bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphodiesterase Inhibition Increases Kv7.5 (KCNQ5) Potassium Currents in Vascular Smooth Muscle Cells by Activation of Protein Kinase A
Authors: Robakowski Christina , Brueggemann Lyubov , Mani Bharath , Byron Kenenth
Published: June 2021

Kv7 (KCNQ) potassium channels are essential for maintaining resting membrane voltage in smooth muscle cells. Functional Kv7 channels in rat mesenteric artery myocytes are formed as heteromers of Kv7.4 and Kv7.5 α‐subunits, whereas cultured A7r5 rat aortic smooth muscle cells natively express only homomeric Kv7.5 channels. Here we provide evidence that a selective inhibitor of phosphodiesterase‐4 (PDE‐4), rolipram (100nM), increases Kv7.5 currents in A7r5 cells in a protein kinase A‐ (PKA‐) dependent manner. The increase in Kv7.5 currents in A7r5 cells was concentration‐dependent, reaching a maximum of 3.4±0.1‐fold increase at 100nM rolipram. Direct activation of adenylyl cyclase with 1µM forskolin, in combination with the PDE inhibitor isobutylmethylxanthine (IBMX, 500µM), also effectively enhanced endogenous Kv7.5 currents in A7r5 cells. Pretreatment with a PKA inhibitor (KT5720, 1µM) completely prevented the rolipram‐induced enhancement of Kv7.5 current relative to its vehicle control. In mesenteric artery myocytes, rolipram did not increase native Kv7 currents. Using A7r5 cells as an expression system for human (h) Kv7 subunits, we found that rolipram did not increase currents through exogenous homomeric hKv7.4 or heteromeric hKv7.4/h7.5 channels. In contrast, rolipram robustly increased the current amplitude through exogenous homomeric hKv7.5 channels or through retigabine‐insensitive hKv7.5 mutant channels, KCNQ5W235L. The latter result suggests an absence of direct binding of rolipram to the site implicated in the binding of many pharmacological Kv7 channel activators. Our results suggest differential regulation of homomeric Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels by PKA.
Omega‐3 Polyunsaturated Fatty Acid Epoxide Metabolites Are Potent Vasodilators via Potassium Channel Activation
Authors: Zehr Kayla , Wiest Elani , Walsh Mary , Schunck Wolf‐Hagen , Walker Mary
Published: June 2021

The cardiovascular protective benefits of omega 3 polyunsaturated fatty acids are derived, in part, from cytochrome P450‐generated vasodilatory epoxyeicosatetraenoic (EEQ) and epoxydocosapentaenoic (EDP) acids. However, the potencies and mechanisms that mediate this vasodilation have not been fully elucidated. Thus, our study objectives were to compare the potencies of EEQs and EDPs to arachidonic acid (20:4n‐6)‐derived epoxyeicosatrienoic acids (EETs) and to elucidate the degree to which vasodilation was mediated via potassium channels. Using a wire myograph mouse thoracic aortic rings were preconstricted with U46619 (TP receptor agonist) and dose‐dependent relaxation was assessed to R,S and S,R enantiomers of 17,18‐EEQ and 19,20‐EDP; and to 11,12‐EET and 14,15‐EET. In addition, studies with EEQs and EDPs were conducted in the presence of iberiotoxin (IBX, BK‐channel inhibitor), tetraphenylphosphonium (TPP, K‐ATP channel inhibitor), and glibenclamide (GBN, K‐ATP channel inhibitor and TP receptor antagonist). The EEQ and EDP enantiomers were significantly more potent (log EC50, ‐11.3 to ‐11.8 ±0.2 M) than the EETs (log EC50, ‐8.0±0.1 M). In addition, TPP exhibited the greatest inhibition of relaxation of the EEQ and EDP enantiomers, ranging from 57‐100%, while IBX inhibited 20‐50% and GBN inhibited 0‐64% depending on the enantiomer. These data demonstrate that epoxide metabolites of omega‐3 PUFAs are more potent vasodilators than EETs in some vascular beds and that they mediate a significant portion of vasodilation via potassium channels.
Ca2+‐dependent and Ceramide‐mediated Membrane Repair with Annexin V Recruitment and Aggregation in Mouse Endothelial Cells
Authors: Chen Yang , Xia Min , Boini Krishna M. , Li Pin‐Lan
Published: June 2021

Annexin V is able to interact with biological membranes and may repair damaged cell membrane. It remains unknown whether this annexin V‐associated cell membrane repair can protect coronary endothelial cells (CECs) from a variety of injuries and whether is attributed to a Ca2+‐dependent, ceramide‐mediated membrane repair in CECs. Using high energy Laser gun installed in confocal microscope, a tiny hole was made in CECs bathed with FM1‐43, and entry of FM1‐43 fluorescence was monitored to measure the extent of membrane injury. The cells exhibited a Ca2+ dependent, ceramide‐mediated membrane repair, as shown by reduction of FM1‐43 fluorescence within CECs if they are bathed with a Ca2+‐solution or pretreated by acid sphingomyelinase (ASM) inhibitor or siRNA. In CECs of ASM‐/‐ mice, addition of ceramide also rescued the membrane repair response. By introduction of RFP‐annexin V into CECs, it was found that RFP‐annexin V were recruited and aggregated on the edge of a hole and accompanied with less FM1‐43 into cells. This ceramide‐mediated membrane repair was also studies in mouse streptozotocin (STZ) diabetic model. Ceramide and annexin V decreased in the coronary arterial endothelium by confocal and undetectable in ASM‐/‐ coronary endothelium. Using YOYO‐1 and PI co‐staining with a time lag i.v. injection, confocal microscopic analysis of endothelial layer injury showed that more YOYO‐1 and PI staining on the arterial wall in STZ group, indicating less membrane resealing. These data show that Annexin V participated in a Ca2+‐dependent and ceramide‐mediated membrane resealing process in CECs. In diabetes, this membrane resealing may be damaged leading to endothelial dysfunction, injury and ultimate sclerosis.
Targeting Kir4.2 in the Treatment of Type 2 Diabetes
Authors: Kharade Sujay , Guan Michael , Denton Jerod
Published: June 2021

Inward rectifier potassium (Kir) channels play fundamental roles in diverse physiological processes, and in some cases, represent novel drug targets for human diseases. KCNJ15, which encodes the non‐KATP, β‐cell inward rectifier channel Kir4.2, was recently identified as a type 2 diabetes mellitus susceptibility gene. Kir4.2 mRNA and protein expression levels are upgregulated in rat insulinoma (INS1) cells under high‐glucose conditions, and knock‐down of Kir4.2 expression in vivo enhances glucose‐stimulated insulin secretion (GSIS). Taken together, these data suggest that Kir4.2 upregulation may contribute to the impairment of GSIS observed in diabetics, and that pharmacological inhibition of the channel may restore glucose responsiveness in diabetics. In an effort to develop selective inhibitors of Kir4.2, we established a fluorescence‐based, thallium (Tl+) flux assay for interrogating large small‐molecule libraries in high‐throughput screens. The wild type channel failed to generate robust currents or Tl+ flux required for HTS, leading us to systematically explore a series of Kir4.2 mutations believed to increase its cell surface expression. A single point mutation of Lys110Asn or C‐terminal truncation at site 354 (Gln354st) resulted in ~4 and ~6 fold increase, respectively, in whole cell current density. Interestingly, the Lys110Asn and Gln354st dual mutation increased the whole cell current density by >10 fold and also showed a robust TI+ flux. We are currently validating the Kir4.2 mutant assays to screen ~10,000 small molecules for novel inhibitors of Kir4.2. Our long‐term goal is to develop potent and selective inhibitors to explore the physiological roles and therapeutic potential of Kir4.2 in diabetes.
Tumor suppression activity of bioactive components from Phomopsis sp. endophyte isolated from Ligustrum liukuensis Koidz
Authors: Cheng Jing‐Jy , Lu Chung‐Kuang
Published: June 2021

Endophytic symbionts are involved in the production of bioactive secondary metabolites in medicinal plants. In this study, EN‐032601, an endophyte from seeds of Ligustrum liukuensis Koidz, was indicated to Phomopsis sp. after 18s rDNA sequence. Cultivated metabolites and isolated compounds were used to evaluate their tumor suppression effect. The broth extracts (YHC1‐200‐2) and anthraquinones (YHC2‐81‐X) from EN‐032601 showed differential cancer cell cytotoxicity. The tumor suppression effect was observed after YHC1‐200‐2 treatment using H460 xenograft model. Body weight loss was not observed. Blood cell composition, hematopoietic parameters, bone marrow cells and spleen/total BW were not altered indicating the safety of this component. Among those anthraquinones, compound YHC2‐81‐3 arrested cell cycle in G2/M phase and gradually induced apoptosis. Cell‐cycle related proteins including induction of cyclin B1, pMPM‐2, p Cdk1 and pCdc25C, and suppression of cyclin E, cyclin A and p27 (a Cdk inhibitor) were observed. Apoptotic proteins alternation was observed including decrease of Bcl‐2 and Bcl‐xl, increase of Bax, activation of caspase 3 and PARP cleavage. Further, YHC2‐81‐3 enhanced the cytotoxic sensitivity of cispatin on H460 cells. Further, YHC2‐81‐3 showed strong DNA damaging property (induce DNA fragmentation and tailing of comet assay) and also impaired the DNA repair gene expression (ATR and ATM). In summary, these results indicated these metabolites might participate in attenuation of cancer progress toward development of targeting therapeutic agents.
Evaluation of newly developed histone deacetylase inhibitors for the treatment of solid tumors
Authors: Steinemann Karl Gustav , Nitzsche Bianca , Hoffmann Björn , Kuzyniak Weronika , Pries Axel , Hoepfner Michael
Published: June 2021

The inhibition of histone deacetylases (HDACs) with small‐molecule inhibitors is a new and promising approach for innovative cancer treatment. Histones are of importance for the organization of DNA in the form of chromatin. Transcriptional regulation comprises of a complex machinery of histone modifications, DNA‐methylation as well as acetylation and deacetylation of the chromatin. Dysregulation of acetylation and deacetylation of histones plays an important role in the development and progression of cancer and is often associated with an overexpression or upregulated activity of HDACs. HDAC inhibitors (HDACi) are highly versatile, and have effects on cell cycle, DNA‐repair, DNA‐damage, mitosis, apoptosis and angiogenesis. Here we investigated the suitability of newly synthesized HDACi with enhanced antitumor efficacy as compared to the already established HDACi, such as SAHA.

Studies on a set of different cancer cell models (urological cancer cells with or without cisplatin resistance, neuroendocrine gastrointestinal tumor cells and esophageal cancer cells) revealed pronounced growth inhibitory and apoptosis‐inducing effects – starting at nanomolar concentrations (180nM – 1.6µM). Moreover, in vivo tests using the CAM assay method revealed antiangiogenic effects and a concomitant reduction of tumor growth of inoculated urological tumors. Further investigations will focus on the underlying mode of action of the new HDACi in terms of apoptosis, attenuation of subtype‐specific HDAC‐activity and cell cycle regulation.
Pyrrolidine Dithiocarbamate, a Potent NF‐Kappa‐β Inhibitor, Induces Cell Cycle Dysregulation and Suppression of Autophagy in Triple‐Negative Breast Cancer Cells
Authors: Tomblin Justin , Mehmi Inderjit , Salisbury Travis
Published: June 2021

Triple‐negative breast cancer cells (BCCs) lack the estrogen, progesterone, and ERBB2 receptors. As such, these cells do not respond well to standard chemotherapy regimens such as hormone based therapies like tamoxifen and aromatase inhibitors. Triple‐negative cancers require combinatorial therapies, which are often unsuccessful and are associated with marked toxicity. New drug therapy effective against these cancers is, thus, of great importance. Here, for the first time, we show pyrrolidine dithiocarbamate (PDTC), a potent NF‐Kappa‐β inhibitor, dysregulates the cell cycle by decreasing expression of cyclin‐dependent kinases (CDK)‐4 and 5 while simultaneously inducing cyclin D1 (CCND1) in MDA‐MB‐231 and MDA‐MB‐436 BCCs. We correlated this with reduction in activated Retinoblastoma (Rb) protein. Changes in cell morphology were noted by 24hrs, and cell viability following PDTC treatment (1μM‐100μM) was reduced compared to control cells. To observe whether apoptosis was occurring, levels of nuclear protein poly (ADP‐ribose) polymerase (PARP), which undergoes cleavage during apoptosis, were assayed and were not significantly different from control cells following 24hr PDTC treatment. Interestingly, we noted decreased levels of several proteins involved in autophagy, a stress response cells undergo which when inhibited leads to cell death. Currently, we are investigating whether PDTC's tumoricidal effects occur through this potential mechanism.
ROLE OF HSP72 IN THE CARDIAC REMODELING INDUCED BY HIGH FAT DIET
Authors: Moreira Luana , Guedes Elaine , Leal Tábata , Mestril Ruben , Miyabara Elen , Diniz Gabriela
Published: June 2021

Introduction

Previous study demonstrated that HSP72 protects against obesity‐induced insulin resistance. However, the potential role of HSP72 in the cardiac remodeling induced by high fat (HF) diet remains unknown.

Objective

The aim of this study is to investigate the role of HSP72 in the cardiac remodeling induced by HF diet.

Methods

Male wild‐type (WT) and HSP72 transgenic (HSP72 Tg) mice were fed a normal diet or a HF diet for 10 weeks. Body weight was monitored weekly. Fasting glucose levels were measured and intraperitoneal glucose tolerance test (iGTT) was performed. Systolic blood pressure (SBP) was evaluated by tail‐cuff plethysmograph. Western Blot was used to evaluate the cardiac HSP72 and HSP90 levels. Cardiac hypertrophy will be determined by heart weight normalized by tibia length. Histological analysis will be performed to evaluate the cardiomyocyte diameter and the interstitial fibrosis.

Results

HF diet increased the body weight and the fasting glucose levels in WT mice. However, HSP72 Tg were resistant to HF diet‐induced increase in body weight.In addition, HF diet induced glucose intolerance in WT mice. HF diet did not change the SBP in WT mice. The cardiac HSP70 levels were increased in HSP72 Tg when compared to those observed in WT mice. On the other hand, cardiac HSP90 levels were similar between WT and HSP72 Tg mice.

Conclusions

These results demonstrate that HF diet increases the body weight and fasting glucose levels, as well as induces glucose intolerance in WT mice. In addition, cardiac HSP70 levels were increased on HSP72 Tg mice, supporting the use of these mice to investigate a potential role of HSP72 in the cardiac remodeling induced by HF diet.
Wnt3a and Wnt1 Enhance Myogenesis of C2C12 Myoblasts – Potential Mechanisms of Osteocyte to Muscle Cell Signaling
Authors: Huang Jian , Mo Chenglin , Bonewald Lynda , Brotto Marco
Published: June 2021

We have proposed that muscle and bone communicate via biochemical factors that are reciprocally important for optimal function. WNTs are secreted by osteocytes and have a significant potential to directly influence muscle function. We showed that WNT3a enhanced myogenic differentiation at a concentration of as low as 0.5ng/ml, and stimulated β‐catenin translocation to the nucleus of C2C12 muscle cells. Now, we show that Wnts express in MLO‐Y4 osteocytes and 2T3 osteoblasts, and their expression is dramatically enhanced by fluid flow shear stress, suggesting a physiological role with increased activity for example. WNT3a promoted overexpression of myogenic markers (MyoD, MyoG, MHC), and downstream genes of the β‐catenin pathway (Fhl1 and Numb). Furthermore, WNT3a treated C2C12 cells demonstrated higher expression of Axin2, Wnt5a, Gata3 and Adm, while the expression of Hey2, Ifng, Icam1, Tnf and Lrg1 decreased significantly. These genes represent the Wnt signaling pathway (Axin2, Wnt5a), JAK1, 3/Stat6 signaling pathway (Gata3), Hypoxia signaling pathway (Adm), Notch signaling pathway (Hey2), NFκB signaling pathway (Tnf, Ifng, Icam1) and the Stat3 signaling pathway (Lrg1). These results suggest that WNT3a may function through these pathways to regulate C2C12 myoblast differentiation.

Support: NIH‐NIA P01 AG039355 (LB, MB), and the Dale and Dorothy Thompson Endowment Fund (MB).
Conducted Vasodilation in Brain Parenchymal Arterioles is Impaired during Chronic Hypertension
Authors: Chan Siu‐Lung , Cipolla Marilyn
Published: June 2021

Brain parenchymal arterioles (PAs) are high resistance vessels that directly connect the pial surface vessels to the capillaries. We hypothesized that conduction of vasodilatory signals upstream in PAs is impaired during chronic hypertension, a consequence that may influence functional hyperemia and local control of blood flow in the brain. PAs from 18‐week old male stroke‐prone spontaneous hypertensive rats (SHRSP) and age‐matched Wistar rats (n=4‐8/group) were studied isolated and pressurized in an arteriograph system. As K+ is an important dilatory substance released locally during functional hyperemia that activates inward rectifier potassium channel (Kir), 15 mM KCl was applied locally by a micro‐pipette ejection system (400 ms, 20 psi) and lumen diameter of PAs recorded at local and conducted (600 µm proximally) sites. In Wistar rats, KCl dilated PAs at the local site close to their maximum diameter (59±5 in KCl vs. 70±5 µm in zero calcium; p>0.05). Local dilation to KCl was attenuated by the Kir blocker BaCl2 (30 µM), suggesting that KCl elicited vasodilation in PAs through Kir activation. Conduction of the vasodilatory signal was highly effective in PAs from Wistar rats, producing a conducted vasodilation that was 87±5 % of local. In SHRSP, both local and conducted responses to KCl were attenuated. The diameter of PAs from SHRSP in KCl locally was only 35±1 vs. 51±5 µm in zero calcium (p<0.05). Conduction of vasodilation was also less effective in SHRSP with only 64±6 % of the dilation conducted to the proximal site (p<0.05 vs. Wistar). In conclusion, conducted vasodilation in PAs is impaired during chronic hypertension that may adversely impact functional hyperemia and possibly lead to chronic ischemia in the brain. Supported by NIH PO1 HL095488.
Effects of arginine vasopressin (AVP) deficiency on basal mean arterial blood pressure in the rat
Authors: Villanueva Gloria , Organista‐Esparza Alejandro , Viñuela‐Berni Verónica , Huerta‐Carreón Erika , Chavira‐Ramirez Roberto , Tinajero‐Ruelas Manuel , Kovacs Kalman , Quintanar‐Stephano Andrés
Published: June 2021

The role of AVP controlling the mean arterial blood pressure (MBP) in basal conditions remains unclear. We described that neurointermediate pituitary lobectomy (NIL) in rats, induced an early but transient diabetes insipidus and low MBP (8 weeks after lobectomy). To elucidate the chronological effects of NIL, we evaluated the AVP serum levels, water intake and urine output (diabetes insipidus, DI), and MBP immediately (0 to 6 hours post NIL) and 3, 15, 45 and 90 days post NIL. The renin, angiotensin II and aldosterone system (RAAS) was assessed at 3 and 45 days post NIL. Values were compared with the respective SHAM operated groups. Results: NIL caused significant and permanent AVP serum level decrease (10.6 ± 0.8 vs 2.4 ± 0.16 pg/ml (SD), SHAM vs NIL), transient DI (15‐30 days of duration) and permanent decrease of the MBP in 70% of the NIL animals (106.8 ± 3.15 vs 73.8 ± 3.2 mmHg). Hypotension was apparent 20‐30 minutes after NIL. No significant differences in the RAAS were evident.

Conclusions

We propose that in non‐stressed animals, basal serum levels of AVP are required to maintain the hydro‐electrolytic balance and MBP. Further studies are needed to explain why some animals did not develop hypotension, and to determine how cardiovascular, renal and hormone adaptive mechanisms in the NIL group support their survival.

Supported by UAA‐PIFF and CONACYT, México and the Jarislowsky and L. Carr‐Harris Foundations
Improved Signal Quality and Sensitivity Using Intravascular Placement of Solid Tip ECG Leads in Rats
Authors: Simenson Heather , Fine Megan , Bogie Heather , Holliday‐White Kimberly , Lillegard Kathryn , Lindquist Ryan , Doyle Jennifer , Sarazan R , Main Bradley
Published: June 2021

Continuous data recording, using telemetry, is the preferred way of collecting electrocardiographic (ECG) data in conscious freely moving animals. In small animals, however; the resulting recordings can have indistinguishable P, Q, R, S and T segments due to low signal to noise ratios. In large animals, this has been addressed using wireless telemetry devices with an intravascular negative electrode, or solid tip lead (STL). The purpose of this study was to determine if STLs could be implanted in rats and result in improved ECG signal quality and sensitivity to detect changes when compared to subcutaneous (SQ) leads. Three groups of Sprague Dawley rats (SQ lead placement, STL with shallow placement in the jugular vein, STLs with deep placement in the cranial vena cava) were dosed with verapamil to induce predictable interval changes in the ECG. The three types of ECG signals were compared for ease of automated analysis and signal quality through Q, R, S and T match and noise parameters. STLs resulted in improved signal quality and increased PR interval change following dose. Average number of waveform complexes identified as “bad” by the analysis software was substantially lower in the STL placements than the data collected with SQ electrodes. STLs were successfully implanted in rats and necropsy showed variable tip locations with no gross abnormalities. This application could be especially useful in other small animal models such as the guinea pig, to increase ECG quality and improve automated ECG analysis.
P Wave Indices on Electrocardiogram and their Association with Obesity and Metabolic Syndrome
Authors: Vaidean Georgeta , Manczuk Marta , Vansal Sandeep
Published: June 2021

Introduction

The obesity and atrial fibrillation epidemics may be linked. Obesity is a risk factor for atrial fibrillation but whether alterations of the P wave indices are associated with obesity is not fully understood.

Methods

We used cross‐sectional data of 11792 men and women, ages 45 to 64 years, in the PONS cohort in Poland. Data were collected via standardized questionnaires, physical exam and fasting blood samples. The P wave duration and PR interval were recorded on a 12‐lead standard ECG. Lack of sinus rhythm, third degree heart block and PR intervals <= 80ms or >=320 ms were excluded.

Results

There were 3893 men (33.01%) and 7899 women with a mean age of 55.67 years (SD 5.37). The mean (SD) for the P wave duration and PR interval were 106.86 ms (16.44) and 156.37 ms (24.52) respectively. For both indices, there were progressive increases from the lowest (< 25 kg/m2) to the highest (> 30 kg/m2) BMI quartile (p for linear trend < 0.0001), after adjusting for age, gender, smoking , heart rate and heart disease. A trend was observed with increasing number of metabolic syndrome components. There was a positive association between the adjusted P wave duration and blood pressure control: SBP/DBP <120/80 : 104.85 ms (95% CI 103.98‐105.72); SBP 120‐140/DBP 80‐90: 106.79ms (106.42‐107.16); SBP/DBP> 140/90: 107.93 ms (95% CI 107.35‐108.51).

Conclusion

P wave indices were significantly associated with obesity, metabolic syndrome and level of blood pressure control; they may represent markers of atrial fibrillation in individuals with obesity and/or metabolic syndrome.
The 4th Window: A Novel, Clinically Relevant Model of Delayed Ischemic Preconditioning in Conscious, Chronically Instrumented Pigs
Authors: Bravo Claudio , Kudej Raymond , Zhao Xin , Pachon Ronald , McNulty Tara , Yan Lin , Vatner Dorothy , Vatner Stephen
Published: June 2021

Most studies on cardiac ischemic preconditioning (IPC) focused on the 1st or 2nd window, where the preconditioning ischemic stimulus was induced briefly and the cardiac protection all occurred within a few hours or days. More recently 3rd window models have been described with IPC induced over several days. The goal of this investigation was to determine if IPC could be extended for a longer time, making it more clinically relevant. In this new IPC model, which we named the 4th window, pigs, chronically instrumented with coronary blood flow transducers, hydraulic occluders, and ultrasonic wall thickness crystals implanted in potentially ischemic and non‐ischemic zones, underwent IPC induced by 6 episodes of 90 min coronary stenosis (40% reduction in blood flow but no necrosis) every 12 hours over 3 days. 60 min coronary artery occlusion (CAO) followed by 3 days of coronary reperfusion, induced one week after IPC, resulted in infarct size, as a fraction of the area at risk, of 11±2.5%, less (p<0.05) than in the absence of IPC (42±3.9%). Most genes expressed in this model were novel, compared to prior IPC models, included genes mediating angiogenesis. To confirm angiogenesis, we found increased capillary density adjacent to the infarct in the 4th window by 154% (p<0.05), and 38% increased, p<0.05, blood flow (measured with microspheres) during the 60min CAO, compared in the same samples with brief CAO before IPC. In summary, this novel model of 4th window IPC induces angiogenesis and markedly different gene regulation than previous IPC models.
Ischemic Postconditioning during Cardiopulmonary Resuscitation Improves Cardiac Mitochondrial Respiration
Authors: Matsuura Timothy , Riess Matthias , Bartos Jason , Olson Matthew , Rees Jennifer , McKnite Scott , Cheng Qunli , Bienengraeber Martin , Aufderheide Tom , Neumar Robert , Lurie Keith , Yannopoulos Demetris
Published: June 2021

Ischemic postconditioning (IPC) during cardiopulmonary resuscitation (CPR) increases neurologically intact survival in a porcine cardiac arrest model. We investigated the effect of IPC on mitochondrial respiration, an important therapeutic target for resuscitation, during CPR in this model. Blood pressure and ECG were continuously recorded. After 15 min of ventricular fibrillation, animals received standard CPR +/‐ IPC. IPC consisted of 3 cycles of 20 sec compression / 20 sec pause for the first 2 min of CPR. Cardiac mitochondria were isolated after 4 min. Closed‐cell respiration was measured for complex I (pyruvate+malate) and II (succinate+rotenone). In IPC‐treated animals vs. standard CPR, complex I and II respiratory control indices (RCI), calculated as the ratio of state 3:4 respiration, were higher (6.7±0.8 and 3.1±0.6 vs. 4.3±0.7 and 2.4±0.2, respectively) and complex I and II state 4 respiration (µmol O2*hr‐1*mg‐1) were lower (1.7±0.2 and 2.7±0.3 vs. 2.3±0.5 and 3.3±0.5, respectively). Blood pressure during CPR did not differ. In conclusion, IPC improved mitochondrial oxidative phosphorylation as early as 4 min after initiation of CPR, suggesting that CPR is a source of reperfusion injury and a critical time for intervention. Supported by NIH (R01 HL123227) and VA (CARA‐026‐10F).
Role of O‐GlcNAcylation in Regulating Mitophagy in Cardiomyocytes
Authors: Wright JaLessa , Kramer Philip , Darley‐Usmar Victor , Chatham John
Published: June 2021

Acute increases in O‐GlcNAcylation in the heart have been shown to be cardioprotective, which was associated with O‐GlcNAcylation of mitochondrial proteins and increased tolerance of mitochondria to oxidative stress. Mitophagy, a specific type of cargo‐mediated autophagy, is critical in removing damaged mitochondria and has also been shown to contribute to protection against ischemia/reperfusion injury. Therefore, the goal of this study was to determine whether O‐GlcNAcylation contributed to the regulation of mitophagy. Mitophagy was induced in AC16 cardiomyocytes, using 2,3‐dimethoxy‐1,4‐napthoquinone (DMNQ, 20 µM), a redox cycling agent which increases mitochondrial reactive oxygen species (ROS). Protein O‐GlcNAc levels were increased by treatment with the O‐GlcNAcase inhibitor, Thiamet‐G (3 µM). DMNQ treatment had no significant effect on either O‐GlcNAc levels or protein levels of PINK1, which recruits Parkin to damaged mitochondria to initiate mitophagy; however, Thiamet‐G significantly increased total PINK1 levels 2‐fold. Preliminary studies showed that PINK1 is O‐GlcNAcylated under basal conditions, which is markedly decreased by DMNQ; whereas Thiamet‐G prevents the loss of PINK O‐GlcNAcylation.Treatment of AC16 cells with either DMNQ or 4‐Hydroxynonenal (HNE, 30 µM) resulted in significantly decreased mitochondrial maximal oxygen consumption and reserve capacity, which was partially improved if thiamet‐G was added following treatment. Surprisingly pre‐treatment with Thiamet‐G had no protective effect. These results support the concept that protein O‐GlcNAcylation may play a role in regulating mitophagy and protect mitochondria against oxidative stress.
oxLDL induce endothelial cell stiffening through Rho/ROCK signaling pathway
Authors: Zhang Chongxu , Oh Myung‐Jin , Levitan Irena
Published: June 2021

Our lab demonstrated that Oxidized LDL (OxLDL) induce endothelial cell stiffening. The signal transduction pathways leading to this increase in stiffness remain poorly understood. We hypothesize that the Rho/ROCK pathway plays a role in oxLDL induced endothelial cell stiffening. To test our hypothesis, human aortic endothelial cells (HAEC) were treated with 50µg/ml oxLDL and Rho/ROCK signaling was probed by western‐blot. First, our results show that RhoA is significantly activated by 50ug/ml oxLDL stimulation. The activated RhoA level increased 1.9 times vs control after 15 minutes incubation with oxLDL, and activated RhoA level return to control level after 60 minutes of incubation with oxLDL. Second, incubation with oxLDL increase phospho‐myosin phosphatase target subunit 1 (p‐MYPT1 (Thr853)) and phosphor‐Myosin ligh chain 2 (p‐MLC2 (Thr18/Ser19) expression level vs control. We further test that if oxLDL could affect myosin phosphorylation. Our data showed p‐MLC2 increased 1.7 times vs control after 15 minutes incubation with oxLDL, and increased 2.6 times vs control after 60 minutes incubation. The level of p‐MYPT1, which indicate the inhibition of myosin phosphatase, increased to 1.7 times vs control after 15 minutes incubation with oxLDL. Third, to further test our hypothesis, we treated HAECs with RhoA inhibitor (C3) or ROCK inhibitor (Y27632) before oxLDL incubation. Our data showed that both C3 and Y27632 attenuate oxLDL induced increase of p‐MLC2 and p‐MYPT1. Lastly, our data showed that increase of cell stiffening is also attenuated by C3 or Y27632 when treated with oxLDL.

Conclusion

oxLDL induce cell stiffening through increase RhoA activity, active Rho/ROCK pathway, inhibition myosin phosphatase, thereby increasing phosphorylation of myosin and leading to cell stiffening.
Effect of Renal Denervation on Blood Pressure and MicroRNA 181a in Hypertensive Schlager Mice
Authors: Head Geoffrey , Gueguen Cindy , Marques Francine , Jackson Kristy , Eikelis Nina , Stevenson Emily , Lambert Gavin , Charchar Fadi , Davern Pamela
Published: June 2021

Hypertensive Schlager mice (BPH) are hypertensive due to an exaggerated contribution of the sympathetic nervous system (SNS) and renin angiotensin system (RAS). The latter was associated with reduced expression of the renin regulatory micro RNA 181a. We therefore determined the effect of bilateral renal denervation (Rx) on blood pressure (BP) in BPH compared to normotensive BPN. BP was measured by radiotelemetry and Rx performed by surgery and 10% phenol. After 3 weeks, BP was 8±2 mmHg lower than sham in Rx BPH mice but Rx had no effect in BPN. Following Rx, enalapril (RAS contribution) decreased BP more in BPH mice compared to sham group but had no effect in BPN. The depressor response to pentolinium (SNS contribution) was greater in BPN mice following Rx compared with the sham group but the response was unaffected by Rx in BPH. Rx reduced renal norepinephrine levels in both strains but more so in BPH. Rx normalised both miR‐181a and renin mRNA in BPH to levels comparable to the control strain. We suggest that renal sympathetic activity is essential in maintaining hypertension in BPH mice partly by overexpression of renal renin as a result of inhibiting miR‐181a. Thus the antihypertensive effects of Rx may be dependent on the form of hypertension, whether it involves neuronal or renal mechanisms.
Effect of acute lower leg heating on aortic pulse wave reflection in postmenopausal women
Authors: Kosaki Keisei , Sugawara Jun , Akazawa Nobuhiko , Matsubara Tomoko , Zempo‐Miyaki Asako , Maeda Seiji
Published: June 2021

Postmenopausal women exhibit greater aortic pulse wave reflection, a cardiovascular disease risk, compared with premenopausal women. This might be partly attributed to the elevated peripheral vascular resistance. In this study, we examined in postmenopausal women whether aortic pulse wave reflection was decreased by acute lower leg heating (LLH) which expectedly reduced local vascular tone. Using a chamber incorporated with far infrared radiation heater, 13 postmenopausal women (64±1 years) underwent 20 minutes of supine resting with and without LLH (at ∼44ºC and room temperature, respectively) on separate days by cross‐over design. Blood pressure, aortic and leg pulse wave velocity (PWV) and aortic augmentation index (AI) were measured before, 30 min and 60 min after each intervention. AI and leg PWV decreased 30 min after LLH (P<0.05 for both) and returned to the baseline 60 min after LLH, whereas blood pressure and aortic PWV did not change significantly. No significant changes were observed in these parameters in the control trial. Changes in the difference between leg PWV and aortic PWV (Δleg PWV – aortic PWV) correlated with corresponding changes in AI (r=0.686, P<0.05), suggesting that LLH‐induced change in impedance mismatch between central elastic and peripheral muscular arteries might be associated with the decrease in aortic AI. In postmenopausal women, LLH might be available for the acute reduction of left ventricular afterload.
The Role of AMPK‐SIRT1 Pathway in High Glucose‐Induced Insulin Resistance in Rat Cultured Podocytes
Authors: Rogacka Dorota , Piwkowska Agnieszka , Audzeyenka Irena , Angielski Stefan , Jankowski Maciej
Published: June 2021

Podocytes insulin sensitivity is critical for glomerular function and the loss of correct insulin signaling in consequence leads to alterations and disorders featuring diabetic nephropathy. The energy sensing pathways, like AMP‐dependent protein kinase (AMPK), and class III protein deacetylase SIRT1 have been shown to play an important role in insulin resistance.

The aim of this study was determination of the role of AMPK and SIRT1 in insulin resistance of podocytes exposed to high glucose concentration. Experiments were performed in primary rat podocytes cultured with normal (NG, 11.1 mM) or high (HG, 30 mM) glucose concentrations for 5 days. Insulin‐stimulated changes in glucose uptake were used to detect insulin resistance.

HG decreased the phosphorylation of AMPK by about 20% (P<0.05) and abolished insulin‐stimulated glucose uptake into podocytes. Knockdown of SIRT1 protein expression in NG‐cultured podocytes decreased AMPK phosphorylation by about 30% and abolished insulin‐stimulated glucose uptake. The stimulating effect of resveratrol (RSV) on AMPK phosphorylation was observed in cells cultured in NG or HG medium, by about 50% and 65%, respectively. Glucose uptake was also increased in HG‐cultured podocytes treated with RSV.

We found that impairment of insulin induction of glucose uptake into podocytes cultivated in the presence of high glucose concentrations for long periods of time is associated with decreased AMPK phosphorylation in an SIRT1‐dependent manner. This work was supported by grant from the National Science Centre (Grant 2012/05/N/NZ5/01448).
Hydrogen Sulfide Inhibits Ca2+‐induced M itochondrial Permeability Transition Pore Opening in Type‐1 Diabetes
Authors: Kundu Sourav , Pushpakumar Sathnur , Sen Utpal
Published: June 2021

Hydrogen sulfide (H2S) attenuates N‐Methyl‐D‐Aspartate receptor (NMDA‐R) and mitigates diabetic renal damage but the molecular mechanism is not known. While NMDA‐R facilitates Ca2+ permeability, H2S is known to inhibit L‐type Ca2+ channel. High Ca2+ activates cyclophilin D (CyPD), a gatekeeper protein of mitochondrial permeability transition pore (mPTP), thus facilitating molecular exchange between matrix and cytoplasm causing oxidative outburst and cell death. We tested the hypothesis that NMDA‐R mediates Ca2+ influx causing CyPD activation and mPTP opening leading to oxidative stress and diabetic renal injury. H2S treatment blocks Ca2+ channel and thus inhibits CyPD and mPTP opening and reduces renal damage. C57BL/6J and Akita (C57BL/6J‐Ins2Akita) mice were treated without or with H2S donor (GYY4137, 0.25mg/Kg/day) intra‐peritoneally for 4 weeks. In vitro studies were performed using mouse glomerular endothelial cells. Low levels of H2S and increased expression of NMDA‐R1 in diabetes induced Ca2+ permeability, which was ameliorated by H2S treatment. We observed cytosolic Ca2+ influx in hyperglycemia along with mt‐CyPD activation, increased mPTP opening and oxidative outburst which were reversed after H2S treatment. Renal injury biomarker, KIM‐1 was up regulated in diabetes and decreased after H2S treatment. Inhibition of NMDA‐R by pharmacological blocker, MK‐801 revealed similar results. We conclude that NMDA‐R1 mediated Ca2+ influx in diabetes induces mPTP opening via CyPD activation leading to increased oxidative stress and renal injury. We provide further evidence that H2S protects diabetic kidney by blocking mitochondrial Ca2+ permeability through NMDA‐R1 pathway.
A Non‐Linear Career Path
Authors: Bailey Cheryl
Published: June 2021

Learning about “self” theories (self‐efficacy, self‐determination, self‐worth) can shed light on skills and motivation for career satisfaction and opportunity. Working in industry, a funding agency and academia have taught me skills that I value. There have been people with whom I've worked that have helped me learn how to work better with others. As teachers and researchers, much of our work focuses on developing others, which is laudable. This presentation focuses on self‐awareness of our own development and ways we can use the “self” theories for intentional development of intrinsic motivation in areas that we may avoid. The outcomes of the presentation is for each of us to identify a skill or insight we learned in a recent situation, identify a skill we would like to learn with a plan to learn that skill, and identify a growth opportunity for someone else that we can provide.
Spontaneous Hypertension and Cardiorenal Pathologies in the African Green Monkey
Authors: Rhoads M , Goleva S , Beierschmitt A , Osborn J
Published: June 2021

The African Green monkey (AGM), Chlorocebus aethiops sabaeus, is a highly translational non‐human primate model of spontaneous hypertension. Using forearm plethysmography under ketamine sedation (~15mg/kg), AGMs were categorized as hypertensive (HT, SBP >140mmHg), borderline hypertensive (BHT, 120mmHg < SBP < 140mmHg), or normotensive (NT, SBP < 120 mmHg). Of the 264 animals phenotyped, 32% (85 of 264) were HT (average MAP 108.2±2.2mmHg), 18% (49 of 264) were BHT (average MAP 87.5±2.0mmHg), and 49% (130 of 264) were NT (average MAP 68.6±1.3mmHg). HT animals tend to be older than NT animals (12.4±0.7 yrs vs. 8.7±0.6 yrs; p<0.05) with elevated heart rates (134.3±2.1 bpm vs. 121.6±1.9 bpm, p<0.05), similar to human essential hypertension. HT animals have increased cellular myocyte cross‐sectional area in left ventricles (1658.4±194.7 μm2) compared to NT (791.0±168.9 μm2;p<0.05, n=8) AGMs. Wall/lumen (W/L) ratios of renal arterioles (>100µm diameter) were greater in HT (0.15±0.02 vs. 0.11±0.04; p<0.05, n=8) animals. HT glomeruli have increased Bowman's capsular space (44.4±3.1% area vs. 30.9±1.9% area; p<0.05, n=5). Elevated heart rates and left ventricular hypertrophy suggest that hypertension in AGMs is driven by increased sympathetic nerve activity (SNA). Increased W/L ratios and Bowman's capsular space indicate renal vascular remodeling and altered glomerular function. This data postulates that the AGM is a model of spontaneous hypertension with elevated SNA, increased renal vascular resistance and altered glomerular capillary function. Supported by Biomedical Sciences Research Group, LLC and the Behavioural Sciences Foundation
Telemetric Recording of Renal and Carotid Blood Flow Velocity and Arterial Blood Pressure Simultaneously in Rats
Authors: Koeners Maarten , Lewis Kirsty , Paton Julian
Published: June 2021

Hypertension‐induced barotrauma without or with concomitant vasoconstriction, tissue ischemia and hypoxia has been postulated to initiate and drive the pathogenic cascades that lead to hypertension induced tissue injury. Yet the precise temporal relationship between arterial pressure and tissue blood flow over time as causative mechanisms for barotrauma remain unresolved and controversial. A major technical limitation has been the absence of methods allowing simultaneous high fidelity long‐term measurement of blood flow and arterial pressure in conscious animals. Transonic has developed a cutting edge technology to continuously evaluate arterial blood pressure and two blood flow velocity's simultaneously in conscious rats. Here, we report the first demonstration of recording renal and carotid blood flow and arterial pressure using this new multi‐variable telemetric system. A solid state pressure sensor was placed in the abdominal aorta and 0.8mm and 0.5mm in diameter flow probes were sutured on the renal and carotid artery, respectively, in Wistar rats. Both flow velocity signals correlated with the arterial pressure signal and exhibited constant lag times. After 5 min of carbogen breathing carotid vascular resistance decreased by 31±4% while renal vascular resistance decreased by 7±1%. These experiments demonstrate the feasibility of the technique for high fidelity temporal profiling of both blood pressure and blood flows to renal and carotid vascular beds simultaneously in conscious rats. British Heart Foundation funded research.
A Fructose Enriched Diet (20%) Induces Salt‐Sensitive Hypertension and Prevents Salt‐Induced Decrease in Plasma Renin
Authors: Beierwaltes William , Ismail Alexander , Szandzik David , Garvin Jeffrey , Ortiz Pablo
Published: June 2021

Dietary Fructose is implicated in the development of hypertension, diabetes and metabolic syndrome. Rats supplemented with 20% fructose develop hypertension, hypertriglyceridemia, and hyperglycemia after 8 weeks (Mamikutty N,et al. Biomed Res Int. '14). We hypothesized rats given a fructose‐enriched diet mimicking the upper range consumption in the US would cause salt‐sensitive hypertension prior to the onset of metabolic abnormalities. Groups of 8‐11 rats were given a normal rat chow diet, supplemented with either 1% NaCl, 20% fructose, or 1% NaCl plus 20% fructose in their drinking water over 4 weeks. Systolic blood pressure and body weights were monitored and 24 hour urine and blood collected at 4 weeks. After 4 weeks, systolic blood pressure was significantly increased by 10 ±4 mmHg (p<0.05) only in the fructose plus salt group. Growth rates were similar in all four groups (5.4 ± 0.4 g/day). Plasma electrolytes and glycemic indices were also similar. Plasma renin activity (PRA) was similar in control vs fructose rats (2.51±0.72 vs 2.71 ±0.90 ng AngI/ml/hr, respectively) but was suppressed by 80% (0.43 ±0.07 ngAng1/ml/hr) in rats fed 1% NaCl. However, fructose feeding prevented high salt induced‐suppression of PRA (1.89 ±0.43 ngAng1/ml/hr). Sodium excretion was elevated in both groups receiving NaCl. We conclude addition of salt to a fructose‐enriched diet induced salt‐sensitive hypertension prior to other pathologies. The blunting of salt‐induced suppression of PRA by fructose suggests it alters the sensitivity of renin secretion to salt intake and this may contribute to the increase in blood pressure.
Effects of Norepinephrine on Blood Pressure and Intra‐renal Perfusion and Oxygenation in Ovine Hypotensive Sepsis
Authors: May Clive , Kosaka Junko , Evans Roger , Bellomo Rinaldo , Lankadeva Yugeesh
Published: June 2021

Background and Aims

In sepsis, norepinephrine (NE) is a primary treatment to maintain arterial pressure, but its effects on intrarenal oxygenation and perfusion are unclear. We therefore examined the effects of NE on mean arterial pressure (MAP), renal blood flow (RBF), urine flow (UF) and renal cortical and medullary perfusion and oxygenation during hypotensive sepsis in conscious sheep.

Methods

Sheep were instrumented with a renal artery flow probe, and fiber‐optic probes in the renal cortex and medulla to measure tissue PO2 and perfusion. Sepsis was induced by infusion of E coli for 32 h. NE (0.4 μg/kg/h) was infused intravenously from 25‐30 h of sepsis (n=5).

Results

Sepsis was characterized by hypotension (‐10 mmHg), increased RBF and oliguria. At 24 h of sepsis, cortical tissue perfusion (1163±37 to 1607±200 mmHg) and PO2 (30.8±3.9 to 37.4±5.3 mmHg) had increased, but medullary perfusion (1423±113 to 621±100 BPU) and oxygenation (31.3±1.7 to 16.1±3.8 mmHg) were reduced. Infusion of NE restored MAP and UF, did not change cortical perfusion and oxygenation, but further reduced medullary perfusion (to 540±200 BPU) and oxygenation (to 12.3±3.1 mmHg).

Conclusions

Hypotensive sepsis was associated with reduced medullary perfusion and oxygenation, which may play a pivotal role in development of AKI. Administration of NE reversed sepsis‐induced hypotension and oliguria, but caused a further decline in medullary perfusion and oxygenation. Although NA treatment restored blood pressure in sepsis, it exacerbated medullary hypoxia, which may worsen kidney injury in the long term.
In C57Bl/6N Female Mice 17β‐Estradiol Leads to Cardiac Mass and Function Loss via a β‐Catenin Mechanism
Authors: Kararigas Georgios , Zelarayan Laura , Toischer Karl , Hasenfuss Gerd , Jarry Hubertus , Regitz‐Zagrosek Vera
Published: June 2021

We recently found that 17β‐estradiol (E2) induced physiological hypertrophic growth in the heart of C57Bl/6J but not of C57Bl/6N female mice. We aimed at characterizing the E2 effects in C57Bl/6N mice hypothesizing that β‐catenin mediates these. Two‐month‐old female C57Bl/6N wild‐type (WT) and cardiac‐specific β‐catenin‐deleted (β‐catΔex2‐6) mice were ovariectomized and randomized to an E2‐containing or soy‐free (CON) diet (n = 7‐13/group). The 3‐month physiological dose of E2 led to a higher relative uterus weight compared with CON (P < 0.001) in both WT and β‐catΔex2‐6 mice. The relative heart weight and cardiomyocyte cross‐sectional area were significantly decreased by E2 compared with CON in WT mice (P < 0.001), while there was no significant effect in β‐catΔex2‐6 mice. Echocardiographic measurements revealed a significant decrease in septum width (P < 0.001), posterior wall thickness (P < 0.01) and fractional shortening (P < 0.05) in E2 treated WT mice compared with CON, while there was no significant change in β‐catΔex2‐6 mice. Immunoblotting analysis of the muscle‐specific RING finger protein 1 (MuRF1) revealed a significant increase in its levels by E2 compared with CON in WT mice (P < 0.05), while there was no significant effect in β‐catΔex2‐6 mice. In conclusion, our surprising findings indicate that in C57Bl/6N mice E2 leads to cardiac mass and function loss via a β‐catenin mechanism.