bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Prolonged Adenosine Infusion on Resting Forearm Vasodilatory Responses and Exercise Hyperemia
Authors: Ranadive Sushant , Shepherd John‐Roger Mohan , Dinenno Frank , Curry Timothy , Joyner Michael
Published: November 2023

High doses of adenosine (ADO) infused over a prolonged period (3 hours) produces marked vasodilation. However, this vasodilation wanes over time in an isolated canine skeletal muscle preparation. To our knowledge the waning of vasodilation with high doses of ADO has not been shown in humans. In addition, ADO plays a controversial role in exercise hyperemia. Therefore, our primary hypothesis was that in humans during a continuous three‐hour intra‐arterial infusion of a high dose of ADO (23 μgrams ·100 mL forearm vol−1·min−1) skeletal muscle blood flow would wane over time. Our secondary hypothesis was exercise hyperemic responses during ADO administration would be similar compared to control. Nine healthy young participants (29 ± 2 years) performed rhythmic handgrip (HG) exercise bouts (20% of maximum) during saline and 180 minutes of continuous ADO infusion. Forearm vascular conductance (FVC; ml min−1 (100 mmHg)−1) was calculated from measured forearm blood flow and mean blood pressure. FVC during minute 5 versus minute 175 during ADO infusion were 24.22 ± 3.2 versus 23.24 ± 1.95 ml min−1 (100 mmHg)−1 respectively (p>0.05). FVC during exercise increased significantly from baseline (4.78 ± 0.63 to 31.13 ± 4.17 ml min−1 *100 mmHg−1) during saline infusion and from (24.22 ± 3.23 to 56.33 ± 4.82 ml min−1 *100 mmHg−1) at five minutes and 21.99 ± 1.25 to 59.16 ± 5.21 seen at 150 minutes during ADO infusion. There was no significant difference in the delta FVC (exercise FVC – baseline FVC) between saline exercise bout versus ADO exercise bout (26.34 ± 3.75 versus 32.11 ± 5.55 ml min−1 *100 mmHg−1). Our findings show that contrary to the animal model, ADO vasodilatation does not wane in humans. Also, the vasodilator responses to exercise are similar during saline and prolonged ADO infusion compared to control condition. These findings suggest a minimal role of ADO in exercise hyperemia.

Support or Funding Information

Supported by National Institutes of Health Research Grants HL‐119337 (to M.J. Joyner and F.A. Dinenno) and by CTSA UL1 TR000135. The Caywood Professorship (M.J. Joyner) via the Mayo Foundation also supported this research.
Effect of partial denervation mitochondrial ROS generation in skeletal muscle
Authors: Pollock Natalie , Staunton Caroline Amy , Vasilaki Aphrodite , McArdle Anne , Jackson Malcolm
Published: November 2023

Dramatic changes in mitochondrial generation of hydrogen peroxide (H2O2) are observed in the skeletal muscle of rodents following experimental nerve transection. The consequences of this denervation are well documented. We have hypothesised that denervation of even a limited number of fibers, such as that observed in muscles during ageing, could influence mitochondrial H2O2 generation by both denervated and innervated fibers throughout the muscle.

Thy1‐YFP mice (expressing YFP in neuronal cells only) underwent surgical transection of one of the 3 nerve branches entering the anterior tibialis (AT) muscle. Generation of H2O2 by mitochondria in permeablised fibers with intact mitochondria from 4 different regions of the AT muscle was assessed using Amplex Red. Confocal microscopy of the excised muscle allowed us to visually track and map the morphological changes in both muscle and nerve which accompanied the post‐surgical changes in H2O2 generation.

Following partial denervation, oxidation of Amplex red was increased by 40–70 fold in the bundles of fibers from all regions of the AT muscle. This increase was comparable to those observed in fully denervated muscles in our previous experiments.

Imaging techniques have demonstrated that NMJ morphology following partial denervation is variable and undergoes rapid changes. At 24 hrs following surgery, the site of surgical denervation is clearly identifiable by the loss of nerve endplates, with no evidence of post‐synaptic changes. By 7 days post‐surgery, the morphological status of the NMJ's is highly variable across the entirety of the muscle.

Cross‐sectional area analysis has revealed that even partial denervation is sufficient to cause muscle fibre atrophy, with no clear correlation between the site of initial injury and fibre cross‐sectional area.

Taken together, these data show that mitochondria in innervated muscle fibers are affected by denervation of adjacent fibers and contribute to the increased H2O2 generation. Thus data support our hypothesis that loss of innervation in some muscle fibres is sufficient to disrupt the regulation of mitochondrial H2O2 generation with consequences for the entire muscle, an effect which may be mirrored in sarcopenia.

Support or Funding Information

Supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC).
Aldosterone Acutely Enhances Coronary Artery Contractile Responsiveness without Inducing Endothelial Dysfunction
Authors: Cleeton Hannah M. , Meuth Alexander I , Bender Shawn B
Published: November 2023

Inappropriate activation of the renin‐angiotensin‐aldosterone system (RAAS) contributes to increased contractility of the vasculature in many disease states. Activation of vascular mineralocorticoid receptors (MR) has recently been implicated as an important regulator of vascular function. In vascular smooth muscle, genomic mechanisms of MR activation have been implicated in vascular contractility, however, little is known regarding non‐genomic MR signaling as a modulator of vascular contractility. Based on available evidence, we hypothesized that acute aldosterone incubation of murine coronary arteries ex vivo would enhance coronary contractility via non‐genomic MR activation in smooth muscle cells (SMC). Wire myography experiments were conducted on isolated coronary arteries from C57BL/6J mice. Contractile responses to KCl (10mM–45mM) were determined by repeated measures design before and following one hour aldosterone treatment (10nM or 100nM). KCl induced dose‐dependent coronary vasoconstriction that was enhanced by aldosterone incubation in a dose dependent fashion. Vasoconstriction to 20mM KCl was doubled following incubation with 10nM aldosterone and increased >3‐fold following incubation with 100nM aldosterone. Pretreatment with the selective MR antagonist eplerenone (10μM) did not significantly reduce enhanced KCl vasoconstriction in response to 100nM aldosterone. Furthermore, endothelium‐dependent coronary vasodilation to acetylcholine was unchanged following one hour aldosterone incubation regardless of dose. In conclusion, acute aldosterone incubation enhances coronary vasoconstrictor responses to KCl that, at least in response to high dose aldosterone (100nM), is not dependent on MR activation. Importantly, this effect appears to be smooth muscle‐specific and not secondary to aldosterone‐induced endothelial dysfunction as acute aldosterone incubation did not impair endothelium‐dependent coronary vasodilation. These results highlight important vascular cell‐specific effects of aldosterone that may underlie enhanced coronary contractility in disease states associated with inappropriate RAAS activation.

Support or Funding Information

Funded by VA CDA‐2 IK2 BX002030 and the American Physiological Society.
Effect of chronic stress and aerobic exercise on glycemic control in rats
Authors: Marcondes Fernanda Klein , Sanches Andrea , Costa Rafaela , Casarini Dulce Elena , Cunha Tatiana Sousa
Published: November 2023

We have previously shown that chronic mild and unpredictable stress (CMS) induced endothelium dysfunction and insulin resistance in rats. Physical exercise is recognized to provide better glycemic control. Thus, the aim of this study was to evaluate the effect of moderate exercise on the response to oral glucose tolerance test (OGTT) of rats submitted to CMS. Male Sprague‐Dawley rats were divided into four groups: 1) Control, 2) Exercise, 3) CMS and 4) Exercise plus CMS (n=7–9). CMS protocol consisted on application of different stressors, 7 days per week (wk) during 3 wk (5, 6 and 7th wk of protocol). The physical exercise was performed after a period of adaption on the treadmill and consisted of an intensity of 50–70% of the maximal exercise test, 5 days per wk, and 1 hour per day (during 8 wk). Data were analyzed by ANOVA bifatorial (p<0.05). All procedures were approved by Institutional Ethics Committee. Exercise, CMS and Exercise plus CMS presented higher corticosterone plasma level in comparison with Control group (p < 0.05). Exercise (781.1 ± 17,2 mmol × min/L) and Control (842.9 ± 15.5 mmol × min/L) group presented lower area under the curve of glucose during OGTT in comparison with Exercise plus CMS (872.0 ± 12,9 mmol × min/L) and CMS (879.5 ± 11.5 mmol × min/L) group, respectively (p<0.05). These data show that aerobic training has not been able to abolish the impairment of glucose tolerance induced by CMS.

Support or Funding Information

FAPESP, CAPES, CNPq, FAEPEX‐UNICAMP.
Aged islets are refractory to Nkx6.1 mediated β‐cell proliferation
Authors: Wright Brent , Garland Kevin , Tidwell Chad , Kang Sean , TESSEM JEFFERY Sivert
Published: November 2023

Type 1 and Type 2 diabetes ultimately end in β‐cell loss. Islet transplantation could have widespread use in order to restore a diabetic patients functional β‐cell mass. A major impediment is the availability of transplant ready islets, therefore manipulation of molecular pathways that permit β‐cell expansion are critical to wider use of this treatment. As most islets available for transplant will be from aged donors, the ability to manipulate functional β‐cell mass must be applicable to islets from aged sources. We have demonstrated that expression of Nkx6.1 in islets isolated from young rats can enhance β‐cell proliferation, function and survival. Interestingly, islets isolated from aged rats are refractory to Nkx6.1 mediated enhancement. Here we present data characterizing the differences between aged and young islets in terms of morphology, islet mass, insulin secretion, transcriptome and function of Nkx6.1 overexpression. Our data demonstrate that aged β‐cells are significantly different from young β‐cells, and suggest potential applications to expanding β‐cell mass ex vivo for islet transplantation.
Mouse SERCA2b is Critical in Compensatory Response to High Fat Diet by Maintaining Pancreatic β Cell Proliferation
Authors: Tong Xin , Kono Tatsuyoshi , Anderson‐Baucum Emily , Shull Gary , Evans‐Molina Carmella
Published: November 2023

Type 2 diabetes (T2D) affects over 380 million individuals worldwide and is characterized by peripheral insulin resistance and inadequate insulin secretion from the pancreatic β cell. During the development of T2D and in the face of increasing peripheral insulin resistance, the β cell undergoes a compensatory response to both increase insulin production and secretion. Here, we tested the hypothesis that intact activity of the sarco‐endoplasmic reticulum calcium ATPase (SERCA) pump, which transports 2 calcium molecules into the ER lumen during each catalytic cycle and is responsible for maintaining a robust ER calcium pool, is a critical determinant of the β cell's adaptive response to diet‐induced obesity. To this end, mice with a total body heterozygous deletion of SERCA2 (S2HET) and wild‐type littermate controls (WT) were placed on high fat diet (HFD) containing 45% of kilocalories from fat for 16 weeks. Compared to WT controls, S2HET mice showed equivalent body weight gain and fat content. Interestingly, S2HET mice demonstrated fasting hyperglycemia, hypoinsulinemia, and significantly impaired glucose tolerance. Insulin sensitivity and insulin signaling in liver, skeletal muscle, and adipose tissue were not different between S2HETs and WT controls, suggesting a primary defect at the level of the β cell. Histologic analysis of pancreatic sections obtained after 16 weeks of HFD revealed significantly reduced β cell mass as well as reduced β cell proliferation in S2HET mice. To define mechanisms underlying these observations, a SERCA2 deficient rat insulinoma cell line (S2KO INS‐1) was created using CRISPR/Cas9 technology. At baseline, thymidine incorporation was reduced in S2KO INS‐1 cells. Cell cycle analysis revealed a dramatic shift towards decreased proliferation and a G1/S block, while SERCA2b restoration by adenoviral transduction was able to fully rescue these defects. In the meantime, we observed the expression of adaptive and terminal ER stress markers including Bip, Dnajc3, hsp90b1, Pdia4 and spliced Xbp‐1 were elevated in both S2KO INS‐1 cells and isolated S2HET islets. Consistent with this, further analysis by polyribosomal profiling analysis revealed a translational initiation block in both S2KO cells and islets isolated from S2HET mice. In aggregate, these data suggest that impaired SERCA2 activity and altered ER calcium homeostasis lead to increased β cell ER stress and impaired proliferation, thereby limiting the β cell compensatory response to diet‐induced obesity.

Support or Funding Information

NIH R01 DK 093954 and VA Merit Award 1I01BX001733
Paracrine GABA and insulin restrain pancreatic α‐cell proliferation in islets of Langerhans
Authors: Feng Allen L. , Xiang Yun‐Yan , Gui Le , Feng Qingping , Lu Wei‐Yang
Published: November 2023

In type 1 diabetes loss of pancreatic β‐cells is followed by a rapid increase of α‐cells. However, the underlying mechanisms are not fully understood. β‐cells express glutamic acid decarboxylase (GAD) and produce a large amount of γ‐aminobutyric acid (GABA), which, together with insulin, inhibits α‐cell secretion of glucagon. We investigated the role of GABA and insulin in the regulation of α‐cell proliferation in vitro and in vivo. Results showed that treatment with insulin and the type‐A GABA receptor (GABAAR) agonist muscimol significantly decreased intracellular [Ca2+], phosphorylated mammalian target of rapamycin (p‐mTOR) and proliferation rate in cultured αTC1‐6 cells. Administration of streptozotocin (STZ) to mice caused a substantial decrease of GAD and GABA as well as insulin in β‐cells at 12 hours, which was followed by an upsurge of p‐mTOR in α‐cells at day 1 and a significant increase of α‐cell mass at day 3. Remarkably, treatment with GABA largely restored the level of insulin and GAD in pancreatic β‐cells, and significantly decreased α‐cell mass and hyperglucagonemia in the STZ‐injected mice. These results show that decreases in GABA and insulin in β‐cells increase α‐cell proliferation in type 1 diabetes. We conclude that the paracrine GABA and insulin restrain α‐cell proliferation in islets of Langerhans.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)
Beta Cells Deletion of the Angiotensin‐II Type 1a Receptor Differentially Influences Insulin Levels in Male and Female Mice
Authors: Chodavarapu Harshita , Lazartigues Eric
Published: November 2023

Renin‐angiotensin system (RAS) blockade with either Angiotensin Converting Enzyme (ACE) inhibitors or Angiotensin‐II type 1 receptor blockers improves type 2 diabetic (T2D) symptoms. We have shown that up‐regulation of islet AT1a receptors (AT1aR) may be associated with T2D in male mice. However, the role of islet AT1aR on the glucose regulating hormone “insulin”, is yet to be elucidated in a gender specific manner. Therefore, we hypothesized that β‐cell specific AT1aR loss of function positively regulates insulin in male and female C57BL/6 mice. To address this hypothesis, we used male and female 2 months old wildtype (WT) and transgenic β‐AT1a KO (β‐cell specific AT1aR knock out) mice. This new strain was generated by breeding AT1aR flox/flox mice with animals expressing Cre recombinase under the control of a mouse insulin 2 promoter. Basal non‐fasted plasma insulin levels were assessed using an ELISA kit. Plasma insulin levels were significantly increased in β‐AT1a KO male mice compared to gender‐matched WT mice (β‐AT1a KO: 1.24±0.17 vs. WT: 0.9±0.07 ng/mL, P<0.05). However, plasma insulin levels did not differ among β‐AT1a KO and WT females. In addition, β‐AT1a KO males displayed significantly higher plasma insulin levels compared to β‐AT1aKO females (β‐AT1a KO: 1.24±0.17 vs. WT: 0.73±0.09 ng/mL, P<0.05). Further, basal islet ACE2 activity levels were significantly (p<0.05) higher in females compared to males. In addition, we previously showed that, after 16 weeks of high fat diet (HFD) feeding, WT males had significantly increased body weight compared to animals on regular diet (RD) (HFD: 41±0.9 vs. RD: 30.3±0.6 g). Additional features included elevated plasma insulin levels (HFD: 7±0.1 vs. RD: 2.6±0.8 ng/mL) that were associated with hyperglycemia (HFD: 137.2±0.48 vs. RD: 90±3.2 mg/dL) and glucose intolerance (HFD: 44898±2297 vs. RD: 37928±1273 arbitrary units). These changes in glucose homeostasis were associated with an upregulation in islet AT1aR gene (−1.5 fold vs. RD) expression levels but not ACE2 gene expression and activity. Altogether, our results demonstrate that despite having higher basal islet ACE2 activity, islet AT1aR loss of function positively impacts plasma insulin levels in male but not female mice. Therefore, islet AT1aR plays an important role in the regulation of basal insulin levels. Although islet AT1aR levels are positively correlated to impaired glucose homeostasis as well as hyperinsulinemia in a diet induced obese mouse model, further studies are required to better our understanding about the role of islet AT1aR loss of function on blood glucose levels and glucose regulating hormone “insulin” in a diabetic milieu.

Support or Funding Information

American Heart Association: 12EIA8030004 and 14PRE18830012COBRE: P30GM106392
Calcium‐dependent sliding of actin thin filaments isolated from hearts with obstructive hypertrophic cardiomyopathy and donor controls does not differ
Authors: Snyder Julia P. , Previs Samantha Beck , Day Sharlene M. , Previs Michael J.
Published: November 2023

Cardiac contraction and relaxation results from the calcium‐dependent sliding of actin‐thin filaments along myosin‐thick filaments. Nearly 1 in 500 individuals are affected by familial hypertrophic cardiomyopathy (HCM) which impairs relaxation. Genetic mutations to myosin‐binding protein C (MyBP‐C) are commonly found in patients with HCM, but this disease is also associated with non‐sarcomeric etiologies. To investigate whether increases in thin filament calcium sensitivity impair relaxation in HCM from differing origins, we examined the calcium‐dependent motion of thin filaments isolated from human hearts with MyBP‐C mutations, non‐sarcomeric etiologies and donor controls. Thin filaments containing their calcium regulator proteins, troponin‐tropomyosin, were isolated from flash frozen myocardium by mechanochemical dispersion and fluorescently labeled. Myosin molecules were isolated from a single control heart and thin filament sliding on myosin was visualized using epifluorescence microscopy. At low calcium levels (<100 nM free Ca2+), 95% of the thin filaments were immobilized on the surface in all 3 groups. Addition of calcium (1 nM – 0.1 mM) resulted in sigmoidal increases in the fraction of filaments moving and their sliding velocities. Neither calcium sensitivity (EC50 0.25 μM) nor maximal sliding velocities (1.2 μm/sec) differed between the groups (1 mM ATP and 30°C). These results suggest that changes in thin filament calcium sensitivity do not contribute to impaired relaxation.

Support or Funding Information

Funded by APS UGSRF.
Rewarming Shock Disrupts Intracellular Ca2+ Homeostasis and Contractility in Cardiomyocytes
Authors: Hoang Thuy , Schaible Niccole , Han Young Soo , Arteaga Grace , Tveita Torkjel , Sieck Gary
Published: November 2023

Hypothermia/Rewarming (H/R) induces myocardial dysfunction although the underlying intracellular mechanisms remain elusive. We hypothesized that in cardiomyocytes, H/R disrupts intracellular Ca2+ ([Ca2+]cyt) homeostasis via an effect on both release and clearance of cytosolic Ca2+ leading to a prolongation of evoked [Ca2+]cyt transients associated with activation with downstream impact on contractility. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were loaded with the Ca2+ indicator, Fura‐2AM (0.5 μM), and exposed to H/R with or without electrical pacing at 0.5 Hz. In both groups, evoked [Ca2+]cyt and contractile (sarcomere length (SL) shortening) responses were simultaneously measured at 35°C before and after hypothermia using an IonOptix system. H/R experiments were conducted as follows: experimental temperature was cooled from 35°C to 15°C in 30 min, maintained at 15°C for 2 h, and then rewarmed back to 35°C in 30 min. Time‐matched controls were implemented for 3 h at 35°C. With electrical pacing throughout hypothermia, basal [Ca2+]cyt increased and the kinetics of the evoked [Ca2+]cyt responses were prolonged. In addition, the extent and velocity of SL shortening was reduced in cardiomyocytes that were paced during hypothermia. In contrast, in cardiomyocytes that were not electrically paced during hypothermia, basal [Ca2+]cyt was maintained and the kinetics of evoked [Ca2+]cyt transients were unaffected. In addition, in the absence of pacing during hypothermia, contractility of cardiomyocytes was unaffected. These results suggest that H/R affects the release and clearance of evoked [Ca2+]cyt transients (e.g., RyR mediated Ca2+ release and SERCA mediated clearance) such that basal [Ca2+]cyt increases. This effect of H/R on [Ca2+]cyt homeostasis is associated with reduced cardiac contractility.
Jagged‐1 Enhances CaSR‐Induced Increase in [Ca2+]cyt by Promoting Recruitment of STIM2 to ER‐PM Junctions
Authors: Song Shanshan , McDermott Kimberly M. , Makino Ayako , Yuan Jason X.‐J.
Published: November 2023

Calcium‐sensing receptor (CaSR) is a G protein‐coupled receptor (GPCR) that can be activated by extracellular Ca2+. Activation of CaSR results in an increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) due to inositol l,4,5‐trisphosphate (IP3)‐dependent Ca2+ release and diacyglycerol (DAG)‐mediated Ca2+ influx. STIM2 is a Ca2+ sensor protein located in the endoplasmic reticulum (ER) that forms clusters at ER‐plasma membrane (PM) junctions when [Ca2+] is reduced in the ER and has been proposed to regulate Ca2+ entry at low concentration of agonist stimulation. Jagged‐1 (Jag‐1) is a ligand of Notch receptors and has been reported to enhance store‐operated Ca2+ entry (SOCE) in human pulmonary arterial smooth muscle cells (PASMC). Here, we examined whether activation of Notch promotes recruitment of STIM2 to ER‐PM junctions and thus enhances extracellular Ca2+‐mediated increase in [Ca2+]cyt through CaSR. Addition of extracellular Ca2+ induced increases in [Ca2+]cyt in HEK cells transiently transfected with CaSR. Treatment of CaSR‐transfected HEK cells with Jag‐1 (for 30 min) significantly increased the amplitude of CaSR‐mediated increase in [Ca2+]cyt. Furthermore, the extracellular Ca2+‐induced increase in [Ca2+]cyt was also significantly enhanced in HEK cells transfected with STIM2. Passive depletion of intracellularly stored Ca2+ in ER by cyclopiazonic acid (CPA), an ER Ca2+ pump inhibitor, induced the formation of STIM2 clusters in ER‐PM junctions in STIM2‐transfected HEK cells. Activation of CaSR by extracellular Ca2+ in CaSR‐overexpressed HEK also resulted in STIM2 clustering. Together, these results suggest that activation of Notch functionally enhances CaSR‐mediated increases in [Ca2+]cyt by promoting recruitment of STIM2 to the ER‐PM junctions and enhancing store‐operated Ca2+ entry.

Support or Funding Information

This study was supported by NIH grants (HL115014, HL066012 and HL098053).
Effects of Gender, Aging, and Training Mode on Muscular Heat Shock Protein and Contractile Adaptations in Mice
Authors: Huey Kimberly , Drake Ty , Dillon Gabrielle , Wilhelmi Alec
Published: November 2023

Aging is associated with reductions in muscle strength and may also blunt heat shock protein (Hsp) responses to various stressors. Previous studies have shown that exercise is associated with increased Hsp levels in active muscles. Further evidence suggests that Hsp responses to exercise may be blunted in females compared to males and that estrogen has shown the potential to attenuate Hsp responses in males. We tested the hypothesis that aging reduces the Hsp response to voluntary running wheel (RW) or treadmill exercise (TM), with lower responses expected in female than male mice. It was also hypothesized that both RW and TM exercise can attenuate age‐associated reductions in muscle force. Gastrocnemius muscle levels of Hsp25 protein (measured with Western blot) and in vivo plantarflexor maximal force were measured in young (4 mo. old) and aged (24 mo. old) sedentary (SED) male and female mice and following 2 weeks of RW (24 hour unlimited access) or TM training (45 min/day, 5 day/wk). In males and females, RW activity was associated with similar significant increases in muscle Hsp25 compared to SED, independent of age, despite greater RW activity in young vs. aged groups (p<0.05). Further, daily RW activity over 14 days was greater in young and aged female mice compared to respective male groups (74707 vs. 29393 AUC for females and 41096 vs. 7272 AUC for males, respectively, p<0.05). TM was associated with increases in Hsp25 compared to SED in aged mice only (0.11±0.02 vs. 0.16±0.01 for males and 0.11±0.01 vs. 0.15±0.02 AU for females, p<0.05). Maximal muscle force was lower in aged than young SED mice in both genders (1.1±0.04 vs. 1.6±0.07 g/kg body mass for males and 1.0±0.03 vs. 1.5±0.01 g/kg body mass for females, respectively, p<0.05). In young groups of both genders, neither TM nor RW increased force over SED. In aged groups, TM was associated with 26% and 30% higher maximal forces than SED mice for males and females respectively (p<0.05), but was still lower than respective young groups. In aged RW groups, increases in muscle force were only observed in males (19% increase over SED), with no change in females. These findings suggest that independent of gender, overall increases in daily activity (RW) has a greater impact on muscle Hsp25 up‐regulation than a daily acute exercise bout (TM), especially in young mice, while Hsp25 up‐regulation was independent of training type in aged mice. Treadmill training was more effective in attenuating age‐associated reductions in muscle force than RW activity, especially in females.

Support or Funding Information

Supported by NIH R15AR060469
Heat Shock Proteins as Markers of Contraction‐Induced Muscle Damage
Authors: Pollock‐Tahiri Evan , Locke Marius
Published: November 2023

Angular contraction velocity has been proposed as a determinant in the severity of lengthening contraction‐induced muscle damage, yet empirical evidence is equivocal. We measured heat shock protein (HSP) content as a marker of muscle damage severity following lengthening contractions (LCs) at multiple angular velocities (slow; 63°/sec, medium; 127°/sec, fast; 190°/sec) to determine the relationship between angular velocity and muscle damage. To do this, male sprague‐dawley rats were anaesthetized and one tibialis anterior (TA) muscle was subjected to 60 LCs (3 sets of 20 LCs with 5 minutes rest between sets) at three velocities, while the contralateral TA served as a control. Twenty‐four hours after the last LC, intramuscular Hsp72 content, as assessed by western blotting, was significantly (p<0.001) increased in the stimulated TA regardless of the angular velocity. Similarly, Hsp25 content was also elevated but only considered significant (p<0.05) after LCs at the slow velocity (63°/sec). Interestingly, TA muscle function (ability to generate torque) was affected greatest following slow LCs. When compared to fast LCs (190°/sec), slow LCs caused significantly (p<0.05) greater loss of muscle torque with each set of 20 LCs, suggesting muscle damage and/or fatigue was greater after slow LCs. However, histological assessment of TA cross‐sections suggest the severity of muscle damage (measured as necrotic fibers and inflammatory infiltrates) and metabolic fatigue (measured as glycogen depletion) was similar between velocities. Taken together, the results suggest Hsp72 content may be a novel marker for determining the severity of LC‐induced muscle damage.

Support or Funding Information

No external funding
Myostatin Inhibition as an Adjuvant Therapy in Type 1 Diabetes Mellitus
Authors: Hawke Thomas James , Coleman Samantha , Rebalka Irena , D'Souza Donna , Deodhare Namita
Published: November 2023

While Type 1 Diabetes Mellitus (T1DM) is characterized by hypoinsulinemia and hyperglycemia, persons with T1DM also develop insulin resistance and recent studies have demonstrated that insulin resistance is a primary mediator of the micro and macrovascular complications that invariably develop in this chronic disease. Therefore, we hypothesized that a reduction in circulating myostatin (a TGF‐beta family member known to regulate muscle mass) in a T1DM mouse model would improve skeletal muscle health, resulting in an increased insulin sensitivity and a reduction in blood glucose. To that end, we crossed Akita diabetic mice with the MyostatinLn/Ln mouse line (which displays a ~60% reduction in myostatin levels) to generate a novel mouse line (MyostatinLn/Ln; Akita+/−). Our data support the hypothesis that reducing circulating myostatin levels prevented the loss of skeletal muscle mass observed in T1DM, as well as significantly increasing Glut1 and Glut4 transporter densities with the end result being an increased glucose uptake in response to an insulin tolerance test (ITT). These positive changes in T1DM skeletal muscle health resulted in significant reductions in resting blood glucose levels and other diabetic symptoms (hyperphagia, polydypsia), even in the absence of exogenous insulin. Taken together, these studies provide a foundation for considering pharmacological myostatin inhibitors as an adjuvant therapy in T1DM as a means to delay the development of diabetic complications.

Support or Funding Information

This work is funded by a grant from Natural Sciences and Engineering Research Council of Canada to TJH
RNAseq analysis of exercise, heat, and dehydration stress in a Kona Ironman World Championship
Authors: Lee Elaine Choung‐Hee , Aizik Shlomit , Haddad Faddia , Zaldivar Frank P , Casa Douglas J , Armstrong Lawrence E , Stearns Rebecca L , Munoz Colleen X , Bartley Jenna M , Maresh Carl M
Published: November 2023

Exercise and environmental stressors shift genome‐wide, transcript‐level expression in a tissue‐specific manner to upregulate stress‐specific protective genes and downregulate genes required for normal growth and function. The dynamic transcriptional landscape can inform us of the effect that whole body stress (e.g., exercise, environmental heat/humidity exposure, dehydration) has on sub‐cellular health. Studying global genome expression shifts may allow us to elucidate mechanisms of stress resilience, discover new stress biomarkers, identify targets for optimizing performance using supplementation or training paradigms, and better define rest and recovery post‐exercise stress during training. In this study we designed experiments to characterize the transcriptome level changes that occur before and after exercise and environmental stress in conditions that put individuals at high‐risk for exertional heat illness. At the Kona Ironman World Championship (2012) we recruited 8 competitors for participation in this study. Pre‐race (PRE) and immediately post‐race (IP), we collected physiological data (e.g., core body temperature IP (38.51±0.66°C, p<0.05 vs. PRE) and percent body mass change (−3.5±2.0%, p<0.05). Samples for blood analyses were collected two to three days prior to race day (BASE), at IP, and 1 day post‐race (24HPOST) on all subjects. For RNAseq experiments, we analyzed PBMC samples isolated from whole blood at BASE and IP; 4 subjects were the most dehydrated (−6.1±1.1% body mass IP vs. BASE) and 4 were the least dehydrated at IP (−2.0±1.3% IP vs. BASE). mRNAs were sequenced on the HiSeq2500. All samples had a minimum of 43.5 million passed‐filter reads and aligned at >81% efficiency to the reference genome (Ensembl Biomart 72 hsa37). Effect of time point and hydration state at IP was assessed via ANOVA (Tukey post hoc comparisons) (R 3.0). FDR were calculated from p‐values using Benjamini and Hochberg methods. Of the pathways enriched (p<0.05) in the most dehydrated subjects at IP included ribosome, lysosome, and ion channel functions, while pathways significantly different in the less dehydrated subjects included genes primarily in macronutrient metabolic pathways. These findings suggest that great dehydration enhances cell organelle and membrane activities whereas mild dehydration enhances metabolism in response to exercise‐heat stress.

Support or Funding Information

Timex, Inc.
The Circadian Clock Protein Per1 and Aldosterone Mediate Opposing Action on Endothelin‐1 Expression in Murine Collecting Duct Cells
Authors: Michel Michelot , Solocinski Kristen , Cain Brian D. , Wingo Charles S. , Gumz Michelle L.
Published: November 2023

Hypertension is a major risk factor for cardiovascular disease, the leading cause of death worldwide. The epithelial sodium channel (ENaC) plays a role in sodium reabsorption, which affects blood volume and therefore blood pressure. The rate limiting alpha subunit of ENaC (αENaC) is positively regulated by the circadian clock protein Period 1 (Per1) and the mineralocorticoid hormone aldosterone but negatively regulated by the peptide hormone endothelin‐1 (ET‐1). Interestingly, ET‐1 and Per1 are both acutely induced by aldosterone. Full‐length Per1 is known to have a molecular weight of 140 kDa but we have consistently observed a 50 kDa nuclear fragment in many tissues and cell lines in addition to full length Per1 in the cytosol. However, it is unknown if this nuclear form of Per1 is directly regulated by aldosterone. The goals of the current study were to 1) test the hypothesis that the 50 kDa nuclear Per1 is induced by aldosterone and 2) determine the interaction of Per1 and aldosterone in the regulation of ET‐1 in mouse collecting duct cells. For the first time, we show that the ~50 kDa nuclear Per1 is induced by aldosterone treatment (100 nM for four hours) in a model of the mouse inner medullary collecting duct (mIMCD‐3 cells). Using Per1‐specific siRNA in conjunction with aldosterone treatment, we also show that the response of ET‐1 to aldosterone is enhanced in the absence of Per1. Thus, Per1 and aldosterone mediate opposing effects on Endothelin‐1 (ET‐1), but the same effects on αENaC. These findings suggest a coordinated regulation of ENaC by Per1, ET‐1, and aldosterone in the mouse inner medullary collecting duct. These findings may have implications for our understanding of ENaC‐dependent mechanisms underlying hypertension.

Support or Funding Information

This work was supported by NIH DK085193 and DK098460, the ASN Foundation for Kidney Research to MLG, 2T32HL083810 to KS and NIH DK082680 to BDC and CSW.
Histone Deacetylase Inhibitors Exhibit Enhanced Kidney Functions in Guanylyl Cyclase‐A/Natriuretic Peptide Receptor‐A Gene‐disrupted Mice: Role of Epigenetic Mechanisms
Authors: Kumar Prerna , Periyasamy Ramu , Subramanian Umadevi , Pandey Kailash N.
Published: November 2023

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA)/cGMP signaling plays a well‐defined role in the regulation of blood pressure and blood volume. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit hypertension, kidney disease, and heart failure. The objective of the present study was to determine the combined effect of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all‐trans retinoic acid (ATRA) on enhanced renal functions and attenuation of renal fibrosis in Npr1 gene‐disrupted mutant mice. Adult (18–20 week old) male Npr1 gene‐disrupted heterozygous (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+), and gene‐duplicated (3‐copy; Npr1++/+) mice were treated by injecting ATRA‐NaBu hybrid drug (1.0 mg/kg/day) intraperitoneally for 2‐weeks. A marked attenuation in tubulo‐interstitial fibrosis was observed in Npr1+/− mice after treatment with ATRA‐NaBu (50%, p < 0.001). Western blot analyses exhibited reduction in renal expression of collagen type I alpha 2 and transforming growth factor‐beta in ATRA‐NaBu‐treated Npr1+/− mice compared with vehicle‐treated control animals. A significant decrease in systolic blood pressure was observed in ATRA‐NaBu‐treated Npr1+/− mice. The ATRA‐NaBu treatment enhanced plasma cGMP levels in Npr1+/, Npr1+/+, and Npr1++/+ mice. Creatinine clearance (CCr) was significantly reduced by 62% in Npr1+/− mice and 2‐fold higher in Npr1+/++ mice compared with wild‐type control mice. Treatment with ATRA‐NaBu hybrid markedly enhanced CCr in Npr1+/− mice comparable with vehicle‐treated group. Proteinuria was detected in Npr1+/− mice as evidenced by significantly (p < 0.01) high urinary albumin content compared with Npr1+/+ mice. A complete reversal of proteinuria was observed in ATRA‐NaBu hybrid‐treated Npr1+/− mice group compared with Npr1+/− control mice. Moreover, the increased histone deacetylase activity in Npr1+/− mice was significantly reduced by ATRA‐NaBu treatment compared with untreated Npr1+/− control mice. The present results provide direct evidence that ATRA‐NaBu acts as a potent antifibrotic agent and repairs the renal pathology in Npr1+/− mice, which will have important implications in prevention of hypertension‐related renal pathophysiological conditions.

Support or Funding Information

This work was supported by the NIH grants R01HL057531 and R01HL062147.
RNA‐seq analysis of paired servo‐controlled kidneys suggests molecular pathways affected by pressure
Authors: Dayton Alex , Evans Louise C , Yang Chun , Liu Pengyuan , Ahn Kwang Woo , Bukowy John D , Stingo Francesco , Vannucci Marina , Laud Purushottam , Liang Mingyu , Cowley Allen W
Published: November 2023

We have established that administration of a 4% salt diet to Dahl Salt‐sensitive (SS) rats induces increased blood pressure (BP), as well as significant gene expression changes in the renal medulla. It has been unclear, however, which of these changes were a consequence of changes in salt diet, versus those which were a consequence of increased renal perfusion pressure (RPP) per se. We have developed a preparation that uses a chronic servo‐control system that maintains the RPP of the left kidney at normotensive levels during the salt‐induced increase in pressure to separate the pathological effects of salt from the effects of pressure in the same animal.

8 SS rats were surgically prepared with carotid and femoral arterial catheters implanted for continuous measurement of BP. An inflatable cuff was placed around the aorta between the left and right renal arteries and attached to a syringe driven by a motor, which continuously adjusted the level of inflation to regulate RPP of the left kidney. After recovery, baseline carotid (right RPP) and femoral (left RPP) BP were measured on control 0.4% salt diet for three days (the set‐point for left RPP). The rats were then fed a 4% salt diet for 7 days, after which the kidneys were flushed and renal medullas collected for RNA‐seq analysis (16 renal medullas; 8 left‐right pairs).

RNA was prepared from each sample for RNA‐seq measurement. Significant difference in gene expression was measured by paired t‐test on the log2FPKM dataset. 1613 genes were found to be differentially expressed (q<0.05) between the high pressure right kidney and the servo‐controlled left kidney.

The 1613 genes were analyzed by three methods: DAVID, Ingenuity Pathway Analysis (IPA) and a Bayesian discriminant analysis. DAVID uses only the identities of the 1613 genes to find overrepresented ontologies. IPA uses the identities and the binary‐coded up‐ or down‐regulated status of the genes to identify up‐ or down‐regulated pathways and predict potential regulators. The Bayesian analysis directly uses the expression values as well as the measured RPP data to find pathways which discriminate the groups on the basis of pressure. DAVID and IPA results were considered significant at p<0.05. The probabilities generated by the Bayesian analysis are not directly comparable to p‐values, but the top ten pathways were considered significant.

Pathways that are differentially regulated between the high pressure kidneys and the servo‐controlled kidneys can be grouped into four broad categories: 1) leukocyte trafficking and cytokine production, 2) metabolism of TCA cycle intermediates, 3) oxidative phosphorylation, and 4) cell‐cycle processes. This analysis therefore suggests that these pathways are regulated by RPP, and are targets for future study for further dissection of the effects of salt from the effects of pressure.
Independent and Pleiotropic Gene Effects on Hypertension and Metabolic Syndrome in Lyon Hypertensive Rats
Authors: Kwitek Anne E , Pettus Janette M , Jakoubek Jessica A , Ma Man Chun John
Published: November 2023

The Lyon Hypertensive (LH) rat is an inbred selection model of spontaneous hypertension. However through the selection and inbreeding of the LH strain for high blood pressure, genes causing salt‐sensitivity and several features of the Metabolic Syndrome (MetS) including obesity and dyslipidemia were also fixed; thus the LH rat is a genetic model of MetS associated hypertension. Conversely, the Lyon Normotensive (LN) control strain is normotensive, salt‐resistant and displays no features of MetS. Both strains were selectively bred from the same outbred Sprague Dawley founders, based on divergent blood pressures, yet their genomes are >99.98% identical. Our overall goal is to identify novel genes and pathways linking disparate phenotypes in MetS. We performed comprehensive systems genetic studies in LH rats and determined that rat chromosome (RNO) 17 contributes not only hypertension but also obesity and dyslipidemia. Genetic mapping studies identified overlapping quantitative trait loci (QTL) for blood pressure, body weight, and plasma lipids on RNO17 in the LH rat. Whole genome sequencing of LH and LN strains revealed large blocks of RNO17 are of shared ancestry; haplotype fine‐mapping refined a list of 26 candidate causal genes. Consomic and congenic studies determined that the whole chromosome substitution of LH RNO17 with that of the LN was sufficient to reduce blood pressure, lipid levels, and body weight. Interestingly, a congenic strain substituting only the distal end of LH RNO17 with the LN allele reduces blood pressure and serum lipids independent of body weight, suggesting both independent and pleiotropic gene effects in LH hypertension. We used RNA‐seq to identify transcriptome differences between LH and LN strains, and to map genetic regulation of gene expression (eQTL). Proximal RNO17 was found to regulate the expression of over 60 genes located elsewhere in the genome (a trans‐eQTL hotspot). A single candidate gene in this locus (RGD1562963), is cis‐regulated, has sequence variation between LH and LN rats, is differentially expressed in multiple tissues between LH and LN, and controls the expression of downstream genes mapping to the trans‐eQTL hotspot that are associated with hypertension and MetS. Our integrative studies suggest that genetic variation in LH RNO17 has independent and pleiotropic effects on hypertension and features of MetS.

Support or Funding Information

This work is supported by NIH grants R01HL089895 and R21DK089417, AHA grant 14GRNT20410043, and the Fraternal Order of Eagles Diabetes Research Center at the University of Iowa.
The Hypercapnic Ventilatory Response and Behavior in Ca2+‐activated K+ (BK) Channel knock Out Mice
Authors: Putnam Robert W , Patrone Luis G.A. , Ratliff Christine A. , Gargaglioni Luciane H.
Published: November 2023

In locus coeruleus (LC) neurons, the firing rate response of CO2/pH chemosensitive neurons has been suggested to be modulated by the activity of BK channels. Hypercapnic acidosis (HA) activates Ca2+ channels which lead to increased intracellular Ca2+ and activation of BK channels. When BK channels are inhibited, the firing rate response to HA increases (Imber et al. 2014. Biochim. Biophys. Acta 1842:2569–2578). In the current study, we examined the hypercapnic ventilatory response in mice with the BK channel knocked out (BK KO mice). We also studied the firing rate response of LC neurons to hypercapnia in wild type (WT) and BK KO mice. Finally, we studied the phenotype of these mice with respect to overall locomotion and anxiety responses. Elevated inspired CO2 (7%) reversibly increased ventilation in both WT and KO mice (increase about 3 fold in WT, n=8, and 4–5 fold in KO mice, n=6; P<0.05). The increased ventilation was due to increased tidal volume and respiratory frequency in both WT and KO mice, but the larger ventilatory increase induced by inspired CO2 in KO vs. WT mice was entirely due to a larger tidal volume response. Hypercapnia‐induced increases in ventilation were not due to increased metabolism, which was the same in both WT and KO mice in normocapnia and in hypercapnia (about 5 ml/100g‐min). The firing rate response to 10% CO2 (from a control level of 5% CO2) increased about 74% (from 0.42–0.73 Hz) in LC neurons from male WT mice while it increased by greater than 3 fold (from 0.30–1.26 Hz) in LC neurons from male BK KO mice. BK KO mice showed an increased activity level and an unusual hopping behavior compared to WT mice. When total distance traveled and average velocity were measured over a 30 minute period, BK mice showed significantly greater distance traveled (P<0.02; n=6) and faster average velocity (P<0.02; n=6) compared to WT mice. However, at room air there was no evidence for anxiety behavior in the performance of BK mice in either the open field test or the elevated plus maze test. Our data show that the lack of a BK channel in LC neurons from mice results in increased firing rate in response to hypercapnia. Further, the lack of BK channels in KO mice leads to an increased hypercapnic ventilatory response. These findings support the hypothesis that BK channels act as a brake on the hypercapnic response of chemosensitive neurons. Our findings also suggest that there is an activity phenotype in BK KO mice although we find no evidence of increased anxiety, at least under normocapnic conditions, in BK KO mice.

Support or Funding Information

Supported by a Wright State University Emerging Science Seed Grant (to RWP) and CNPq and Fapesp grants (to LGAP and LHG).