bluuebunny/crossref_metadata_2025 huggingface dataset:
p70S6K signaling induces load‐mediated myofiber hypertrophy in humans in an mTOR‐ and RPS6‐indedpendent manner
Authors: Mayhew David , Roth Christopher , Cross James , Bamman Marcas
Published: June 2021

We studied translation initiation signaling in two clusters of humans on the extremes of myofiber hypertrophy capacity during resistance training (RT). Sixty‐six subjects underwent 16 wk of RT with muscle biopsies at baseline (T1) and 24 h after the first (T2) and last (T3) bouts. K‐means cluster analysis on changes in mean fiber area yielded 3 response clusters: non‐ (NR, n = 17), modest (MR, +1111 μm2, n = 32), and extreme (XR, +2475 μm2, n = 17) responders. Signaling was assessed at T1 and T2 by Western blot in 14 NR and 12 XR in order to associate the response at T2 with hypertrophy at T3. Only XR increased p(T421/S424)‐p70S6K; however, p(S2481)‐, p(S2448)‐, and total mTOR, and p(S240/244)‐RPS6 increased in both groups similarly. Because, unlike p‐p70S6K, p‐mTOR and p‐RPS6 increased irrespective of hypertrophy cluster, we suggest 1) p70S6K did not mediate the increase in p‐RPS6; 2) the primary target of mTOR was likely RPS6, presumably through S6K2; and 3) increased p‐mTOR and p‐RPS6 alone were insufficient to induce hypertrophy. Therefore, targets of p70S6K other than RPS6 may have mediated the observed p70S6K‐induced myofiber hypertrophy. Other putative regulators of p70S6K function, such as p(T202/Y204)‐Erk1/2, p(T180/Y182)‐p38, p(S241)‐PDK‐1, p(Y397)‐FAK, and ILK, were not altered from baseline in NR or XR. Support: R01 AG017896 and VA Merit.
Nerve‐derived agrin cooperates with neural activity to potentiate translational control of skeletal muscle growth via p70 S6K
Authors: Michel Robin Nicholas , Kulig Ewa M.
Published: June 2021

The trophic mechanisms by which motor nerves impart growth of skeletal muscle targets are rudimentary. We have shown that muscles exposed to increased neural activation and contractile loading fail to hypertrophy if not provided with suitable trophic chemical support from their motor nerves. When trophically‐challenged and overloaded muscles are treated with recombinant neural agrin, hypertrophic growth is rescued via p70 S6K signaling. Agrin therapy induced extensive phosphorylation of p70 S6K, but not via Akt or PDK‐1 as upstream modulators, suggesting neural agrin binding to its receptor activates p70 S6K, either directly, or indirectly via other yet to be identified signaling elements. We also assessed the contribution of neural activity and nerve trophic factors on p70 S6K phosphorylation in muscles either denervated or paralyzed by TTX‐inactivation of the sciatic nerve. We found p70 S6K to be fully dephosphorylated after denervation but not after TTX, suggesting nerve trophic factors to be major modulators of this signaling pathway. Considering muscle growth is regulated in part by translational mechanisms downstream of Akt/mTOR and by transcriptional mechanisms associated with muscle activation, our data suggest agrin may cooperate with these signaling pathways to converge upon p70 S6K to potentiate translational control of muscle growth. Supported by NSERC and the CRC to RNM.
INTERMITTENT HYPOXIA IN RATS INHIBITS ENDOTHELIUM‐DEPENDENT VASODILATION
Authors: Youngblood Victoria Marie , Kanagy Nancy L.
Published: June 2021

Rats exposed to eucapnic intermittent hypoxia (E‐IH) to mimic sleep apnea have elevated mean arterial pressure (MAP) and increased vascular production of superoxide anion (O2−). We hypothesized that increased arterial O2− in rats exposed to E‐IH quenches NO to inhibit acetylcholine‐induced (ACh) dilation. To test this hypothesis, aortas were dissected from rats exposed to E‐IH or air cycling (Sham) for 14 days. Aortic segments were suspended in tissue baths containing physiological saline solution in the presence of the superoxide dismutase mimetic, tiron (10 μM), the eNOS inhibitor, nitro‐L‐arginine (L‐NNA, 100 μM) or vehicle. Increasing concentrations of ACh were added to endothelium‐intact aortic rings constricted to 50% of maximal tension with phenylephrine. ACh‐induced dilation was diminished in E‐IH aortas compared to Sham (75.5±4.0 vs 97.1±1.5, p<0.05). L‐NNA diminished dilation in both groups but dilation in E‐IH arteries was still less than that in Sham arteries (16.06±7.7 vs. 41.5±11.4, p <0.05). Tiron treatment appeared to increase ACh‐induced dilation in E‐IH aortic segments but had no effect on dilation in Sham segments. Consistent with our hypothesis, E‐IH appears to cause endothelial dysfunction that may be caused in part by loss of a non‐NOS dilator and in part by increased O2− scavenging of NO.
Mechanisms of Mitochondrial Complex 1 Inhibition by Intermittent Hypoxia
Authors: Khan Shakil A , Nanduri Jayasri , Kumar Ganesh K , Prabhakar Nanduri R
Published: June 2021

We previously reported that intermittent hypoxia (IH) inhibits the mitochondrial complex I but not the complex III. IH has been shown to activate NADPH oxidases. The present study tests the hypothesis that activation of NADPH oxidases and the resulting increase in reactive oxygen species (ROS) mediate complex I inhibition by IH. To test this possibility, experiments were performed on rat PC12 cells exposed to IH or normoxia. IH increased NADPH oxidase activity in a stimulus‐dependent manner and the effects could be reversed following re‐oxygenation for 3h. Apocynin (1mM), an inhibitor of NADPH oxidase as well as ROS scavengers prevented IH‐evoked inhibition of complex I. ROS are known to mobilize intracellular Ca2+. 2APB (75μM; inhibitor of IP3 receptors) but not nifedipine (3μM; L‐type calcium channel blocker) restored IH‐evoked complex I inhibition. Furthermore, ruthenium red (20μM; inhibitor of mitochondrial Ca2+ uniporter) or CGP37157 (10μM; inhibitor of mitochondrial Na+/Ca2+ exchanger) prevented complex I inhibition by IH. Taken together these observations suggest that ROS generated by NADPH oxidases mobilize Ca2+ flux from cytosol to mitochondria during IH resulting in inhibition of complex I. (Supported by NIH HL‐25830).
ROLE OF CAROTID BODIES IN CHRONIC INTERMITTENT HYPOXIA‐EVOKED AUGMENTED LTF OF PHRENIC NERVE ACTIVITY
Authors: Peng Ying‐Jie , Prabhakar Nanduri R
Published: June 2021

Previous studies have shown that chronic intermittent hypoxia (CIH) induces sensory long‐term facilitation (sLTF) of the carotid body and augments LTF of phrenic nerve activity (pLTF). The present study was designed to examine whether sLTF plays a role in CIH‐induced potentiation of pLTF. Experiments were performed on male adult rats exposed to 10 days of normoxia or CIH. The effects of repetitive hypoxia (10 episodes of 15s hypoxia followed by 5min normoxia) were examined on efferent phrenic nerve activity and/or carotid body sensory activity in anesthetized, vagotomized, paralyzed, and mechanically ventilated rats. In the first series of experiments on CIH exposed rats (n=5), carotid body sensory activity was recorded along with efferent phrenic nerve activity. Repetitive hypoxia evoked robust pLTF and time course of the response paralled with carotid body sLTF. In the second series of experiments (n=6–8 rats each group), pLTF was examined in control, and CIH exposed rats with and without carotid body innervation. The magnitude of pLTF was significantly greater in CIH compared with control rats (p<0.05). Bilateral sectioning of the carotid sinus nerves immediately after terminating repetitive hypoxia attenuated pLTF in CIH rats (p<0.05). These results suggest that the carotid body sLTF contributes to augmented pLTF in CIH exposed rats. (Supported by NIH‐HL‐25830).
Endurance enhancement activities of CordyMax‐5 in mice weight‐loaded swimming model
Authors: Chen Wei , Yao Yisang , Zhang Lijuan , Gao Ling , Lu Jihong , Zhu Jia‐Shi
Published: June 2021

CordyMax (CM), a fermented product of Cordyceps sinensis for anti‐aging and disease rehabilitation, has shown anti‐fatigue and endurance enhancement activates (FASEB J 16:A628, 2002; 18:A931, 2004). We also showed the enhanced muscle contractility and anti‐fatigue proprieties of CM5 and exercise metabolic and cardiovascular benefits in elite athletes (FASEB J 21:A939, 2007; Chin J Clin Pharm 16:299–302, 2007). We further examined the endurance enhancement activity of CM5 in mice with use of a weight‐loaded swimming protocol. CM5 was given to mice at doses of 0.6, 1.2 and 1.8 g/kg by gavage (n=13 each). Swimming time to exhaust was recorded as a measure of endurance capability. After 30‐day oral supplementation, mice received CM5 appeared to swim longer than placebo controls with a possible dose‐dependent response. Particularly, mice in the 1.8 g/kg group swam 5 times longer (p<0.001). In conclusion, we demonstrate possible endurance enhancement activities of CM5 in this study, indicating potential anti‐fatigue benefits.
Prevention of Emphysema‐Induced Reductions in Diaphragm Muscle Mass
Authors: Mattson John Paul , Hinkle Richard T , Isfort Robert J , Ferreira Leonardo F , Musch Timothy I , Poole David C
Published: June 2021

Chronic hypercapnic COPD patients have an unfavorable prognosis. Respiratory muscle atrophy and weakness may lead to CO2 retention and ultimately respiratory failure. Therefore, the purpose of this investigation was to determine whether PG‐873637, a corticotrophin‐releasing factor 2 receptor‐selective agonist, would ameliorate diaphragm atrophy and/or induce hypertrophy.

To establish the time course of emphysema induced diaphragmatic atrophy, costal diaphragm mass was measured at 4 and 8 months following saline (control) or elastase (emphysema) instillation in hamsters. Subsequently, after demonstration of atrophy, control and emphysema hamsters were treated with either saline (vehicle) or PG‐873637 (treatment) for 1 or 5 months prior to sacrifice.

Excised lung volume increased (>140%) with emphysema at all time points. PG‐873637 treatment increased diaphragm muscle mass at both time points in emphysema animals (1‐month = 13% and 5‐month = 12%). These data demonstrate that PG‐873637 is efficacious as a treatment to preserve mass or induce hypertrophy in the emphysematous diaphragm.
Intracellular Critical PO2 Measured in Human Skeletal Muscle In Vivo
Authors: Lanza Ian R , Befroy Douglas E , Tevald Michael A , Kent‐Braun Jane A
Published: June 2021

Under some conditions, intracellular oxygen tension (PO2) may decrease to the point where oxygen availability limits respiration. Information about this critical PO2 and its determinants in vivo is sparse. PURPOSE: To determine the critical PO2 of human skeletal muscle, and its relationship to cellular energy consumption. METHODS: Interleaved proton and phosphorus magnetic resonance spectroscopy were used to quantify deoxymyoglobin and phosphorous metabolites in the tibialis anterior muscle of 6 subjects during 15 minutes of leg ischemia. Critical PO2 was determined as the PO2 at the phosphocreatine inflection point during ischemia. The rate of ATP consumption was determined from the linear slope of PCr depletion following the inflection point. RESULTS: The time to PCr inflection point was 282 ± 112 s (mean±SE). The corresponding critical PO2 was 0.47±0.27 torr, with a range of 0.21–0.96. PO2crit was not associated with the resting rate of ATP consumption (r =0.17), but was directly correlated to the rate of myoglobin desaturation (r =0.93) and inversely correlated to the time required to reach the PCr inflection point (r = −0.74). CONCLUSION: These data suggest that the relatively wide range of PO2crit in these subjects is attributable to inter‐subject differences in the ability of the muscle to maintain ATP production in the face of an ischemic insult.

Support NIH AG21094, K02 AG023582
TNF induces atrogin‐1 expression via AKT‐independent activation of Foxo4
Authors: Moylan Jennifer Stevenson , Smith Jeffrey D , Smith Melissa A , McLoughlin Thomas J , Reid Michael B
Published: June 2021

Atrophy of murine skeletal muscle during starvation is regulated by atrogin‐1, a muscle‐specific ubiquitin ligase. Starvation decreases AKT activity which removes inhibition of the forkhead transcription factors Foxo1 and 3a and thereby stimulates atrogin‐1 gene expression. Atrogin‐1 is also regulated by tumor necrosis factor (TNF). We tested the hypothesis that TNF increases atrogin‐1 via AKT and Foxo1/3a. Data from C2C12 myotubes indicate that the AKT/Foxo1/atrogin‐1 pathway is intact in these cells. Wortmannin inhibits AKT phosphorylation, increasing atrogin‐1 mRNA. Overexpression of Foxo1 also increases atrogin‐1 mRNA. Paradoxically, TNF increases phospho‐AKT as it increases atrogin‐1 mRNA. Even in the presence of insulin‐like growth factor stimulated AKT, TNF retains the ability to increase atrogin‐1. In addition, TNF is unable to induce nuclear translocation of Foxo1/3a. Instead, TNF increases protein levels and stimulates nuclear translocation of Foxo4, an alternate Foxo isoform. This isoform appears to promote TNF‐induced atrogin‐1 expression since Foxo4 siRNA partially inhibits the TNF effects. In conclusion, we propose that TNF activates atrogin‐1 expression independent of AKT via Foxo4. These results suggest a mechanism by which inflammatory catabolic states persist in the presence of adequate growth factors and nutrition. Supported by NIH grant HL59878 (MBR).
O‐GlcNacylation of phospholamban induces cardiac dysfunction
Authors: Asahi Michio , Yokoe Shunichi , Suzuki Keiichiro , Taniguchi Naoyuki , Miyoshi Eiji
Published: June 2021

Cardiac type sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA2a) plays a major role in cardiac muscle contractility. Phospholamban (PLN) regulates the function via its Ser16‐phosphorylation. Since it has been proposed that the Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O‐linked N‐acetylglucosamine (O‐GlcNAc), we examined the effect of O‐GlcNAc modification (O‐GlcNAcylation) on the function of PLN. In rat adult cardiomyocytes treated with O‐(2‐acetamid‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate (PUGNAc), O‐GlcNAcylation of PLN was significantly increased compared to non‐treated cardiomyocytes. Simultaneously, Ser16‐phosphorylation and pentamer formation of PLN were reduced. The same results were observed in heart homogenates from diabetic rats. The measurement of SERCA activity showed that the inhibitory function of PLN was increased in PUGNAc‐treated cardiomyocytes, resulting in a lower peak and a prolonged relaxation time in mechanical properties and a lower peak and a prolonged decay time in the profiles of calcium transient. These data suggest that PLN is O‐GlcNAcylated, resulting in the deterioration of cardiac muscle function. This might define a novel mechanism by which PLN regulation of SERCA2a is altered under conditions where O‐GlcNAcylation is increased, such as those occurring in diabetes.
Electrical stimulation activates myocyte mitochondrial MMP
Authors: Metreveli Naira , Moshal Karni S , Tyagi Suresh C
Published: June 2021

Cardiac arrhythmias, instigated by mechanical and electrical remodeling, are associated with activation of extracellular matrix metalloproteinase (MMP). However the connection between intracellular MMPs activation and arrhythmogenesis is not well established. We tested the hypothesis that electrical pacing induces the activation of mitochondrial MMP (mtMMP) and causes myocyte mechanical dysfunction. Myocytes were isolated and field stimulated at 1 and 4 Hz. Myocyte mechanics and calcium transient was studied using IonOptix system. Localization of MMP‐9 in the mitochondria was determined by confocal microscopy. Mitochondrial MMP‐9 activation was evaluated using zymography. We observed an increase in mtMMP‐9 activation with increase in electrical pacing compared to control (Fig. 1). Field stimulation at 4 Hz decreased cell shortening, maximal rate of relaxation (+dL/dt) and contraction (‐dL/dt) compared to 1 Hz and control group. Furthermore, field stimulation at 4 Hz increased the rate of calcium decay compared to 1 Hz and control. We conclude that electrical stimulation activates mtMMP‐9 and causes myocyte mechanical dysfunction. Study was supported by AHA grant to KSM and NIH to SCT. NM and KSM contributed equally.
Rho‐kinase‐mediated suppression of KDR in cerebral arteries requires an intact actin cytoskeleton
Authors: Luykenaar Kevin D , Welsh Donald G
Published: June 2021

This study examined the role of the actin cytoskeleton in Rho‐kinase‐mediated suppression of the delayed rectifier K+ (KDR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated and whole cell KDR currents monitored using conventional patch clamp electrophysiology. At +40 mV, the KDR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by uridine triphosphate (UTP; 50 μM). Consistent with past observations, KDR suppression was blocked by the Rho‐kinase inhibitor Y‐27632 (30 μM). Likewise, disruption of the actin cytoskeleton using cytochalasin D (10 μM) or latrunculin A (10 nM) abolished the ability of UTP to suppress KDR. Interestingly, these agents inhibited the current when used at higher concentrations, further suggesting a mechanistic link between the actin cytoskeleton and channels underlying the KDR current. These preliminary findings suggest that an intact cytoskeletal framework is a requisite for Rho‐kinase‐mediated suppression of KDR current and the subsequent depolarization and constriction of cerebral arteries. Supported by CIHR and HSFC.
Mechanically Stretching Microvascular Smooth Muscle Cells (VSMC) Induces Three‐dimensional Reorganization of Vinculin in Focal Adhesions
Authors: Sun Zhe , Li Zhaohui , Huang Shaoxing , Meininger Gerald A.
Published: June 2021

Focal adhesions (FA) are hypothesized to mediate cell mechanotransduction by forming a conduit linking the cytoskeleton with extracellular matrix. This enables transmission of force and biochemical signals across the cell membrane. In order to gain insight into the nature of force transmission within and across the cell, a hybrid system combining confocal and atomic force microscopy (AFM) was used to apply pulling force on the apical cell surface while simultaneously monitoring FA changes at the cell substrate interface. VSMCs were isolated from rat skeletal muscle arterioles (70~100 μm Dia), and were transfected with GFP‐tagged vinculin. The tips of AFM probes were fused with a FN‐coated 5 μm bead. The bead was placed on the VSMC surface (20 min) to form an integrin‐mediated cell adhesion. Controlled pulling force was applied through the bead adhesion site by AFM and the GFP‐vinculin signal at FAs was recorded in time‐lapse by confocal microscopy. Collectively, FAs on any given cell were found to either increase, decrease or remain unchanged in size in response to the AFM pulling force. Also, the directionality of FA movement varied with respect to the site of AFM pulling. These data suggests the transmission of force from the apical surface to the lower cell surface is complex. Further analyses will require knowledge of actin distribution and the forces experienced at individual FA sites. (Supported by NIH HL‐062863 to GAM)
Orai1 expression and function in vascular smooth muscle cells
Authors: Li Jing , Jairaman Amit , Sukumar Piruthivi , Porter Karen , Beech David
Published: June 2021

Recent studies suggest TRPC5 is a component of calcium‐entry channels that help drive vascular smooth muscle cell migration (Xu et al 2006, Circ Res 98, 1381–9). Here we investigated another type of channel protein (Orai1) which is distinct from the TRP proteins and has been suggested to have pivotal roles in calcium‐entry of lymphocytes (Hogan & Rao 2007, Trends Biochem Sci 32, 235–45). Our experiments focused on proliferating vascular smooth muscle cells of the human saphenous vein. Quantitative RT‐PCR revealed high levels of mRNA encoding Orai1 and its homologues Orai2 and Orai3. Calcium‐entry was measured in the cells after store‐depletion with thapsigargin and using fura‐2 indicator dye on a real‐time 96‐well plate reader. RNAi knock‐down of Orai1 suppressed calcium‐entry, while knock‐down of Orai2 or Orai3 had no effect. Functional relevance was studied using modified Boyden chamber assays to quantify cell migration (chemotaxis) and invasion (through basement membrane matrix) in the absence of store‐depletion. Knock‐down of Orai1 markedly suppressed migration and invasion. Invasion is facilitated by secretion of matrix metalloproteinases (eg MMP‐2) but knock‐down of Orai1 had no significant effect on secretion of MMP‐2. The data suggest Orai1 is a novel calcium channel protein of vascular smooth muscle cells with important roles in cell motility.

Supported by the British Heart Foundation and Wellcome Trust.
Increased Cytochrome P450 2C9 signaling does not contribute to vascular endothelial dysfunction in healthy older adults
Authors: Jablonski Kristen L , Gano Lindsey B , Lawson Brooke R , Pierce Gary L , Eskurza Iratxe , Seals Douglas R , Donato Anthony J
Published: June 2021

Oxidative stress impairs endothelium‐dependent vasodilation (EDV) with aging in healthy sedentary adults. Increased cytochrome P450 2C9 (CYP450) signaling can contribute to oxidative stress‐mediated suppression of EDV, but its role in aging is unknown. We hypothesized that inhibition of CYP450 signaling with Sulfaphenazole (Sulfa) would improve EDV in older, but not young, healthy sedentary adults. At baseline, the increases in forearm blood flow (FBF; venous occlusion plethysmography) in response to incremental brachial artery infusions of acetylcholine (ACh; 1, 2, 4, and 8 μg 100mL forearm volume min−1), an endothelium‐dependent dilator, were smaller in older (n=16, 61±1 yr; mean±SE) vs. young (n=8; 22±1 yr) adults (P<0.05), with a reduction in peak FBF of 38% (10.4±0.97 vs.16.7±2.3 mL 100mL tissue−1min−1). Co‐infusion of ACh with Sulfa at doses previously shown to block CYP450 signaling in humans did not significantly affect the FBF responses to ACh in either the older (peak FBF: 11.8±3.9 mL tissue−1min−1, P>0.05) or the young (peak FBF: 16.8±2.0 mL 100mL tissue−1min−1, P>0.05) group. Endothelium‐independent vasodilation, assessed with sodium nitroprusside, was similar in the young and older subjects and was not affected by Sulfa co‐infusion (P>0.05). These results indicate that increased CYP450 signaling does not contribute to impairments in EDV with aging in healthy adults.
The involvement of norepinephrine, neuropeptide Y and nitric oxide in the cutaneous vasodilator response to slow local heating
Authors: Hodges Gary J , Kosiba Wojciech A , Zhao Kun , Johnson John M
Published: June 2021

Adrenergic blockade abolishes the axon reflex (AR) during slow local heating and reduces the final vasodilator response. We tested whether the neurotransmitters, norepinephrine (NE) and/or neuropeptide Y (NPY) were involved and whether they acted via nitric oxide synthase (NOS). In 7 subjects, 4 forearm sites were instrumented with microdialysis fibers, local heaters and laser‐Doppler probes. Part 1: treatments were untreated control, propranolol (PRO) and yohimbine (YOH) to antagonize a‐ and ß‐adrenergic receptors, BIBP to antagonize Y1‐receptors and combined PRO, YOH and BIBP. Sites were locally heated from 33 to 40°C over 70 min. PRO+YOH or BIBP raised the temperature at which AR occurred or abolished it. The combination treatment consistently eliminated it. Overall vasodilation at the PRO+YOH and BIBP sites was less than at the control site. To test whether NE and NPY were acting via NOS, treatments were: untreated control, bretylium (BT) to stop neurotransmitter release, L‐NAME to inhibit NOS and BT+L‐NAME. The AR was absent at all treated sites. The decrease of the final vasodilation was not different among sites. These data suggest that NE and NPY are important for the AR and achieving complete vasodilation. Additionally, vasoconstrictor nerve and NOS blockade does not have an increased inhibition, suggesting that NE and NPY promote vasodilation via an NO‐dependent mechanism.

Supported by NIH, HL‐059166
Anti‐inflammatory treatment restores endothelium‐dependent dilation in older mice by increasing nitric oxide bioavailability
Authors: Connell Melanie L , Durrant Jessica R , Folian Brian J , Russell Molly J , Seals Douglas R , Lesniewski Lisa A
Published: June 2021

Chronic low‐grade inflammation may contribute to impaired endothelium‐dependent dilation (EDD) with aging, but evidence is limited. To gain further insight, we studied isolated carotid arteries from young (Y, 4–7 mo) and older (O, 29–34 mo) B6D2F1 mice (n=6–8/group). Maximal carotid artery dilation to acetylcholine (ACh), an endothelium‐dependent dilator, was 24% lower in O (76±3 vs. 99±1%, mean±SE, P=0.001). Administration of the nitric oxide (NO) synthase inhibitor NG‐nitro‐L arginine methyl ester (L‐NAME) decreased ACh dilation less (P=0.02) in O (−20% to 56±8%) vs. Y (−51% to 48±8%). Treatment with the anti‐inflammatory compound sodium salicylate (200 mg/kg/d via diet for 5 d, n=5/group) resulted in similar ACh dilation (P=0.98) in O and Y mice before (O: 97±2 vs. Y: 99±1%) and after (O: 45±15 vs. Y: 43±9%) L‐NAME. Inhibition of superoxide with TEMPOL markedly improved EDD in untreated, but not in sodium salicylate treated O mice or in Y mice. Carotid artery dilation to the endothelium‐independent dilator sodium nitroprusside was not different in Y and O and was not affected by sodium salicylate (P=0.93). These preliminary results support the hypothesis that the development of vascular inflammation plays a key role in impaired EDD with aging in B6D2F1 mice through an oxidative stress‐mediated reduction in NO bioavailability. Supported by NIH AG006537, AG013038 and AG022241
High‐salt intake up‐regulation of AT1 and AT2 protein expression in the left ventricle is independent of blood pressure
Authors: Ferreira Daniele Nunes , Coelho Michella Soares , Oliveira Ivone Braga , Rosa Kaleizu T. , Heimann Joel C.
Published: June 2021

Objective:

To evaluate the mechanisms of cardiac hypertrophy (CH) induced by salt overload.

Methods:

Eighteen‐week‐old Wistar rats received a normal (NS: 1.3% NaCl), a high 1 (HS1 4%), a high 2 (HS2 8%) salt diet, or HS2+hydralazine (HZ). Tail cuff blood pressure (BP), myocyte transverse diameter (TD) and interstitial fibrosis (IF) were evaluated in the left (LV) and right ventricle (RV). The angiotensin II type 1 (AT1) and 2 (AT2) receptors, collagen I (COL 1) were measured in the LV by Western blot.

Results (mean±SEM – p<0.05, n=4–8/group):

BP (mmHg) was higher on HS1 (150±2) and HS2 (154±3) than on NS (123±2). BP on HS2+HZ (126±2) did not differ from NS. LV TD was higher on HS1 (15±0.2), HS2 (16±0.7) and HS2+HZ (15±0.2) compared to NS (13±0.6). Similar results were observed by echocardiographic LV mass determination. RV TD was higher on HS2 (16±0.7), HS2+HZ (17±0.4) than on NS (12±0.4). IF (%) and COL 1 (AU) were higher on HS1, HS2 than on HS2+HZ and NS (COL I: 11319±625, 9603±185, 3666±920, and 5653±134, respectively). IF did not differ among the four groups in the RV. AT1 and AT2 were higher on HS2, HS2+HZ than on NS (AT1: 19276±722, 20421±602, and 17392±338, respectively).

Conclusions:

High salt diet leads to CH independently from BP. IF in HS2 is due to a direct effect of salt overload and an indirect effect through a higher BP. It can be speculated that increased AT1 and AT2 may be associated with the mechanism of salt induced CH.

FAPESP
CNS blockade of aldosterone synthase prevents sympathetic hyperactivity and hypertension by central Na+ loading in Wistar rats
Authors: Huang Bing S. , Ahmad Monir , White Roselyn A. , Jeng Arco Y. , Leenen Frans H. H.
Published: June 2021

In Wistar rats increasing CSF [Na+] by intra‐cerebroventicular (icv) infusion of hypertonic saline induces sympathetic hyperactivity and hypertension, which are prevented by blockade of brain mineralocorticoid receptors (MR). We postulated that locally in CNS produced aldosterone (aldo) is involved. Wistar rats instrumented with icv cannula were chronically infused icv with artificial CSF (aCSF), Na+‐rich (0.8 M) aCSF, aCSF plus aldo synthase inhibitor FAD286 (100 μg/kg/d), or Na+‐rich aCSF plus FAD286, using osmotic minipumps. After 2 wks of infusion, resting BP, HR & renal sympathetic nerve activity (RSNA) in response to air‐stress, & baroreflex function as well as hypothalamic and plasma aldo and corticosterone (corti) were examined.

In another set of Wistar rats, via telemetry 24 h BP and HR were recorded 3 days before and 14 days after icv infusion of Na+‐rich aCSF with or without FAD286. Icv infusion caused sustained increases (~10 mmHg) in 24 h average MAP, which were absent in rats with FAD286 treatment. These data suggest that in Wistar rats a small increase in CSF [Na+] may increase biosynthesis of corticosterone and aldosterone in the hypothalamus, and mainly aldosterone activates CNS MR, leading to sympathetic hyperactivity and hypertension.

Supported by a grant from Canadian Institutes of Health Research.
Serum and glucocorticoid‐regulated kinase mediates hypertension and end organ damage in DOCA‐salt hypertension
Authors: Schnackenberg Christine G. , Costell Melissa H. , Hoang Bao , Duddy Graham , Willette Robert N.
Published: June 2021

Serum and glucocorticoid‐regulated kinase 1 (SGK1) plays an important role in mediating mineralocorticoid receptor stimulated sodium reabsorption. We have previously shown that SGK2 can compensate for renal sodium reabsorption in the absence of SGK1. Therefore, we tested the hypothesis that SGK1 and SGK2 contribute to the hypertension and end organ damage during DOCA‐salt hypertension. The systolic blood pressure, cardiac, and renal responses to DOCA‐salt hypertension or SHAM were determined in sgk1−/−, sgk2−/−double knockout (DKO, n = 20) and homozygous wildtype littermates (WT, n = 19). During baseline conditions DKO had lower body weight and systolic blood pressure, and higher water intake and heart rate compared to WT. After 11 weeks of DOCA‐salt hypertension, genetic ablation of SGK1 and SGK2 significantly attenuated the hypertension (192 ± 8 vs. 158 ± 6 mmHg), microalbuminuria (9.9 ± 2.1 vs. 5.0 ± 1.7 mg/day), left ventricular hypertophy, renal fibrosis, protein deposition, and glomerular hypertrophy (3.3 ± 0.2 vs. 1.7 ± 0.3 total renal score), and morbidity and mortality (6/11 vs. 1/9 deaths). Absence of functional SGK1 and SGK2 shifted the pressure‐natriuresis curve to the left and increased the slope, indicating improved renal function and reduced salt‐sensitivity. These results suggest that SGK1 and SGK2 contribute to the cardiovascular and renal pathophysiology of DOCA‐salt hypertension.
The Wilms Tumor Suppressor Protein (WT1) splice variants WT1(‐KTS) and WT1(+KTS) show distinct influences on different control levels of the Renin gene expression
Authors: Steege Andreas , Fähling Michael , Martinka Peter , Patzak Andreas , Brinkmeier Thomas , Persson Pontus B. , Scholz Holger , Mrowka Ralf
Published: June 2021

Clinical studies indicate that Renin gene expression is controlled by the WT1 splice variants WT1(‐KTS) and WT1(+KTS). We investigated the underlying mechanisms through which they influence the Renin gene expression. Over expression of both WT1 variants in human kidney cells decrease the Renin protein level. Reporter gene studies demonstrate that the WT1 splice variants modulate the Renin promoter activity either directly (+KTS form) or through interactions within a regulatory region [hRENc] (both variants). WT1(+KTS) has an activating influence mediated by the Renin promoter. The WT1(+KTS) variant is able to interact with the Renin mRNA‐UTRs in contrast to the WT1(‐KTS) form. We show that the Renin mRNA undergoes a translocation into a translational inactive compartment after over expression of the WT1(+KTS) variant. The posttranscriptional influence of WT1(+KTS) may be responsible for a translational arrest of Renin synthesis, which explain the decrease of the Renin protein following WT1(+KTS) over expression. Both variants of the Wilms Tumor Suppressor Protein inhibit the Renin synthesis through distinct and different mechanisms and thus may participate in a hyperreninism in patients suffering from Wilms Tumor.

Research support: Pontus B. Persson, Institut für Vegetative Physiologie, Charité‐Universitäts medizin Berlin, Tucholskystr. 2, 10117 Berlin, Germany
The PPARgamma Agonist Rosiglitazone Decreases Blood Pressure and Renal Injury in a Female Mouse Model of Systemic Lupus Erythematosus
Authors: Venegas‐Pont Marcia , Maric Christine , McLemore Gerald R. , Glover Porter , Ryan Michae J.
Published: June 2021

Women with the inflammatory autoimmune disorder systemic lupus erythematosus (SLE) exhibit a high incidence of hypertension and renal injury. Peroxisome proliferator activated receptor gamma (PPARγ) activation has anti‐inflammatory and anti‐hypertensive effects. We tested whether the PPARγ agonist rosiglitazone (Rosi) prevents hypertension and renal injury in a female mouse model of SLE (NZBWF1). 30 week old SLE and control (NZW/LacJ) mice (n≥ 7 per group) were fed either Rosi (5mg/kg/day in standard chow) or unsupplemented chow for 4 weeks. Renal injury was assessed using the glomerulosclerosis index (GSI) and a positive urinary albumin test ≥100 mg/dL, Albustix). MAP, measured by indwelling catheters, was increased in SLE mice compared to controls (139±4 vs. 112±4, p<0.05). Rosi treatment significantly lowered MAP in SLE mice (127±4, p<0.05) but had no effect in control mice (111±4 mmHg). Anti‐DNA auto‐antibodies, a marker for SLE, did not differ in vehicle or Rosi treated SLE mice (756±157 vs 762±251 ng/ml). Only 18% of Rosi treated SLE mice exhibited albuminuria compared to 42% of vehicle treated SLE mice. Mice with SLE had increased GSI compared with controls (1.9±0.6 vs 0.6±0.1, p<0.05). Preliminary data indicate that GSI is reduced in Rosi treated SLE mice (1.0±0.2) with no effect in control mice (0.7±0.1). These data implicate PPARγ as a potential therapeutic target for the treatment hypertension and renal end‐organ damage associated with SLE. (supported by R01HL085907 MJR)
Inhibition of FosB in the lamina terminalis (LT) blocks sustained hypertension in intermittent hypoxia (IH)
Authors: Cunningham J. Thomas , Toney Glenn M. , Hinojosa‐Laborde Carmen , Chakravarty Sumana , Nestler Eric J. , Mifflin Steven W.
Published: June 2021

FosB expression is increased in the LT after IH. To test the role of FosB in IH hypertension, rats were injected in the ventral LT (n = 6) with 400 nl of an adeno‐associated virus (AAV) vector expressing a dominant negative construct for FosB, (AAV‐ JunD) plus GFP, or a control construct expressing only GFP (n=4). All rats were instrumented with radio telemetry transmitters for continuous monitoring of blood pressure and heart rate. After a 7 day control period, rats were exposed to IH (3 min of periods of 21% O2 with 10% O2 for 8 h/day) for 7 days. At the end of IH, rats were anesthetized and perfusion fixed. Their brains were sectioned and processed for GFP fluorescence to verify the location of viral injections and to confirm expression of the AAV transgenes. During the control period, rats injected with the AAV constructs displayed blood pressures and respiratory rates that were comparable to untreated controls. When exposed to IH, both groups showed a normal increase in respiratory rate and blood pressure while being exposed to changes in O2. However, during the dark phase of the light dark cycle while breathing room air, rats injected with the FosB dominant null failed to show the sustained increase in blood pressure as seen in GFP‐construct injected controls. These findings suggest that the LT contributes to IH hypertension and that FosB may mediate changes in gene expression that contribute to IH hypertension. (HL62579 & HL41894)
The role of the renal sympathetic nerves in hypertension
Authors: Barrett Carolyn J , Malpas Simon C , McBryde Fiona D , Guild Sarah‐Jane
Published: June 2021

The development of hypertension is hypothesized to involve an inappropriate increase in renal sympathetic nerve activity (RSNA), and a concomitant blunting of the reflex response to blood volume changes. We tested this hypothesis using a rabbit model of hypertension, where hypertension was induced using a high salt diet coupled with an infusion of angiotensin II (20ng/kg/min). RSNA, arterial pressure and heart rate were measured via telemetry in rabbits living in their home cages continuously for a period of one week prior and 3 weeks after inducing the hypertension. The model of hypertension resulted in a slow increase in arterial pressure, with a mean increase in pressure of approximately 15mmHg. Over the first few days of angiotensin II infusion RSNA was suppressed, but as pressure was maintained RSNA increased to above baseline levels, thus nerve activity was inappropriately high for the given level of arterial pressure. In a second group of animals, using the same model of hypertension, we examined both the lumbar and renal sympathetic responses to a number of acute stimuli. While the lumbar and renal nerves responded similarly to rapid changes in arterial pressure, the responses to volume infusion were very different. Our results suggest that it is specifically RSNA control of blood volume control that is impaired in hypertension. Supported by the Health Research Council and Auckland Medical Research Foundation.
Mechansim of reduced vascular relaxation in aorta from Dahl salt‐sensitive rats on elevated dietary fat
Authors: Spradley Frank Travis , Kang Kyu‐Tae , Sullivan Jennifer C. , Pollock David M. , Pollock Jennifer S.
Published: June 2021

Dahl salt‐sensitive (DS) rats on 4 weeks high fat diet (HF; 35%) become hypertensive compared to SS.BN13 controls. This study tested the hypothesis that 4 weeks of HF increases oxidative stress and blunts relaxation in arteries from DS compared to SS.BN13 rats. Thoracic aortae and third order mesenteric arteries are isolated and concentration response curves are performed to acetylcholine (Ach) and sodium nitroprusside (SNP). Ach‐induced relaxation is blunted in aorta from DS compared to SS.BN13 (% max. relaxation: 86 ± 3 vs 66 ± 5; p<0.05). Treatment with L‐NAME (100 μM; NOS inhibitor), abolished relaxation in aorta, and tempol (10 mM; superoxide dismutase mimetic) blunted vasorelaxation in aorta from SS.BN13 only (% max. relaxation: 69 ± 7; p<0.05). Aorta from DS displayed decreased sensitivity to SNP compared to SS.BN13 (logEC50: −8.5 ± 0.1 vs −7.8 ± 0.1; p<0.05). Ach‐ and SNP‐induced relaxations are similar in small mesenteric arteries from both strains. L‐NAME produced a comparable and significant decrease in sensitivity to Ach in arteries from both strains (logEC50: SS.BN13: −6.9 ± 0.2 vs −7.8 ± 0.1, p<0.05; DS: −7.3 ± 0.1 vs −8.2 ± 0.2, p<0.05). Tempol did not alter relaxation in mesenteric arteries. Therefore, HF induces aortic dysfunction in DS which is likely related to reduced NO responsiveness of smooth muscle and decreased dilatory reactive oxygen species, with no dysfunction in small mesenteric arteries.
Transmyocardial laser revascularization and phosphorylation of endothelial adherens junction and cytoskeletal elements
Authors: Tinsley John H , Beyer Erik A
Published: June 2021

Transmyocardial laser revascularization (TMLR) is a technique whereby channels are made, using lasers or hypodermic needles, through ischemic tissue of the left ventricle. TMLR has been reported to be highly effective in relieving angina in patients who are unsuitable candidates for bypass surgery or angioplasty. However, the treatment remains somewhat controversial because the mechanisms by which TMLR lead to apparent benefits are unknown. Others have shown that within one to two weeks’ post‐TMLR treatment, areas of the infarcted myocardium exhibit significant increases in the levels of transforming growth factor‐β and basic‐fibroblast growth factor. In addition, the treated areas exhibited an increase in microvessel concentration, suggesting that the beneficial effects of TMLR may be connected with angiogenesis. Because angiogenesis is rooted in endothelial cell migration and growth, we wanted to determine any possible impact that TMLR might have on well‐known endothelial processes. We were able to determine that in rats, 30–120 min. following TMLR, the left ventricular tissue surrounding TMLR channels exhibited increases in the phosphorylation levels of both β‐catenin and myosin light chain. Phosphorylation of these junctional and cytoskeletal elements is known to occur concomitant with alterations in the barrier function of the endothelium, which also can lead to angiogenic responses.
Short axis inferior vena cava measures are more sensitive than long‐axis measures in defining acute volume loading in normal human volunteers.
Authors: Chuen M. Jennyfer Ng Kam , Lip Gregory Y. H. , MacFadyen Robert J.
Published: June 2021

Introduction

Change in inferior cava diameter (IVC) during respiration is a potentially non invasive measure of blood volume in haemodialysis (HD) and heart failure (HF) patients. We tested its sensitivity to acute volume loading in normal volunteers (NV).

Methods

30 NV (16 male; 48.7 ± 8.6 years, range 48–63) received intravenous 0.9% saline (IVS; 1.5 L over 20 minutes). IVC was measured by M‐mode echocardiography in long‐(LAX) and short‐axis (SAX) views during expiration (IVCe), inspiration (IVCi) and maximal inspiration (IVCmi) at 0, 1, 2 and 4 h. IVC was corrected for body surface area (IVCD) and IVC collapsibility index (IVCCI) was defined as IVCCIi=[(IVCe‐IVCi)/IVCe] × 100.

Results

There was good linear correlation between LAX and SAX IVC [IVCe: r=0.809; p<0.0001, IVCi: r=0.660; p<0.0001 and IVCmi: r=0.498; p=0.005]. Parameters increased following IVS, with greater change from baseline in SAX. Only IVCe and IVCDe in SAX increased significantly following IVS (Table 1).

Conclusions

Despite good correlation between LAX and SAX, SAX inferior vena cava indices are more responsive to IVS in NV. Further studies to assess the utility of these indices in the routine care of HF and HD patients are indicated.

Funding

Haemostasis Thrombosis and Vascular Biology Unit charitable funds.
In the isolated perfused heart phenylephrine‐induced activation of ERK is attenuated by glucosamine
Authors: Zhu Xiaoyuan , Marchase Richard B , Chatham John C
Published: June 2021

We have shown that in the heart increased hexosamine biosynthesis pathway (HBP) flux inhibits phenylephrine (PE)‐induced inotropy; however, the mechanisms underlying this are unclear. Therefore, the goal of this study was to determine the effect of increased HBP flux on α‐adrenergic signaling pathways in the heart. Isolated rat hearts were perfused with or without 5mM glucosamine (GlcN) and were treated with or without 10μM PE for 15 mins and p38, Akt and ERK phosphorylation assessed by immunoblot. PE increased rate‐pressure product ~2‐fold and this was significantly attenuated with GlcN (p<0.05). There was no effect of either PE or GlcN on p38 phosphorylation and although PE increased p‐Akt ~2‐fold (p<0.05) this was unaffected by GlcN. However, in the absence of GlcN, PE increased ERK phosphorylation 6.5‐fold (p<0.05), but only ~2‐fold with GlcN (p<0.05 vs. no GlcN). Surprisingly, basal ERK phosphorylation by was increased 2.4‐fold by GlcN (p<0.05 vs. no GlcN); however, even correcting for this ERK phosphorylation was still significantly lower with GlcN + PE compared to PE with no GlcN (4.8±0.4 vs. 6.5±0.4; p<0.05). These data demonstrate that increasing HBP flux with GlcN alters PE‐induced activation of ERK but not p38 or Akt. Further studies are needed to determine if this contributes to the blunted inotropic response to PE.
HIV GP120 Mediates Negative Inotropic Effect through CXCR4 Receptor on Cardiac Myocytes
Authors: Yuan Youxi , Kan Hong , Fang Qiujuan , Chen Fangping , Xie Sherry , Beto Robert J. , Warden Bradford E , Jain Abnash C. , Finkel Mitchell
Published: June 2021

The prognosis for patients with human immunodeficiency virus (HIV) infection has improved remarkably as a result of effective antiretroviral therapy. This has resulted in an increased awareness of cardiac complications from HIV infection, including cardiomyopathy and overt heart failure. However, the mechanisms responsible for HIV cardiomyopathy and heart failure are unknown. We have previously reported that the HIV gp120, has a p38 MAP kinase dependent negative inotropic effect on adult rat ventricular myocytes (ARVM). This signaling pathway presumably results from the binding of gp120 to a specific receptor on the surface of cardiac myocytes. HIV gp120 has been shown to bind to CD4, CXCR4 and CCR5 receptors on lymphocytes and macrophages. Accordingly, we sought to determine if HIV gp120 regulated its negative inotropic effect through activation of one of these binding sites on cardiac myocytes. AMD3100, a highly selective CXCR4 receptor antagonist, reversed HIV gp120 induced negative inotropic effect on ARVM. AMD3100 also blocked HIV gp120 phosphorylation of both p38 MAP kinase and Troponin I. The binding of gp120 to the CXCR4 receptor on ARVM was confirmed by co‐immunoprecipitation. We conclude that the negative inotropic effect of HIV gp120 is mediated by a novel signaling pathway that begins with binding to a cardiac myocyte CXCR4 receptor, followed by phosphorylation of both p38 MAP kinase and Troponin I.
hhLIM Is a Novel F‐Actin Binding Protein Involved in Actin Cytoskeleton Remodeling
Authors: Zheng Bin , Wen Jin‐kun , Han Mei
Published: June 2021

The human heart LIM protein (hhLIM) is a newly cloned protein. In vitro analyses showed that green fluorescent protein (GFP)‐tagged hhLIM protein accumulated in the cytoplasm of C2C12 cells and colocalized with F‐actin indicating that hhLIM is an actin binding protein in C2C12 cells. Overexpression of hhLIM‐GFP in C2C12 cells significantly stabilized actin filaments and delayed depolymerization of the actin cytoskeleton induced by cytochalasin B treatment. Expression of hhLIM‐GFP in C2C12 cells induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that hhLIM functions as an actin bundling protein. This hypothesis was confirmed by low‐speed cosedimentation assays and direct observation of F‐actin bundles that formed in vitro in the presence of hhLIM. hhLIM has two LIM domains. To identify the essential regions and sites for association, a series of truncated mutants were constructed and showed that LIM domain 2 has the same activity as the full length of hhLIM. To further characterize the binding sites, the LIM domain was destructed by replacing cysteine with serine in domain 2, and results showed that the second LIM domain plays a central role in bundling of F‐actin. Taken together, these data identify hhLIM as an actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.
Ranolazine inhibits hypoxia‐stimulated late sodium current
Authors: Ma Jihua , Song Yejia , Hu Liangkun , Yan Xisheng , Zhang Peihua , Shryock John C , Belardinelli Luiz
Published: June 2021

Ranolazine (Ran) has been suggested to attenuate cardiac arrhythmic activity associated with hypoxia. The objective of this study was to determine whether Ran inhibits hypoxia‐induced, arrhythmogenic late sodium current (INa). Both whole‐cell and single‐channel recordings were performed on guinea pig ventricular myocytes exposed to normoxic or hypoxic medium. Late INa was measured 200 ms after the onset of a 500‐ms depolarizing pulse. Hypoxia (PO2 = 3 KPa) caused an increase of the amplitude of inward late INa from −0.47±0.01 to −3.99±0.07 pA/pF. The stimulatory effect of H2O2 on late INa was attenuated by Ran in a concentration‐dependent manner. Ran at 3 and 9 μmol/L significantly reduced the hypoxia‐induced late INa by 16±3% and 55±3%, respectively, without altering the voltage‐dependence of the current. Hypoxia increased the mean open probability and open time of late sodium‐channel openings from 0.016±0.001 to 0.063±0.006 and from 0.693±0.042 to 1.081±0.097 ms, respectively. Ran at 3 and 9 μmol/L significantly attenuated the increase of open probability by 19±7% and 61±1%, and the increase of open time by 26±19% and 74±21%, respectively. The effects of hypoxia on whole‐cell and single‐channel properties of late INa were also significantly inhibited in the presence of tetrodotoxin (2 μmol/L). In conclusion, Ran significantly inhibits hypoxia‐stimulated late INa. The decrease by Ran of late INa is due to reduction of the open probability and open time of late channel openings. The inhibitory effect of Ran on late INa may provide cardiac protection in hypoxia.