bluuebunny/crossref_metadata_2025 huggingface dataset:
The ER/SR Ca2+ sensing protein STIM1 is essential for normal cardiomyocyte function (1150.8)
Authors: Collins Helen , He Lan , Pat Betty , Zou Luyun , Qu Jing , Zhou Lufang , Litovsky Silvio , Yang Qinglin , Young Martin , Chatham John
Published: June 2021

Store‐operated Ca2+ entry (SOCE) and its molecular mediator, the endoplasmic reticulum (ER) Ca2+ sensor, Stromal interaction molecule 1 (STIM1) have been implicated in hypertrophic signaling. However, since the physiological role of STIM1 in the adult heart remains unknown, we developed a cardiomyocyte‐restricted STIM1 knockout (crSTIM1‐KO) mouse. Using serial echocardiography and histological analysis, we observed a progressive decline in contractile function from 20 weeks of age, which was associated with marked left ventricular dilatation by 36 weeks. Immunoblot analyses revealed that crSTIM1‐KO hearts exhibit increased ER stress prior to contractile dysfunction (i.e., 12 weeks old) as indicated by increased levels of CHOP. Electron microscopy revealed ER dilatation, mitochondrial disorganization and smaller mitochondria in crSTIM1‐KO hearts, which was associated with a decrease in mitofusin 2 and an increase in Drp1 consistent with increased mitochondrial fission. Interestingly, perfused hearts from 20 week crSTIM1‐KO mice exhibited significant reductions in both glucose oxidation and net lactate release. These data demonstrate for the first time that STIM1 plays an essential role in normal cardiomyocyte function in the adult heart and may be mediated through regulation of ER/ mitochondrial function and/or metabolism.

Grant Funding Source: Supported by NIH grants HL101192 and HL110366 and William W Featheringill Postdoctoral Fellowship
Matrix metalloproteinase 2 increases ROS in aortic tissue (1153.10)
Authors: Prado Alejandro , Pernomian Laena , Azevedo Aline , Castro Myrella , Bendhack Lusiane , Tanus‐Santos Jose , Gerlach Raquel
Published: June 2021

So far ROS has been shown to activate MMP‐2, a key protease in cardiovascular diseases. This study tested whether MMP‐2´s effect on vascular dysfunction was mediated by ROS. The cDNA of the catalytic domain of the human MMP‐2 was cloned in fusion with GFP, and the activity of the hMMP‐2cat/GFP was assayed. The rhMMP‐2/GFP protein was catalytically active (both were expressed and purified from E coli). Both proteins were injected into the lumen of New Zealand rabbit aortas, and filled vessels (MMP‐2‐FA) were incubated for 30 min. Concentration used: 1.2 ug/ml or 16 mmol/L, 4 times the level of MMP‐2 found in healthy humans. The exogenous MMP‐2 increase in aortic wall was tracked by direct observation of GFP, immunostaining and gelatinolytic activity. This last parameter doubled in the MMP‐2‐FA, as well as in MMP‐2‐FA+PMSF (a serine‐proteinase inhibitor), but displayed normal values in MMP‐2‐FA+ Doxycycline (an MMP inhibitor). ROS activity was increased by 85% (±4%) in MMP‐2‐FA, while there was only a slight increase in the MMP‐2‐FA co‐infused with the MMP inhibitor Doxycycline (15 ±8%). Apocinin and PEG Catalase decreased the levels of ROS in MMP‐2‐FA to basal levels (P<0.001), while Tiron only reduced ROS levels by 17%(±8%)(p<0.05). This is the first study to show that MMP‐2 at levels described in plasma of patients are sufficient to increase ROS in vessels.

Grant Funding Source: FAPESP
Oxidative stress increases pulmonary capillary permeability in lean Zucker rats with chronic hyperglycemia (1153.6)
Authors: Lu Silu , Xiang Lusha , Clemmer John , Mittwede Peter , Hester Robert
Published: June 2021

Chronic hyperglycemia in obesity and diabetes is associated with increased vascular oxidative stress and endothelial dysfunction, including an increase in endothelial permeability. Increases in pulmonary capillary permeability associated with hyperglycemia and oxidative stress may contribute to lung edema in obesity and diabetes following trauma. We hypothesized that chronic hyperglycemia‐induced oxidative stress increases pulmonary capillary filtration coefficient (Kf). Lean Zucker rats (LZ) (n=12, STZ 50 mg/kg, ip) were hyperglycemia for 4 wks. A subgroup (n=6) was treated with apocynin (NADPH oxidase inhibitor, 2 mM in drinking water for 4 wks). Kf was measured in isolated lungs. Superoxide was measured in aortas using DHE. PMS (superoxide donor, 1 µM) was added to the isolated lung perfusate to determine the effect of an increased superoxide on Kf. Aortas from control LZ (LC) were treated with PMS to verify an increase in superoxide levels. STZ‐treated LZ (LS) exhibited significantly increased vascular superoxide levels and Kf (31 ± 4 ml/mmHg/g/min) compared to LC (7 ± 3 ml/mmHg/g/min). Apocynin treatment significantly reduced the superoxide levels and Kf (18 ± 3 ml/mmHg/g/min) in LS. PMS significantly increased Kf (37 ± 7 ml/mmHg/g/min) in isolated lungs from LC. These results suggest that, during chronic hyperglycemia, elevated vascular superoxide increases pulmonary permeability.

Grant Funding Source: Supported by AHA‐12POST12060126, NIH HL‐51971, HL‐89581, and AHA‐12SDG12050525
Contractile function in myocytes from aged hearts is altered with glucose compared to fat as the substrate (1154.7)
Authors: Barton Gregory , Diffee Gary , Aiken Judd
Published: June 2021

Substrate utilization generally favors fatty acid oxidation over glucose utilization in normal healthy hearts. Aging is associated with attenuated myocardial contractile function; however age related changes in substrate utilization are less understood. Most studies of metabolic aspects on cardiac function have been done in whole hearts and little information is available about cellular contractile responses in the presence of different substrates. We determined substrate effects on contractile properties in isolated myocytes from young (6 months) and old (33 months) FBN rats. We measured percent shortening and rate of shortening at time 0 (T0) and after 10 minutes (T10) of continuous stimulation at 2 Hz in myocytes isolated from healthy hearts and bathed in Ringers solution containing either 11mM glucose (G) or 0.4 mM oleate (O). Both percent shortening and rate of shortening at T10 were lower than at T0 regardless of age or substrate. However, in old myocytes there was a significantly (p < 0.05) larger decrease over time (∆ = T10 ‐ T0) when G (∆ % shortening = ‐2.87) was the substrate compared to O (∆ % shortening = ‐1.37). In young myocytes there were no substrate differences (∆ % shortening = ‐1.36 and ‐1.42 in G and O, respectively). Similar results were seen with the rate of shortening. These data suggest that aging results in altered substrate utilization that affects myocyte contractile properties.

Grant Funding Source: Supported by NIH AG 030423
Inverse relationship between cutaneous vascular conductance and augmentation index after administration of nitroglycerin (1156.9)
Authors: Pearson James , Schlader Zachary , Rivas Eric , Crandall Craig , McDonnell Barry
Published: June 2021

Augmentation index (AIx) is a marker of wave reflection and arterial stiffness, derived from a central blood pressure waveform. AIx is acutely reduced with sublingual nitroglycerin (NG) via endothelium independent dilation. In this way NG may also influence the cutaneous vascular conductance (CVC), which has a high capacity to influence central hemodynamics and blood pressure. The relationship between cutaneous vascular conductance and AIx is unknown. We examined alterations in AIx and CVC in ten older individuals (62 ± 8 yrs, 169 ± 11 cm, 71 ± 9 kg) following 3 min sublingual administration of NG (400 µg). AIx and vascular hemodynamics were measured at baseline and every 20 seconds for 20 minutes following NG administration. In the 5 min period following NG administration AIx was reduced by 45% from 27 ± 10 % at baseline to 15 ± 14 % (P = 0.000), while CVC increased 32% over the same period from 27 ± 6 AU/mmHg at baseline to 36 ± 12 AU/mmHg (P = 0.014). Mean arterial pressure was unchanged throughout (P > 0.05). Throughout the 20 minute period following NG administration, alterations in AIx and CVC were inversely related (r = ‐0.76; P < 0.001). A relationship between AIx and CVC may suggest an interplay between the cutaneous vasculature and surrogate measures of arterial stiffness that warrants further investigation. Such findings may have implications for our understanding of cutaneous vascular control and cardiovascular risk.
Skeletal muscle proteomic signature in pulmonary arterial hypertension (1158.8)
Authors: Pflieger Aude , Potus François , Malenfant Simon , Breuils‐Bonnet Sandra , Bonnet Sebastien , Provencher Steeve
Published: June 2021

Introduction: Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular (RV) hypertrophy and ultimately failure. Numerous observations suggest that exercise limitation in pulmonary arterial hypertension (PAH) is not solely due to cardio‐pulmonary impairment, but that other determinants such as skeletal muscle abnormalities are involved. To better understand the exercise limitation origins in PAH, we studied the proteomic signature of skeletal muscle impairment in idiopathic PAH (iPAH) patients.

Methods and results: Muscle proteins from 4 iPAH patients and 4 sex and age matched controls were extracted following quadriceps muscle biopsy. Fractioned peptides were then tagged using isobaric Tags for Relative and Absolute Quantitation (iTRAQ, ABsciex) specific for each patient. Tagged peptides were then mixed, fractioned, identified and quantified using mass spectrometry quantitative method. Over 900 proteins were identified, among them, 9 were under‐expressed and 7 were over‐expressed in all iPAH patient quadriceps. More than 75% of the under‐expressed proteins are involved in oxidative metabolism and most other highlighted proteins play a role in myogenesis. These findings were structurally associated with decreased mitochondrial density (citrate synthase assay) and impaired mitochondrial structure measured by electronic microscopy, while muscle fibers distribution was unchanged. Functionally, all these abnormalities resulted in decreases in oxidative metabolism (pyruvate dehydrogenases assay).

Conclusions: For the first time in humans, we provide evidences that, as in lungs and the right ventricle, the impaired mitochondrial metabolism is also present in skeletal muscle suggesting that PAH might be considered as a global mitochondrial/metabolic disease.
Regulation of LNCaP respiration by plasma membrane and mitochondrial angiotensin II receptors (1159.11)
Authors: Duerr Jeffrey , Reister Anna
Published: June 2021

Prostate cancer is diagnosed in over 186,000 men in the United States and accounts for more than 28,000 deaths annually. Angiotensin II (AngII) is a major effector peptide of the renin‐angiotensin system (RAS), and is an important factor in hypertension. Correlations between ACE therapy and improved prostate cancer outcomes led to in vitro studies the action of AngII in cultured prostate cancer cell lines. Investigators have reported that exogenous application of Ang II to LNCaP cells results in the activation of Akt signaling, increased activity of SOD, and glutathione peroxidase. As the mitochondrion is a major contributor of total cellular ROS, we investigated the effect of AngII on mitochondrial respiratory function directly. AngII can bind to either of two receptors: the losartan‐sensitive AT1‐R and the PD123319‐sensitive AT2‐R. We present Western‐blot evidence of the presence of AT1‐R at the plasma membrane and both AT1‐R and AT2‐R receptor at the mitochondrion in LNCaP cells. Using fluorescent respirometry, we show AngII stimulates LNCaP whole‐cell respiration over a 24‐hour period with maximal stimulation at 5 hours post‐application. Increased respiratory rates are abolished using either losartan or PD123319 at time points up to 5 hours post‐application implicating the participation of both receptor subtypes in mediating this effect. At 24 hours post‐application respiratory rates remain elevated, though to a lesser degree, and appear to be sustained by AT1‐R activity only. Given the differential contribution of these two receptor sub‐types to modulation in respiratory activity, there may be two independent pathways activated by their respective AT‐R subtype.
HDL and lipid components of HDL inhibit oxysterol induced macrophage differentiation by altering cellular lipid status and mechanisms involving oxidative stress (1161.2)
Authors: Hayden John , Martinez Didio , Franco Daniel
Published: June 2021

Background and Objectives: we have reported that monocytes treated with the proatherogenic oxysterol 7‐ketocholesterol (7KC) form macrophages and become foam cells in vitro. HDL has been shown to possess many antiatherogenic properties; however, its effect on modulating oxysterol‐induced differentiation has not been studied. Thus, we tested the hypothesis that HDL and components of HDL inhibit oxysterol induced macrophage formation. Methods: serum deprived THP‐1 monocytic cells were treated with 7KC for up to 7d and treated with various physiologic concentrations of HDL, recombinant HDL and phospholipids. Formation of adherent macrophages was determined, cellular ROS and glutathione status were assessed, and cell lysate were prepared for the analysis of varied lipid components by HPLC. Results: native HDL (2.5‐100 μg protein/ml), lipid components of HDL and recombinant HDL containing phosphatidylcholine (PtC) markedly inhibited (P<.001) macrophage formation. Moreover, the exogenous addition of PtC, with varying fatty acid composition, also inhibited (P<.001) macrophage formation. Upon further examination, 7KC treatment resulted in reduced concentration of cellular PtC which was associated with a increase in reactive oxygen species (ROS) and marked alteration of glutathione status (GSH:GSSG ratio ~10). In contrast to PtC, cellular levels of ceramide and sphingomyelin were increased during cellular stress by 7KC treatment. HDL treatment restored PtC and sphingomyelin levels to non‐oxysterol treated cells; and exogenous PtC supplementation, or HDL treatment, restored ROS and GSH status to levels similar to non‐treated controls. Conclusions: these results demonstrate that HDL inhibited macrophage differentiation induced by 7KC treatment. In addition, HDL possesses the capability to alter lipid status and mechanisms controlling oxidative stress that are involved, in part, with monocyte‐macrophage differentiation.

Grant Funding Source: Supported by grants from the Veterans Affairs VISN18 and Carl T. Hayden Medical Research Foundation
Trans IL‐6 signaling and suppressed muscle protein synthesis with cancer (1163.11)
Authors: Puppa Melissa , Narsale Aditi , Rose‐John Stefan , Carson James
Published: June 2021

Systemic inflammation is associated with many types of cancer‐induced cachexia, and often includes increased IL‐6 levels. Tissue STAT3 activation can occur by classical or trans IL‐6 signaling. Inhibition of IL‐6 or muscle STAT3 can attenuate cachexia muscle mass loss in tumor bearing mice. The purpose of this study was to determine if inhibition of either trans IL‐6 or systemic STAT3/NfκB signaling could improve cancer‐induced suppression of muscle protein synthesis in ApcMin/+ (Min) mice. After the initiation of cachexia Min mice were randomized to receive sgp130Fc (150ug/wk) to inhibit trans IL‐6 signaling, PDTC (10mg/kg/day) to inhibit STAT3/NfκB signaling, or PBS for 2‐wks. At 16 weeks of age Min mice had lost 7% body weight (BW). Administration of PDTC or sgp130Fc attenuated further BW loss, while PBS mice lost an additional 8% BW. Additionally, PDTC and sgp130Fc attenuated the cachexia‐mediated loss of gastrocnemius muscle mass. While sgp130Fc was unable to attenuate cachexia suppression of muscle protein synthesis, PDTC administration increased muscle protein synthesis. In summary, trans IL‐6 signaling inhibition was sufficient to attenuate cancer‐induced muscle mass loss independent of effects on suppressed muscle protein synthesis, while global STAT3 and NfκB inhibition rescued both muscle mass and protein synthesis during cancer cachexia.

Grant Funding Source: Supported by R01CA121249
An investigation of kinases responsible for site‐specific phosphorylation of p70S6K1 in skeletal muscle following chronic functional overloading (1163.13)
Authors: Martin Tony , Dennis Michael , Gordon Bradley , Kimball Scot , Jefferson Leonard
Published: June 2021

The present project was designed to investigate the phosphorylation of p70S6K1 in an animal model of induced hypertrophic growth of skeletal muscle. Within 24 h of chronic functional overloading of the plantaris muscle by unilateral tenotomy in male Sprague Dawley rats, phosphorylation of p70S6K1 on Thr389 and Thr421/Ser424 was significantly increased. Treatment of rats with inhibitors of mTOR and c‐Jun N‐terminal Kinase (JNK) provided evidence of a role for the JNK signaling pathway in mediating regulation of p70S6K1. To investigate further a role for JNK in p70S6K1 phosphorylation, a cell culture model was employed. In HEK293E cells deprived of serum, treatment with IGF1 enhanced phosphorylation of Thr389 and Thr421/Ser424 sites. Inhibition of mTORC1 and/or JNK using selective inhibitors revealed a complex interaction suggesting that mTORC1 signaling pathway mediates the IGF1‐induced phosphorylation of both Thr389 and the Thr421/Ser424 sites, whereas JNK is responsible for basal phosphorylation (i.e. phosphorylation in serum‐deprived cells). Further support for this suggestion is provided by the finding that anisomycin, a JNK activator specifically stimulated phosphorylation of Thr421/Ser424 but not Thr389. Taken together, these results demonstrate that both the mTORC1 and JNK signaling pathways contribute to the stimulation of cell growth by mediating site specific phosphorylation of p70S6K1.

Grant Funding Source: Supported by NIH Grant DK15658
Myostatin and TGF‐β signaling in overload‐induced skeletal muscle hypertrophy (1163.14)
Authors: Mack Gary , Affleck Paige
Published: June 2021

Myostatin (GDF‐8) negatively controls adult skeletal muscle growth. The role of myostatin and TGFB signaling pathways in overload‐induced hypertrophy of adult muscle is unclear. We tested the hypothesis that overloaded adult rodent skeletal muscle would reduce skeletal muscle myostatin levels and thereby lower pSMAD2/3 levels and increase pAKT levels. Overload‐induced hypertrophy was induced by unilateral tenotomy of the gastrocnemius tendon in 11 male adult Sprague‐Dawley rats followed by a 2‐week period of compensatory overload of the plantaris and soleus muscles. Western blot analysis was performed to evaluate changes in active and latent complex myostatin protein levels and the proteinase BMP1/TLD. The TGFβ signaling pathway was evaluated using a Milliplex magnetic bead multiplex assay (TGFβ receptor, pSMAD2, pSMAD3, SMAD4, pERK, and pAKT). Significant hypertrophy was noted in both the soleus (494 ± 29 vs 405 ± 15 mg, p<0.05) and plantaris (289 ± 12 vs 179 ± 37 mg, p<0.05) muscles following overload. Overloaded skeletal muscle reduced active myostatin protein (range 32.7 ± 9.4% to 28.5 ± 8.5%, p<0.05), latent myostatin protein (range 15.0 ± 5.9% to 70.0 ± 2.3%, p<0.05), and the BMP‐1/TLD proteinase (range 40.4 ± 12.9% to 32.9 ± 6.9%, p<0.01). Changes in TGFβ signaling were muscle type dependent. In the predominantly fast twitch plantaris muscle there was an increase in TGFβ‐r, pSMAD2, pSMAD3, SMAD4, and pAKT (p<0.05). In the predominantly slow twitch soleus muscle there was an increase in pERK, pSMAD2, and pSMAD3 (p<0.05). The paradoxical increase in pSMAD2/3 and pSMAD4 with a decrease in active myostatin in either muscle type may be related to cross talk between an ERK‐mediated phosphorylation of SMAD2/3 and/or other TGFβ stimulated MAPK pathways.
G protein coupled receptor 56 is a target gene of the PGC‐1α4 isoform and regulates overload‐induced muscle hypertrophy (1163.15)
Authors: White James , Wrann Christiane , Rao Rajesh , Wu Zhidan , Glass David , Spiegelman Bruce
Published: June 2021

The PGC‐1α isoform referred to as PGC‐1α4 has recently been shown to promote muscle hypertrophy and attenuate muscle wasting. Although it has been shown that PGC‐1α4 can regulate the expression of IGF‐1 and myostatin, two potent regulators of muscle mass, its detailed mechanism of action remains unclear. Gene array analysis has identified the G‐protein coupled receptor 56 to be a transcriptional target of PGC‐1α4. GPR56 is a newly defined adhesion receptor which can mediate both G protein coupled signalling and integrin function, although little in know about its function in skeletal muscle. We have found the anabolic effect of PGC‐1α4 is, in part dependent on GPR56 signalling as knockdown of GPR56 attenuates PGC‐1α4‐induced muscle hypertrophy in vitro. Forced expression of GPR56 in primary myotubes results in mild hypertrophy. However, when forced expression of GPR56 is accompanied by the addition of its ligand, Collagen III, it drives robust myotube hypertrophy. In vivo models of overload‐induced muscle hypertrophy increase GPR56 expression while GPR56 loss of funtion attenuates the hypertrophic response to mechanical overload. Lastly, we show muscle GPR56 expression is increased during human resistance exercise. In conclusion, we have discovered GPR56 is a PGC‐1α4 transcriptional target and could play a role in muscle hypertrophy induced by resistance/loading‐type exercise.
Docosahexaenoic acid attenuates palmitate‐induced unfolded protein response in myotubes (1163.21)
Authors: Woodworth‐Hobbs Myra , Hudson Matthew , Rahnert Jill , Zheng Bin , Price S.
Published: June 2021

Accumulation of saturated fatty acids in skeletal muscle results in dysregulation of protein metabolism and muscle atrophy. In cultured myotubes, the saturated fatty acid palmitate (PA) stimulates protein degradation and decreases cell size, while co‐treatment with the omega‐3 polyunsaturated fatty acid docosahexaenoic acid (DHA) prevents the response. It has been shown that PA also induces endoplasmic reticulum stress and activates the unfolded protein response (UPR) in myotubes, resulting in decreased protein synthesis. The objective of this study is to test whether DHA protects against palmitate‐induced endoplasmic reticulum stress and activation of the UPR. C2C12 myotubes were treated with 500μM PA and/or 100μM DHA for 24h and protein markers of the UPR were evaluated by western analysis. PA induced activation of protein kinase R‐like protein kinase (PERK), as indicated by an increased phospho‐PERK:total PERK ratio. PA also induced phosphorylation and inactivation of eukaryotic initiation factor 2α (eIF2α). While co‐treatment with DHA attenuated the effects of PA on PERK ratio, eIF2α phosphorylation was similar in myotubes treated with PA and PA+DHA. These results indicate that DHA attenuates the effects of PA on activation of the UPR but does not restore global protein synthesis; this suggests that DHA restores myotube diameter primarily by inhibiting palmitate‐induced protein degradation.

Grant Funding Source: Supported by NIH RO1DK95610, AHA GRNT7660020, and VA MERIT X01BX001456 awarded to SR Price.
Lewis lung carcinoma cells regulation of stretch‐induced mTOR activation in C2C12 myotubes (1163.8)
Authors: Gao Song , Carson James
Published: June 2021

Imbalances in rates of protein synthesis and degradation are responsible for cancer‐induced skeletal muscle wasting. Lewis lung carcinoma (LLC) cell implantation is a widely used explant mouse model for examining cancer cachexia and muscle wasting. We have demonstrated that trans‐signaling molecules from LLC cells resulted in mTOR suppression in C2C12 myotubes and muscle from mice implanted with LLC tumors. Mechanical overload activates mTOR signaling and increases protein synthesis rate, serving as a potential therapeutic strategy for skeletal muscle mass maintenance. The purpose of this study is to investigate the effect of mechanical stretch on C2C12 myotube protein synthesis regulation in the presence of trans‐signaling molecules from LLC cells. C2C12 myotubes were differentiated and then stretched on collagen‐coated silastic membranes. Chronic uniaxial stretch (5% for 24h) induced ERK (T202/Y204) and S6 ribosomal protein (S235/236) phosphorylation as well as rate of protein synthesis in control medium (DMEM with 2.5% FBS). However, the addition of LLC medium blocked the stretch induction of S6 ribosomal protein phosphorylation. These results suggest LLC can directly suppress mechanical activation of signaling that regualtes of skeletal muscle protein synthesis.
Effects of exercise and body temperature on eNOS, SIRT1, SIRT3 and Hsp70 expression in rat plantaris muscles (1164.6)
Authors: Hodge Taylor , Starnes Joseph , Feger Bryan , Hixson Lindsay , Harris M. Brennan
Published: June 2021

Previously, we have demonstrated a dramatic increase in mitochondrial biogenesis in skeletal muscle of rats exercise trained while maintaining a constant core temperature. In this study, we explored the role of body temperature during exercise on the expression of multiple proteins (eNOS, SIRT1, SIRT3, and Hsp70) believed to play a role in mitochondrial biogenesis. Female, Sprague‐Dawley rats (5 mos of age) were divided into three groups sedentary (S), exercise in 22°C room (ET), and exercise while maintaining core temperature (E). Exercised animals trained for 5 weeks on a motor‐driven treadmill at 30 m/min, 60 min/day, 5 days/wk during final 2 wks. Core temperature was held constant in E by reducing room temp to 6‐8°C. Plantaris cytochrome oxidase activity (μmoles O2/min/g wet wt) was significantly (P<0.05) elevated in both ET (37.00±2.10) and E (47.14±4.11) versus S (24.15±1.37) and significantly greater in E versus ET. Preliminary results reveal no significant differences in SIRT1, SIRT3, or eNOS expression in the soleus muscles of S, E, or ET. Hsp70 was significantly (P<0.05) elevated in the ET group. These data suggest that although mitochondrial biogenesis increases in this model, eNOS, SIRT1 and SIRT3 do not parallel this increase and may not play a role in this process.
Bone density is maintained during forced disuse in hibernators throughout both the active and hibernation seasons (1166.1)
Authors: Bogren Lori , Johnston Erin , Van Tets Ian , LeBlanc Adrian , Drew Kelly
Published: June 2021

Long‐term space flight produces a decrease in bone density that poses a hazard for astronauts during and after missions in space. Bone loss is due in part to disuse since long‐term bed rest immobilization in many animals mimics the effects of space flight on bone mineral density. Decreases in metabolism associated with aging and hormones that signal energy status are also thought to contribute to a loss of skeletal mass. Hibernation is a unique metabolic state where some species of mammals remain inactive for periods of up to 8 months and metabolism decreases to 1 to 2 percent of basal metabolic rate. Hibernating ground squirrels maintain bone strength despite long periods of disuse and decreased metabolism. The present study sought to delineate changes in bone density that occur during natural disuse (torpor) and forced disuse (sciatic neurectomy) in the hind limbs of the arctic ground squirrel over the winter and summer seasons. We found that arctic ground squirrels retained bone density with both natural and forced disuse and that this persisted throughout both the summer/active and winter/hibernations season.

Further research is needed to better understand the relationship between skeletal load (use), metabolism and energy homeostasis and bone density that may lead to therapeutics or strategies to maintain bone density in space.

Grant Funding Source: Supported by the Alaska Space Grant Program
Activation of peripheral υ‐opioid receptors suppresses ischemic mediator‐induced cardiac excitatory reflex responses (1169.11)
Authors: Fu Liang‐Wu , Longhurst John
Published: June 2021

Myocardial ischemia evokes cardiac sympathoexcitatory reflex responses through stimulation of cardiac spinal afferents. We have demonstrated that a P2X2/3 agonist stimulates ischemia‐sensitive, but not ischemia‐insensitive cardiac sympathetic afferents, while blockade of peripheral opioid receptors with naloxone enhances the responses of these afferents to P2 receptor activation with ATP. In the present study we tested the hypothesis that opioids, through stimulation of peripheral µ‐opioid receptors, inhibit excitatory reflex responses induced by ATP. Hemodynamic parameters were recorded in six anesthetized rats after bilateral vagotomy. Injection of the P2X2/3 receptor agonist α,β‐meATP (125‐250 nmol), into the pericardial sack increased mean blood pressure (BP) from 85±6 to 115±7 mmHg, a response that was reduced by 19% following activation of peripheral opioid µ‐receptors with intrapericardial DAMGO, a selective µ‐receptor agonist. In contrast, blockade of opioid µ‐receptors with intrapericardial CTOP, selective µ‐receptor antagonist, exaggerated the α,β‐meATP‐induced BP response by 22% in five other rats. Single‐unit cardiac afferent recordings showed that the responses of five ischemia‐sensitive cardiac spinal afferents to α,β‐meATP were enhanced by 51% after epicardial CTOP. Together, these data suggest that peripheral µ‐opioid receptor stimulation suppresses P2X2/3 receptor evoked cardiac excitatory reflex responses through inhibition of ischemia‐sensitive cardiac sympathetic afferents. (Supported by NIH Grant HL66217)

Grant Funding Source: NIH
Tonic arterial chemoreceptor activity contributes to cardiac, but not renal, sympathetic activation in heart failure (1169.2)
Authors: Xing Daniel , May Clive , Ramchandra Rohit
Published: June 2021

In heart failure (HF) cardiac sympathetic nerve activity (CSNA) is increased, which has detrimental effects on the heart. The arterial chemoreceptor activation plays an important role in stimulating renal SNA (RSNA) and muscle SNA in HF. We recorded CSNA and renal SNA (RSNA) in conscious normal sheep and in sheep with mild HF induced by rapid ventricular pacing (ejection fraction <40%). Tonic arterial chemoreceptor function was evaluated by deactivating chemoreceptors by supplementing room air with intranasal 100% oxygen (2‐3 L/min) for 20 min. In HF, chemoreceptor deactivation induced by hyperoxia reduced CSNA (90±2 vs. 75±5 bursts/100 beats, P<0.05; 86±5 vs. 66±7 bursts/min, P<0.001; n=10; hyperoxia vs. room air) and HR (96±4 vs 85±4 beats/min, P<0.001; n=12), but not RSNA burst incidence (93±4 vs 92±4 bursts/100beats; n=7), although due to the bradycardia RSNA burst frequency was decreased (90±8 vs 77±7 bursts/min; P<0.001). In normal sheep, chemoreceptor deactivation reduced HR without a significant effect on CSNA and RSNA. In HF, the response of CSNA, but not RSNA, to sodium cyanide injection was enhanced compared with normal sheep. There was no change in baroreflex control of HR, CSNA and RSNA either HF or normal sheep. In an ovine model of HF, tonic arterial chemoreceptor activation plays a critical role in stimulating the increased sympathetic activity to the heart.

Grant Funding Source: Supported by NHMRC 628573, 07M 3293, 566819
ASP+ reveals potential function of the serotonin transporter in perivascular adipose tissue (1170.8)
Authors: Ayala‐Lopez Nadia , Jackson William , Wilson James , Watts Stephanie
Published: June 2021

Perivascular adipose tissue (PVAT) can modulate vascular tone. Norepinephrine transport in PVAT may serve as a buffer system to reduce adrenergic activation of vascular contraction. We hypothesized that the norepinephrine transporter (NET) is present in PVAT and used the NET selective fluorescent dye 4‐(‐4‐(dimethylamino)‐styryl)‐N‐methylpyridinium (ASP+) to probe for NET in mesenteric PVAT from normal male Sprague Dawley rats. PVAT was incubated ex‐vivo with ASP+ and/or inhibitor and imaged by confocal microscopy. A concentration‐response curve for ASP+ staining of PVAT was constructed and used to select 2 μM as the optimum concentration. Vehicle‐incubated ASP+ signal was 137.5±8.6 AFU (arbitrary fluorescence units, n=18). Nisoxetine, a NET specific inhibitor, reduced ASP+ binding at 10 μm but not 100 nM [96.0±14.5 AFU and 115.9±4.1 AFU respectively (p<0.05; n=6‐7)]. Similarly, citalopram (100 nM), a specific serotonin transporter (SERT) inhibitor, reduced ASP+ binding to 97.7±14.5 AFU (p<0.05; n=11). ASP+ binding was not reduced by GBR 12935 (100 nM), a specific dopamine transporter specific inhibitor (138.0±8.6 AFU; n=12). These data suggest that SERT mediates monoamine uptake into PVAT. Dysfunction of the monoamine transport systems may in part explain altered vascular tone in hypertensive states.
Augmented hemodynamic response to insufficient sleep in women: response to a novel repetitive sleep restriction challenge (1171.4)
Authors: Yang Huan , Haack Monika , Surette Renata , Jabri Ingy , Mullington Janet
Published: June 2021

Recent epidemiological studies demonstrate a consistent association between short sleep (i.e. self‐reported) and higher risk of hypertension. While this association seems to be stronger in women compared to men, the evidence from laboratory‐controlled studies is lacking. Thirty‐four healthy subjects (age 32±2 yrs; BMI 24±1 kg/m2; 18 men and 16 women) participated in a 22‐day in hospital study. Following 3 nights of adaptation sleep of 8h/night, subjects were randomly assigned to 4 cycles of repeated sleep restriction (4h of sleep/night for 3 nights followed by a night of 8h of sleep, repeated 4 times), or an 8h sleep control group. Blood pressure (BP) and heart rate (HR) were measured every morning at 8:30am and evening at 10pm in a seated position for 5 times over a 15min period. Morning BPs were not significantly different between sleep restriction and control groups in men or women (p>0.05). In contrast, women had significantly increased evening BP (i.e. up to 10±2mmHg in systolic BP, 5±2mmHg in diastolic BP) on the sleep restriction days compared to control (p=0.003 and 0.023 for systolic and diastolic BP, respectively). Moreover, repeated sleep restriction induced an increase in morning HR compared to control in women (p=0.005). Women show a pronounced pressor response to repeated challenges of sleep restriction, in a well‐controlled study.

Grant Funding Source: Supported by NIH (HL‐106782)
Solubleform of (pro)renin receptor is excreted from mesothelial cells to peritoneal dialysis effluent of peritoneal dialysis patients (1173.5)
Authors: Oba Ikuko , Mori Takefumi , Takahashi Chika , Ohsaki Yusuke , Sato Emiko , Koizumi Kenji , Furusho Masahide , Chida Makiko , Naganuma Eri , Ito Sadayoshi
Published: June 2021

Renin angiotensin system (RAS) is associated with peritoneal injury in peritoneal dialysis (PD). Angiotensin II dependent and independent role of (pro) renin receptor ((P)RR) to the cellular injury has been demonstrated. Thus, the present study determined the expression of (P)RR in drained PD effluent (PDE) and human peritoneal mesothelial cells (HPMCs) of the PD patients.

PDE drained after 2.5% glucose PD solution during peritoneal equivalent test (0hours, 2hours, 4hours) was collected from PD patients. Human soluble from of (P)RR level was measured by ELISA. The expression of (P)RR on cultured HPMCs and media isolated from PDE were investigated by Western blot analysis and/or flow cytometry. HPMCs was specified by cytokeratin positive cells in flow cytometry. Full length or soluble (P)RR was identified by the molecular weight compared to the full length and soluble (P)RR synthesized in vitro from the expression vectors.

Soluble (P)RR was detected in both plasma and PDE of PD patients. Time dependent increase in soluble (P)RR was observed in PDE. Expression of (P)RR was observed in cytokeratin positive mesothelial cells. Full length (P)RR was detected in HPMCs while soluble from of (P)RR was detected in concentrated cultured medium.

These results indicate that full length (P)RR is expressed in HPMCs and excrete soluble (P)RR in drained PDE. The role of full length (P)RR in HPMCs and soluble (P)RR in plasma and PDE of PD patients requires further investigation.
Cardiomyocyte differentiation of mouse iPSCs regulated by polypeptide multilayer films (1180.21)
Authors: Fu Pei‐Wen , Wang Shih‐Ying , Chen Yu‐Ru , Lo Chun‐Min
Published: June 2021

Polyelectrolyte multilayer film is an emerging method for substrate coating to modulate cellular behaviors. This material allows for modulation of thickness, stiffness, and adhesiveness. In this study, mouse induced pluripotent stem cells (iPSCs) were cultured on polypeptide multilayer films built up by the alternate deposition of cationic poly‐L‐lysine and anionic poly‐L‐glutamic acid, cross‐linked with EDC/sulfo‐NHS, and coated with gelatin. The characteristics of film thickness, roughness, and shear modulus were measured by AFM and QCM. Cardiomyocyte differentiation was evaluated by RT‐PCR analysis and immunostaining of cardiac markers, and cardiomyocyte beating was monitored by polypeptide multilayer‐coated electrodes using impedance sensing technique. The results showed that mouse iPSC colonies demonstrated better cell spreading and differentiation on even number layer of films, where the top layers are poly‐L‐glutamic acid films. In addition, the cardiomyocyte differentiation of mouse iPSCs showed a significant increase in the differentiation rate when higher concentration of EDC/sulfo‐NHS cross‐linkers was used to form the films. Our observations indicate a possible way to promote iPSCs toward cardiomyocyte differentiation on polypeptide multilayer films by changing the outermost polypeptide layer, the concentration of cross‐linkers, and the number of layers in the film.

Grant Funding Source: Supported by the National Science Council in Taiwan
Pressure applied in vitro increases proliferation of renal proximal tubular cells (1180.7)
Authors: Diaz Bianca , Chen Jie , Gonzalez Juana , Poppas Dix , Felsen Diane
Published: June 2021

Increased pressure in the kidney is a common finding in kidneys with obstructed ureters [UO]. In animal models of UO, increased cellular proliferation may contribute to renal damage and/or repair. We investigated the direct effect of pressure on proliferation of renal proximal tubular cells in vitro. Human HKC‐8 or rat NRK‐52E proximal tubule cells were grown at either ambient pressure, or at pressures of 60 or 90 mm Hg for 24 or 48 hr. Cells were grown in complete medium [CM; DMEM with 10% FBS] or in starvation media [SM; DMEM]. At 24 or 48 hr, cells were lysed and counted using the Cyquant assay. Western blot for proliferating cell nuclear antigen [PCNA] was determined, as were cell cycle measurements using flow cytometry. Blood gases were determined in an iSTAT system. HKC‐8 grown in CM for 48 hr at 60 mm Hg, demonstrated a 16.5 ± 5.8% increase in cell number compared to cells grown at ambient pressure. After 48 hr of 60 mm Hg pressure in SM, there was a 39.2 ± 5.1 % increase in cell number. HKC‐8 grown at 90 mm Hg in SM also demonstrated a significant increase in cell number. Similar results were found with NRK‐52E. Western blot confirmed increased expression of PCNA in pressurized cells. Cell cycle measurements demonstrated an increase in HKC‐8 in S phase, with a corresponding decrease in cells in G1. Pressure did not significantly affect pH, PCO2 or PO2 of either CM or SM. We previously showed [AJP Renal 297:F114, 2009] that pressure applied to HKC‐8 cells induces cytokine signaling. Here we demonstrate direct transduction of this mechanical stimulus into a proliferative response in HKC‐8 and NRK‐52E cells, measured by cell number, PCNA or increase in cells in S phase. Effects on proliferation were more pronounced in SM, suggesting that pressure may be able to [re‐]activate pathways normally turned off during starvation. Understanding the signaling pathways activated by pressure should lead to a better understanding of how pressure contributes to renal damage in UO, or in other diseases characterized by high pressure.

Grant Funding Source: supported by CTSA, RUCCTS Grant#8 UL1 TR000043 Natl Ctr for Advancing Translational Sciences
Inhibition of cAMP stimulated VAMP2 trafficking in renal epithelial cells by small molecule inhibitors (1181.13)
Authors: Nahle Lana , Caceres Paulo , Ortiz Pablo
Published: June 2021

cAMP stimulates exocytosis in many cell types in the kidney. In juxtaglomerular cells, cAMP stimulates renin exocytosis. In thick ascending limbs and collecting ducts, cAMP stimulates exocytic delivery of NKCC2 and aquaporin 2, respectively. The mechanism by which this occurs includes the vesicle associated membrane protein VAMP2. New compounds aimed at inhibiting VAMP2‐mediated exocytosis could be useful to target these renal processes. We hypothesized that delphinidin, a small molecule that binds VAMP2, and 4‐AD, an inhibitor of aquaporin 2 trafficking, block cAMP‐stimulated VAMP2 trafficking to the surface in renal epithelial cells. We expressed C‐terminus GFP tagged VAMP2 (GFP faces vesicle lumen or surface) in LLC‐PK1 cells. Confocal imaging showed that in polarized cells, VAMP2‐GFP localized to the apical surface, primarily in small clusters. We then measured cAMP‐stimulated surface VAMP2 delivery by surface biotinylation. We used forskolin/IBMX to maximally stimulate cAMP. In vehicle treated cells cAMP enhanced surface VAMP2‐GFP by 173±57% in 8 min (p<0.02). In cells pre‐treated with delphinidin chloride (50 µM) cAMP did not increase surface VAMP2‐GFP (baseline= 100, cAMP= 95±8%). Similarly, when cells were pretreated with 4‐AD (40 µM), cAMP failed to increase surface VAMP2‐GFP (baseline= 100, cAMP=108±14%). We concluded that cAMP stimulates VAMP2‐GFP trafficking to the surface in renal epithelial cells, most likely as part of the exocytic process. The small molecule delphinidin, and the V‐ATPase inhibitor 4‐AD block cAMP‐stimulated VAMP2 trafficking.

Grant Funding Source: Supported by NIH, AHA
Ion channel modulation of chondrocyte cell volume regulation (1182.1)
Authors: Lewis Rebecca , Barrett‐Jolley Richard
Published: June 2021

Healthy chondrocytes exist with depolarised resting membrane potentials (Vm); critical for volume regulation. Chondrocytes express several cell volume regulation channels, including Transient Receptor Potential (TRP) V4, V5 and V6 and epithelial sodium channels. We investigated mechanisms of cell volume regulation in healthy chondrocytes. Patch clamp electrophysiology was used to measure canine chondrocyte Vm (Vm=‐12±3mV; n=21). Hypotonic challenge caused a significant Vm hyperpolarisation of 11±1mV (n=9; p<0.05). We hypothesised this was due to Ca2+ influx activating a Ca2+‐activated potassium channel (KCa). Studies propose TRPV4 as a possible component of osmotically‐activated Ca2+ influx so we investigated this channel. However, the TRPV4 inhibitor, RN1734, failed to prevent hypotonicity‐induced hyperpolarisation (ΔVm=‐19±4mV, n=4; p<0.05). Application of a TRPV4 agonist, 4αPDD, caused no significant change in Vm (n=12), whole cell current (n=7) or cell volume (n=8). Furthermore, application of iberiotoxin or 2‐APB completely abolished the hypotonicity‐induced hyperpolarisation. This leads us to conclude that Ca2+ increase in response to hypotonicity is likely to result from an, as yet, unidentified range of ion channels and/or Ca2+ stores and the KCa channel responsible for the hyperpolarisation is a large‐KCa channel.

Grant Funding Source: Supported by European Union Seventh Framework Programme(FP7/2007‐2013) under grant agreement 305815
Molecular pathways for water fluxes in mouse choroid plexus epithelial cells (1182.3)
Authors: Cha David , Crum Jeannine , Alvarez‐Leefmans Francisco
Published: June 2021

Regulation and maintenance of cerebrospinal fluid (CSF) composition is crucial for brain function. Choroid plexus epithelial cells (CPECs) secrete most of the CSF and regulate its ion and water composition. However, the mechanisms of secretion and absorption of water across choroid plexus epithelium (CPE) are poorly understood. Secretion of CSF entails net water movement across CPECs from the basolateral membrane (blood‐facing) through the apical membrane (CSF‐facing). Water fluxes across cell membranes occur through aquaporin channels and cotransporters like the Na+‐K+‐2Cl‐ cotransporter 1 (NKCC1). Both NKCC1 and aquaporin‐1 (AQP1) are expressed in the CPECs apical membrane but are undetectable in the basolateral membrane. The inward water flux across the basolateral membrane of CPECs may be explained by the expression of other members of the gene families of AQPs and/or the cation‐coupled chloride cotransporters (slc12a). Using RT‐PCR, we detected transcripts for several AQPs in whole choroid plexus tissue. Transcripts for AQPs 1, 4, 7, but not 9, have so far been found. Immunolabeling studies in process will reveal the location of these proteins in the various components of the choroid plexus. Explaining the mechanisms of CSF production and absorption is key for understanding pathologies like hydrocephalous and increased intracranial pressure that cause brain ischemia and/or edema.

Grant Funding Source: Supported by 226119 Seed Grant Proteomics Program 2012 Boonshoft School of Medicine
In‐cell cross‐linking and identification of the nuclear actor of activated T‐cells 5 protein interactome using the mass spectrometry‐cleavable covalent cross‐linker DC4 (1182.7)
Authors: Ramkissoon Kevin , DuMond Jenna , Izumi Yuichiro , Williams Chester , Gucek Marjan , Wang Guanghui , Burg Maurice , Ferraris Joan
Published: June 2021

The Nuclear Factor of Activated T‐cells 5 (NFAT5) transcription factor undergoes hypertonicity‐induced nuclear localization and regulation of the transcription of osmoprotective genes. Its structure, function, compartmentalization, DNA‐, and protein‐protein interactions are influenced by the various cellular proteins that interact with it, including a number of kinases and phosphatases. Prior in vitro NFAT5 studies and ongoing in vitro peptide affinity chromatography indicate that NFAT5 is involved in a large number of protein‐protein interactions. To validate these in vitro results and to ascertain which interactions are physiologically relevant, we performed in‐cell protein crosslinking experiments. We used the collision induced dissociation (CID)‐cleavable cross linker DC4 in NFAT5‐transfected HEK 293 cells. Protein crosslinking was followed by column and/or gel purification of NFAT5 complexes, protease digestion, LC‐MS‐MS and protein identification analyses. The proteins identified by in‐cell analysis were cross‐referenced with the results of our in vitro experiments to help gain domain‐specific information on NFAT5 protein interactions.

Grant Funding Source: Supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute