ATPase Protein Levels Research Articles

Involvement of plasma membrane H+-ATPase in the nitrate-nutrition uptake and utilization in indica rice

Utilization of nitrogen by crops is essential for sustainable agriculture. The transport of nitrate (NO3−) across the plasma membrane is a critical gateway for N uptake and subsequent utilization. This process requires proton (H+) coupled cotransport, which is driven by proton motive force, provided by plasma membrane (PM) H+-ATPase. In this report, two indica rice varieties [Meixiangzhan 2 (MXZ) and Jifengyou 1002 (JFY)] in South China were selected and cultivated in hydroponic solution with 0.5 mM or 2.0 mM NO3− as the N source. The JFY exhibited stronger growth with higher biomass than MXZ under both 0.5 mM and 2.0 mM NO3−. PM H+-ATPase activity of JFY roots was significantly higher than that of MXZ. The higher PM H+-ATPase activity in JFY was consistent with a higher abundance of PM H+-ATPase protein and higher transcription levels of OSAs, such as OSA2, OSA7 and OSA8 in roots, OSA3, OSA7 and OSA8 in leaves. The expression of nitrate transporters (OsNRT1;1b, OsNRT2.1, OsNRT2.2, and OsNAR2.1) were also higher in roots or shoots of JFY than those in MXZ. Under 0.5 mM and 2.0 mM NO3−, the NO3− absorption and translocation rate, nitrate content, as well as nitrate reductase (NR) activity were all significantly higher in JFY, as compared to those in MXZ. Taken together, in JFY and MXZ, a higher level of PM H+-ATPase protein and higher activity coupled with greater efficiency in nitrate uptake, translocation and assimilation, suggesting the existence of a close correlation between PM H+-ATPase and nitrate utilization in indica rice. PM H+-ATPase may one of the elite genes that can contribute to nitrate use efficiency in rice.

Treatment of Refractory Mucosal Leishmaniasis Is Associated with Parasite Overexpression of HSP70 and ATPase and Reduced Host Hydrogen Peroxide Production (Brief Report).

Mucosal leishmaniasis (ML) is a deforming type of American Tegumentary Leishmaniasis caused by Leishmania (Viannia) braziliensis that frequently does not respond to treatment. Despite its relapsing clinical course, few parasites are usually found in mucosal lesions. Host and parasite factors may be responsible for this paradox in the pathogenesis of the disease, allowing for both a low parasite burden and the inability of the host to clear and eliminate the disease. In this work, we present a clinical case of relapsing ML that was treated for 25 years without success with SbV, N-methyl glucamine, sodium stibogluconate, amphotericin B deoxycholate, gabromycin, antimonial plus thalidomide, liposomal amphotericin B, Leishvacin (a vaccine made in Brazil) and miltefosine. In a comparative analysis using nanoscale liquid chromatography coupled with tandem mass spectrometry of protein extracts of L. (V.) braziliensis promastigotes isolated from the patient and from the reference strain (MHOM/BR/94/M15176), we observed increases in ATPase and HSP70 protein levels in the parasite. We also observed an impairment in the production of hydrogen peroxide by peripheral mononuclear blood monocytes (PBMCs), as assessed by the horseradish peroxidase-dependent oxidation of phenol red. We hypothesise that these parasite molecules may be linked to the impairment of host parasiticidal responses, resulting in Leishmania persistence in ML patients.

Activation of farnesoid X receptor retards expansion of renal collecting duct cell-derived cysts via inhibition of CFTR-mediated Cl- secretion.

The transcription factor farnesoid X receptor (FXR) regulates energy metabolism. Specifically, FXR functions to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion in intestinal epithelial cells. Therefore, this study aimed to investigate the role of FXR in CFTR-mediated Cl- secretion in renal tubular cells and to further elucidate its effects on renal cyst formation and growth. CFTR-mediated Cl- transport was evaluated via short-circuit current (ISC) measurements in Madin-Darby canine kidney (MDCK) cell monolayers and primary rat inner medullary collecting duct cells. The role of FXR in renal cyst formation and growth was determined by the MDCK cell-derived cyst model. Incubation with synthesized (GW4064) and endogenous (CDCA) FXR ligands reduced CFTR-mediated Cl- secretion in a concentration- and time-dependent manner. The inhibitory effect of FXR ligands was not due to the result of reduced cell viability and was attenuated by cotreatment with an FXR antagonist. FXR activation significantly decreased CFTR protein but not its mRNA. In addition, FXR activation inhibited CFTR-mediated Cl- secretion in primary renal collecting duct cells. FXR activation decreased ouabain-sensitive ISC without altering Na+-K+-ATPase mRNA and protein levels. Furthermore, FXR activation significantly reduced the number of cysts and renal cyst expansion. These inhibitory effects were correlated with a decrease in the expression of protein synthesis regulators mammalian target of rapamycin/S6 kinase. This study shows that FXR activation inhibits Cl- secretion in renal cells via inhibition of CFTR expression and retards renal cyst formation and growth. The discoveries point to a physiological role of FXR in the regulation of CFTR and a potential therapeutic application in polycystic kidney disease treatment.NEW & NOTEWORTHY The present study reveals that farnesoid X receptor (FXR) activation reduces microcyst formation and enlargement. This inhibitory effect of FXR activation is involved with decreased cell proliferation and cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion in renal collecting duct cells. FXR might represent a novel target for the treatment of autosomal dominant polycystic kidney disease.

Effects of Salacia Oblonga on aluminium induced changes on membrane ATPases of various organs in Wistar albino rats

Background: Aluminium, one of the abundant elements present in nature enters human body through diet, food packaging material and medication. Aluminium can induce toxicity by causing oxidative damage and by altering the function of membrane bound ATPases, Salacia oblonga, a woody climbing plant with vast geographical distribution in India possess various biological functions. Previous studies have shown a protective effect of S. oblonga on Al induced visceral toxicity. Our aim is to evaluate the effect of S. oblonga extract on aluminium induced oxidative organ damage and haematological changes Methods: The experimental animals, Wistar albino rats, were divided into four groups of 6 per group. The four groups are divided into distilled water, Aluminium chloride, Salacia oblonga extract and aluminium and Salacia oblonga. On 37th day animals were sacrificed and organs and blood were collected for assessment of ATPase activity, protein levels and haematological parameters. Results: Salacia oblonga increased the ATPase activity in the brain, heart and lungs, improved the protein levels and haematological parameters which were deteriorated by Al toxicity. Though statistically insignificant S. oblonga improved the ATPase enzymes and haematological parameters. Conclusions: Our study demonstrated that Aluminium causes membrane damage in various organs by altering the membrane ATPases level as a result of oxidative stress and altered the haematological parameters. We demonstrated that S. oblonga ameliorates Al induced membrane toxicity and also confers hemoprotective effect.

Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen.

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

Anti-VEGF therapy prevents M\xfcller intracellular edema by decreasing VEGF-A in diabetic retinopathy

BackgroundAlthough vascular endothelial growth factor A (VEGF-A) is known to play a key role in causing retinal edema, whether and how VEGF-A induces intracellular edema in the retina still remains unclear.MethodsSprague-Dawley rats were rendered diabetic with intraperitoneal injection of streptozotocin. Intravitreal injection of ranibizumab was performed 8 weeks after diabetes onset. rMC-1 cells (rat Müller cell line) were treated with glyoxal for 24 h with or without ranibizumab. The expression levels of inwardly rectifying K+ channel 4.1 (Kir4.1), aquaporin 4 (AQP4), Dystrophin 71 (Dp71), VEGF-A, glutamine synthetase (GS) and sodium-potassium-ATPase (Na+-K+-ATPase) were examined using Western blot. VEGF-A in the supernatant of the cell culture was detected with ELISA. The intracellular potassium and sodium levels were detected with specific indicators.ResultsCompared with normal control, protein expressions of Kir4.1 and AQP4 were down-regulated significantly in diabetic rat retinas, which were prevented by ranibizumab. The above changes were recapitulated in vitro. Similarly, the intracellular potassium level in glyoxal-treated rMC-1 cells was increased, while the intracellular sodium level and Na+-K+-ATPase protein level remained unchanged, compared with control. However, ranibizumab treatment decreased intracellular sodium, but not potassium.ConclusionRanibizumab protected Müller cells from diabetic intracellular edema through the up-regulation of Kir4.1 and AQP4 by directly binding VEGF-A. It also caused a reduction in intracellular osmotic pressure.

Topical nerve growth factor attenuates streptozotocin-induced diabetic cataracts via polyol pathway inhibition and Na+/K+-ATPase upregulation

The purpose of this study was to investigate whether and how topical nerve growth factor (NGF) attenuates streptozotocin (STZ)-induced diabetic cataracts in vivo. Rats were randomly divided into three groups, including the normal control rat group, STZ-induced diabetic cataract rat group (DM group), and STZ-induced diabetic cataract rat group treated with 200 μg/mL recombinant rat β-NGF (DM + NGF group). Cataract formation was evaluated by portable slit lamp biomicroscopy following pupil dilation at 8 weeks. The expression levels of NGF, aldose reductase (AR), and Na+/K+-ATPase in the lens epithelial cells (LECs) of the three groups were measured in the presence or absence of topical NGF. TUNEL-positive LECs were quantified to determine if hyperglycemia caused LEC apoptosis. At 8 weeks, the mean cataract score in the control group was significantly lower than that in DM and DM + NGF groups, and the score in the DM + NGF group was significantly lower than that in the DM group. At the equatorial zone and anterior central zone of lens, NGF and Na+/K+-ATPase expression levels were significantly decreased in the DM group; however, they were partially restored in the DM + NGF group. At the equatorial zone and anterior central zone of lens, AR expression and TUNEL-positive apoptotic LECs were significantly increased in the DM group compared with the control group, however, they were significantly decreased in the DM + NGF group. In conclusion, topical NGF could delay the progression of diabetic cataracts by attenuating polyol pathway activation and increasing Na+/K+-ATPase protein levels.

Abstract 1786: High expression of NCX-1 in triple negative breast cancer cell lines identifies a potential biomarker for sensitivity to cardiac glycosides

Abstract Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are defined by lack of the estrogen and progesterone receptors and HER2 gene amplification. Therefore, TNBC patients do not benefit from available therapies targeting these receptors. The significant heterogeneity of TNBC precludes the expectation that a single molecular target will be identified for this disease. Our goal is to identify compounds with selective cytotoxic effects in subsets of TNBC cells to identify new therapeutic targets for TNBC subgroups. Our studies show that the TNBC cell lines BT-549 and Hs578T are distinct from other TNBC cells in that they are highly sensitive to 9 cardiac glycosides/cardenolides isolated from Calotropis gigantea as well as digoxin. BT-549 cells are 15-times more sensitive to the isolated cardiac glycoside calotropin and 9-times more sensitive to digoxin as compared to MDA-MB-231 TNBC cells. Cardiac glycosides bind to the Na+/K+ ATPase and inhibit its activity, leading to increased intracellular Na+, which results in higher intracellular Ca2+ through reversal of the Na+/Ca2+ exchanger (NCX). Notably, among the 10 cardiac glycosides/cardenolides, there was a significant correlation between potency for inhibition of purified Na+/K+ ATPase and cytotoxic potency and selectivity for BT-549 cells. These data suggest that inhibition of Na+/K+ ATPase is critical for the selective cytotoxic effects of these compounds. BT-549 and Hs578T cells express high levels of non-specific TRPC1/4 cation channels1 and low levels of SERCA2 Ca2+ pumps2 compared to other TNBC cells, suggesting that they have impaired ability to handle intracellular Ca2+. Consistent with this hypothesis, within 3 h, concentrations of calotropin and digoxin that are selectivity cytotoxic each initiated a significant increase in intracellular Ca2+ in BT-549 cells. To test whether sensitivity to cardiac glycosides was due to higher Na+/K+ ATPase expression, message and protein levels were evaluated in 10 TNBC cell lines. The results show that there is no correlation between levels of Na+/K+ ATPase and sensitivity to cardiac glycosides. In contrast, NCX-1 expression was found to be 120 and 60-fold higher in the sensitive BT-549 and Hs578T cells, respectively, as compared to the resistant TNBC cell lines. Ongoing studies are evaluating whether genetic manipulation of NCX-1 is sufficient to modulate sensitivity to cardiac glycosides. These findings demonstrate that NCX-1 expression is associated with the selective sensitivity of a subgroup of TNBC cell lines to the cytotoxic effects of cardiac glycosides. Our results suggest that NCX-1 could be a biomarker to identify TNBC patients that could ultimately benefit from use of a cardiac glycoside for anticancer indications. 1. Grant CV, et al. Breast Cancer Res Treat. 2019;177(2):345. 2. Varga K, et al. BMC Cancer. 2018;18(1):1029. Citation Format: Petra E. Pederson, Shengxin Cai, Chase M. Carver, Tanja Grkovic, Barry R. O'Keefe, April L. Risinger, Robert H. Cichewicz, Susan L. Mooberry. High expression of NCX-1 in triple negative breast cancer cell lines identifies a potential biomarker for sensitivity to cardiac glycosides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1786.

Comparison of the efficacy of a Tabernaemontana divaricata extract and of biosynthesized silver nanoparticles in preventing cataract formation in an in-vivo system of selenite-induced cataractogenesis

Abstract The putative anticataractogenic efficacy of silver nanoparticles (AgNPs), biosynthesized with a Tabernaemontana divaricata (T. divaricata) extract, was evaluated in-vivo in four groups of Wistar rat pups: Group I (normal [control]); Group II (selenite-challenged [19 μmol/kg body weight], untreated); Group III (selenite-challenged, T. divaricata extract-treated [350 mg/kg body weight]); and Group IV (selenite-challenged, biosynthesized AgNPs-treated [200 mg/kg body weight]). Mean levels/activities of lenticular reduced glutathione (GSH), malondialdehyde (MDA), calcium, Ca2+ and Na+/K+-ATPase and total calpain were measured. The expression pattern of genes at mRNA transcript (by reverse transcription-PCR) and protein (by immunoblot analysis) levels for Ca2+ transporters, calpain isoforms and lenticular proteins (αA- and βB1-crystallins, and vimentin) were evaluated. In extract-treated and AgNPs-treated rats, relatively higher mean lenticular levels/activities of GSH, Ca2+ and Na+/K+-ATPase, total calpain, and higher mRNA transcript and protein levels of calpain isoforms and lenticular proteins were observed. However, relatively lower mean lenticular levels of MDA, calcium and also mRNA transcript and protein levels of plasma membrane Ca2+-ATPase-1 (PMCA-1) and sarco/endoplasmic reticulum Ca2+-ATPase-2 (SERCA-2) were recorded. The prevention of such lenticular alterations was more pronounced in AgNPs-treated rats than in plain extract-treated rats. These observations suggest that AgNPs biosynthesized with T. divaricata extract have the potential to prevent selenite-induced cataractogenesis in-vivo by maintaining normal lenticular calcium homeostasis and calpain cascade activities and by preventing alterations in key lenticular proteins.

EXTH-21. DIFFERENTIAL PROTEIN EXPRESSION LEVELS OF ABC AND SOLUTE CARRIER TRANSPORTERS AT THE BLOOD-BRAIN BARRIER OF HUMAN BRAIN AND GLIOBLASTOMA

Abstract BACKGROUND Mechanistic understanding and quantitative prediction of drug penetration across the human blood-brain barrier (BBB) is critical to rational drug development and treatment for brain cancer especially glioblastoma. However, prediction of drug brain/tumor penetration has been significantly hindered mainly due to the lack of quantitation data on transporter protein expression levels at the human BBB. This study was to determine protein expression levels of major transporters and markers at the BBB of human brain and glioblastoma. METHOD The absolute protein expression levels of major transporters and markers were determined in isolated microvessels of human brain (N=30), glioblastoma (N=47), rat (N=10) and mouse brain (N=10), using liquid chromatography with tandem mass spectrometry (LC-MS/MS) based targeted proteomics. RESULTS In isolated microvessels of 30 human brain specimens, the median protein abundances for ABCB1, ABCG2, GLUT1, GLUT3, LAT1, MCT1, Na/K ATPase, and Claudin-5 were 3.38, 6.21, 54.51, 7.17, 3.42, 5.71, 32.14, and 1.15 fmol/µg protein, respectively. In glioblastoma microvessels, ABCB1, ABCG2, MCT1, GLUT1, Na/K ATPase, and Claudin-5 protein levels were significantly reduced, while LAT1 was increased and GLU1 remained the same. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in isolated microvessels of both human brain and glioblastoma. There was species difference in transporter protein expression levels in isolated microvessels of human, rat and mouse brain. Specifically, rodent BBB expressed significantly higher ABCB1 but similar ABCG2, as compared to human BBB. CONCLUSION The physical and biochemical barriers of the BBB in glioblastomas are largely disrupted, as indicated by the loss or significant reduction in protein expression of the tight junction marker (claudin-5), brain endothelial cell marker (GLUT1), and major efflux transporters (ABCB1 and ABCG2) as compared to normal human BBB. Differential BBB transporter protein expression levels provides mechanistic and quantitative basis for the prediction of heterogeneous drug penetration into human normal brain and glioblastoma.

Modulation of exercise training related adaptation of body composition and regulatory pathways by anabolic steroids

Anabolic steroids have a long history of abuse in amateur and professional athletics. However, their interaction with training and the resulting effects on body composition and tissue adaptation, relying on a concert of factors and pathways, remain under investigation. This study aims at investigating the changes of body composition and the expression of selected genes and pathways essential for this adaptation process. Therefore, male wistar rats were treated with the anabolic steroid metandienone in two groups (n = 16; metandienone, metandienone + exercise) alongside with control groups (n = 16; control, exercise). Following a 6-week steep-angle treadmill training protocol, weight of organs, visceral fat and muscles was determined. M. gastrocnemius was histologically assessed by ATPase staining, mRNA and protein levels of factors of regeneration, hypertrophy and myogenesis and selected master regulators and markers were determined. Results show additive effects of anabolic steroids and exercise on body, tibia and reproductive organs weight. Mm. gastrocnemius and soleus weight was increased by training but not anabolic steroids. Muscle fiber diameter and composition remained unchanged. Visceral fat mass and fat cell size was affected by training and anabolic steroids but no additive effects could be observed. Exercise and anabolic steroids result in a complex regulation of the expression of genes in M. Gastrocnemius involved in skeletal muscle metabolism, hypertrophy, inflammation and regeneration. In summary, our data suggests distinct molecular mechanisms involved in the adaptation of the skeletal muscle to anabolic androgenic steroids and exercise. Metandienone treatment neither results in skeletal muscle hypertrophy nor liver-toxic effects but in an induction of skeletal muscle regeneration and an activation of endocrine negative feedback. Moreover our study demonstrates that visceral fat and bone responds with higher sensitivity to ASS and exercise than the skeletal muscle. This apparent plasticity of adipose and bone tissue rather than skeletal muscle could indicate a potentially superior future role of fat rather than muscle related parameters to detect and AAS abuse in a biologic passport strategy in professional athletes.

Root plasma membrane H+-ATPase is involved in low pH-inhibited nitrogen accumulation in tea plants (Camellia sinensis L.)

Tea is a perennial leaf crop and grows in acidic soil with a pH usually ranging from 4.0 to 5.0. Tea leaves are rich in free amino acids that are strongly associated with nitrogen accumulation. Previous studies mainly have focused on NH4+ nutrition on tea growth and development; however, studies of the impact of NO3− nutrition on tea root plasma membrane (PM) H+-ATPase activities are limited. In our study, tea plants (cv. Shu chazao) were grown using NO3− as the nitrogen (N) source at pH 4.0 and 5.0. We found that compared with tea plants grown at pH 5.0, significantly lower N accumulation was found in tea plants grown at pH 4.0. Also, more depolarized PM potentials, and a lower net H+ fluxes, PM H+-ATPase activities, Km, Vmax and PM H+-ATPase protein levels were found at pH 4.0 than at pH 5.0. Furthermore, the PM H+-ATPase inhibitor, vanadate, significantly reduced nitrogen accumulation in tea roots and plants at pH 4.0 and 5.0, further confirming the role of PM H+-ATPase in nitrogen accumulation in tea plants. Exogenously applied ATP induced increased PM H+-ATPase activity in tea roots at both pH 4.0 and 5.0. Low pH impaired root respiration and decreased ATP concentration in tea roots grown under nitrate nutrient. Taken together, our results suggest PM H+-ATPase is involved in the lower accumulation of N in tea roots cultivated in nitrate nutrient at pH 4.0 than 5.0, and this is related to lower respiration rate and ATP concentrations.

Involvement of plasma membrane H+‐ATPase in the ammonium‐nutrition response of barley roots

AbstractBarley (Hordeum vulgare L.) is a typical ammonium ()‐sensitive crop species and exhibits a futile‐ efflux from root cells. Plasma membrane (PM) H+‐ATPase is responsible for pumping H+ out of plant cells and providing the driving force for the transport of various substances. We hypothesized that PM H+‐ATPase is involved in this efflux process of barley roots. Barley plants were cultivated in hydroponic solution with 1.0 mM or 1.0 mM nitrate () as the sole nitrogen source. Plasma membrane vesicles were isolated from the root tips for analyses of the activity. Plasma membrane H+‐ATPase was tested by Western Blot. The transcription of PM H+‐ATPase genes was analyzed using qRT‐PCR. Proton and efflux from barley roots was analyzed by scanning ion‐selective electrode technique. The results show that root and shoot growth was repressed by nutrition when compared to nutrition. Plasma membrane H+‐ATPase activity of barley roots under nutrition was significantly higher than that under nutrition. Methyl‐ammonium (MeA), a non‐metabolizable analog of , had no significant effect on PM H+‐ATPase activity. The enhanced PM H+‐ATPase activity by nutrition was consistent with a higher abundance of PM H+‐ATPase protein and higher transcription levels of six PM H+‐ATPase genes. By using the pharmacological agents vanadate and fusicoccin, a close linkage was observed between H+ efflux and efflux from barley roots. Taken together, nutrition stimulates PM H+‐ATPase activity, and this stimulation is possibly triggered by assimilation in barley roots. efflux from barley roots is associated with PM H+‐ATPase activity which could provide the driving force for efflux. Ammonium efflux might be considered as a disposal strategy in barley roots which prevents overloading the root cytoplasm.

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca2+ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca2+ transient larger. Ca2+ cycling was modified with a RyR2 mediated Ca2+ leak from the sarcoplasmic reticulum and impaired Ca2+ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca2+. The Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca2+ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic “adaptations” of Ca2+ cycling with complex compensative interactions between Ca2+ handling partner and regulatory proteins.

Choroid plexus aquaporin 1 and intracranial pressure are increased in obese rats: towards an idiopathic intracranial hypertension model?

Idiopathic intracranial hypertension (IIH) is a condition of increased intracranial pressure (ICP) without identifiable cause. The majority of IIH patients are obese, which suggests a connection between ICP and obesity. The aim of the study was to compare ICP in lean and obese rats. We also aimed to clarify if any ICP difference could be attributed to changes in some well-known ICP modulators; retinol and arterial partial pressure of CO2 (pCO2). Another potential explanation could be differences in water transport across the choroid plexus (CP) epithelia, and thus we furthermore investigated expression profiles of aquaporin 1 (AQP1) and Na/K ATPase. ICP was measured in obese and lean Zucker rats over a period of 28 days. Arterial pCO2 and serum retinol were measured in serum samples. The CPs were isolated, and target messenger RNA (mRNA) and protein were analyzed by quantitative PCR and western blot, respectively. Obese rats had elevated ICP compared to lean controls on all recording days except day 0 (P<0.001). Serum retinol (P=0.35) and arterial pCO2 (P=0.16) did not differ between the two groups. Both AQP1 mRNA and protein levels were increased in the CP of the obese rats compared to lean rats (P=0.0422 and P=0.0281). There was no difference in Na/K ATPase mRNA or protein levels (P=0.2688 and P=0.1304). Obese Zucker rats display intracranial hypertension and increased AQP1 expression in CP compared to lean controls. The mechanisms behind these changes are still unknown, but appear to be unrelated to altered pCO2 levels or retinol metabolism. This indicates that the increase in ICP might be related to increased AQP1 levels in CP. Although further studies are warranted, obese Zucker rats could potentially model some aspects of the IIH pathophysiology.

Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression.

Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg-1 day-1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na+/K+-ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na+/K+-ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na+/K+-ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na+/K+-ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na+/K+-ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy.

Leucine-Rich Repeat Kinase 2 (Lrrk2)-Sensitive Na+/K+ ATPase Activity in Dendritic Cells

Leucine-rich repeat kinase 2 (Lrrk2) has been implicated in the pathophysiology of Parkinson’s disease. Lrrk2 is expressed in diverse cells including neurons and dendritic cells (DCs). In DCs Lrrk2 was shown to up-regulate Na+/Ca2+-exchanger activity. The elimination of Ca2+ by Na+/Ca2+ -exchangers requires maintenance of the Na+ gradient by the Na+/K+ -ATPase. The present study thus explored whether Lrrk2 impacts on Na+/K+ -ATPase expression and function. To this end DCs were isolated from gene-targeted mice lacking Lrrk2 (Lrrk2−/−) and their wild-type littermates (Lrrk2+/+). Na+/K+ -ATPase activity was estimated from K+ induced, ouabain sensitive, current determined by whole cell patch clamp. Na+/K+ -ATPase α1 subunit transcript and protein levels were determined by RT-qPCR and flow cytometry. As a result, the K+ induced current was significantly smaller in Lrrk2−/− than in Lrrk2+/+ DCs and was completely abolished by ouabain (100 μM) in both genotypes. The K+ induced, ouabain sensitive, current in Lrrk2+/+ DCs was significantly blunted by Lrrk2 inhibitor GSK2578215A (1 μM, 24 hours). The Na+/K+ -ATPase α1 subunit transcript and protein levels were significantly lower in Lrrk2−/− than in Lrrk2+/+ DCs and significantly decreased by Lrrk2 inhibitor GSK2578215A (1 μM, 24 hours). In conclusion, Lrrk2 is a powerful regulator of Na+/K+ -ATPase expression and activity in dendritic cells.

Characterization of sarcoplasmic reticulum Ca2+ ATPase pumps in muscle of patients with myotonic dystrophy and with hypothyroid myopathy

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca2+ from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca2+ homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca2+-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca2+ ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy.

Increased stiffness is the major early abnormality in a pig model of severe aortic stenosis and predisposes to congestive heart failure in the absence of systolic dysfunction.

BackgroundIt remains unclear whether abnormal systolic function and relaxation are essential for developing heart failure in pathophysiology of severe aortic stenosis.Methods and ResultsYorkshire pigs underwent surgical banding of the ascending aorta. The animals were followed for up to 5 months after surgery, and cardiac function was assessed comprehensively by invasive pressure–volume measurements, 3-dimensional echocardiography, echocardiographic speckle-tracking strain, and postmortem molecular and histological analyses. Pigs with aortic banding (n=6) exhibited significant left ventricular hypertrophy with increased stiffness compared with the control pigs (n=7) (end-diastolic pressure–volume relationship β: 0.053±0.017 versus 0.028±0.009 mm Hg/mL, P=0.007); however, all other parameters corresponding to systolic function, including ejection fraction, end-systolic pressure–volume relationship, preload recruitable stroke work, echocardiographic circumferential strain, and longitudinal strain, were not impaired in pigs with aortic banding. Relaxation parameters were also similar between groups. Sarcoplasmic reticulum calcium (Ca2+) ATPase protein levels in the left ventricle were similar. There were significant increases in 3-dimensional echocardiographic left atrial volumes, suggesting the usefulness of these indexes to detect increased stiffness. Right atrial pacing with a heart rate of 120 beats per minute induced increased end-diastolic pressure in pigs with aortic banding in contrast to decreased end-diastolic pressure in the control pigs. Histological evaluation revealed that increased stiffness was accompanied by cardiomyocyte hypertrophy and increased perimysial and perivascular fibrosis.ConclusionIncreased stiffness is the major early pathological process that predisposes to congestive heart failure without abnormalities in systolic function and relaxation in a clinically relevant animal model of aortic stenosis.

Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation–contraction (E–C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E–C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.