core temperature (Tc) 0.8–1.0°C (35–45 min). In protocol 2 subjects were equipped with 4 microdialysis fibers on the forearm and each randomly assigned as: 1) lactated Ringer's (control); 2) 5mM NPLA (nNOS inhibition); 3) 10mM L‐NIO (eNOS inhibition); and 4) 20mM L‐NAME (non‐selective NOS inhibition). At the end of protocol 2 all sites were locally heated to 43°C and infused with SNP to elicit maximal dilation. Mean arterial pressure (MAP), skin blood flow via laser‐Doppler flowmetry (LDF), and Tc via ingestible telemetric pill were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to maximum. In protocol 2 there was no significant difference between control (62±5 %CVCmax) and NPLA (61±6 %CVCmax). L‐NIO (38±4 %CVCmax) and L‐NAME (41±7 %CVCmax) significantly attenuated CVC compared to control and NPLA (p<0.001 all conditions). There was no difference between L‐NIO and L‐NAME. We conclude eNOS, not nNOS, contribute to reflex cutaneous vasodilation during dynamic exercise.
Hypercapnia does not improve hyperthermic simulated hemorrhagic tolerance
Authors: Lucas Rebekah A.I. , Pearson James , Ganio Matthew S. , Crandall Craig G.
Published: June 2021

This study tested the hypothesis that hypercapnia induced increases in cerebral perfusion improves tolerance to a simulated hemorrhagic challenge in heat stressed individuals. Seven individuals (32±9 y; 73±15 kg) underwent passive heat stress followed by a hemorrhagic challenge on two separate days (randomized). Hemorrhage was simulated via progressive lower body negative pressure (LBNP) to pre‐syncope after body core (intestinal) temperature was raised ~1.5 °C using a water perfused suit. From 30 Torr LBNP to pre‐syncope, subjects inhaled either (blinded) a hypercapnic gas mixture (5% carbon dioxide [CO2], 21% oxygen, balance nitrogen) or normal room air (SHAM). Middle cerebral artery blood velocity (MCAv, transcranial Doppler) and end tidal CO2 (PETCO2, capnograph) were measured. 5% CO2 increased PETCO2 at 30 Torr LBNP (15±3 mmHg) and pre‐syncope (19±3 mmHg), compared to SHAM (P<0.01). Subsequently, MCAv was higher in the 5% CO2 trial at 30 Torr (24±10 cm.s−1, ~33%) and presyncope (21±14 cm.s−1, ~30%) relative to the SHAM (P<0.01). However, hypercapnia did not alter LBNP tolerance time (5% CO2: 7.5±3.5 min; SHAM: 9.1±3.6 min; P=0.33). These data indicate that inhaling a hypercapnic gas mix and circumventing hyperventilatory‐induced hypocapnia does not improve an individualˈs tolerance to a hyperthermic hemorrhagic challenge, despite improving cerebral perfusion.

Supported by NIH Grant HL061388
Simulation of apneas at the end of argon inhalation, using seven parallel regions of the lung
Authors: Caucha Luis Jhony , Cruz Julio Cesar , Melendrez Jose Maria
Published: June 2021

Jeng et al. (Ann. Biomed. Eng, 28:453, 2000) could not simulate apneas after argon (Ar) inhalation because the convection‐diffusion equation, solved by diluting the inhaled Ar with FRC, obtained too much Ar in the alveoli. Using the model of 7 parallel regions to simulate a normal breath of O2, and CO2, new concepts were introduced in mixing the inhaled gas with the expiratory reserve volume (ERV), by the mechanism of convection‐diffusion during inspiration. During the time of expiration, gases diffused from RV to ERV for CO2 and from ERV to RV for O2 (Caucha et al. Am. J. Respir. Crit. Care Med. 183: A5180, 2011). These concepts are now applied to forced inspiratory (2s) and expiratory (3s) maneuvers by inhaling 1.9 L of 21% O2, 79% Ar from FRC and exhaling to RV. The breath was held at the end of inspiration for 5s, 10s and 20s. The simulated expired curves are shown in the Figure. The last 8, 9 and 10 generations of Weibel's anatomical model were used at the base, middle and apical regions, respectively, at 5s a 10s of apnea. For 20s it was necessary to use the last 10th and 11th generations for the base and apical regions. In conclusion, the model described is capable of simulating the experiments of Cruz (Respir. Physiol. 86:1, 1991).
Sudomotor activity and heat balance with serial cooling and heating of thermosensors in the esophagus and stomach
Authors: Jay Ollie , Bain Anthony R , Lesperance Nicole C
Published: June 2021

This study assessed heat balance parameters and characterized transient sweating responses on the forehead following graded changes in local temperature of the esophagus and stomach from ingesting water of different temperatures. Seven male participants cycled for 75‐min in a temperate environment while ingesting four 3.2 mL·kg−1 boluses of either 1.5°C, 10°C, 37°C or 50°C water, 5‐min before, and 15, 30 and 45‐min after the start of exercise at 50% VO2max. Whole‐body sweat loss (WBSL), rectal temperature (Tre), mean skin temperature (Tsk) and local sweat rate on the forehead (SRhead) were measured throughout. Evaporative heat loss from sweating (Esk), respiratory heat loss, dry heat losses and heat exchange with ingested water were subsequently calculated to estimate net body heat storage (S). Despite no differences in Tre or Tsk, the onset time for sudmotor activity on the forehead was significantly greater with declining fluid temperature (P<0.05), and both mean SRhead throughout exercise and WBSL was significantly lower with 1.5°C compared to 50°C water ingestion (P=0.002). Values for S were significantly greater following 1.5°C water ingestion relative to 50°C (P=0.033), indicating a disproportionate reduction of thermoregulatory sweating, and therefore Esk, relative to the internal heat transfer with 1.5°C water. It is proposed that these data present evidence of independent thermoafferent processes arising from changes in local temperature of the esophagus and/or stomach.

Funding for the study was provided by a NSERC discovery grant.
Activation of hemostatic pathways by exercise induced hyperthermia
Authors: Hopman Maria , Eijsvogels Thijs , Veltmeijer Matthijs , van Geffen Mark , Thijssen Dick , van Heerde Waander
Published: June 2021

BACKGROUND

Retrospective studies indicated that patients with heat stroke have a sustained activation of coagulation, fibrinolysis and platelets. However, little is known about changes in haemostatic parameters in hyperthermic athletes without exertional heat stroke. Therefore, the aim of this study was to determine changes in thrombin and plasmin generation after exercise induced hyperthermia.

METHODS

Core body temperature was measured before and at the finish line of a 15 km run (n=16, 51±8 years). Venous blood samples were collected pre‐ and post‐race. The Novel Haemostasis Assay was used to measure thrombin and plasmin generation simultaneously. This allows insight in the interaction between coagulation and fibrinolysis.

RESULTS

Core body temperature increased from 37.7±0.5°C to 39.4±0.9°C. The time to thrombin peak decreased from 9.2±2.3 min to 8.1±1.0 min (p<0.05), while the thrombin peakheight increased from 178±63 nM to 190±67 nM (p<0.001). The plasmin peak‐time increased from 28.4±9.5 min to 32.3±10.9 min (p<0.05), with a decrease in plasmin peakheight from 4.3±4.0 nM to 2.7±2.1 nM (p=0.07).

CONCLUSION

An increase of 1.7°C in core body temperature stimulated coagulation by enhancing thrombin generation and reducing plasmin generation. These innovative results suggest that exercise induced hyperthermia leads to activation of the coagulation pathway.
Hypothalamic gene expression profiles during heat stroke recovery in a conscious mouse model
Authors: Biedenkapp Joseph , Helwig Bryan G , Leon Lisa R
Published: June 2021

Heat stroke (HS) induces a dynamic thermoregulatory response that consists of a progression from hyperthermia (Tc,Max=42.7°C), to hypothermia (29.3±0.4°C) followed by a delayed fever (37.8±0.3°C) that develops within ~24h. This response is accompanied by behavioral and metabolic changes that suggest these are tightly regulated responses. The physiological mechanisms underlying these responses are unknown. We hypothesized that distinct hypothalamic gene expression profiles of heat shock protein (HSP)‐72, cytokines (IL‐1β, IL‐6, TNFα, MIP‐1β), cyclooxygenase (COX‐2) and microglia activation markers (MHCII, CD200) would associate with each stage of the response in a conscious mouse model. HSP‐72 showed a 7.7‐ and 15.8‐fold increase from Tc,Max to hypothermia, with return to baseline by 24h (P<0.001). MIP‐1β showed a 9.5‐fold increase at hypothermia only (P<0.001). Peak expression of IL‐6 (4.4‐fold) and COX‐2 (12.8‐fold) occurred at hypothermia, but these cytokines remained elevated above controls through 24h. IL‐1β (3.6‐fold) and TNFα (3.6‐fold) showed peak expression during fever (P<0.001). No significant changes in microglia activation markers were observed. These data suggest that distinct changes in hypothalamic cytokines and COX products may regulate the different thermoregulatory responses to HS. Author views not official US Army or DoD policy.
Activation of Human Sweat Glands using Intradermal Electrical Stimulation
Authors: Mack Gary W. , Gifford Jayson , Heal Cory , Goldthorpe Scott
Published: June 2021

A model for examining sweat gland function using intradermal electrical stimulation to activate sudomotor nerves (small sympathetic cholinergic C‐fibers) was examined in the dorsal aspect of the forearm in 10 healthy adults. We monitored the local sweat rate (SR) response of a small area of skin using small capsule (0.7 cm2) containing a humidity sensor and thermocouple flushed with dry gas at 100 ml • min−1. Two small stainless steel needles were place into the skin immediately outside of the capsule and stimulated at frequencies of 0.2, 1, 2, 4, 8, 16, 32, and 64 Hz at two current intensities (1.5 and 2.5 mA) for 30 s. The stimulus‐response characteristics of local SR were evaluated using a non‐linear four‐parameter logistic curve fitting equation. The increase in local SR reached a plateau at a stimulus frequency of 32 Hz. The peak local SR response was significantly greater at 2.5 than 1.5 mA (0.21 ± 0.08 and 0.40 ± 0.12 mg • min−1 • cm−2, p<0.05). The stimulus frequency that produced 50% of the increase in local SR was similar for both current intensities and averaged 11.2 ± 1.3 Hz. Subjective ratings of pain (0 to 100 scale) during intradermal electrical stimulus increased in a linear manner starting at 6 ± 2 (“weak”) and rising to 34 ± 7 (“strong”) at 64 Hz and 2.5 mA. Delivery of a 1% atropine sulfate solution to the skin by iontophoresis (2 mA x 10 min) completely blocked the sweating response to intradermal electrical stimulation providing strong evidence that the local SR response was mediated by acetylcholine released in response to depolarization of the small sympathetic cholinergic C‐fibers. Intradermal electrical stimulation in combination with measurement of local SR can be used to quantify small C‐fiber function in human skin with minimal pain.
Influence of 10 days of unilateral lower limb suspension and combined exercise training on human vastus lateralis and soleus muscles
Authors: Cotter Joshua Allan , Haddad Fadia , Yu Alvin M , Hoang Theresa N , Kreitenberg Arthur , Baker Michael J , Tesch Per A , Baldwin Kenneth M , Caiozzo Vincent J , Adams Gregory R
Published: June 2021

The unilateral lower limb suspension (ULLS) model is used to study disuse conditions, such as spaceflight, which typically induce a slow‐to‐fast phenotype transition. The vastus lateralis (VL) and soleus (SOL) are both antigravity muscles that are very sensitive to changes in loading states. We investigated whether fiber type changes associated with 10 days of ULLS could be ameliorated with combined aerobic and resistance exercise (EX) on a gravity independent device. Total RNA concentration was increased only in EX by 14% and 19% in both the VL and SOL respectively. In the VL, exercise increased type IIa mRNA expression by 25% while exhibiting a 58% decline in type IIx mRNA. Type IIx mRNA expression in the SOL was increased from 5% to 10% for ULLS and from 3% to 9% for EX. No significant changes were seen in protein expression. Increases in VL IGF1 and COL3A mRNA were seen for EX only and in both groups for SOL. These data show that EX can produce favorable MHC transitions during unloading in the mixed muscle VL but not the slow type I dominant soleus. Supported by NIH Postdoctoral Training grant 2T32AR047752 to VJC. Funded by NSBRI grant NCC9‐58‐70 to GRA.
Leucine preserves muscle and strength and enhances recovery following bed rest
Authors: English Kirk L. , Mettler Joni , Mamerow Madonna M. , Mathers Charles H. , Pattarini James M. , Paddon-Jones Douglas
Published: June 2021

Reduced mechanical loading is a hallmark of hospitalization and space flight, and contributes to a rapid decrease in muscle mass and functional performance. Mechanistically, these changes are mediated by a 30% reduction in the anabolic response to regular meals. If the ability to exercise is compromised, nutritional interventions represent one of the few viable alternatives. To this end, we added leucine (0.06 g · kg lean mass · meal−1; LEU) to the meals (containing 0.9 g protein · kg · day−1) served to adults (45–60 y) during 14 d bed rest. Our goal was to reduce the loss of muscle mass and function and facilitate recovery during 7 d of rehabilitation. During bed rest, control subjects (CON) lost 3‐ times more total lean body mass than LEU (CON: −3021 ± 400 g vs. LEU: −966 ± 264 g; p=0.001). Following rehabilitation, lean mass deficits persisted in CON while LEU returned to near pre‐bed rest values (CON: −1746 ± 303 g; LEU: −140 ± 367 g). The loss of isometric knee extensor peak torque was also three times greater in CON (−20.3 ± 6.8%) vs. LEU (−7.0 ± 2.5%; p=0.07). Changes in knee extensor total work during a 20 repetition isokinetic endurance test at 180° · s−1 followed a similar pattern (CON: −18.0 ± 7.4% vs. LEU: −5.3 ± 2.8%; p=0.08). These preliminary findings indicate that supplementing regular meals with a small amount of leucine attenuates the loss of muscle mass and function typically associated with bed rest and facilitates recovery.

Grant Funding Source : NSBRI
Skeletal muscle fatigue and neuromuscular activation during bed rest
Authors: Mettler Joni A , English Kirk L , Doucet Barbara M , Mamerow Madonna M , Paddon-Jones Douglas
Published: June 2021

Physical inactivity results in muscle atrophy, conversion to faster muscle fibers, decrements in oxidative capacity and increased muscle fatigability. These changes may alter motor unit activation and recruitment strategies utilized by the central nervous system in controlling sustained muscular force. The aim of this study was to determine the effect of 14 days of bed rest on muscular endurance and neuromuscular activation during an isometric sub‐maximal fatigue task in the soleus muscle in healthy middle‐aged adults. Surface electromyography (EMG) and torque were recorded from the soleus muscle during isometric maximal voluntary contractions (MVC) and during an isometric, 50% MVC fatigue task pre‐post 14 days of bed rest. MVC decreased by 8.7% and maximal root mean square (RMS) EMG amplitude declined by 6.4% after bed rest. Muscular endurance time decreased by 49.7% after bed rest. Mean RMS EMG amplitude during the fatigue task increased by 7.9% following bed rest and median frequency of the EMG signal during the fatigue task decreased by 17.9%. Neural adaptations during inactivity may contribute to increased muscle fatigability. Some physical activity during periods of prolonged bed rest may be necessary to preserve motor pathways, and thereby may attenuate muscle fatigue. Funded by NSBRI grant NNJ08ZSA002N (Paddon‐Jones) and by grant 1UL1RR029876‐01 from the National Center for Research Resources, NIH.
Influence of hypergravity environment on interaction between baroreflex and vestibulo‐sympathetic reflex during head‐up tilt
Authors: Morita Hironobu , Abe Chikara , Kawada Toru , Sugimachi Masaru
Published: June 2021

Because arterial pressure (AP) decreased during rear‐up in rats with baroreceptor denervation or vestibular lesion (VL), baroreflex and vestibulo‐sympathetic reflex cooperate to maintain AP during rear‐up. Functional interaction between these two reflexes was examined. Isolated carotid sinus pressure (CSP) was increased stepwise from 60 to 180 mmHg, while rats were placed prone flat position or at 60° head‐up tilt (HUT). HUT shifted the CSP‐sympathetic nerve activity (SNA) relationship (baroreflex central arc) to a higher SNA, shifted the SNA‐AP relationship (baroreflex peripheral arc) to a lower AP, and consequently moved the operating point to high SNA while maintaining AP. In VL rats, the HUT‐induced central arc shift was completely abolished; the operating point moved to a lower AP. Thus, the vestibular system elicits sympathoexcitation upon HUT and shifts the baroreflex central arc to a higher SNA. However, the vestibular system is known to be highly plastic, i.e., if rats were maintained under different gravitational environment, vestibular sensitivity might be reduced. Thus, influence of hypergravity environment on functional interaction between baroreflex and vestibulo‐sympathetic reflex was further examined.