bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased renal glucose‐6‐phosphatase gene expression and activity in mice lacking insulin receptors in the renal proximal tubule cells
Authors: Tiwari Swasti , Singh Ravi Shankar , Pandey Gaurav , Li Lijun , Ecelbarger Carolyn M , Godbole Madan M
Published: June 2021

Enhanced gluconeogenesis hasbeen demonstrated in renal proximal tubules of insulin resistant Zucker rats. We have demonstrated reduced insulin receptor expression in renal‐tubular‐epithelial cells of insulin‐resistant rats. Since insulin has been shown to inhibit gluconeogenesis, we tested whether reduced insulin receptor in the proximal tubule affects renal gluconeogenesis. We analyzed gluconeogenesis components in the kidney tissues from mice with targeted deletion of insulin receptor from the proximal tubule (n=7/genotype). For glucose‐6‐ phosphatase (G6Pase) activity, male mice were fasted for 4 hours prior to study followed by intraperitoneal injection of insulin (0.5 U/kg·bw) in 300 μl saline plus 300 μl 25% dextrose in saline in order to amplify the difference between KO and WT (wild type) littermates. After 20 minutes, mice were euthanized and kidneys were removed for G6Pase activity and mRNA levels. Real time RT‐PCR revealed 2.5 fold higher expression of G6Pase mRNA in KO relative to WT littermates (p=0.02, n=7/genotype). In addition, renal G6Pase activity was trending higher (20%) in KO relative to WT; G6Pase activities (in μmolPi/min/mg of protein at 37°C) were 70.1 ± 7.4 in WT versus 84.5 ± 6.3 in KO). Furthermore, the KO mice were mildly hyperglycemic in the fasting state. These results support a physiologic role for the insulin receptor in renal proximal tubule to affect renal gluconeogenesis; and suggest that downregulation of the insulin receptor in renal proximal tubule in the insulin‐resistant state may further contribute to hyperglycemia through enhanced gluconeogenesis. Department of Biotechnology (Government of India) grant to S.T. (BT/HRD/35/02/17/2008).
High Glucose Differentially Influences Endocannabinoid CB‐1 and CB‐2 Receptors in Renal Epithelial Cells
Authors: Hakuda Debroah , de Lima Rariane , Pirro Nancy , Diz Debra , Chappell Mark
Published: June 2021

Endocannabinoids recognize at least two receptor subtypes expressed in central and peripheral tissues. Within the kidney, CB‐1 and CB‐2 receptors are localized to glomerular and proximal tubule elements. CB‐1 receptor blockade is renoprotective in type 1 and 2 diabetic models, while a CB‐2 agonist may be beneficial as well. Indeed, the renoprotective actions of CB‐1 blockade may reflect a greater role for stimulated CB‐2 receptors. To define the regulatory mechanisms of CB receptors within the kidney, we assessed CB‐1 and CB‐2 protein expression in the rat kidney NRK52E epithelial cell line under normal and high glucose conditions. Confluent NRK52E cells were placed in serum‐free DMEM/F12 media and maintained in normal (5mM) or high (30mM) glucose conditions. Immunoblot and immunocytochemical staining revealed the intracellular expression of both CB‐1 and CB‐2 receptors to the nuclear, mitochondrial and plasma membrane fractions of the NRK52E cells. High glucose increased CB‐1 receptor protein expression 10‐fold [0.34 ± 0.10 vs. 3.99 ± 0.66; p = 0.002]; however, CB‐2 receptor expression was not changed [0.94 ± 0.31 vs. 0.88 ± 0.29]. Moreover, the AT1 receptor antagonist candesartan attenuated the increase in CB‐1 receptor expression to high glucose. We conclude that regulation of the CB‐1 receptor may reflect an interaction of the Ang II‐AT1 receptor axis of the renin‐angiotensin system.
Volatile anesthetics produce both vasodilation and vasoconstriction of the aorta by different regulation of calcium, K2P channels and sarcoplasmic reticulum activity.
Authors: Bychkov Rostislav , Cubano Luis , Gonzales Maria Teresa , Mayol Noel , Maldonado Hector
Published: June 2021

We demonstrated that halothane is capable to contract and dilate mouse aorta. To elucidate the mechanism of action of halothane, we performed whole‐cell and perforated‐patch voltage clamp experiments. Halothane inhibited dose‐dependently and voltage‐independently Ba2+‐currents through voltage gated Ca2+‐channels, with half maximal inhibitions at 0.87 ± 0.21 μM. Current inhibition by halothane was prominent over the whole voltage range without changes in the peak of the current voltage‐relationship. Neither intracellular infusion of the GDPβS (100 μM, 5 min) nor strong conditioning membrane depolarization prevented the inhibitory effect of halothane. At a holding potential of −80 mV, 5 μM halothane induced 83 % initial block of Ca2+ channel currents. Halothane increased the amplitude of the K+‐currents in the presence of cocktail of K+‐channels blockers and increased ATP‐elicited release of Ca2+ from sarcoplasmic reticulum (SR). We conclude that halothane contracts aorta by promoting release of Ca2+ from SR and dilates it by inhibiting L‐type Ca2+ channels and opening of K2P channels. This work was made possible by the Grant Number HL107237.
RGS5 is crucial for vascular smooth muscle cells during arterial remodeling in mice
Authors: Arnold Caroline , Feldner Anja , Genové Guillem , Wieland Thomas , Hecker Markus , Korff Thomas
Published: June 2021

G protein‐coupled receptors (GPCR) are major regulators of contractility of vascular smooth muscle cells (SMCs) thereby controlling vascular tone, blood pressure and flow. GPCR signal transduction is modulated by the regulators of G protein signaling (RGS) which terminate G protein activation. Although RGS5 is known to inhibit Gαq activation in arterial SMCs, its role in arterial remodeling is unclear.

To this end, we induced arteriogenesis – the flow and wall stress‐induced growth of collateral arterioles – in mice. Immunofluorescence analysis of remodeling arterioles showed increased RGS5 abundance in wild type medial SMCs while remodeling in rgs5−/− arterioles was disturbed. Exposing cultured human arterial SMCs to cyclic stretch, mimicking elevated wall stress, also led to increased RGS5 levels. Over‐expressing RGS5 in human arterial SMCs prevented Gαq/Ca2+‐mediated constriction upon sphingosine‐1‐phosphate (S1P) stimulation whereas S1P‐treated isolated rgs5−/− arteries showed enhanced contractility. This shifted S1P signaling from Gαq towards Gα12/13‐mediated RhoA kinase activation.

In summary, elevated wall stress elicited RGS5 expression which blunts Ca2+‐mediated vasoconstriction leading to Gα12/13‐dependent RhoA kinase activation. This appears to be critical for arteriogenesis. This work was supported by the Deutsche Forschungsgemeinschaft (SFB TR23, projects B6/C5/C6).
Purinergic modulation of smooth muscle intracellular Ca2+ concentration
Authors: Olson Marnie L , Kennedy Charles , McCarron John G
Published: June 2021

Basal, steady‐state Ca2+ levels are determined by the balance between Ca2+ release/influx and uptake/extrusion mechanisms. Extrinsic factors can alter this balance to affect smooth muscle excitability. Extracellular ATP acts as an inhibitory neurotransmitter in gastrointestinal and vascular (portal vein) smooth muscle. This study examined the effect of extracellular ATP on the steady‐state intracellular Ca2+ concentration ([Ca2+]c). Extracellular ATP (1 mM; applied via picopritzer) reversibly decreased steady‐state [Ca2+]c (measured using fluo 4). To assess whether ATP functioned by altering influx or extrusion [Ca2+]c and inward currents were measured simultaneously in voltage‐clamped myocytes during a depolarizing pulse. ATP decreased the peak amplitude without altering the voltage‐dependence of the inward current. The P2 purinoceptor inhibitor, suramin, and the P2Y1 specific inhibitor, MRS2179, each prevented ATP inhibition of steady‐state [Ca2+]c and inward current. Interestingly, suramin and MRS2179 each also increased the magnitude of the inward current per se, indicating tonic ATP inhibition of Ca2+ channels by endogenous ATP. ATP did not alter Ca2+ uptake/extrusion mechanisms. ATP appears to influence Ca2+ influx mechanisms in smooth muscle via P2Y receptors to exert a negative regulation of excitability.
Extracellular UDP potentiates bladder purinergic signaling and smooth muscle contractility via P2Y6 activation of PLC/IP3 pathway
Authors: Yu Weiqun , Sun Xiaofeng , Robson Simon C. , Hill Warren G.
Published: June 2021

Abnormal bladder smooth muscle (BSM) contractility is critical to the disease process in overactive bladder, incontinence and spinal cord injury. Purinergic signaling comprises a key pathway in modulating BSM contractility but mechanisms remain unclear. We now demonstrate that P2Y6, a specific receptor for UDP, is present on mouse BSM cells by immunolocalization. Activation of P2Y6 by either UDP or a specific agonist (MRS2693) induced a sustained increase in BSM tone (up to 2 mN) in a dose‐dependent manner. Significantly, activation of P2Y6, enhanced ATP‐mediated BSM contractile force by up to 45%, indicating synergistic crosstalk between P2X and P2Y signaling. P2Y6‐activated responses were abolished by PLC and IP3 receptor antagonists U73122 and Xestospongin C, demonstrating involvement of the PLC/IP3 pathway. Mice lacking Entpd1, an ectonucleotidase on BSM, demonstrated increased P2Y6 expression (140%), and increased force generation upon P2Y6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y6 activity and bladder contractility. Therefore, P2Y6 regulates BSM tone and in doing so selectively maximizes P2X1‐mediated contraction forces. This novel purinergic receptor pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility. Supported by NIH Grants DK083299 to WGH and P01‐HL076540 & R01‐HL094400 to SCR.
Ceramide initiates PP2A colocalization with eNOS at the cell membrane by dissociating PP2A from I2PP2A in the cytosol
Authors: Panneerseelan Leena , Ruan Ting , Ravindran Anindita , Li Youyou , Zhang Quan‐Jiang , Abel E Dale , Symons J David
Published: June 2021

In whole cell lysates we showed that palmitate disrupts the interaction between inhibitor 2 (I2) of protein phosphatase 2A (PP2A) and PP2A and that this event initiates PP2A colocalization with eNOS. Both responses were negated when ceramide synthesis was inhibited. We sought to confirm this palmitate evoked, ceramide‐mediated effect using immunofluorescent approaches. Bovine aortic endothelial cells (BAECs) were treated for 3 h with BSA (V), V + 500 μM palmitate (P), V + ceramide inhibition (10 uM myriocin; M), or P+M (PM). P‐induced increases (p<0.05) in ceramide were prevented (p<0.05) in cells exposed to P+M (n=8). P increased PP2A association with eNOS (70±9% vs. V) and decreased p‐eNOS(S)1177 to eNOS (35±5% vs. V) in isolated membrane fractions (both p<0.05, n=8), and these responses were prevented in cells exposed to P+M. We then permeabilized cells that were treated as above, and incubated them with 1° antibodies for eNOS, PP2A, I2PP2A, or ceramide, followed by incubation with fluorescent 2° antibodies to assess subcellular localization in intact cells using confocal microscopy. Relative to V‐treatment, P: (i) increased ceramide association with I2PP2A in the cytosol by 70±6%; (ii) decreased PP2A association with I2PP2A in the cytosol by 45±5%; and (iii) increased PP2A association with eNOS in the membrane by 80±9% (all p<0.05; n=6 per combination). All responses were negated in P+M –treated cells. Endogenous ceramide disrupts the cytosolic interaction between I2PP2A and PP2A and causes translocation of PP2A to the membrane where it associates with eNOS. ADA1–12‐BS‐208, 2R15HL091493
Subarachnoid Hemorrhage‐Induced Activation of Protein Kinase C and Suppression of Voltage‐Gated Potassium Channels
Authors: Rudy Robert F , Koide Masayo , Wellman George C.
Published: June 2021

Subarachnoid hemorrhage (SAH) caused by aneurysm rupture is often associated with decreased cerebral blood flow and the development of severe neuronal deficits in patients. One cause of this disability is intracerebral (parenchymal) artery constriction. This constriction may be due in part to suppression of voltage‐gated potassium (KV) channels causing membrane potential depolarization, enhanced Ca2+ influx and vasoconstriction. Previous studies have shown that protein kinase C (PKC) is activated following SAH and that KV currents are suppressed in myocytes of larger diameter cerebral arteries from SAH model animals. Here, we examined PKC involvement in SAH‐induced KV current suppression in parenchymal arterioles using the conventional whole cell patch clamp technique. We found that KV currents were suppressed in myocytes obtained from SAH model rats compared to cells obtained from control animals. We also observed that the PKC activator 1,2‐dioctanoyl‐glycerol suppressed KV currents in control but not SAH myocytes, indicating PKC may be maximally active following SAH. In summary, our data indicate that PKC activity is increased following SAH and contributes to suppression of KV channels. Supported by the National Institutes of Health (NIH; P01‐HL095488), the Totman Medical Research Trust, the University of Vermont Summer Internship Grant and the Peter Martin Fund.
Angiotensin type 1A receptors transfected into the nucleus tractus solitarii of AT1a−/− mice increase blood pressure and cardiovascular responses to aversive stress
Authors: Head Geoffrey A , Abegaz Beza , Jackson Kristy L , Bassi Jaspreet K , Connelly Angela , Choong Yan‐Ting , Allen Andrew M , Davern Pamela J
Published: June 2021

We examined the effect of expression of angiotensin type 1A (AT1A) receptors in neurons in the nucleus tractus solitarii (NTS) of AT1A−/− mice on basal blood pressure (BP) and cardiovascular responses to stress. Bilateral microinjections of lentivirus with the phox2a‐responsive promoter PRSx8 of either green fluorescent protein (GFPv, n=8) or AT1A receptors (AT1Av, n=6) were made into the NTS of AT1A−/− mice fitted with telemetry devices for BP recording. Expression of AT1A receptors increased BP by 10±4 mmHg (P<0.05 at day 21) but there was no change in GFPv mice. Depressor responses to ganglion blockade were similar in both groups. There was a greater pressor response to cage‐switch stress following expression of AT1A receptors (18±2 mmHg pre virus to 24±2 post virus, P<0.05) but no difference in GFPv mice. Activation of the paraventricular hypothalamus, amygdala and brainstem regions including the NTS, rostral and caudal ventrolateral medulla following stress was 3–8 fold greater than GFPv mice. These results suggest that expression of AT1A receptors on neurons in the NTS of AT1A−/− mice increases basal BP through a mechanism independent of the sympathetic nervous system as well as through forebrain projections to the amygdala/hypothalamus involved in the response to aversive stress. Supported by grants from National Health and Medical Research Council of Australia.
Enhanced CB1 Cannabinoid Receptor Tone Contributes to Impaired Baroreflex Sensitivity in Hypertensive (mRen2)27 Transgenic Rats
Authors: Schaich Chris , Nautiyal Manisha , Howlett Allyn , Chappell Mark , Diz Debra
Published: June 2021

We demonstrated that activation of CB1 cannabinoid receptors within the solitary tract nucleus (NTS) of the brainstem via bilateral microinjection of the CB1 agonist CP55,940 modulates baroreflex sensitivity (BRS) for control of heart rate, an index of vagus nerve function, in a dose‐related biphasic manner. The CB1‐selective antagonist SR141716 (36 pmol) prevented or reversed these changes, and there was no effect of the antagonist alone. We now determine the effects of CB1 receptor blockade in (mRen2)27 (Ren2) rats, a model of angiotensin (Ang) II‐dependent hypertension with impaired baseline BRS. Unlike in Sprague‐Dawley (SD) rats, SR141716 dose‐dependently improved BRS when microinjected into the NTS of Ren2 rats. Microinjection of 0.36 pmol SR141716 (n = 5) increased BRS by 59% (p < 0.001), while 36 pmol SR141716 (n = 5) increased BRS by 124% (p < 0.001), to a level similar to baseline BRS of SD rats. There was no change in BRS by 1.2 pmol CP55,940 (n = 3) in Ren2 rats. Immunoblots revealed a trend for higher CB1 expression in brainstem of the Ren2 strain compared to SD rats. Thus, elevated endocannabinoid actions in the NTS contribute to impaired BRS in Ang II‐dependent hypertension. It is not yet known whether this results from elevated receptors or endogenous levels of the cannabinoids, but CB1 receptor blockade may positively influence BRS in the context of hypertension. Supported by P01‐HL051952 and R01‐DA03690.
Repetitive Electroacupuncture Attenuates Cold‐Induced Hypertension and Simultaneously Enhances rVLM Preproenkephalin mRNA Expression
Authors: Li Min , Chi Seung Yoon , Tjen‐A‐Looi Stephanie C , Longhurst John C
Published: June 2021

Acupuncture lowers elevated blood pressure in clinical and experimental hypertension, likely through an opioid mechanism. To mimic human studies, we performed repeated electroacupuncture (EA) on unanesthetized male SD rats (150g) exposed to cold (5¡ãC) for 11 weeks. Systolic tail cuff blood pressures (SBP) were measured weekly. Rats were randomly divided into EA‐hypertension (n=9), sham (n=6) and untreated hypertension groups (n=6) after 6 weeks of cold exposure. Blood pressures measured outside the cold room (25¡ãC) stabilized at an elevated level in the three groups. EA and sham groups received either repetitive EA (1–4 mA, 2 Hz, 0.5 ms) at ST36–37 acupoints (overlying deep peroneal nerve) or sham (needle placement without electrical stimulation) for 30 min twice weekly for 5 weeks. The elevated SBP in the EA group was reduced after 5 sessions and remained low throughout EA treatment (155 ¡À 4 vs. 187 ¡À 3 mmHg EA vs. sham). The relative ratio of preproenkephalin mRNA (normalized to 18s) in rVLM 72 hr after the terminal EA treatment was increased in the EA (14.3¡À1) compared to the sham group (4.3¡À0.5), which was similar to values in the untreated hypertensive rats (4.3¡À0.6). These data suggest that the prolonged inhibitory effect of EA is related to prolonged increases of opioid mRNA expression in the rVLM
Intermittent induced change in gene expression in the median preoptic nucleus (MnPO) of rats
Authors: Saini Naharika , Shell Brent , Nedungadi T. Prashant , Cunningham J. Thomas
Published: June 2021

Our previous work indicated that changes in MnPO gene expression are necessary for the sustained increase in blood pressure produced by chronic intermittent hypoxia (CIH), an animal model of the hypoxemia associated with sleep apnea. This study tested the effects of a 7d CIH exposure on the expression of additional genes in the MnPO that could contribute to changes in the excitability of neurons in this region: Angiotensin receptor 1a (AT1a), Angiotensin receptor 1b (At1b), delta FBJ murine osteosarcoma viral oncogene homolog B (ΔFosB), transient receptor potential channel 4 (TRPC4), potassium‐chloride cotransporter 2 (KCC2), and sodium‐potassium‐chloride cotransporter 1 (NKCC1). The brain was harvested from adult male rats anesthetized with inactin (100 mg/kg ip) after 7 d of CIH or 7 d of normoxia. Laser Capture Microdissection of MnPO was carried out from 10μ sections. MnPO samples were run through RNA purification, cDNA synthesis and PCR using S18 as the housekeeping gene. PCR results revealed significant increases in the expression of TRPC4, ΔFosB, NKCC1 and AT1a in the MnPO of CIH treated rats relative to normoxic controls. KCC2 and AT1b gene expression in MnPO were not significantly affected by CIH. Increased expression of these genes, especially the marked upregulation of the AT1a receptor, could participate in the contribution of the MnPO to the sustained hypertensive effects of CIH. P01 HL88052
Voluntary physical activity versus inactivity reduces dendritic field complexity in putative RVLM cardiovascular neurons
Authors: Mischel Nicholas Ara , Llewellyn‐Smith Ida J , Mueller Patrick J
Published: June 2021

Blood pressure regulation relies on neurons in the RVLM and is influenced by regular physical activity. We hypothesized that the morphology of RVLM neurons differs in active vs inactive rats. Rats were housed with and without running wheels for 12 weeks and then cholera toxin B subunit (CTB) was injected into cord segment T10. Double immunoperoxidase labeling for CTB and tyrosine hydroxylase identified bulbospinal C1 neurons. RVLM neurons from 4 active (196 neurons) and 3 sedentary (150 neurons) rats were reconstructed and analyzed using Neurolucida software. Active rats ran 373±90 km. Compared to inactive rats, physically active rats had shorter dendrites (995±54μm vs. 1453±21 μm, p=0.0010), fewer dendritic branches (2.4±0.1 vs. 5.1±0.3, p<0.001) and fewer intersections in Sholl analyses (84±4 vs. 109±1, p<0.01) but cell body area was similar (245±6μm2 vs. 258±2μm2). These results suggest that physical activity decreases dendritic complexity but does not affect the cell body size of putative RVLM cardiovascular neurons. This morphological plasticity may contribute to differences in cardiovascular regulation in physically active vs sedentary individuals. (F30‐HL105003; NHMRC 1025031; R01‐HL096787).
Cardiac Sensory Consequences of Low‐Amplitude Autonomic Regulation Therapy
Authors: Ardell Jeffrey L , Beaumont Eric , Kenknight Bruce H , Gibbons David , Southerland Marie , Armour John Andrew
Published: June 2021

Objective

To determine if vagus nerve stimulation (VNS) or spinal cord stimulation (SCS) affects the capacity of cardiac afferent neurons to transduce myocardial ischemia (MI).

Methods

Using extracellular recordings in anesthetized canines, cardiac‐related dorsal root (DRG; T1‐T3 spinal levels) or nodose ganglia neurons were identified by touch/chemical activation in their epicardial sensory fields located adjacent to the left anterior descending (LAD) coronary artery. Neuronal responses to 1 min LAD coronary artery occlusion (CAO) were then evaluated prior to and following SCS [T1‐T3 spinal level; 50Hz, 90% motor threshold; 20 min] or left cervical VNS [20 Hz; current amplitudes (CA): CA1 (1–1.9 mA) or CA2 (2–3.5 mA); 3 min].

Results

LAD CAO activated cardiac afferent neurons in nodose and DRG ganglia, enhancement that persisted for up to 30 min into reperfusion. Following SCS or CA2 VNS, LAD CAO failed to alter their neural activity. VNS at CA1 did not mitigate MI‐induced activation of nodose neurons.

Conclusions

Transient myocardial ischemia activates primary afferent inputs to the central nervous system (CNS), responses that persist into reperfusion. Pre‐emptive VNS or SCS obtunds sensory transduction of the ischemic myocardium to the CNS, in part accounting for the anti‐anginal and broader cardioprotective effects of such therapy being evaluated clinically. (HL71830)
Autonomic cardiac modulation in acute ischemic stroke: potential relationship with neurogical impairments
Authors: Wu Maddalena Alessandra , Fiorelli Elisa Maria , Tobaldini Eleonora , Cantoni Flavio , Mandelli Clara , Torgano Giuseppe , Montano Nicola
Published: June 2021

Acute ischemic stroke (IS) is associated with alterations of autonomic cardiovascular control (ACC). However, evidence on the role of autonomic nervous system in the acute phase of IS is still lacking. Aim of the study was to investigate the correlation between ACC and changes of neurological impairment after 72 h from presentation to the Emergency Department, measured by the difference in NIHSS score (Δ NIHSS). ECG and respiration were recorded from 13 consecutive subjects with acute IS, using the BT16 acquisition system. Linear and nonlinear analysis of heart rate variability was performed. Spectral analysis was applied to identify the main oscillations embedded in the signal: low‐frequency (LF, 0.04 – 0.15 Hz) and high‐frequency (HF, 0.15 – 0.4 Hz) components, markers of sympathetic and parasympathetic activity respectively. Symbolic analysis allows to assess different patterns: 0V% (sympathetic modulation), 1V% (uncertain meaning), 2LV% and 2ULV% (vagal modulation). A linear regression revealed a significant relationship between Δ NIHSS and sympathetic indices. An increase of LFnu, LF/HF and 0V% were positively associated with an increase in Δ NIHSS (R = 0.58, 0.69 and 0.71 respectively; p< 0.05). Increasing Δ NIHSS was associated with decreasing HFnu (R=0.56; p< 0.05). We speculate that maintained oscillatory sympathetic modulation in the acute phase of IS might be a predictor of a better outcome.
The Effects of Sympathetic Nerve Stimulation on Ventricular Electrophysiology in Long QT Syndrome‐1
Authors: Brack Kieran , Winter James , Gupta Ayushman , Ng G. Andre
Published: June 2021

Congenital long QT syndrome 1 (LQTS1) results from reduced slow activating delayed rectifier potassium channel (IKs) activity. Ventricular arrhythmias occur with increases in sympathetic tone which can cause sudden cardiac death (SCD). Underlying mechanisms are not understood but may include changes in electrical restitution (RT) – the relationship between action potential duration and diastolic interval. We aimed to examine the effects of sympathetic nerve stimulation (SNS) on effective refractory period (ERP), RT slope and inducibility of ventricular fibrillation (VF) in a pharmacological model of LQTS1. The innervated heart preparation from adult male guinea pigs (n=6, 450–550g) were used. ERP and LV apical and basal RT were measured using an extrastimulus protocol. Inducibility of VF was assessed with ventricular fibrillation threshold (VFT). Parameters were measured during baseline (BL) and SNS (3Hz, 1V) in control (Ctrl) and in the presence of the IKs blocker HMR1556 to mimic LQTS1 (HMR). Data are mean±SEM, analysed using Student's T‐Test (*vs BL, **vs Ctrl). During control, SNS significantly reduced ERP and VFT and increased RT slope but only at LV base, (Table). In the presence of HMR1556, the effects of SNS on ERP and VFT were augmented whilst the effect on RT was abolished. In conclusion, SNS increases susceptibility to VF which appears to be augmented in this pharmacological LQTS1 model and related to changes in ERP but not RT.

Research support from the University of Leicester
Low frequency power spectrum is associated with baroreceptor sensitivity in obese individuals during paced breathing
Authors: Baynard Tracy , Goulopoulou Styliani , Sosnoff Ruth F. , Fernhall Bo , Kanaley Jill A
Published: June 2021

Recent data suggests that the low frequency (LF) power spectrum from heart rate variability analyses is indicative of baroreceptor sensitivity (BRS). We aimed to determine to the association between LF and BRS in obese persons and to evaluate the effect of 4 mo of aerobic exercise training on these associations. ECG recordings were obtained in obese individuals (n=55) while supine for 5 min while they performed paced breathing (12 breaths/min), prior to and after 4 mo of exercise training (4 d/wk, 65% VO2peak). HRV was analyzed in the spectral domain and log transformed (ln). BRS was assessed using the sequence technique using finger photoplethysmography. Data were further analyzed by creating low and high parasympathetic modulation (PM) groups. LFln did not change with training during paced breathing (Low PM: 5.15 ±0.17 vs. 5.35±0.18 ms; High PM: 5.07±0.17 vs. 5.21±0.17 ms). LFln was associated with BRS both pre and post‐training, with stronger relationships observed for the high PM group vs. the low PM group. LF was associated with BRS in this obese population and this relationship was consistent after a 4 mo exercise training program, suggesting LF may be a surrogate for BRS during paced breathing. The association was stronger among obese individuals who had higher baseline levels of parasympathetic modulation. These findings may help further elucidate the role of LF in the HRV spectrum.

Pre‐training Log LF and BRS
Post‐training Log LF and BRS

Whole group
0.60*
0.53*

Low Parasympathetic Modulation
0.54*
0.55*

High Parasympathetic Modulation
0.69*
0.70*

p<0.05
Effect of low‐dose dopamine on cardio‐respiratory physiology in heart failure patients
Authors: Edgell Heather , McMurtry M. Sean , Paterson Ian , Ezekowitz Justin A. , Haykowsky Mark J. , Dyck Jason R.B. , Stickland Michael K.
Published: June 2021

Previous work has shown that carotid chemoreceptor (CC) inhibition increases cardiac output at rest in dogs with heart failure (HF). To translate this knowledge to humans, we recruited clinically stable HF patients (n=7; EF: 36±15%) and risk‐matched controls (CON; n=9; EF: 64±4%) from the Heart Function Clinic at the University of Alberta hospital, the Alberta HEART study, and from the general population. Ventilation, blood pressure, ECG, and aortic ultrasound were measured. Dopamine (DA) was infused at 2μg/min/kg to inhibit the CC. At rest, we found higher mean arterial pressure (98±14 vs 78±9mmHg; p=0.003) and higher total peripheral resistance index in CON (0.05±0.03 vs 0.04±0.01mmHg/mL/min/m2; p=0.01). These differences could be due to more frequent anti‐hypertensive treatment in HF. The DA decreased ventilation in HF (7.6±2.1 vs 6.8±1.7L/min; p=0.04) but not in CON (6.7±2.0 vs 6.7±2.0L/min; p=0.96). While there was no difference in cardiac output index (Q) at rest between groups (p=0.15), after DA infusion HF patients had higher Q (CON:1.7±0.5 vs HF:2.6±0.5L/min/m2; p=0.02). In HF, low‐dose DA inhibits the CC and increases resting cardiac performance. Funded by Heart and Stroke Foundation of Canada.
ROLE OF NITRIC OXIDE SYNTHASE AND SERCA ON THROMBOXANE RELEASE IN THE VASCULAR HYPERREACTIVITY INDUCED BY DIABETES MELLITUS
Authors: Morales‐Loredo Humberto , Espinosa‐Tanguma Ricardo
Published: June 2021

The molecular mechanisms that lead to the diabetes mellitus (DM) vascular hyper‐reactivity still are a matter of debate. Unpublished results from our laboratory showed that aortic rings from streptozotocin (STZ)‐DM induced rats with 10 weeks of evolution have: a) an endothelium‐dependent vascular hyper‐reactivity, b) a similar response to phenylephrine (Phe, 10μM) when incubated with the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) blocker thapsigargin (Thaps, 1μM) which is also a sarcoendoplasmic reticulum stress inducer, and c) a decreased hyper‐reactivity when incubated with indomethacin or the TXA2 receptor blocker SQ 28548 (1μM). In order to further investigate the mechanism involved in DM induced vascular hyper‐reactivity, in the present work we evaluated: a) the effect of nitric oxide synthase (NOS) blockade with L‐NAME (100 μM) on DM‐induced vascular hyper‐reactivity, b) the SERCA expression in aorta, and c) the thromboxane release from aortic tissue stimulated with: Phe (10 μM), Phe + Thaps (1μM), and L‐NAME (100 μM) + Thaps (1μM) + Phe (10 μM).

RESULTS

SERCA protein expression was decreased in aortic tissue from DM compared with CTRL. The ELISA assay for TXB2 showed that thromboxane release was increased in: 1) diabetic tissue with vehicle, 2) diabetic tissue stimulated with Phe, and 3) diabetic tissue with L‐NAME + Phe. Both, the tissue from CTRL rats treated with Thaps + Phe or with L‐NAME + Thaps + Phe, released TXB2 at levels comparable to those from DM. Nevertheless, L‐NAME decreased the releasing of TXB2 and the vascular hyper‐reactivity as well.

CONCLUSIONS

Our results suggest that the decrease in the expression of SERCA pump in DM could increase the cytosolic Ca2+ concentration, which in turn increases the vasoactive agent's release, mainly TXA2. Also, our data suggest that NO is involved in the release of TXA2 which produces vascular hyper‐reactivity.
Chronic reductions in synaptic inputs to the phrenic motor nucleus increases GluA2‐containing AMPA receptors
Authors: Strey Kristi A , Baertsch Nathan A , Shires Joel A , Baker‐Herman Tracy L
Published: June 2021

An acute disruption in descending synaptic inputs to the phrenic motor nucleus, elicits a form of spinal plasticity called inactivity‐induced phrenic motor facilitation (iPMF). iPMF is manifested as a rebound increase in phrenic amplitude that is apparent when synaptic inputs are restored. Here we tested the hypothesis that chronic withdrawal of phrenic synaptic inputs also elicits plasticity. Rats received a unilateral injection of aCSF or botulinum toxin A (BoNT/A;10ng) into the C4 phrenic motor pool. Three days post‐unilateral BoNT/A, cleaved SNAP‐25 was localized to ipsilateral C4 spinal segments, suggesting impairment of synaptic inputs. In a separate group of rats, ventral spinal segments (C3‐C5) were harvested and membrane proteins were biotinylated to determine membrane expression of GluA1/A2 receptors. No change in GluA1 was detected in spinal segments ipsilateral or contralateral to BoNT/A (ipsi: 14±15; contra: 19±10% sham; p<0.05). By contrast, membrane GluA2 expression was significantly increased in ventral segments ipsilateral, but not contralateral to BoNT/A (ipsi: 70±24; contra: 36±26% sham; p<0.05). Collectively, our data suggest that chronic withdrawal of phrenic synaptic inputs increases ipsilateral membrane expression of GluA2‐containing AMPA receptors. We hypothesize that iPMF is a compensatory mechanism to maintain respiratory motor output throughout life. NIH‐HL105511.
Selective blockades of agonist‐induced reflex and afferent responses of vagal lung C‐fibers by perivagal antagonist treatment in rats
Authors: Lin Yu‐Jung , Ruan Ting , Kou Yu Ru
Published: June 2021

It has been known that several types of pharmacological receptors are expressed on the terminals of vagal lung C‐fibers (VLCFs). These receptors play crucial roles in triggering airway reflexes and in the development of airway hypersensitivity. This study was carried out to use perivagal treatment with antagonists (PAT) targeting the TRPV1, P2X and 5‐HT3 receptors to block reflex and afferent responses of VLCF to intravenous injections of agonists in anesthetized rats. Capsaicin, α,β‐methylene‐ATP, and phenylbiguanide were used as the agonists, and capsazepine, iso‐PPADS, and tropisetron were used as the antagonists of TRPV1, P2X, and 5‐HT3 receptors, respectively. Perivagal capsazepine treatment prevented both VLCF‐mediated reflex apneic and afferent firing response to intravenous injections of capsaicin but not to α,β‐methylene‐ATP and phenylbiguanide. Similar patterns of the selective blockade by PAT to corresponding agonists were also found in iso‐PPADS and tropisetron. Our results indicate that PAT selectively abolished the stimulatory action of corresponding agonists on the VLCF terminals, which suggest that PAT has the possibility to become a novel intervention for studying the role of pharmacological receptors in the functioning of VLCFs (NSC 98–2628‐B‐040–011‐MY3 and NSC 101–2320‐B‐010–042‐MY3).
Direct projection from the dorsomedial hypothalamus to the rostral medullary raphe drives brown adipose tissue thermogenesis
Authors: Kataoka Naoya , Hioki Hiroyuki , Kaneko Takeshi , Nakamura Kazuhiro
Published: June 2021

In the central regulation of thermogenesis in brown adipose tissue (BAT), sympathetic premotor neurons in the rostral medullary raphe (rMR) play a pivotal role by integrating inputs from upper brain sites and by providing an output to the spinal cord. One of the candidate brain sites that provide direct excitatory inputs to these premotor neurons is the dorsomedial hypothalamus (DMH). Using an optogenetic technique, we examined the functional role of the DMH‐rMR projection in the efferent mechanism driving BAT thermogenesis. Virus‐mediated delivery of channelrhodopsin‐2 (ChR2) gene into rat DMH resulted in localization of ChR2 proteins not only in DMH neurons but also in their axon terminals in the rMR. In vivo photostimulation of ChR2‐containing axons in the rMR elicited increases in BAT thermogenesis, blood pressure and heart rate. Photostimulation of ChR2‐expressing cell bodies in the DMH also elicited similar physiological responses, which were eliminated by antagonizing glutamate receptors in the rMR. The responses to photostimulation of ChR2‐containing terminals in the rMR were inhibited by warming the preoptic area (POA). These results indicate that the DMH‐rMR projection provides a glutamatergic input to sympathetic premotor neurons to drive thermogenic and cardiovascular responses and that this activation of sympathetic premotor neurons is influenced by thermal information from the POA.
Epigenetic Regulation of Cerebrovascular Remodeling Mediated Through Exosomes in Hyperhomocysteinemia: Role of Folic Acid
Authors: Kalani Anuradha , Narayanan Nithya , Qipshidze Natia , Givvimani Srikanth , Kamat Pradip K. , Tyagi Suresh C. , Tyagi Neetu
Published: June 2021

An elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with neurovascular diseases. HHcy induces oxidative stress that may epigenetically mediate cerebrovascular remodeling. It has been recently shown that exosomes may induce epigenetic changes and affect brain functions. However, the mechanisms behind such alterations remain unclear. The present study examined whether cerebrovascular remodeling is mediated through exosomes and improved epigenetically with folic acid (FA) treatment. To test this hypothesis we employed 8 weeks old male wild type (WT), cystathionine‐beta‐synthase heterozygote knockout (CBS+/‐), WT + FA, CBS (+/‐) +FA (0.0057 μg/g/day dose in drinking water/4weeks) mice. Microvascular permeability in pial vessels was measured by intravital microscopy. The brain tissues were analyzed for matrix metalloproteinases, tissue inhibitor of metalloproteinases, oxidative stress, Hcy metabolic enzymes by western blot, qRT‐PCR, IF and HPLC. Microvascular permeability, MMP‐2–9 and gp‐91(oxidant) were increased while TIMP‐1,‐4, tight junction proteins, MTHFR, SAHH and DNA methyltransferases were decreased in CBS (+/‐) mice as compared to WT. Interestingly, cerebrovascular remodeling/epigenetic markers were also altered on exosomes from CBS (+/‐) mice as compared to WT. Thus, cerebrovascular remodeling, mediated through exosomes, is significantly improved with FA treatment in CBS (+/‐) mice showing its potential therapeutic role against HHcy related pathologies.
Calcium‐related channels as determinants of the excitability of dopaminergic neurons in rodent substantia nigra
Authors: Chung Jun‐mo , Noh Jihyun
Published: June 2021

Dopaminergic neurons in rat substantia nigra (SN) are excited or inhibited by Zn2+ depending on how to chelate intracellular Ca2+; Zn2+ enhances tonic activity in the depolarizing state by intracellular dialysis with EGTA, whereas Zn2+ attenuates activity by employing BAPTA as an intracellular Ca2+ stabilizer. Here, we show that Zn2+‐induced augmentation of firing activity at the depolarized state is mediated by the prevention of small‐conductance Ca2+‐activated K+ channels (SKCs) and by the activation of transient receptor potential channels (TRPMCs) with contribution of voltage‐gated L‐type Ca2+ channels (VGCCs) and ryanodine receptors (RyRs). The increase of firing activity in the presence of Zn2+ was abolished by the application of VGCC antagonists, nifedipine and the RyR blocker, dantrolene. The Zn2+‐mediated potentiation of firing activity was eliminated after blockade of TRPMCs with flufenamic acid and 9‐phenanthrol. Apamin, an SKC inhibitor, enhanced firing activity. This effect was attenuated by intracellular dialysis with BAPTA. Notably, the enhancement of firing activity in the presence of Zn2+ and also after washout of Zn2+ was abolished by the pre‐incubation of apamin. Zn2+ may modulate the intrinsic activity of rat SNc dopaminergic neurons depending on the involvement of cytosolic Ca2+‐dependent signaling processes. (Supported by the SBIE Grant from GIST & the NRF Grant from KMEST to JMC)
Beneficial effect of melatonin and treadmill exercise on remodeling of neural circuit after focal cerebral ischemia in rats
Authors: Lee Seunghoon , Hong Yunkyung , Hong Yonggeun
Published: June 2021

The promotion of post‐ischemic motor recovery remains a major challenge in clinical neurology. The present study was performed to investigate the effect of melatonin (MT) and/or treadmill exercise (Ex) on remodeling of neural circuit after focal cerebral ischemia. Following the MCAo, the 8‐week‐old Sprague‐Dawley (SD) rats were treated twice daily with MT (10 mg/kg) at 7:00 and 19:00 hr and control group were treated with physiological saline (vehicle) instead of MT. Also, the rats were trained twice daily with Ex (20 m/min) at 17:00 and 22:00 hr. We clarified that MT could be induced the reorganization of brain structure both hemispheres via NeuN and NG2 positive immunostaining. Furthermore, we determined that ischemic brain injury promotes to wallerian degeneration from brain to spinal cord, and melatonin is not only attenuated to wallerian degeneration after MCAo but also the rescue of neural cells, including neurons and oligodendrocytes. In conclusion, we identified that ischemic brain injury promoted to destroy of brain structure as well as breakdown of corticospinal tract for transduction of motor impulse to peripheral nerve. We established that melatonin has a neuroprotective effect on acute phase after MCAo. Moreover, there has a synergistic effect to treadmill exercise dual application for structural and functional recovery after focal cerebral ischemia.
Hypothalamo‐pituitary axis dysfunction in animals that exhibit a maladaptive response to a traumatic stressor
Authors: Whitaker Annie M. , Gilpin Nicholas
Published: June 2021

Post traumatic stress disorder (PTSD) often co‐exists with substance use disorders including alcohol abuse. Our lab utilizes predator odor exposure, an established model of traumatic stress, allowing rats to be divided into groups based on persistent avoidance an odor‐paired chamber, a phenotype similar to humans with PTSD. Preliminary studies in our lab show that avoider rats consume significantly more alcohol than non‐avoider rats and unstressed controls. Dysregulation in hypothalamo‐pituitary axis (HPA) function occurs in both PTSD patients and alcohol‐dependent individuals. We hypothesize that HPA dysregulation following exposure to a traumatic stressor contributes to increases in alcohol consumption in avoider rats. The purpose of this study was to examine HPA activity in avoider and non‐avoider rats following a traumatic stress. Male Wistar rats (300g) underwent a place conditioning procedure to assess avoidance of the odor‐paired chamber. Fifty percent of the rats exhibited avoidance of the odor‐paired chamber. Non‐avoider rats show an increase adrenocorticotropin releasing hormone (ACTH) post‐odor exposure compared to unstressed controls (769±133 vs. 1204±379pg/ml). ACTH levels are attenuated in avoider rats compared to non‐avoider (1204±379 vs. 879±85pg/ml; p=NS). Whether this contributes to the excessive drinking in avoider animals is unknown and the focus of current studies. This work was supported by NIH grant AA018400 (NWG).
Psycho‐Physiological Changes in Mouse Strain Submitted to Social Stress
Authors: Jibitu Meskerem
Published: June 2021

Three different mouse strains of C57BL/6j, BABLB/cj and DBA/2j were submitted to 10 day and 5 day aggressor exposed (Agg‐E) stress. Mice were kept inside an aggressor (Agg ) home cage and randomly exposed to the Agg three times a day for one minute or up to ten attacks while the controls never experiences the Agg. The ethogram of the Agg‐E stress evaluated by a situation reminder study was analyzed immediately post stress and after a pre‐determined rest period.

C57BL/6j, BABLB/cj and DBA/2j displayed noticeable changes in behavioral parameters such as jumping, fighting‐back, grooming, tail rattling and freeze motion when exposed to (Agg ) mice for 5‐ or 10‐day. Strain‐wide physiological alteration was evaluated in body weight, body temperature changes and urine marking; a sign of territorial behavior.

Situation reminder evaluated a cluster of traits simulating post‐traumatic stress disorder. Three strains responded differently to a contextual stress 1day and 1.5 weeks post 5‐day social stress, and 1 day and 6 weeks post 10 day social stress. Final result indicate, that DBA/2j and BALB/cj is most resilient and vulnerable to Agg‐E stress, respectively. C57BL/6j displayed a modest change in phenotype character primarily after 10‐day stress.

Data analyses of psychological and physiological results were calculated using EthoVision and Graph Pad prism software.
Leptin knockdown reduces innate immune function in zebrafish
Authors: Dalman Mark R , Mustafa Ahmed , Liu Qin , Londraville Richard L
Published: June 2021

We investigated innate immune function in developing zebrafish with reduced leptin expression (via antisense morpholino technology). Leptin is a pleiotropic, adipose derived hormone known to be involved in mammalian lipid mobilization and storage, as well as the maintenance of many peripheral systems such as immune function. Despite many well‐described effects in mammals, leptin's function(s) in lower vertebrates are still poorly resolved. A fluorescent strain of P. aeruginosa was used to challenge the immune system of developing zebrafish and to visualize the progression of infection. Leptin knockdown significantly reduced the ability to fight invading pathogens, which was reflected in both reduced survivability and increased bacterial load of leptin morphants. Overall, these data suggest that leptin signaling influences innate immune function in fishes as it does in mammals, and promotes zebrafish as another vertebrate model for studying leptin function.
Chronic metformin treatment induces beneficial adaptations in dystrophic skeletal muscle
Authors: Ljubicic Vladimir , Couvrette Jean‐Francois , Renaud Jean‐Marc , Megeney Lynn A , Jasmin Bernard J
Published: June 2021

DMD is the most severe inherited neuromuscular disorder for which there is no effective treatment. Our pre‐clinical studies in mdx mice demonstrated that pharmacological activation of PPARβ/γ or AMPK elicits a fiber type shift towards the slower, more oxidative (SO) phenotype together with utrophin A upregulation thereby attenuating the dystrophic pathology (HMG, 18:2640, 2009; HMG, 20:3478, 2011; AJP Cell 302:C110, 2012). The challenge now is to identify agonists of these pathways that have more immediate clinical relevance. The purpose of this study was to investigate whether AMPK stimulation via metformin (MET), a front‐line drug in the treatment of type 2 diabetes, induces beneficial muscle remodeling. In C2C12 cells, MET augmented expression of PGC‐1α, a master regulator of the SO myogenic program, as well as utrophin A levels. MET treatment of mdx mice for 6 weeks resulted in an increase in PGC‐1α in fast skeletal muscle with a concomitant reduction of the transcriptional corepressor RIP140. Expression of utrophin A was also augmented after MET administration in vivo. The data clearly suggest that MET‐induced reciprocal changes in PGC‐1α and RIP140 expression are linked to elevations in utrophin A content. As these adaptations are expected to be highly beneficial to dystrophic fibers, we propose that MET represents a novel and promising therapeutic avenue for DMD.

Supported by MDA, CIHR, and Jesse's Journey.
Lysophosphatidylcholine is not a paracrine factor following stretch in C2C12 myotubes
Authors: Hsu Chia , Burkholder Thomas J.
Published: June 2021

Lysophospholipids, including lysophosphatidylcholine (LPC) are generated through the action of phospholipase A2. LPC can act as a paracrine factor through G2A receptors and by altering Transient receptor potential channel conductance, but also has non‐specific detergent effects. We have previously reported that stretch‐induced LPC production may mediate mechanotransduction in skeletal myotubes. The purpose of this study was to investigate the mechanism of LPC in MAPK activation in C2C12 myotubes and to examine the influence of LPC structure. Exogenous LPC induced a dose‐dependent increase in MAPK phosphorylation, however this was closely correlated with membrane permeabilization, indicated by Trypan blue uptake, and with cell death. These effects were independent of acyl chain length or saturation. Likewise, incubation with bee venom PLA2 was accompanied by increasing MAP Kinase phosphorylation and cell death. These results suggest that exogenous LPC acts primarily as a detergent and induces MAPK phosphorylation only at cytotoxic concentrations. If LPC participates in mechanotransduction, it must be through a highly localized reaction pathway.