bluuebunny/crossref_metadata_2025 huggingface dataset:
High Fat, High Sugar Diet Induces Metabolic Changes in Articular Cartilage that are Reversed with Low Intensity Exercise and Genistein Treatment
Authors: Lopez Janelle , Castro Monica , Al‐Nakkash Layla , Broderick Tom L , Plochocki Jeffrey H
Published: June 2021

High fat, high‐sugar diet (HSFD) has been shown to induce obesity, metabolic dysregulation, and inflammation in peripheral tissues. Inflammation within cartilage tissue is associated with metabolic changes that lead to arthrosis. In this study, we examine the effects of HSFD on expression of col10a and MMP‐13 in articular cartilage of the knee joint. Both of these proteins are markers for inflammation in chondral tissue. In the presence of proinflammatory cytokines, col10a expression by chondrocytes decreases, while MMP‐13 increases. We also examined the effects of low intensity exercise and genistein treatment on cartilage metabolism. Both of these treatments have been shown to improve bone health, but their effects on diet‐induced cartilage inflammation are unclear. We randomly assigned 100 mice to five treatment groups of equal sizes: lean controls, HFSD fed, HFSD + exercise, HFSD + genistein, and HFSD + exercise + genistein. HFSD included 60% fat in chow and 42 g/L sugar in the drinking water. Exercise training included treadmill running for 180 min/week. Genistein was given in the diet at 600 mg/kg. Duration of treatment was 12 weeks. Col10a expression was significantly reduced in mice fed HFSD versus lean controls (P < 0.05). Mice treated with exercise and genistein in combination exhibited decreased col10a expression that was similar to levels expressed in cartilage of lean controls. MMP‐13 expression did not differ significantly among the treatment groups (P > 0.05). Examination of articular cartilage stained with H&E revealed the thickness of the uncalcified zones was significantly reduced in mice fed HFSD in comparison to lean control mice (P < 0.05). Staining in this zone also reflected reduced proteoglycan content in the cartilage of mice in the HFSD treatment group. These findings suggest articular cartilage of mice fed HFSD exhibit signs of inflammation and reduced capacity to resist compression. Treatment with genistein and exercise in combination resulted in articular cartilage that more closely resembles cartilage of lean mice fed a standard diet. Low‐intensity exercise and genistein intake may prove beneficial to joint health in the obese condition.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lapatinib Metabolism in CYP3A5‐Genotyped Primary Human Hepatocytes
Authors: Bissada Jennifer , Abouda Arsany , Jackson Klarissa
Published: June 2021

Drug‐induced liver injury is associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by cytochrome P450 (CYP) 3A4 and CYP3A5 to yield an O‐debenzylated metabolite (Lap‐OH), which can undergo further oxidation to a reactive quinoneimine. We hypothesized that polymorphic expression of CYP3A5 may contribute to individual differences in lapatinib bioactivation and influence hepatic exposure to reactive, potentially toxic metabolites. The objective of this study was to assess the metabolism of lapatinib in CYP3A5‐genotyped human hepatocyte cultures, and evaluate whether CYP3A5 genotype and activity lead to differences in lapatinib metabolite formation. Commercially available human hepatocytes were obtained from XenoTech (pooled) and BioIVT (individual). CYP3A5 genotype (*1/*1, *1/*3, and *3/*3) and donor characteristics (sex, age, ethnicity) were provided by the companies. Lapatinib (10 μM) was incubated with genotyped human hepatocytes in suspension for 2 h. Separate incubations were carried out to measure CYP3A and CYP3A5‐selective activity using probe substrates, midazolam (2.5 μM) and T‐5 (5 μM), respectively. Metabolite formation was analyzed by liquid chromatography‐tandem mass spectrometry. The results from experiments with genotyped human hepatocytes from pooled donors showed that formation of Lap‐OH was higher in CYP3A5 expressers (CYP3A5 *1/*1 and CYP3A5*1/*3 donors) compared to CYP3A5 non‐expressers (CYP3A5*3/*3 donors). CYP3A activity, as measured by midazolam 1’‐hydroxylation, and CYP3A5‐selective activity, as measured by T‐5 N‐oxidation, were also higher in CYP3A5 expressers (*1/*1 and *1/*3) compared to non‐expressers (*3/*3). In incubations with single‐donor human hepatocytes (n = 15), the levels of Lap‐OH generated were 2.2‐fold higher in CYP3A5 expressers compared to non‐expressers (p = 0.0497). Moreover, formation of Lap‐OH was 2.5‐fold higher in hepatocytes from female donors compared to males (p = 0.0390). Collectively, these results indicate that CYP3A5 genotype and donor sex influence lapatinib bioactivation in human hepatocyte cultures. Future studies will examine the cytotoxicity of lapatinib in primary human hepatocytes to determine the impact of CYP3A5 genotype, sex, and other individual factors on lapatinib‐induced hepatocellular injury.

Support or Funding Information

This research is funded by the National Cancer Institute of the National Institutes of Health (K01CA190711). Research reported here is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Short‐Term and Long‐Term Dexamethasone Action on Expression of Glucocorticoid and Mineralocorticoid Receptors in the Gastric Mucosa
Authors: Filaretova Ludmila , Sudalina Maria
Published: June 2021

Glucocorticoids may have dual action on the stomach: gastroprotective and proulcerogenic one. Our previous study was designed to investigate how physiological gastroprotective action of glucocorticoids can be transformed to pathological ulcerogenic effect. The results obtained demonstrate that single injection of dexamethasone at the dose of 1 mg/kg may attenuate or aggravate indomethacin‐induced gastric erosions depending on the time of its injection before indomethacin (prolongation of dexamethasone action). At the present study to elucidate the mechanisms of transformation of gastroprotective action of dexamethasone into ulcerogenic one we tested the hypothesis that disbalance between the glucocorticoid and mineralocorticoid receptors (GR and MR) may contribute to this transformation. To test the hypothesis, effect ща dexamethasone at the dose of 1 mg /kg, (i.p.) on the expression of GR and MR in the gastric mucosa was studied 1 h (a short‐term action when the gastroprotective action was observed) and 24 h (long‐term action when the proulcerogenic effect was observed) after its administration. The expression of GR and MR was detected by a immunohistochemical staining technique using α‐glucocorticoid (ab3580; 1:500. Abcam) and mineralоcorticoid (ab41912; 1:500. Abcam) receptor antibodies. The results of the staining were assessed by a morphometric study of microscopic images in five fields of view using the ImajeJ software. A distribution of cytoplasmic staining was estimated with relative stained area (%) in gastric glands and smooth muscle cells of muscularis mucosae. We found that GR are presented in the parietal cells, whereas MR are ubiquitous. Dexamethasone provoked translocation of both GR and MR into nuclei. The short‐term effect of dexamethasone resulted in a decrease of cytoplasmic GR and their translocation into the cell nuclei. The long‐term dexamethasone exposure increased cytoplasmic GR expression not accompanied by nuclear translocation. The short‐term dexamethasone action caused an increase of cytoplasmic MR level, but did not lead to nuclear translocation of these receptors. The long‐term action of dexamethasone induced nuclear translocation of MR, without significant changes of MR expression in cytoplasm. The results suggest that disbalance between GR and MR may contribute to transformation of gastroprotective action of dexamethasone to proulcerogenic effect and support the hypothesis.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) N 14‐15‐00790.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stable Gastric Pentadecapeptide BPC 157 Antagonized Local Anesthetic Effect of Lidocaine
Authors: Lozic Marin , Stambolija Vasilije , Kovacevic Josip , Zivanovic‐Posilovic Gordana , Suran Jelena , Duplancic Bozidar , Mirkovic Ivan , Kokot Antonio , Drmic Domagoj , Seiwerth Sven , Sikiric Predrag
Published: June 2021

We documented that the stable gastric pentadecapeptide BPC 157 antagonized local anaesthetic effect of lidocaine. BPC 157 (LD1 not achieved) was implemented as an anti‐ulcer peptide in inflammatory bowel disease trials and now in a multiple sclerosis trial (Curr Pharm Des. 2018;24(18):1990–2001). Stable gastric pentadecapeptide BPC 157 was previously shown as a cardioprotective compound in a model of arrhythmia induced by bupivacaine toxicity where it counteracts arrhythmias and prevents lethal outcome (Eur J Pharmacol. 2016 Dec 15;793:56–65) much like in other cardiotoxicity mainly related to potassium disturbances, both hyperkalemia and hypokalemia, in vivo and in vitro (HEK293 cells) (Curr Pharm Des. 2018;24(18):1990–2001). We used Wistar Albino male rats, underwent regional blocks with lidocaine (spinal intrathecal block (lidocaine 6 mg/kg, 0.1 ml/rat, 550 g b.w.) or axillary block (lidocaine 15 mg/kg, 0.3 ml/rat, 220 g b.w.). Rats received BPC 157 (10 μg, 10 ng, 10 pg/kg intraperitoneally or intragastrically) or an equivolume of saline (5 ml/kg), either immediately or at 10 min when local anesthesia was fully established. Consistently, while lidocaine application produced a prolong function failure, all BPC 157 regimens significantly shortened time to full function recovery, either given immediately after lidocaine application or later, in the conditions of full local anesthesia. In other experiments, using a hot plate (55°C for 3 minutes) when rat hind paws were infiltrated with 2% lidocaine (0.1 ml/paw), a subsequent infiltration with BPC 157 (10 μg, 10 ng, 10 pg/kg) results in the faster feet lifting and much less edema (Figure 1). Finally, ECG recording documented that the mentioned regimens of BPC 157 markedly counteracted the lidocaine‐induced arrhythmias as well. Therefore, it may be possible that pentadecapeptide BPC 157 acts as the missing antidote to local anesthethics, and potentially deleterious and even life threatening adverse effects of toxic doses of local anesthethics would be markedly attenuated or even abolished.

Support or Funding Information

This work was supported by the University of Zagreb scientific support fund [grant number BM099].

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Small GTPase Rab7 Mediates Aquaporin‐2 Recycling and Apical Trafficking
Authors: WANG WEI‐LING , YANG CHAN‐WEI , WONG KIT YEE , YU MING‐JIUN
Published: June 2021

Water permeability of the kidney collecting ducts is regulated by the abundance of the molecular water channel protein aquaporin‐2 (AQP2) in the apical plasma membrane of the collecting duct cells. The apical AQP2 abundance in turn is regulated by trafficking of AQP2‐containing intracellular vesicles to and from the apical membrane by the peptide hormone vasopressin. Here we used confocal microscopy to follow how AQP2 trafficked among biotin‐labeled apical membrane, Rab5+ early endosome, Rab7+ late endosome, and Rab11+ recycling endosome in response to vasopressin. Before vasopressin stimulation, most AQP2 stayed in Rab5+ early endosome and Rab11+ recycling endosome. In response to vasopressin, AQP2 in the Rab11+ recycling endosome trafficked first to the apical membrane followed by AQP2 in the Rab5+ early endosome. Upon vasopressin withdrawal, apical AQP2 returned to the Rab5+ early endosome before joining the Rab11+ recycling endosome. Rab5 knockdown slowed APQ2 internalization upon vasopressin withdrawal. Rab7 knockdown resulted in AQP2 accumulation in the Rab5+ early endosome upon vasopressin withdrawal, consistent with a role of Rab7 in AQP2 sorting to the late endosome. However, Rab7 knockdown also reduced AQP2 in the Rab11+ recycling endosome upon vasopressin withdrawal, suggesting that Rab7 participates in AQP2 sorting from the early endosome to the recycling endosome. In fact, in the Rab7 knockdown cells vasopressin‐stimulated apical AQP2 trafficking was greatly impaired. These results indicate that internalizing AQP2 first arrives at the early endosome where Rab7 sorts it to the recycling endosome where it waits for the next vasopressin signal for apical trafficking. Rab11 knockdown did not affect vasopressin‐induced apical AQP2 trafficking as reported previously. In contrast, Vps35 (vacuolar protein sorting‐associated protein 35) knockdown trapped AQP2 in the Rab11+ recycling endosome and reduced apical AQP2 trafficking. We conclude that Rab7 plays an important role in AQP2 sorting from the early endosome to the recycling endosome where vasopressin excites its apical trafficking mediated by Vps35.

Support or Funding Information

This work was supported by the Ministry of Science and Technology, TAIWAN (MOST 107‐2320‐B‐002‐057‐MY3).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and characterization of novel small‐molecule inhibitors of SLC26A3
Authors: Park Jinhong , Namkung Wan
Published: June 2021

SLC26A3 (downregulated in adenoma, DRA), an anion exchanger, is highly expressed in the luminal membrane of intestinal epithelial cells, and loss‐of‐function mutations in this gene result in congenital chloride diarrhea caused by detect of chloride absorption and bicarbonate secretion in the intestine. Here, we report development of the selective small‐molecule inhibitors of DRA for the investigation of the effect of DRA inhibition on constipation and metabolic acidosis. A screen of ~3,000 compounds using a cell‐based high‐throughput screening (HTS) assay revealed four novel compounds of DRA inhibitors that fully blocked Cl−/I− exchange activity of DRA with IC50 < 10 μM. The hit compounds strongly inhibited both Cl−/I− and Cl−/HCO3− exchange activity of DRA. The most potent inhibitor significantly blocked Cl−/I− exchange activity of DRA (IC50 of ~3 μM) but it did not affect the activity of SLC26A4 (pendrin), SLC26A7, SLC26A9, CFTR and ANO1. The small‐molecule DRA inhibitors identified here may be useful tools for pharmacological dissection of DRA and drug discovery for treating constipation and metabolic acidosis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ROLE OF CHLORIDE AND ANO1 FOR THE CONTRACTILITY OF MESENTERIC SMALL ARTERIES IN RATS WITH STREPTOZOTOCIN INDUCED DIABETES
Authors: Aalkjaer Christian , Kamaev Dmitry , Jensen Anders Bisgaard , Matchkov Vladimir
Published: June 2021

Diabetes is associated with altered contractile responses of small arteries. In the streptozotocin (STZ) induced rat model of diabetes enhanced contractility several groups have reported an increased contractility. In this study we tested the hypothesis that chloride is important for the enhanced contractility in the STZ rats.

Diabetes was induced in 6 weeks old rats by a single intraperitoneal injection of 60 mg/kg STZ, control rats received vehicle injection. Eight weeks later blood glucose was significantly increased in the STZ rats compared to control rats (24.6±1.2 mmol/L and 6.7±1.1 mmol/L, STZ (n=9) and control (n=9), respectively). The rats were killed and mesenteric small arteries were isolated. The contractile response to increasing concentrations of norepinephrine of mesenteric small arteries was assessed in isometric myographs in physiological salt solution and in physiological salt solution where Cl− was substituted with aspartate. Expression of the Ca2+ activated Cl− channel ANO1 was assessed with qPCR and Western blotting in mesenteric small arteries.

The maximal contraction to norepinephrine in physiological salt solution was higher in arteries from STZ rats compared to control rats (5.12±0.33 N/m and 4.07±0.24 N/m, STZ (n=14) and control (n=13), respectively). In Cl− free solutions the maximal response to norepinephrine was reduced and no difference between STZ and control rats was found (3.76±0.26 N/m and 3.23±0.22 N/m, STZ (n=14) and control (n=13), respectively). Furthermore the effect of Cl− free solution on the area under the NE concentration response curve was bigger in arteries from STZ rats (2.47±0.42 and 1.39±0.26 STZ (n=14) and control (n=13), respectively). Expression of ANO1 mRNA was higher in arteries from STZ rats (181±10% and 100±18%, STZ (n=4) and control (n=4), respectively). Expression of ANO1 protein was higher in arteries from STZ rats (195±24% and 100±18%, STZ (n=9) and control (n=8), respectively).

This study provides evidence that the enhanced contractility of mesenteric arteries from STZ rats is dependent on the chloride gradient over the cell membrane, and suggests that this may be consequent to enhanced expression of the Ca2+ activated Cl− channel ANO1.

Support or Funding Information

Danish Council for Independent Research (7016‐00013B)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatial Localization Determines Preferential Calcium Signal‐Target Coupling in Microvascular Endothelium
Authors: Ottolini Matteo , Marziano Corina , Cope Eric L , Sonkusare Swapnil K
Published: June 2021

Localized, spatially restricted Ca2+ signals promote endothelium‐dependent vasodilation in systemic and pulmonary microcirculation. Unitary Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, promote endothelium‐dependent vasodilation in systemic and pulmonary resistance arteries. TRPV4 sparklets dilate small mesenteric arteries (MAs) via activation of intermediate and small conductance Ca2+‐activated K+ (IK and SK, respectively) channels, and small pulmonary arteries (PAs) via endothelial nitric oxide synthase (eNOS) activation. Although endothelial Ca2+ is known to activate different downstream signaling targets, the mechanisms for preferential Ca2+ signal‐target coupling remain undefined. We hypothesized that spatial co‐localization determines Ca2+ signal‐target linkage. High‐speed Ca2+ imaging experiments showed that endothelial TRPV4 sparklet activity per site is similar between MAs and PAs. Moreover, perforated whole‐cell patch clamp recordings showed that specific IK/SK channel activator NS309 induced comparable currents in freshly isolated endothelial cells (ECs) from MAs and PAs. While TRPV4 channel agonist GSK1016790A (GSK101) elicited IK and SK currents in the ECs from MAs, it was not able to activate IK and SK currents in ECs from PAs. Consistent with the presence of IK/SK currents in MAs and PAs, NS309 dilated both MAs and PAs in pressure myography experiments. GSK101 dilated MAs in a IK/SK channel‐dependent and eNOS‐independent manner. On the contrary, GSK101 dilated PAs in a eNOS‐dependent and IK/SK channel‐independent manner, further supporting differential TRPV4 sparklet‐target coupling. Immunostaining experiments indicated that TRPV4 channels were localized at endothelial projections to smooth muscle or myoendothelial projections (MEPs) in both MAs and PAs. Whereas IK and SK channels were localized at the MEPs in MAs, they were not concentrated at the MEPs in PAs. These results suggest that functional IK/SK channels exist in ECs from MAs and PAs, and support our hypothesis that spatial proximity is necessary for TRPV4 sparklet‐IK/SK channel coupling. Future studies will address the mechanisms for eNOS activation by TRPV4 sparklets in PAs. Understanding the differential coupling mechanisms may lead to novel therapeutic approaches to selectively dilate pulmonary versus systemic vasculature.

Support or Funding Information

NIH (HL121484‐01, R56HL138496, S.K.S.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stress Strain Speckle‐Tacking Segmental Analysis Reveals Early Indications of Diastolic Dysfunction in a Type 2 Mouse Model of Diabetes Mellitus
Authors: Durr Andrya J , Hathaway Quincy A , Pinti Mark V. , Shepherd Danielle L , Kunovac Amina , Fink Garrett K. , Hollander John M
Published: June 2021

Type 2 diabetes mellitus (T2DM) is becoming increasingly common, as there are currently 23.1 million people diagnosed and another 84.1 million adults diagnosed with pre‐diabetes in the United States alone. While many long‐term consequences are associated with T2DM, the leading cause of mortality is cardiovascular complications. Though reduction in ejection fraction and fractional shortening are well‐described indicators of cardiovascular dysfunction, these indices are usually present late in the progression of the pathology. Identification of predictors that can be observed earlier in the disease progression may aid in diagnosing and predicting future health outcomes for the patient. The goal of the current study was to evaluate cardiac contractile changes in a rodent model of type 2 diabetes mellitus using the well characterized db/db model mouse model. Db/Db FVB mice and wild‐type (WT) littermate controls were utilized for the study (WT = 22 and db/db = 14). Echocardiography, using the Vevo 2100 Imaging System, was implemented to evaluate cardiac function in both groups at 12 weeks of age. M‐mode was used for conventional measures of cardiac function, while B‐mode short and long axis images were used for stress strain speckle‐tracking analyses. In addition, Pulse Wave Doppler analyses were utilized to examine changes in flow across the mitral valve. At 12 weeks of age, db/db mice exhibit lower ejection fractions (P ≤ 0.05) and increased stroke volumes (P ≤ 0.0018) when compared to WT. Concomitantly, db/db mice presented with significant increases in both systolic and diastolic ventricular diameters and volumes, along with an increased LV mass (P ≤ 0.0001). To determine whether spatial differences in cardiac dysfunction was associated with T2DM, we assessed regional changes in cardiac function. Speckle‐tracking strain analyses revealed significant changes in radial displacement at systole in the short axis (P ≤ 0.0024) and diastole at both the short axis (P ≤ 0.033), and long axis (P ≤ 0.0001). Segmental analysis revealed that the lateral wall portion of the left ventricle, in all axes, was significantly altered during diabetic stress. In summary, echocardiography and specifically speckle‐tracking strain analysis has the potential to indicate early dysfunction of the left ventricle during the progression of type 2 diabetes mellitus through segmentally isolated cardiac function, potentially aiding in diagnosis and future outcomes.

Support or Funding Information

R01 HL‐128485 (JMH), AHA‐17PRE33660333 (QAH), AHA 17PRE33660333 (QAH), and the Community Foundation for the Ohio Valley Whipkey Trust.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Type‐Specific Ephrin expression in the Diabetic Coronary Circulation
Authors: McCallinhart Patricia , Trask Aaron J.
Published: June 2021

Ephrin signaling between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is crucial during normal vascular development and homeostasis, primarily by influencing cell behavior, resulting in alterations of the cell cytoskeleton and cell adhesion. Ephrins are re‐expressed in vascular injury and disease states such as cancer and chronic inflammation; therefore the goal of this study was to determine the impact of type 2 diabetes on ephrin expression in coronary VSMCs and ECs. Low‐passage primary coronary VMSCs were isolated from normal (n=6) and type 2 diabetic db/db mice (n=4) and were either cultured alone or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively, and RNA was collected for PCR analysis. Normal (n=5) and type 2 diabetic (T2DM) (n=4) human coronary vascular smooth muscle cells (CVSMCs) were also cultured and protein lysates collected for Western Blot analysis. Ephrin B2 ligand gene expression was significantly increased in diabetic CRM VSMCs in both mono‐ and co‐culture conditions (1.00 ± 0.13 vs. 1.57 ± 0.24, p=0.049 and 1.00 ± 0.14 vs. 1.60 ± 0.22, p=0.044, respectively). There were no significant differences in ephrin B4 receptor levels in normal vs. diabetic coronary VMSCs. In human coronary VSMCs, ephrin B2 ligand protein expression was also significantly increased in diabetic coronary VSMCs over control VSMCs (normal 1.00±0.05 vs. T2DM 2.91±0.29, p=0.019). Interestingly, there was a different trend in the diabetic hCMECs. Ephrin B2 gene expression was significantly reduced in diabetic hCMECs in both mono‐ and co‐culture conditions (1.00 ± 0.05 vs. 0.75 ± 0.07, p=0.02 and 1.00 ± 0.10 vs. 0.50 ± 0.07, p=0.007, respectively). Ephrin B4 receptor gene expression was significantly reduced in diabetic hCMECs in both mono‐ and co‐culture conditions (1.00 ± 0.07 vs. 0.55 ± 0.05, p=0.002 and 1.00 ± 0.03 vs. 0.77 ± 0.03, p=0.0008, respectively). Collectively, these data demonstrate that, in both mice and humans, diabetes alters ephrin expression in VSMCs and ECs in a cell‐specific manner. Further studies will address how alterations in ephrin expression influence coronary cell behavior in the setting of diabetes.

Support or Funding Information

NIH R00 HL116769, NIH R21 EB026518, and Nationwide Children's Hospital to AJT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diastolic Function at Rest and During Handgrip is Impaired in Patients with Peripheral Arterial Disease
Authors: Gao Zhaohui , Cui Jian , Kuroki Marcos T. , Kim Danielle Jin‐Kwang , Blaha Cheryl , Cauffman Aimee , Pai Samuel , Sinoway Lawrence I.
Published: June 2021

Peripheral arterial disease (PAD) patients have larger increases in systolic BP with exercise. Thus it is not surprising that patients with PAD may have diastolic dysfunction. This link has also been suggested by an observed association between elevated BNP levels and the transmitral E‐wave/early diastolic mitral annular velocity (E/E′) ratio in PAD. The characteristics of diastolic function at rest and with exercise in patients with PAD has not been examined. In this study, we used isometric handgrip exercise echocardiography to noninvasively assess left ventricular diastolic function. The aim of this study is to determine whether left ventricular diastolic function at baseline and during isometric handgrip exercise (at 30% of their maximal voluntary contraction) differ in patients with PAD and age, gender‐matched healthy controls. We recruited 15 PAD patients (10 M, 5 F, 66 ± 6 yrs), and 11 control subjects (7 M, 4 F, 65 ± 6yrs). The ankle‐brachial pressure index (ABI) in the most affected leg in the patients was (0.58 ± 0.15). Beat‐by‐beat blood pressure (BP, Finometer) and ECG were recorded at baseline and during handgrip. Conventional non‐invasive methods including pulsed wave Doppler and tissue Doppler imaging were used to measure early mitral inflow diastolic wave velocity (E), late mitral inflow diastolic wave velocity (A), E/A ratio, early diastolic myocardial wave velocity (E′) and late diastolic myocardial wave velocity (A′). E/ E′ was also calculated. Systemic, hemodynamic values at rest and during isometric handgrip (blood pressure and heart rate) were not different in the two groups. At rest: mitral inflow E and A were significantly higher in patients than in controls (E: 78.7 ± 4.2 vs. 64.5 ± 4.9 cm/s P value <0.05; A:82.2 ± 3.9 vs. 65.5 ± 4.4 cm/s P value <0.01); E/A ratio was similar. E/E′ ratios were in the normal range, however, this ratio was significantly higher in PAD patients (8.9 ± 0.6 vs. 6.7 ± 0.5 P value <0.05) than in controls indicating higher left ventricular filling pressures in PAD. During handgrip, hemodynamic responses increased in both groups. Of note, late mitral inflow: increased much less in PAD than in controls (mitral inflow A wave increase 10% ± 4% vs. 25% ± 4% in controls, P value <0.05). This suggests that with exercise atrial contraction contributes less to filling in PAD than it does in controls. Collectively, these data indicate that PAD patients have impaired diastolic function at rest and a reduced left atrial contractile reserve.

Support or Funding Information

Supported by National Institutes of Health Grants P01 HL134609 (Sinoway) and UL1 TR002014 (Sinoway).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Protein Degradation Machinery at the Desmosome Reveals Novel Triggers of the Desmosomal Disease, Arrhythmogenic Right Ventricular Cardiomyopathy
Authors: Liang Yan , Lyon Robert C. , Pellman Jason , Mezzano Valeria , Gu Yusu , Dalton Nancy , Bradford William , Bogomolovas Julius , Lee Mong‐Hong , Iwakuma Tomoo , Nigam Vishal , Scheinman Melvin , Peterson Kirk , Sheikh Farah
Published: June 2021

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic‐based cardiac disease which is characterized by right/bi‐ventricular dysfunction, fibro‐fatty replacement of the myocardium and ventricular arrhythmias, leading to sudden death in young people and athletes. ARVC is termed a “disease of the desmosome” (mechanical cell‐cell junction) as 40% of ARVC patients harbor mutations and/or loss of desmosomal components. However, limited information exists on how cardiac desmosomal protein loss/degradation triggers ARVC. Through a yeast‐two‐hybrid screen in an adult human heart cDNA library, we uncovered a new interaction between the desmosomal protein, desmoplakin (DSP, N terminus) and a protein degradation machinery component, CSN6 (COP9 signalosome subunit 6). Traditionally, CSN proteins are thought to prevent proteins from being degraded; however, a role for CSN6 in the heart is undefined. We show that CSN6 localizes to desmosomal junctions in cardiac muscle and structurally complex with desmoplakin, suggesting that the novel hypothesis that CSN6 may protect the cardiac desmosome from degradation and loss/destablization this pathway may lead to ARVC. Using two novel cardiac‐specific CSN6 knockout (CSN6‐KO) mice, we further show that CSN6 loss accelerates desmosome destruction and triggers ARVC, as CSN6‐KO hearts exhibit selective loss of desmosomal proteins and recapitulate the key cardiomyopathy features and sudden death associated with ARVC, similar to classic mouse model of ARVC (DSP‐cKO). CSN6‐KO and DSP‐cKO hearts also harbor underlying hyper‐accumulation of protein degradation machinery specifically at cell‐cell junctions. Restoration of CSN function by the protein degradation inhibitor (MLN4924) could rescue the loss of desmosomal protein expression associated with CSN6 loss in vitro, further highlighting that the cardiac desmosome is regulated by protein degradation. CSN6 pathways are also relevant to human ARVC as a N‐terminus desmoplakin mutation (R315C) identified in a human ARVC patient was sufficient to abrogate DSP‐CSN6 binding and result in loss of CSN6 localization at the cell‐cell junction in cardiac biopsies. DSP R315C knock‐in mice also exhibited cardiac CSN6 reduction and early electrical defects found in ARVC. We altogether identify CSN6 as a novel “brake” to prevent desmosomal protein degradation, and reveal that disruption of this complex is an important “accelerator” of sudden death and ARVC.

Support or Funding Information

American Heart Association Postdoctoral Fellowship 17POST33670205

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Klotho/FGF23 signaling in cardiorenal syndrome‐induced cardiac hypertrophy
Authors: da Cruz Junho Carolina V , Caio‐Silva Wellington , Ruiz‐Hurtado Gema , Carneiro‐Ramos Marcela Sorelli
Published: June 2021

Introduction

In diseases that generate an inflammatory situation, such as cardiorenal syndrome, different clinical conditions promote cardiac and renal dysfunction. There are type 3 and 4 which are characterized by renal insults that subject the heart to dysfunctions; Among the changes that renal injury can lead, we can mention the modulation in Klotho levels, a gene that is negatively regulated by fibroblast growth factor 23 (FGF23). The objective of this study is to evaluate the participation of this axis in cardiac hypertrophy induced by renal ischemia

Methods

C57BL/6 male mice were subjected to surgical occlusion of left renal pedicle for 60 min followed by reperfusion in acute term (24, 48 and 72h), short term (for 8 and 15 days) and long term (4,6 and 8 weeks). Total heart RNA was extracted for gene expression of Klotho, FGF23 and hypertrophy markers using real‐time PCR.

Results

The renal injury was confirmed by decreasing of left kidney weight and urea/creatinine levels starting at the 15th day of reperfusion. Cardiac hypertrophy was confirmed by increased a‐actin levels at day 15 and 4 weeks of renal reperfusion; however, heart weight has maintained increased until the 8th week of reperfusion. Concerning the FGF23/Klotho axis, both genes were modulated during the cardiorenal syndrome type 3. FGF23 gene expression was mostly increased in heart tissue after 8 days of reperfusion, having its peak during the 4th week. In renal tissue, FGF23 did not present a pattern, although it shows a peak in the first 24 hours, in the 8th day and 15th day (the same timepoints which the heart tissue is affect which electrophysiological and immunological changes). By the other side, Klotho gene expression was mostly reduced in renal tissue after 48h of reperfusion, evidencing even more the renal injury (once some authors consider Klotho is a renal injury biomarker). In heart tissue, klotho expression is reduced since the first 24h, but this is attenuated around the 4th week of reperfusion.

Conclusion

We found that cardiac FGF23/Klotho axis dynamics are affected by renal I/R indicating a possible interaction between these two responses during cardiorenal syndrome.

Support or Funding Information

Financial Support: FAPESP 2015/19107‐5 and 2018/03089‐6.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel MYH6E1503V Variant in a Family with a History of Heart Disease, including Hypoplastic Left Heart Syndrome
Authors: Anfinson Melissa , Kim Min‐Su , Lough John , Geddes Gabrielle , James Jeanne , Geurts Aron , Mitchell Michael , Tomita‐Mitchell Aoy
Published: June 2021

Hypoplastic Left Heart Syndrome (HLHS) is a severe form of congenital heart disease (CHD), requiring prompt surgical intervention and eventual transplant. Despite significant advances in detection and treatment of HLHS, genetic risk factors have been identified in <5% of patients. Our prior studies of 190 HLHS patients identified 19 distinct, rare variants in the gene encoding for a‐myosin heavy chain (MYH6), with variant carriers exhibiting reduced transplant‐free survival (p < 1 × 10−3). In‐depth genetic analysis was performed on the family of one HLHS subject who exhibited a novel E1503V variant in the tail domain of MYH6. The variant was identified in six additional members of the family, all of whom were related to the proband via the maternal grandmother. Four of those individuals also exhibited adult heart disease, with varying ages of onset. Though MYH6 variants have been implicated in a number of heart diseases – including arrhythmias, familial cardiomyopathies, as well as various forms of CHD – to our knowledge this is the first association of a single variant with both adult heart disease and HLHS.

α‐myosin heavy chain (α‐MHC) is a cardiac‐specific protein that functions as a component of the sarcomere, the major contractile unit of the heart muscle. We therefore sought to evaluate functional consequences of MYH6E1503V on a‐MHC in vitro. Using CRISPR‐Cas9 gene editing technology, we successfully introduced the variant into induced pluripotent stem cells (iPSCs) from an unrelated individual, which were then used to generate cardiomyocytes. Subsequent evaluation of these cells revealed key insights into the effect of MYH6E1503V on sarcomere organization and contractile properties in cardiomyocytes, as well as their proliferation and differentiation capacity.

Support or Funding Information

Children's Research Institute at the Children's Hospital of Wisconsin, Little Hearts for Life Foundation, M.A is a member of the Medical Scientist Training Program at MCW, which is partially supported by a training grant from NIGMS T32‐GM080202

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proximity‐Labeling by BioID Reveals Pleiotropic Role of Ski in Cardiac Fibrosis
Authors: Landry Natalie M , Rattan Sunil G , Hnatowich Mark , Dixon Ian MC
Published: June 2021

Despite recent improvements made in cardiac patient treatment and outcomes, current therapies only serve to alleviate the symptoms of cardiac fibrosis, rather than correct the disease itself. Our lab has previously established Ski as a potent inhibitor of TGF‐β signalling in cardiac myofibroblasts, the primary effectors of fibrosis; however our recent investigations suggest that Ski may inhibit a multitude of pro‐fibrotic pathways, including Hippo. We have specifically observed upon Ski overexpression in primary cardiac fibroblasts, a marked reduction in the expression of TAZ (or WWTR1), one of the main nuclear effectors of the Hippo pathway. To further expand the current knowledge on Ski's anti‐fibrotic properties, and provide insight into potential mechanisms of action, we mapped its interactome using enzyme‐catalyzed biotin proximity labelling (BioID2). Using the BioID2 vector, E. coli BirA* biotin ligase was fused to the N‐terminus of human Ski and the fusion protein was then expressed in fibroblasts in the presence of an excess of free biotin. Whole cell lysates were then subject to streptavidin‐mediated protein capture and potential Ski binding partners were identified by tandem time‐of‐flight mass spectrometry. The resulting candidates included several known Ski interactors (eg. RSmad2 and co‐Smad4), but also revealed novel interactors which potentially link Ski's anti‐fibrotic capacity and interaction with the Hippo signaling pathway. We also observed several interactions with cytoskeletal components, including those involved in actin dynamics and stress fiber formation. To confirm results from the affinity capture, candidate interactors were verified using immunoblotting. In addition, the expression of potential Ski interactors was examined in a rat model of cardiac fibrosis, post‐myocardial infarction. Our data suggest that Ski serves not only as a Smad‐dependent TGF‐β inhibitor, but also interacts with the Hippo, Wnt/β‐catenin, and FoxO signaling pathways‐‐all of which are suspected contributors to fibrosis. In addition, Ski's interaction with various points of cytoskeletal organization further indicates that its functions in the cell go beyond simple inhibitory protein‐protein interactions.

Support or Funding Information

NM Landry's research has been funded by studentships from the Canadian Institutes of Health Research (CIHR), Research Manitoba, as well as the Bank of Montreal (BMO). IMC Dixon is grateful to the Heart & Stroke Foundation of Canada for a grant (G‐17‐10018631) in support of this research, as well as the St. Boniface Hospital and Research Foundation for their continued operating patronage.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of Statin on Chemotherapy‐Induced Cardiotoxicity
Authors: Enoma Efosa , Wei Lan , Chen Howard
Published: June 2021

Tyrosine Kinase Inhibitors (TKIs) are chemotherapy agents that can damage cardiomyocytes resulting in cardiotoxicity and heart failure. TKIs target enzymes that control diverse biological pathways such as cell metabolism and apoptosis. Autophagy is a self‐degradative process involving the translocation of light chain 3 (LC3) proteins onto the autophagosome membrane, followed by fusion with lysosome for degradation. In cardiomyocytes, autophagy activation has been shown to be either protective or detrimental. We previously demonstrated autophagy to be cardioprotective of ischemia‐reperfusion injuries, presumably through maintaining cell homeostasis by removing damaged organelles such as mitochondria. Recent studies have shown that statins, HMG‐CoA reductase inhibitors commonly used to lower cholesterol levels, enhance cardiomyocyte autophagy. We hypothesize that TKIs disrupts cardiomyocyte autophagy, and autophagy activation protects cardiomyocytes from TKI‐induced toxicity. As a proof of concept, we chose the well‐established TKI dasatinib due to its high risk for heart failure in patients. We treated H9c2 rat myoblast cells with dasatinib either alone or in combination with commonly used statins: simvastatin, atorvastatin, pravastatin, and rosuvastatin. We performed MTT colorimetric assays on 96‐well plates to quantify H9c2 viability, and dual‐channel flow cytometry to assess autophagy levels with an autophagy detection nanoparticle (ADN) developed in‐house, and cell membrane integrity with propidium. We further confirmed autophagy protein, LC3, expression by western blot. After 24‐hour exposure, dasatinib significantly reduced H9c2 myocyte cell survival and ADN fluorescence. Atorvastatin and dasatinib co‐treatment significantly enhanced H9c2 survival. Interestingly, the reduction in cell death was associated with the restoration of H9c2 autophagy homeostasis, detected by ADN fluorescence. The autophagy increase was confirmed by western blot. Statin treatment thus causes a robust cytoprotective effect in H9c2 myocytes against dasatinib, presumably through autophagy activation.

Support or Funding Information

Funding: R25‐HL007785, R00‐HL121152.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exosome Mediated Globular Adiponectin's Cardioprotective Role via the Adiponectin receptor‐dependent Uptake mechanism
Authors: Wang Yajing , Gan Lu , Xie Dina , Lau Wayne Bond , Liu Jing , Zhang Ling , Gao Erhe , Koch Walter , Ma Xinliang
Published: June 2021

Objectives

Globular domain isotype adiponectin (gAPN) exerts cardioprotective effect against cardiac ischemia injury. T‐Cadherin (TCad) and adiponectin receptors are key molecules mediated adiponectin's cardioprotection. However, whether these receptors mediate the cardioprotective effects of gAPN, the isotype with more potent biological activity, most commonly utilized in metabolic/cardiovascular investigations, remains unclear.

Methods and Results

Adult male WT, AdipoR1KO, and TCadKO mice were subjected to MI, treated with vehicle or gAPN, and observed for 4 weeks. The cardioprotective effects of gAPN were abolished in AdipoR1 Knockout mice, but not Tcad, indicating that AdipoR1, but not Tcad, mediates gAPN cardioprotection. Unexpectedly, we observed that gAPN cardioprotection was significantly attenuated in animals pre‐treated with GW4869 (an exosome generation inhibitor). To further determine whether gAPN regulates exosome biological process, we investigated exosome's biogenesis and/or uptake and the precise involvement of AdipoRs., The effects of gAPN and HMW (high molecular weight APN isoform) upon exosome generation and uptake were determined on cardiomyocytes were isolated from adult AdipoR1KO and TCadKO mice. Surprisingly, cardiomyocyte exosome production increased markedly (8‐fold) when cells were treated with gAPN, but not with HMW. Moreover, gAPN increases cardiomyocyte exosome production in a receptor‐independent fashion, via regulation of the ESCRT‐multivesicular body (MVB) pathway. Furthermore, gAPN‐stimulated exosome uptake and cellular protection in WT cardiomyocytes were abolished in cardiomyocytes derived from AdipoR1KO, but not TCadKO, mice.

Conclusions

These data demonstrate that gAPN and HMW cardioprotection exhibits differential receptor dependence, and is mediated by distinct mechanisms. Whereas HMW protects the heart via TCad‐dependent mechanisms, gAPN promotes exosome biogenesis in a receptor‐independent fashion, and stimulating exosome uptake and cardioprotection in an AdipoR1‐dependent manner. Our results suggest gAPN is superior to HMW with respect to cardioprotection against ischemia heart injury, and represents a promising therapeutic strategy in patients with coronary artery disease.

Support or Funding Information

NIH RO1, ADA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Protective Effects of Phosphodiesterase 5 Inhibitors "Sildenafil & Ordonafil” on Ischemia‐Reperfusion Injury
Authors: Khatib Said Younes
Published: June 2021

Phosphodiesterase type‐5 (PDE‐5) inhibitors, Sildenafil (SILD), Vardenafil (VAR) and Ordonafil (ORD), have been developed for erectile dysfunction. Recently SILD and VAR were shown to have cardioprotective effect by reducing the infarct size in ischemic/reperfusion injury. To support the suggestion that this effect is a generalized "class" effect we decided to test the cardioprotective effect of ORD and to compare it to that of SILD. Isolated rabbit's hearts, perfused using Langendorff system, were subjected to 30 min of global ischemia followed by 2 hrs of reperfusion. At end of perfusion the heart was sliced into 5–6 cuts, 2 mm thick and incubated in 1 % triphenyltetrazolium chloride. The area of infarction and the whole left ventricle (LV) were measured by computer morphometry using image tool software. The infarct size was expressed as % of LV. Creatine kinase enzyme (CK) as a marker of infarction, was determined in the perfusate pre‐ and post‐ischemia by enzyme bioassay. The drugs, SILD or ORD at 1 mg/L, perfused the heart for 5 min before induction of ischemia. The results of both drugs showed significant cardioprotective effect and the infarct size ( as % of LV) was reduced from 38 ± 12 % in the control group to 5±2 and 9±6 %, mean±SD, n=6–8, P<0.004 for ORD and SILD respectively). Similarly the increase in CK was attenuated from 32 times increase in the control group to 20 times and 26 times for ORD and SILD respectively. Theses results support the notation that the class of PDE‐5 inhibitors has protective effect in the ischemic heart beside their effect in the treatment of erectile dysfunction.

Support or Funding Information

Deanship of research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.