bluuebunny/crossref_metadata_2025_split huggingface dataset:
Failure of autophagy maturation in CD38 deficient coronary arterial myocytes due to inhibition of dynein‐mediated autophagosome trafficking (865.3)
Authors: Zhang Yang , Xu Ming , Xia Min , Li Xiang , Boini Krishna , Li Pin‐Lan 
Published: June 2021

Autophagic flux refers to the process that lysosome traffics and fuses with autophagosomes (APs) leading to their maturation to form autophagolysosomes (APLs) and consequent breakdown of autophagic contents by lysosomal proteases. However, the mechanisms controlling autophagic flux, particularly autophagy maturation in vascular cells are poorly understood. Here, we revealed a role of CD38, an enzyme that metabolizes NADP+ into NAADP, in autophagy maturation in coronary arterial myocytes (CAMs). In vivo, CD38‐/‐ fed the Western diet showed increased accumulation of APs in coronary arterial media compared to CD38+/+ mice, suggesting that CD38 gene deletion results in a deranged autophagic process. In primary cultured CAMs, a novel flow cytometric CytoID analysis showed that 7‐ketocholesterol (7‐Ket, an atherogenic stimulus and autophagy inducer)‐induced APs formation was similar between CD38+/+ and CD38‐/‐ CAMs when their autophagic flux was blocked by choloquine, which indicates that CD38 is not involved in APs formation. Tandem RFP‐GFP‐LC3B assay demonstrated that CD38 gene deletion markedly enhanced 7‐Ket‐induced accumulation of APs but decreased the formation of APLs in CAMs, indicating a role of CD38 in autophagy flux. Furthermore, loss of CD38 function was implicated in impaired autophagy maturation rather than in lysosomal alkalization or protease inhibition‐associated blockade of autophagic flux because CD38‐/‐ and CD38+/+ CAMs had similar lysosomal pH and lysosomal protease activities. Importantly, CD38 gene deletion markedly inhibited 7‐Ket‐induced dynein activation and APs trafficking, which were associated with attenuated lysosomal Ca2+ release. Taken together, these results suggest that CD38 plays a critical role in the control of dynein‐mediated APs trafficking and fusion with lysosomes thus determining autophagy maturation in CAMs under atherogenic stimulation.Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316
Expression of Ski induces apoptosis and represses autophagy in cardiac myofibroblasts (868.8)
Authors: Zeglinski Matthew , Davies Jared , Ghavami Saeid , Bathe Krista , Rattan Sunil , Dixon Ian 
Published: June 2021

Following myocardial infarction (MI), relatively quiestent cardiac fibroblasts undergo phenoconversion to active, hyper‐synthetic myofibroblasts. During the late stages of wound healing, myofibroblasts are removed from the infracted region through apoptosis thus decreasing scar cellularity. Apoptosis and autophagy regulate cell fate. The role of apoptosis within the remodeling heart has been well described however the role that autophagy plays following an MI is relatively unknown. Ski is a negative regulator of TGF‐β signaling and has been implicated to play a role in the post‐MI heart. Herein we investigated the regulatory role that Ski has on apoptosis and autophagy using first passage primary rat cardiac myofibroblasts. Using an adenoviral approach for gene delivery, both MTT and Live/Dead viability/cytoxicity assays demonstrated a reduction in the number of viable cells and an increase in the number of dying cells due to the over‐expression of Ski. Additionally, over‐expression of Ski led to distinct morphological changes characteristic of apoptosis. These findings were confirmed via Western blot analysis, and caspase GLO analysis showing induction of executioner caspases‐3 and ‐7, as well as the mitochondrial regulator Bax. Furthermore, evidence of caspase‐9 cleavage, but not caspase‐8, indicated that this was primarily an intrinsic apoptotic response. Markers for autophagosome formation including LC3‐B and ATG‐7 were found to be significantly reduced following Ski over‐expression. In summary, we found that over‐expression of Ski in primary cardiac myofibroblasts leads to an induction of the intrinsic apoptotic pathway and reduction in autophagy leading to cell death.Grant Funding Source: Canadian Institutes of Health Research
Regulation of ENaC and CFTR biogenesis by ERp29 (869.9)
Authors: Bikard Yann , Grumbach Yael , Suaud Laurence , Chanoux Rebecca , Rubenstein Ronald 
Published: June 2021

The Epithelial Na+ Channel (ENaC) plays an important role in blood pressure homeostasis and, in conjunction with the Cystic Fibrosis Transmembrane Conductance regulator (CFTR), regulates airway surface liquid volume. ERp29 is a 29 kD thioredoxin‐homologous protein which primarily resides in the lumen of the endoplasmic reticulum (ER). But unlike the thioredoxins, ERp29 has only a single Cysteine instead of the usual C‐X‐X‐C motif. Our group has previously demonstrated that ERp29 promotes biogenesis of CFTR. As the biogenesis of CFTR and ENaC have a number of similar features, the present studies tested the hypothesis that ERp29 would also regulate ENaC biogenesis and functional expression, as well as further probed the mechanism by which ERp29 promotes CFTR biogenesis. In MDCK epithelial cells expressing epitope tagged αβγ‐ENaC, overexpression of wt ERp29 increased the abundance of the cleaved form of γ‐ENaC, as well as ENaC functional expression (as defined by amiloride‐sensitive Isc in Ussing chamber experiments.) In contrast, siRNA‐mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single Cysteine (C157S ERp29) decreased ENaC functional expression. Interestingly, these observations were not associated with altered expression of β‐ENaC at the apical surface, suggesting that ERp29 may modulate the cleavage state, and therefore open probability (Po) of ENaC at the apical surface. Finally, ERp29 overexpression promotes the interaction of both ENaC and CFTR with the Sec24D cargo recognition component of coat complex II ER exit machinery. Together these findings suggest a key role for ERp29 in the biogenesis of ENaC and CFTR; for ENaC this may include directing channel for cleavage by furin in the Golgi and delivery to the apical membrane in a higher Po form.Grant Funding Source: Supported by R01 DK58046
The role of Pin1 isomerase on neuronal cell death after focal cerebral ischemic in rats (877.14)
Authors: Lee Seunghoon , Min Sang‐Hyun , Hong Yonggeun 
Published: June 2021

Pin1 contains two functional domains, an N‐terminal WW domain and a C‐terminal peptidyl‐prolyl cis/trans isomerase (PPIase) domain. The WW domain is a phosphorylation specific protein interaction module that directs Pin1 to its substrates‐proteins phosphorylated at a certain serine or threonine residue followed by proline (pSer/Thr‐Pro motif). Upon this binding, the PPIase domain catalyzes conformational change of the Pin1 substrates by isomerizing specific pSer/Thr‐Pro bonds. Thus, we hypothesize that Pin1 can regulate neuronal cell death and neurogenesis through Pin1 inhibitor treatment after ischemic brain injury. To test the hypothesis, we elucidated at sub‐acute phases after focal cerebral ischemia to determine the efficacy of Pin1 inhibitor, juglone. Juglone (1mg/kg) was administered once a day during 7 days after focal cerebral ischemia. Histology, protein, and gene analyses were used to determine apoptosis and neurodegeneration at day 7. MCAo+Jug group was significantly led to downregulation of Bcl‐2 and Bax expression on protein level compared to MCAo+Veh group (p<.01). Likewise, juglone treatment was reduced to DAPK1 and Tau5 level on both western blot and immunostaining (p<.01). These results suggest that Pin1 isomerase acts to pro‐apoptotic molecule on ischemic‐induced neuronal cell death after focal cerebral ischemia, as well as Pin1 inhibition might be alleviate to acceleration of apoptotic cell death from acute to subacute after focal cerebral ischemic in rats.Kew words: Focal cerebral ischemia, Pin1, Neuronal cell death.
Voluntary wheel running improves cardiac function in a rodent model of chronic kidney disease (884.15)
Authors: Kuczmarski James , Martens Christopher , Kim Jahyun , Lennon‐Edwards Shannon , Edwards David 
Published: June 2021

The purpose of this study was to determine the effect of voluntary wheel running on cardiac function in a rodent model of chronic kidney disease (CKD). CKD (5/6 ablation‐infarction) or SHAM surgery was conducted on male Sprague Dawley rats. After 4wks of disease development, subsets of CKD animals remained sedentary (CKD‐SED) or underwent a 4wk voluntary wheel running intervention (CKD‐WR). Cardiac output (CO) was measured in all animals at 8wks post‐surgery using an isolated working heart preparation. CKD‐WR rats ran an average of 13.4±1.5km total which resulted in increased soleus muscle citrate synthase activity (p<0.05). Baseline CO was not different from SHAM in CKD‐WR (p>0.05), in contrast to CKD‐SED (p<0.05). CO remained not different from SHAM in CKD‐WR during preload & afterload manipulation (all p>0.05), whereas CKD‐SED CO was impaired (all p<0.05). Left ventricular (LV) tissue analysis revealed that LV hydrogen peroxide (H2O2) was increased and associated with decreased total nitrite + nitrate (NOx) and reduced nitric oxide synthase (NOS)‐3 in CKD‐SED (p<0.05). LV H2O2, NOx, & NOS‐3 were not different from SHAM in CKD‐WR (p>0.05). In conclusion, cardiac function is improved in a rodent model of CKD in response to a modest 4‐week voluntary wheel running intervention. This may be partly mediated by reduced H2O2 and increased NO production and/or bioavailability.Grant Funding Source: Supported by P20‐RR016472‐12 and P20‐GM103446‐12.
Temporal changes in left ventricular mechanics: impact of bed rest and exercise (884.23)
Authors: Scott Jessica , Downs Meghan , Ploutz‐Snyder Lori 
Published: June 2021

BACKGROUND: The use of more sensitive and specific echocardiographic techniques, such as speckle‐tracking imaging, may address the current limitations of conventional cardiac imaging techniques to provide insight into the magnitude and time course of cardiac deconditioning following spaceflight or head‐down tilt bed rest (HDTBR). METHODS: Speckle‐tracking assessment of longitudinal, radial, and circumferential strain and twist was used to evaluate the impact of 70 days of HDTBR (n=7) and HDTBR + exercise (n=11) on temporal changes in left ventricular (LV) mechanics. Echocardiograms were performed before (BR‐2), during (BR31, 70), and following (BR+4h) HDTBR. Repeated measures ANOVA was used to evaluate the effect of HDTBR on cardiac variables in control and exercise subjects. RESULTS: After sedentary HDTBR, longitudinal (‐19.0 ± 1.8% vs. ‐14.9 ± 2.4%) and radial (15.0 ± 1.9% vs. 11.3 ± 2.2%) strain and twist (18.0 ± 4.0° vs. 17.0 ± 3.6°) were significantly impaired. In contrast, exercise preserved LV mechanics, and there were non‐significant improvements from BR‐2 to BR70 in longitudinal strain (‐18.7 ± 1.5% vs. ‐20.4 ± 2.7%), radial strain (13.2 ± 2.4% vs. 14.2 ± 1.6%), and twist (16.3 ± 3.6° vs. 18.6 ± 5.9°). CONCLUSIONS: Speckle‐tracking echocardiography provides important new insight into temporal changes in LV mechanics during disuse and exercise training.Grant Funding Source: NASA Johnson Space Center’s Human Research Program
Effect of hypoxia on nuclear high affinity Ca2+ ‐ATPase activity and nuclear Ca2+ ‐influx in the cerebral cortex of newborn piglets (888.2)
Authors: Delivoria‐Papadopoulos Maria , Wang Andy , Malaeb Shadi , Ashraf Qazi 
Published: June 2021

Nuclear calcium signals control a number of critical nuclear functions including regulation of transcription factors, cell cycle regulation, gene transcription and DNA replication. Previous studies have shown that during hypoxia, there is increase in intracellular Ca2+‐and in nuclear high affinity Ca2+‐ATPase activity in the cerebral cortex of newborn piglets.We test the hypothesis that hypoxia results in increased activity of high affinity Ca2+‐ATPase and increased concentration of Ca2+‐influx in cortical neuronal nuclei of newborn piglets, and this hypoxia‐induced increase is Src kinase mediated.Piglets were divided into normoxic (Nx, n=4), hypoxic (Hx, n=4), and hypoxic pretreated with Src kinase inhibitor (Hx+Srci, n=4). Hypoxia was induced by decreasing FiO2 to 0.07 for 1 hr. Src kinase inhibitor (PP2, 1.0 mg/kg i.v.) was administered 30 min prior to hypoxia. Hypoxia was documented by levels of ATP and phosphocreatine (PCr). Nuclear 45Ca2+ concentration and activity of high affinity Ca2+‐ATPase was determined at 37°C for 30 min.Nuclear 45Ca2+ ‐influx (pmoles/mg protein) was 3.95±0.57 in Nx , 13.36±1.56 in Hx (p<0.05) and 9.61±2.76 in the Hx+Srci group. Ca2+‐ATPase activity increased following hypoxia: from 244.66±73.55 nmol Pi/mg protein/h in the normoxic group to 426.33±94.99 nmol Pi/mg protein/h in the hypoxic group (p<0.05 vs. normoxic group). In the Hx+Srci group, the high affinity Ca2+‐ATPase activity increased to 354.25±62.88 nmol Pi/mg protein/h (p<0.05 vs. hypoxic group). The data show that administration of PP2, a selective inhibitor of Src kinase, prevents the hypoxia‐induced increase in nuclear 45Ca2+ ‐influx concentration and Ca2+‐ATPase activity.We conclude that the hypoxia‐induced increase in nuclear membrane high affinity Ca2+‐ATPase activity is Src kinase‐mediated activation of the high affinity Ca2+‐ATPase, a mechanism of ATP‐dependent Ca2+uptake may lead to increase in intranuclear Ca2+during hypoxia.Grant Funding Source: NIH HD200337
Structural organization of the renal medulla has a significant impact on oxygen distribution (890.11)
Authors: Fry Brendan , Layton Anita 
Published: June 2021

A theoretical model is presented to analyze the impact on oxygen distribution of the heterogeneous organization of the rat outer and inner medulla revealed in anatomical studies.In the inner stripe, tubules and vessels are organized around vascular bundles; in the upper inner medulla, clusters of collecting ducts form the organizing motif. The present study extends a detailed mathematical model of the urine concentrating mechanism of the rat renal medulla developed (Layton, AJP Renal 300: F356‐F371, 2011), which was formulated to represent NaCl and urea, to include transport of red blood cells (RBCs), deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), and free oxygen (O2). Both basal cellular oxygen consumption throughout the whole medulla and active oxygen consumption via active NaCl transport in proximal tubules, thick ascending limbs, and collecting ducts are considered. Model equations are based on conservation of water and solutes, as well as transmural transport, and are solved to steady state. Results from the model suggest that the structural organization of the renal medulla produces significant axial and radial PO2 gradients, and impacts the effectiveness of the medullary urine concentrating mechanism. This research was supported in part by NIH grant DK‐89066.Grant Funding Source: NIH DK089066
CaMKII‐dependent filamin phosphorylation attenuates the MARCKS regulation of ENaC (892.24)
Authors: Alli Abdel , Bao Hui‐Fang , Montgomery Darrice , Ghant Marcus , Al‐Khalili Otor , Eaton Douglas 
Published: June 2021

Filamin A is an actin crosslinking protein regulated by phosphorylation. Several kinases including protein kinase A (PKA), protein kinase B (Akt), protein kinase C (PKC), and Ca(2+)/calmodulin‐dependent protein kinase II (CaMKII) phosphorylate filamin A. Xenopus laevis distal nephron epithelial (2F3) cells pretreated with the specific CaMKII inhibitor, KN‐93, showed an increase in amiloride‐sensitive transepithelial current calculated from transepithelial voltage and resistance measurements obtained with a volt‐ohmmeter, and an increase in ENaC open probability measured by single‐channel patch clamp studies. Whole cell lysates from 2F3 cells pretreated with KN‐93 or vehicle control were used to immunoprecipitate filamin, phosphofilamin, MARCKS, and ENaC before immunoblotting for each protein using specific antibodies. Immunoblot and densitometric analysis showed the level of filamin phosphorylation decreased with KN‐93 treatment compared to vehicle control. The association between ENaC and MARCKS was augmented in cells treated with KN‐93 compared to cells treated with vehicle only. ENaC‐MARCKS interaction is necessary for normal ENaC activity. Sucrose density gradient fractions from KN‐93 or vehicle treated cells were analyzed by immunoblotting. Similar to various actin cytoskeleton proteins, filamin protein expression was observed in non‐lipid raft cytoplasmic fractions. The expression of phosphofilamin between these fractions was different between the KN‐93 treated group compared to the vehicle treated control group. Taken together, these studies indicate filamin is an integral component of the actin cytoskeleton and phosphorylation of filamin by CaMKII disrupts the interaction between MARCKS and ENaC. Grant Funding Source: DK093255‐01
Selective serotonin reuptake inhibitors potentiate anoctamin 6 activation (893.22)
Authors: Nam Joohyun , Kim Yung Kyu , Lee Min Goo 
Published: June 2021

Selective serotonin reuptake inhibitors (SSRIs) are a class of widely used antidepressants. The primary mechanism of action of these drugs involves the inhibition of the serotonin transporter (SERT or SLC6A4). In addition to the inhibition of the serotonin transporter, SSRIs also modulate a variety of ion channels including Ca2+‐activated Cl− channels (CaCCs). Recently, anoctamins (ANOs) have been identified as a family of putative Cl− channels. Several research groups have reported that ANO6 generates outward‐rectifying Cl− currents by increasing intracellular Ca2+ levels, which are activated after a delay of several minutes. However, the reason for the very slow activation of the Cl− current remains unexplained. Using the whole‐cell patch‐clamp technique, we examined the effects of SSRIs, including fluoxetine, paroxetine, and sertraline, on cloned human ANO6 expressed in HEK293 cells. In contrast to their inhibitory effect on ANO1, the SSRIs reduced the latency time for the activation of ANO6 currents in a concentration‐dependent manner and slightly enhanced the magnitude of the peak current resulting from ANO6 Ca2+‐dependent activation. However, when we excised the membrane of the cells by using an inside‐out patch‐clamp technique we found that the ANO6 currents were inhibited by SSRIs. Although the exact mechanism by which the latency time of ANO6 activation is reduced remains unexplained, SSRIs might be involved in this ANO6 activation via an indirect pathway. ANO6 channels are involved in many physiological effects. Therefore, our study might contribute to a better understanding of the therapeutic action of SSRIs and their side effects.Grant Funding Source: This work was supported by a National Research Foundation of Korea (NRF, NO 2011‐0014404)
Anoctamin‐1 channel localization in excitable domains of the heart (893.23)
Authors: Britton Fiona , O'Driscoll Kate , Hatton William 
Published: June 2021

Calcium‐activated chloride (ClCa) currents are widely distributed in cardiac tissues and have been implicated to play a role in cardiac physiology and pathophysiology. Anoctamin‐1 (Ano1) is a membrane protein that functions as a ClCa channel and has been linked to essential physiological roles in a variety of excitable cells. We hypothesized that Ano1 channels in their functional role as ClCa channels are localized in distinct regions of the heart. Our objective was to define the localization and cellular expression of Ano1 channels in heart. Confocal immunofluorescence microscopy of mouse heart indicated that Ano1 channels are clustered at the intercalated discs of cardiac myocytes. We demonstrated strong co‐localization of Ano1 with connexin‐43 (Cx43) gap junction protein in isolated ventricular and atrial myocytes. A direct interaction between Ano1 channels and Cx43 was verified by co‐immunoprecipitation using mouse heart lysates. Our findings reveal that Ano1 channels compartmentalize in the intercalated disc region and suggest that Ano1 channels may play an important role in cell‐cell communication in this excitable membrane domain.Grant Funding Source: Supported by RO1HL091238
Metabolic status changes the interregulation of AMPK and CFTR (893.26)
Authors: Hallows Kenneth , King J Darwin , Rush Elizabeth , Xiong Sheng , Li Hui 
Published: June 2021

A better understanding of CFTR Cl‐ channel regulation could suggest new therapies for cystic fibrosis (CF). We previously found that the α1 catalytic subunit of AMP‐activated kinase (AMPK) binds to the C‐terminal tail of CFTR, and AMPK inhibits PKA‐stimulated CFTR gating via direct phosphorylation of CFTR. Both binding and phosphorylation are required for CFTR regulation by AMPK, which may tune CFTR activity to metabolic and other cellular stresses. Moreover, under basal conditions tonic AMPK activity may limit CFTR activation in the absence of PKA stimulation. Conversely, the loss of normal CFTR apical expression in primary CF human bronchial epithelial cells enhances AMPK expression and activity and disrupts AMPK apical targeting in these cells, suggesting that CFTR reciprocally regulates AMPK function. Here we more closely examined the nature of the AMPK‐CFTR interaction and its potential modulation by cellular AMPK activity and metabolic status. AMPK‐α binding to CFTR was reduced with AMPK activation or chemical ischemia in co‐immunoprecipitation studies performed on HEK‐293 and colonic epithelial T84 cells. Moreover, we found that activated AMPK (AMPK‐α‐pThr172) does not appear to bind to CFTR. Changes in cellular energy status inversely regulated AMPK activity and binding to CFTR in T84 cells. Of note, the CFTR‐binding region in AMPK‐α1 overlaps with the AMPK‐β‐binding region in α1, and AMPK‐α1 subunits bound to CFTR do not appear to be in a heterotrimeric AMPK αβγ complex, suggesting that CFTR may sequester AMPK‐α monomers. In summary, our results suggest that not only does AMPK regulate CFTR function, but that CFTR, via metabolic‐dependent interactions with AMPK, regulates AMPK function and subcellular localization in epithelia. This interregulation may represent a new target for CF therapy, as disruption of AMPK binding to CFTR may prevent untoward CFTR inhibition during treatment with AMPK activators to inhibit excessive inflammation and Na+ reabsorption in the CF airway.Grant Funding Source: Supported by NIH/NIDDK
Aspirin and salicylate are solutes of both sodium monocarboxylate transporters (SMCT1/Slc5a8 and SMCT2/Slc5a12) (896.9)
Authors: Plata Consuelo , Gavi‐Maza Marisol , Vázquez Norma , Romero Michael , Gamba Gerardo 
Published: June 2021

The Na+/monocarboxylate transporters (SMCT1 and SMCT2) are expressed several organ between them intestine and kidney, suggesting that its main physiological function are the absorption of fuel molecules involved in cellular metabolism and have only one ‐COOH group in its chemical structure; (lactate, propionate). Drugs with similar chemical properties also are translocated by SMCTs (B3 vitamin, pyrazinoate). However some propionate‐derivative NSAIDs (ibuprofen) reduce the activity of mammalian SMCT1. Using 22Na+ uptake in Xenopus oocytes injected with zebrafish SMCT’s (zSMCT1, zSMCT2) or human SMCT1 cRNA, we evaluated the effect of aspirin and salicylate on the function SMCT’s. Our results indicate a 22Na+ uptake that is aspirin concentration dependent in all SMCT’s. Similar affinity by aspirin was observed between hSMCT1 (Km ~2.4mM) and zSMCT1 (Km~2.5 mM) in contrast to a lower affinity of zSMCT2 (Km ~5mM). Also the difference of substrate affinity between zSMCT2 and hSMCT1 was evident by salicylate (hSMCT1Km ~4mM vs zSMCT1Km ~30 mM). Interestingly, although zSMCT1 and hSMCT1 shares a similar affinity and transport capacity for several substrates, the salicylate transport by zSMCT1 was not detected and only reduced a small percentage propionate transport induced by zSMCT1 (~10%) in contrast of ibuprofen which drops to 80%. The clear difference of salicylate transporter between SMCT1 could be explained by changes in the structure of proteins because the amino acids sequences are approximately 50% different, so the salicylate could be candidate for studies of structural‐function between SMCT1.Grant Funding Source: CONACYT48709MJ(CP)