bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cardiomyocyte Glucose Handling is Influenced by β‐adrenergic and AMPK Regulation of Glycogen and Autophagy in Metabolic Stress Settings
Authors: Mellor Kimberley M , Varma Upasna , Stevens Ellie , Delbridge Lea MD 
Published: November 2023

Autophagy disturbance and glycogen mishandling in the heart play a role in metabolic stress settings such as diabetes. We have recently demonstrated that an autophagy process specific for glycogen (‘glycophagy’), which is distinct from macro‐autophagy, is modulated by metabolic stress and is an important regulator of glycogen content in the heart. Intracellular glucose flux through these pathways has an important role in glycosylation‐related post‐translational modifications (e.g. O‐GlcNAcylation and advanced glycation end products). The aim of this study was to elucidate β‐adrenergic and AMPK signaling regulation of glycogen handling and autophagy in the heart.Excised hearts (male Sprague Dawley 8 weeks) were perfused with 10−6M isoproterenol (β agonist) or control buffer for 5 or 60 minutes, snap frozen, homogenized and protein extracted. Neonatal rat ventricular myocytes (NRVMs) cultured in 5mM or 30mM glucose were exposed to AICAR (AMPK agonist) for 30 minutes, lysed and frozen. Protein expression was assessed by western blot and glycogen content was assessed by amyloglucosidase assay.Isoproterenol‐induced β‐adrenergic activation was evidenced by an increased phospho(Ser14)‐ to total glycogen phosphorylase ratio after 5min of isoproterenol perfusion (62% increase p<0.05). Glycophagy markers GABARAPL1 and acid α glucosidase were markedly increased by β‐adrenergic signaling activation (3‐fold and 1.5‐fold increase respectively, p<0.05) consistent with depletion of glycogen content (71% lower after 5min and fully depleted after 60min Isoproterenol perfusion, p<0.05). Macro‐autophagy markers, LC3BII:I ratio and p62 protein expression were not changed in the isoproterenol perfusion groups. Inactivation of glycogen synthase (phosphorylation‐Ser641) was only evident after 60min isoproterenol perfusion. AICAR‐induced activation of AMPK attenuated high glucose‐induced glycogen accumulation in NRVMs with no change in glycogen phosphorylase activation, suggesting an independent and parallel role for glycophagy in glycogen management.This is the first study to show that β‐adrenergic signaling positively regulates glycophagy. These findings suggest that β‐adrenergic regulation of glycophagy is distinct from macro‐autophagy, demonstrating that autophagy sub‐types are influenced differentially by signaling modalities. Involvement of AMPK signaling in glycophagy induction is evident and further investigation of glycophagy in cardiac metabolic stress settings is now warranted. These studies seek to understand the relationship between glycophagic throughput and intracellular glucose exposure to functionally detrimental post‐translational modifications.Support or Funding InformationMarsden Fund Project Grant, FRDF University of Auckland Project Grant
Mechanism of Atrial Fibrillation is Related to Changes of Type IV Collagen in Rheumatic Heart Valvular Disease
Authors: Jiang Yi‐Yao , Yang Qin , Liu Xiao‐Cheng , He Guo‐Wei 
Published: November 2023

PurposeAtrial fibrillation (AF) is a common complication in heart valvular disease and is associated with morphology and cell ultrastructure abnormality. We aimed to study the distribution of collagen types and to investigate the relationship between type IV collagen changes in the tissue of the right atrium and the development of permeant AF in patients with mitral valvular disease.MethodsThe atrial appendage obtained from patients with mitral valvular disease associated with either sinus rhythm (SR, n=26, right atrial tissue) or permanent AF (AF ≥6 months, n=36, right and left atrial tissue). One part of the sample was used for histological analysis, including iTRAQ proteomics, immunohistochemistry, immunofluorescence and transmission electron microscope; the other part was analyzed by qRT‐PCR and western blotting to observe the molecular level of type IV collagen.ResultsiTRAQ proteomic study showed that type IV collagen was up‐regulated in right and left atrial tissue. Type IV collagen was located around cardiomyocytes, especially on vascular basement membrane and myocardial cell membrane. The fluorescence intensity of type IV collagen increased in AF patients. Electron microscope revealed that there was a large number of immature fibrocytes among myocytes, likely immature type collagen IV. qRT‐PCR and western blots showed that the mRNA and protein of type IV collagen were significantly elevated in AF patients, especially in the left atrial tissue.ConclusionsOur study identified that the change of type IV collagen in the atrial appendage is related to the development of AF in the mitral valvular disease. The present study suggests that abnormality of the type IV collagen may play an important role in the structure and electrical signal in the heart valvular disease associated with AF.Support or Funding InformationSupported by National Natural Science Foundation of China (No.81170148), Zhejiang Provincial Natural Science Foundation (LY15H020008), Hangzhou Science and Technology Projects (20140633B12), National Science and Technology Major Project (2013ZX09303004‐005), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BH110004), Binhai New Area Health Bureau (2012BWKZ008,2014BWKY002, 2014BWKY010), Tianjin Health Bureau (2013KZ009,2014KZ005).
Cav1.2 channel oligomerization amplifies Ca2+ influx during β‐adrenergic stimulation
Authors: Dixon Rose Ellen , Santana Luis Fernando 
Published: November 2023

L‐type Cav1.2 channels are ubiquitously expressed in the surface membranes of excitable cells where they regulate myriad processes including muscle contraction, neuronal excitability, hormone secretion and gene expression. Recently, we reported that Cav1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activity (NPo) of adjoined channels and occurs through C‐terminal‐to‐C‐terminal interactions. Channel‐to‐channel interactions are initiated by binding of incoming Ca2+ to CaM and proceed through Ca2+/CaM binding to the Cav1.2 pre‐IQ domain. FRET experiments revealed that coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. This creates a ‘primed state’ which may have a role during repetitive stimulations, such as those that occur during periods of increased heart rate – stimulated for instance by exercise or the fight‐or‐flight response. This ‘memory’ could be beneficial in generating a positive inotropic effect. In the present study we used FRET, Bimolecular Fluorescence Complementation (BiFC), electrophysiology and Ca2+ imaging to investigate the effects of β‐adrenergic signaling on Cav1.2 interactions. Optical and electrophysiological examination of single channel activity in mouse ventricular myocytes or transiently transfected tsA‐201 cells revealed increased coupling of Cav1.2 channels after application of the β‐agonist isoproterenol (iso; 100 nM) or the activator of adenylyl cyclase, forskolin (10 μM). BiFC experiments in tsA‐201 cells co‐transfected with Cav1.2 channels tagged with the N‐ or C‐terminal half of the split Venus fluorescent protein (Cav1.2‐VN and Cav1.2 ‐VC) revealed increased channel‐channel interactions stimulated by iso or forskolin and inhibited by the PKA inhibitor (PKI). These data suggest that β‐adrenergic stimulation amplifies Cav1.2 channel activity by increasing/prolonging Cav1.2 channel interactions.Support or Funding InformationThis work was supported by grants from the AHA (15SDG25560035) and NIH (HL085870 and HL085686).
Acid Sensing Ion Channel 1 Contributes to Endothelium‐Dependent Vasodilation in Mesenteric Arteries
Authors: Garcia Selina , Herbert Lindsay , Resta Thomas C , Jernigan Nikki L 
Published: November 2023

Our laboratory has previously shown that acid sensing ion channel 1 (ASIC1) is expressed in pulmonary arterial smooth muscle and contributes to agonist‐induced Ca2+ influx and vasoconstriction. However, the contribution of ASIC1 to vasoreactivity in the systemic circulation is unknown. To address this question, we simultaneously measured changes in arterial wall Ca2+ and inner diameter in response to increasing concentrations of phenylephrine (PE; 10−8‐10−5 M) or endothelin‐1 (ET‐1; 10−11 – 10−8 M) in isolated, pressurized mesenteric resistance arteries. To examine the role of ASIC1 in agonist‐induced vasoconstriction, these experiments were completed in the presence or absence of the selective ASIC1 antagonist, psalmotoxin 1 (PcTX1; 20 nM), applied either abluminally or to both luminal and abluminal surfaces of the artery. Inhibition of ASIC1 did not alter PE‐induced vasoconstriction, but augmented vasoconstriction in response to lower concentrations of ET‐1 when PcTX1 was present in the lumen. These data suggest that, in contrast to vasoconstrictor actions of ASIC1 in the pulmonary circulation, ASIC1 limits agonist‐induced vasoconstriction in the systemic circulation. Therefore, we hypothesized that endothelial expression of ASIC1 contributes to mesenteric arterial dilation. To test this hypothesis, mesenteric arteries were pre‐constricted to ~50% of baseline inner diameter with the thromboxane analog, U‐46619 (10−7 M), and vasodilatory responses to increasing concentrations of acetylcholine Ach (ACh; 10−9 ‐ 10−5 M) were assessed in the presence or absence of PcTX1 in the lumen. ACh‐induced vasodilation was significantly blunted in the presence of PcTX1. We conclude that ASIC1 contributes to endothelium‐dependent vasodilation in mesenteric arteries. These data demonstrate differing roles of ASIC1 in regulation of pulmonary and systemic vasoreactivity.Support or Funding InformationNIH NHLBI ROI HL111084 (N.L. Jernigan)NIH NHLBI T32 HL007736 (B.R. Walker)
Aerobic exercise training increases testosterone production in the testis in OLETF rats
Authors: Kumagai Hiroshi , Zempo‐Miyaki Asako , Myoenzono Kanae , Maeda Seiji 
Published: November 2023

Testosterone is a male sex hormone and low circulating testosterone levels are associated with cardiovascular diseases, type 2 diabetes mellitus. Obesity results in reduced circulating testosterone levels. We have demonstrated that lifestyle modifications (increased physical activity and calorie restriction) can increase circulating testosterone levels in obese men and increased physical activity is important for lifestyle modification‐induced increase in circulating testosterone levels. However, it is unclear the mechanism underlying regular exercise‐induced increase in circulating testosterone levels.The aim of this study was to investigate the effect of aerobic exercise training on testosterone production in the testis in male Otsuka Long Evans Tokushima Fatty (OLETF) rat.OLETF rats (12 weeks of age) were assigned randomly into exercise group (n = 8) and control group (n = 8). In the exercise group, rats ran on the treadmill for 8 weeks (for 60 min at 25 m/min, 5 days/week). After the 8‐week aerobic exercise training, we analyzed circulating and testis testosterone levels by EIA, and the protein expression of 17β‐HSD, enzyme regulating testosterone, in the testis by western blotting.After the 8‐week aerobic exercise training, body mass was significantly lower in the exercise group than control group. Circulating and testis testosterone levels were significantly higher in the exercise group than control group. Also, the protein expression of 17β‐HSD in the testis was higher in the exercise group than control group.In the present study, circulating and testis testosterone levels increased after the 8‐week aerobic exercise training in OLETF rat. Furthermore, the protein expression of 17β‐HSD in the testis in OLETF rat increased after aerobic exercise training. Therefore, the 8‐week aerobic exercise training increases testosterone and 17β‐HSD productions in the testis in OLETF rat, thereby contributing to the increased circulating testosterone level after exercise training.
Autonomic dysfunction precedes cardiometabolic alterations in a metabolic syndrome model: impact of aerobic exercise training
Authors: Dias Danielle , Bernardes Nathalia , Conti Filipe Fernandes , Brito Janaina Oliveira , Irigoyen Maria Claudia , De Angelis Kátia 
Published: November 2023

The aim was to evaluate the effects of exercise training (T) on the time course of cardiometabolic alterations in hypertensive rats (SHR) submitted to fructose overload. Wistar and SHR rats were divided: Control (C), Hypertension (H), Hypertensive + Fructose (HF) and Hypertensive Fructose + Exercise Training (HFT). The fructose overload (100g / L) was initiated 30 days after weaning of the animals. The T was performed on a treadmill (1H / d, 5d / week). The evaluations were performed at 37, 45, 60 and 90 days of life of animals. Blood pressure (BP) and heart rate (HR) were directly recorded through an acquisition system. Cardiovascular autonomic modulation was evaluated in the time and frequency domain. The H and HF groups showed an increase in VAR‐SAP compared to C group at all times. The consumption of fructose induced a further rise of the VAR‐SAP at 45 and 90 days vs. 37 days, which was not observed with the T. The H group showed an increase in LF‐SAP (vascular sympathetic) at 60 and 90 days compared to C group. However, the HF group showed an increase in VAR‐SAP at 45, 60 and 90 days compared to C group and their values in 37 days, with an additional increase in 90 days (40%) vs. H group. The H group showed a decrease in HF‐PI (cardiac parasympathetic) in 37 days and the HF group at 37 and 90 days compared to C group. The HFT group had higher RMSSD and VAR‐PI at 37 and 90 days compared to C group. The spontaneous baroreflex sensitivity was lower in H and HF groups in all times evaluated and the T increased this variable in 60 (79%) and 90 days (72%) vs. HF group. The H and HF groups showed an increase of BP in the all evaluated times compared to C group. (165±3; 179±3 vs. 114±1 mmHg) The fructose induced an additional increase in BP at 90 days and T reduced these values. The HFT group showed a HR reduction in 60 (11%) and 90 (7%) days vs. HF group. There were no significant changes in blood triglycerides and glucose in all evaluated times. The cardiovascular autonomic dysfunction seems to precede hemodynamic and metabolic changes in this experimental model of metabolic syndrome. Moreover, exercise training was effective in attenuate the time course of autonomic and hemodynamic dysfunctions.Support or Funding InformationFinancial Support: FAPESP (2015/11223‐6); CNPq (457200/2014‐6); CAPES (PROSUP).
Modulation of Methylated Arginine Metabolism to Target Exercise‐Induced Fatigue in Duchenne Muscular Dystrophy
Authors: Garbincius Joanne F. , Merz Lauren E. , Cuttitta Ashley J. , Bayne Kaitlynn V. , Schrade Sara , Armstead Emily A. , Whitesall Steven E. , D'Alecy Louis G. , Michele Daniel E. 
Published: November 2023

Duchenne muscular dystrophy (DMD) is an X‐linked disease caused by mutations in dystrophin and characterized by muscle degeneration, cardiomyopathy, and impaired muscle nitric oxide (NO) production that leads to misregulated muscle blood flow and excessive post‐exercise fatigue. Data from our laboratory show that serum levels of the NO synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in dystrophin‐deficient mdx mice and after acute muscle injury, and that infusion of ADMA is sufficient to impair exercise performance in wild‐type mice. Therefore, we hypothesized that excessive circulating ADMA impairs NO production and exaggerates exercise‐induced fatigue in DMD. To determine the impact of ADMA on fatigue resistance in this disease, female mdx mice were crossed to wild‐type males expressing a transgene encoding human dimethylarginine dimethylaminohydrolase 1 (DDAH), an enzyme that degrades ADMA. Voluntary ambulatory activity of transgenic (TG) and non‐transgenic (NTG) offspring was measured in open field assays prior to and following acute bouts of treadmill running to assess exercise‐induced fatigue. DDAH expression lowered serum ADMA concentration in TG offspring, but failed to attenuate the post‐exercise decrease in voluntary activity in mdx males. However, transgene expression resulted in a significant, 20% attenuation of the decrease in voluntary activity following exercise in female mdx carriers. The heart weight/tibia length ratio was significantly reduced in TG versus NTG carriers, although examination of the gastrocnemius and soleus muscles revealed no difference between genotypes in histological markers of dystrophy or ischemic muscle injury, suggesting that improved fatigue resistance was due to cardiac‐specific effects of DDAH expression. We conclude that ADMA contributes to excessive exercise‐induced fatigue in dystrophin heterozygous females, possibly by promoting pathological cardiac remodeling and impairing heart performance. These findings emphasize the importance of NO signaling to cardiac function in dystrophinopathies and reveal a new therapeutic target to mitigate the risk of cardiomyopathy in DMD carriers.Support or Funding InformationSupported by the Muscular Dystrophy Association, the University of Michigan CVRE Training Program, and NIH T32 GM‐08322.
3T3‐L1 Adipocytes Take Up Norepinephrine: No Canonical Norepinephrine Transporters Found
Authors: Ismail Alex , Ayala‐Lopez Nadia , Burnett Robert , Watts Stephanie 
Published: November 2023

Rat perivascular adipose tissue (PVAT) and rat adipocytes contain the organic cation transporter 3 (OCT3), a transporter of norepinephrine (NE). This is physiologically relevant because PVAT reduces vasoconstriction through the uptake of NE. We hypothesized that an accepted model of adipocytes, 3T3‐L1 cells, take up NE through OCT3 or a similar catecholamine transporter. Mouse 3T3‐L1 fibroblasts were induced into adipocytes by adding 0.5 mM IBMX and 0.25 mM dexamethasone for 48 hours. AdipoRedTM assay and Oil Red O staining confirm that induction was successful (15 times more fluorescent than preadipocytes and 95% of cells exhibit lipid droplet staining, respectively) on the 10th day after the induction. NE was added to 3T3‐L1 adipocytes at 0, 1, 10, and 100 mM concentrations in physiological saline solution (PSS) with NE metabolism inhibitors [monoamine oxidase, pargyline (10−5 M), catecholamine‐O‐methyltransferase, Ro 41‐0960 (10−6 M), semicarbazide‐sensitive amine oxidase, semicarbazide hydrochloride (10−3 M)] for 30 minutes at 37 °C. Cells were washed with PSS, collected in tissue buffer, and analyzed for NE content using HPLC. There was an increase in NE, normalized for protein content, from vehicle to 10 mM and 100 mM (0.09 ± 0.01 pg/mg, 5.97 ± 1.48 pg/mg, 18.89 ± 3.37 pg/mg, respectively, p < 0.05, N=6). PCR was performed on cDNA isolated from 3T3‐L1 adipocytes to test for known NE transporters: norepinephrine transporter (NET), OCT3, serotonin transporter (SERT), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and plasma membrane monoamine transporter (PMAT), using beta‐actin as the reference gene. OCT3, SERT, DAT, and VMAT2 did not reach the detectable threshold after 40 amplification cycles (CT value > 40, N=4). NET and PMAT had 1 sample out of 4 with a detected CT value (37.45 ± 0.15 and 37.41 ± 0.05, respectively). The positive controls had detectable amplification (heart: 26.94 ± 0.02 for OCT3, brain: 27.53 ± 0.10 for SERT, 27.90 ± 0.03 for DAT, and 26.97 ± 0.03 for PMAT, adrenal: 25.80 ± 0.15 for VMAT2, pons: 24.59 for NET). These data suggest that NE is entering 3T3‐L1 adipocytes via mechanisms other than the canonical transporters of NE. Learning how NE interacts with adipocytes, and therefore PVAT, is essential for studying the relation between vasoconstriction and obesity.Support or Funding InformationNIH NHLBI P01HL70687IPSTP 5 T32 GM 92715‐4NIH NRSA F31 HL128035‐01
Effect of Vitamin D on Adipogenesis under Hypoxia
Authors: Mutt Shivaprakash Jagalur , Lehtonen Siri , Lehenkari Petri , Leppäluoto Juhani , Järvelin Marjo‐Riitta , Herzig Karl‐Heinz 
Published: November 2023

BackgroundObesity and pandemic vitamin D (vitD) deficiency both share similar pathological associations with adipose tissue chronic inflammation leading to well‐known conditions such as insulin resistance and hypertension. Understanding adipogenesis is of major relevance for human health, as increased adiposity leads to cellular hypoxia followed by inflammation, oxidative damage and adipocyte dysfunction resulting in metabolic diseases. We have shown earlier that vitD supplementation has significant beneficial effects on adipocyte inflammation and improved insulin sensitivity in vitD deficient mice. The modulation of adipogenesis under these circumstances might be beneficial in improving its metabolic functioning. Therefore, we hypothesize that vitD modulates adipogenesis in pathological conditions (hypoxia).MethodsHuman mesenchymal stem cells (hMSCs) were differentiated into adipocytes ± vitD [10−7 M] containing adipogenic induction and maintenance medium under either hypoxic (1% O2) or normoxic (21% O2) conditions. At the end of differentiation cycles the process was assessed by the oil red O staining of the adipocytes. Fully differentiated adipocytes were stimulated with lipopolysaccharide (LPS 10 ng/ml) ± vitD [10−7 M] for 24 hrs and cells and cell culture supernatants were collected from both hypoxic and normoxic basal ± vitD [10−7 M] as well LPS stimulated ± vitD [10−7 M]. The inflammatory marker interleukin‐6 (IL‐6) levels were measured by ELISA. RNA and cell lysate were prepared and analyzed for adipogenic, hypoxic and inflammation regulating proteins.ResultsThe adipogenic differentiation of hMSCs under hypoxia was significantly inhibited in comparison to the normoxia. VitD [10−9 M to 10−7 M] significantly enhanced adipogenic differentiations dose dependently under both hypoxic and normoxic culture conditions. VitD [10−7 M] inhibited basal IL‐6 secretion by 83% in hypoxic and 66% in normoxic hMSCs differentiated adipocytes in comparison to their respective controls (p<0.05). In addition, vitD dose dependently reduced LPS induced IL‐6 secretion from hMSCs differentiated adipocytes under both hypoxic (10−7M:10−8M:10−7M; 66%:40%:19%) and normoxic (10−7M:10−8M:10−7M; 56%:44%:2%) condition in comparison with the LPS 10 ng/ml alone.ConclusionOur data demonstrates that vitD enhances the adipogensis of hMSCs under hypoxic culture condition; it inhibits basal IL‐6 release as well LPS induced IL‐6 release under both hypoxia and normoxia. This suggests that sufficient vitD levels would be able to modulate the hypoxia induced inflammation and adipogenic differentiation potential in obese subjects. These vitD actions might therefore partially mediate the beneficial effect on insulin sensitivity of adipose tissue and reduce the risk of known co‐morbidities of obesity.Support or Funding InformationSupported in part by the Academy of Finland and DynaHEALTH EU No.633595