Further analysis demonstrated that HF-induced atrial electrical remodelling increased susceptibility to atrial alternans mainly due to the increased sarcoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ reuptake, modulated by increased phospholamban (PLB) phosphorylation, and the decreased transient outward K+ current (Ito). The underlying mechanism has been suggested that the increased SR Ca2+ content and prolonged AP did not fully recover to their previous levels at the end of diastole, resulting in a smaller SR Ca2+ release and AP in the next beat. These produced Ca2+ transient alternans and AP alternans, and further caused AP alternans and Ca2+ transient alternans through Ca2+→AP coupling and AP→Ca2+ coupling, respectively. Simulation of a 1D tissue model showed that the combined action of HF-induced ion channel remodelling and a decrease in cell-to-cell coupling due to fibrosis increased the heart tissue’s susceptibility to the formation of spatially discordant alternans, resulting in an increased functional AP propagation dispersion, which is pro-arrhythmic. These findings provide insights into how HF promotes atrial arrhythmia in association with atrial alternans.Heart failure (HF) is associated with an increased propensity for atrial fibrillation (AF), causing higher mortality than AF or HF alone. It is hypothesized that HF-induced remodelling of atrial cellular and tissue properties promotes the genesis of atrial action potential (AP) alternans and conduction alternans that perpetuate AF. However, the mechanism underlying the increased susceptibility to atrial alternans in HF remains incompletely elucidated. In this study, we investigated the effects of how HF-induced atrial cellular electrophysiological (with prolonged AP duration) and tissue structural  remodelling can have an effect on the generation of atrial AP alternans and their conduction at the cellular and one-dimensional (1D) tissue levels. Simulation results showed that HF-induced atrial electrical remodelling prolonged AP duration, which was accompanied by an increased sarcoplasmic reticulum (SR) Ca 2+-ATPase (SERCA) Ca2+ reuptake caused by increased phospholamban phosphorylation and a decrease in transient outward K+ current played significant roles in the genesis of Ca2+ transient alternans and action potential alternans at the single-cell level. The HF-induced decline of cell-to-cell coupling and APD prolongation promoted the genesis of spatially discordant alternans in atrial tissue. This provides insights into how HF facilitates atrial arrhythmia in relation to atrial alternans.Atrial Fibrillation (AF) is the most common arrhythmia in adults, especially in the elderly, with the increased incidence of stroke being a major complication that increases morbidity and mortality. The occurrence of AF is often accompanied by heart failure (HF). AF and HF are also known to have the bidirectional relationship that AF worsens HF and HF promotes AF. HF can induce atrial remodelling, including electrical remodelling, atrial fibrosis, stretch and dilatation, and oxidative stress, in which many factors are associated with arrhythmogenic atrial alternans. HF-induced atrial remodelling varies during various stages and complications of HF, but possible mechanisms underlying their pro-susceptibility to alternans have not been completely elucidated. In this study, we investigated the effects of HF-induced atrial remodelling with prolonged action potential duration (APD) and decreased cell-to-cell coupling on susceptibility to atrial alternans. Simulation results showed that HF-induced an increase in sarcoplasmic reticulum Ca Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular diseases that frequently coexist with each other, resulting in higher mortality than AF or HF alone . For exaet al. showed no significant difference in atrial APD between control and HF patients i half-saturation constant for Ca2+ uptake into the network SR, Kup, was decreased as in . In. In2+ ha90, CaT amplitude and SR Ca2+ content, parameter sensitivity analysis was performed using multivariable regression based on control model at 1 Hz. The results are shown in GCa and Jup(max), and decreasing Gto and Kup had significant contributions to APD90  had the largest contribution to CaT amplitude and SR Ca2+ content. Decreasing Jrel(max) had a stronger contribution to SR Ca2+ content, and increasing Jrel(max) had only a limited effect on APD90 and CaT amplitude. There were minimal effects of GKs and [Csqn]max on APD90, CaT amplitude and SR Ca2+content. Furthermore, we varied each parameter by different degrees in the HF model one at a time to identify which parameters were the main determinants of HF-induced increases in APD90, CaT amplitude and SR Ca2+ content  or GCa remodelling produced a larger increase in APD90, CaT amplitude and SR Ca2+ content; absence of Jrel(max) remodelling produced a smaller increase in SR Ca2+ content, and absence of Gto remodelling produced a smaller increase in APD90 relative to control. These results suggest that HF-induced changes on APD90, CaT amplitude and SR Ca2+ content are a consequence of all remodelling factors, though the individual role of each is different.To identify the relative contributions of the 7 identified parameters  to the Ato APD90 , CaT ampto APD90  and SR C content , of whic content . It was 90  and ICa that led to small Ito and IKur values which contributed to slowing repolarization to increase the APD90.Further simulations were conducted to investigate whether HF-induced atrial ion channel remodelling promotes the susceptibility to alternans. Results are shown in 90 , CaT amp90  and CaT 90  obtained90 . Under t90 ; howeverotential . When th90 restitution curves from the dog atrial cell model under control and HF conditions, as well as their maximal slope. Results are shown in 90 restitution curves under both control and HF conditions were non-monotonic , GKs, Jup(max) and [Csqn]max in Kup, alternans occurred in HF until it was decreased by 78%, which may be related to increased SR Ca2+ content. As the degree of decreased Gto gradually increased, alternans occurred at longer PCLs, which may be caused by an increased APD. On the contrary, of the more we reduced GCa, the shorter PCLs were when alternans was observed, which may result from decreased APD and CaT amplitude. See Further sensitivity analysis of HF-induced remodelling was performed to identify the main determinants for alternans genesis in both control and HF models. Firstly, each HF-induced parameter change was incorporated into the control model one at a time . In eachlternans . These r2+ transient alternans were also investigated at the 1D tissue level. In simulations, both HF-induced ion channel remodelling and changes in intercellular coupling due to fibrosis  were considered. Pacing-induced spatial alternans were studied in the 1D simulation by increasing the pacing rate.Conduction of the AP and its corresponding CaD-PCL parameter space under control and HF conditions, as shown in D = 0.028 mm2/ms, 0.084 mm2/ms, and 0.167 mm2/ms) under control and HF conditions. The curves were flat at long PCLs and produced CVs of 0.60 m/s with D = 0.167 mm2/ms, 0.4 m/s with D = 0.084 mm2/ms, and 0.2 m/s with D = 0.028 mm2/ms. They decreased at short PCLs, producing reduced CVs and alternating large and small CVs under both HF and control conditions. The alternating CVs in D = 0.028 mm2/ms than that when D = 0.084 mm2/ms or 0.167 mm2/ms. These suggest that both HF-induced APD prolongation and decreased CVs caused by decreased PCLs and cell-to-cell coupling contribute to the genesis of discordant alternans at the tissue level.To illustrate the effects of PCLs and cell-to-cell coupling on the genesis of discordant alternans, various AP propagation patterns were investigated in a D = 0.028 mm2/ms at a PCL of 168 ms under HF conditions. The AP and Ca2+ transient propagation along the cable  as time increases  were colour mapped in st cell to 4th cell) as the beat number increased. Along the cable, the alternating areas were out of phase on both sides of the APD node, such as AP traces recorded from the 40th, 100th, and 170th cells in th cell, short-long-short-long-short alternans at the 100th cell, and long-short-long-short-long alternans at the 170th cell, and these represented discordant alternans. Moreover, the AP and Ca2+ transient alternans were in phase  marked by black dashed lines with an arrow was consistent with the APD nodes in 2+ transient alternans , the DI node occurred with no APD or CV node. This DI node then produced APD and CV nodes during the second and third stimuli. As the beat number increased, the spatial dispersions of DI and APD90 were enhanced for two consecutive beats. A shorter distance was required for the DI and APD90 distribution curves to intersect and produce DI and APD nodes. Thus, the nodes gradually moved towards the stimulus site with continued pacing. When D = 0.167 mm2/ms with a CV of ≈ 0.53 m/s at the CV nodes  on the AP and Ca2+ transient alternans. Further simulation results at the 1D tissue level demonstrate the effects of HF-induced atrial electrical and structural remodelling  on spatial alternans. Our major findings follow: (i) The increased SR Ca2+ content caused by increased Ca2+ uptake by SERCA and decreased RyR2 protein expression contributes to the increased Ca2+ transient amplitude, leading to an increase in inward INCX. This combined with the decreased Ito and IKs results in APD prolongation even though with reduced ICa in HF simulations. (ii) The AP and Ca2+ transient alternans can be induced in HF simulations, but could not in control simulations, suggesting that HF-induced atrial electrical remodelling can increase the atrial susceptibility to alternans. HF-induced increased PLB phosphorylation and decreased Ito are the main determinants underlying the occurrence of alternans through [Ca2+]i→AP coupling and AP→[Ca2+]i coupling, respectively. Failure of the increased SR Ca2+ content and prolonged AP recovery at the end of diastole underlies the genesis of AP and Ca2+ transient alternans in HF. (iii) Cell-to-cell coupling plays an important role in discordant alternans patterns. Reduced cell-to-cell coupling caused by HF-induced atrial fibrosis results in a decreased CV and can enhance spatial dispersion of AP propagation to produce discordant alternans. Together with HF-induced APD prolongation, the enhanced spatial dispersion of AP propagation increases susceptibility to spatial discordant alternans in atrial tissue.In this study, the effects of HF-induced atrial remodelling with prolonged APD and decreased cell-to-cell coupling on atrial alternans were investigated z et al.  at the sICa densities in sheep i→AP coupling in HF. Decreasing Kup resulted in an increase in SERCA Ca2+ reuptake  as shown in INa  increases the susceptibility to alternans in HF. Decreasing Gto  in both control and HF conditions (D = 0.167 mm2/ms), additional APD nodes were produced under conditions with weak cell-to-cell coupling (D = 0.028 mm2/ms), and the range of PCLs at which discordant alternans occurred was longer especially in HF conditions  The DI and APD nodes moved towards the stimulus site as pacing continued , which iet al. . With stet al. , produciet al. . This adet al. i. (C) Ito. (D) Junctional SR Ca2+ concentration ([Ca2+]JSR). (E) IKs. (F) Jrel. (G) ICa. (H) Jup. (I) INCX.(A) AP. (B) max were reduced from 0% to 90% relative to the control in the HF model.(A) (TIF)Click here for additional data file.S4 FigJup. (B) [Ca2+]JSR. (C) Jrel. (D) [Ca2+]i. (E) INCX. (F) AP. (G) INa. (H) ICa.(A) (TIF)Click here for additional data file.S5 FigIto. (B) AP. (C) INa. (D) ICa. (E) Jrel. (F) [Ca2+]i. (G) [Ca2+]JSR. (H) INCX.(A) (TIF)Click here for additional data file.S6 Fig90 of each beat, in which black dashed lines with arrows indicate the APD node position as the beat number increases. (B(i)) Time-space plot of the Ca2+ transient in a homogeneous, 1D cable. (B(ii)) The corresponding peak Ca2+ transient of each beat, in which black dashed lines with arrows indicate the peak Ca2+ transient node position as the beat number increases.(A(i)) Time-space plot of the membrane potential of a homogeneous, 1D cable. (A(ii)) The corresponding APD(TIF)Click here for additional data file.S7 FigD = 0.167 mm2/ms. (B) Results with D = 0.028 mm2/ms.(A) Results with (TIF)Click here for additional data file.S8 Fig2+]i. (C) INCX. (D) [Ca2+]JSR. (E) Jrel. (F) Jup.(A) AP. (B) [Ca(TIF)Click here for additional data file.S1 Table90, CaT amplitude, and SR Ca2+ content were computed relative to control, and the bold font demonstrated the HF model.The values of APD(PDF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Sophora alopecuroides L. used in the traditional Uygur medicine, induced a significant increase in cellular glucose uptake of L6 cells, suggesting it has the potential to relieve hyperglycemia. Therefore, we investigated the effects of ALO on type 2 diabetes mellitus (T2DM) through in vitro and in vivo studies. The translocation of glucose transporter 4 (GLUT4) and changes in intracellular Ca2+ levels were real-time monitored in L6 cells using a laser scanning confocal microscope and related protein kinase inhibitors were used to explore the mechanism of action of ALO. Furthermore, high fat diet combined with low-dose streptozotocin (STZ) was used to induce T2DM in rats, and ALO was given to the stomach of T2DM rats for 4 weeks. In vitro results showed that ALO-induced enhancement of GLUT4 expression and translocation were mediated by G protein-PLC-PKC and PI3K/Akt pathways and ALO-enhanced intracellular Ca2+ was involved in activating PKC via G protein-PLC-IP3R-Ca2+ pathway, resulting in promoted GLUT4 plasma membrane fusion and subsequent glucose uptake. ALO treatment effectively ameliorated hyperglycemia, glucose intolerance, insulin resistance and dyslipidemia, alleviated hepatic steatosis, protected pancreatic islet function and activated GLUT4 expression in insulin target tissues of T2DM rats. These findings demonstrated that ALO deserves attention as a potential hypoglycemic agent.Aloperine (ALO), a quinolizidine alkaloid isolated from  Diabetes mellitus is an endocrinal and metabolic disease, caused by the inability of pancreas to produce required insulin and/or an inability of the body to effectively respond to insulin . More thGlucose transporter 4 (GLUT4) is the main carrier of glucose transport. Selective disruption of GLUT4 expression in muscle induce global insulin resistance . GLUT4 h2+ plays a crucial role in the transport of GLUT4 to the plasma membrane. Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ release channel sonthe endo/sarcoplasmic reticulum (ER/SR)  . Studiese canals . RyR chanylation . The effSophora alopecuroides L., a traditional Uygur medicinal plant mainly used in Xinjiang Autonomous region of China to protect against heat and dampness, killing insects and relieving pain  combined with low dose of streptozotocin (STZ) injection, which closely mimicked metabolic characteristics of humans T2DM . The eff2+), U73122 and 2-APB were purchased from Sigma . GLUT4 antibody (#2213), Akt antibody (#9272), phospho-Akt (Ser473) (193H12) antibody (#4058) and phospho-PKC (pan) (Thr410) antibody (#2060) were purchased from Cell Signaling Technology . The Anti-c-myc mouse monoclonal antibody (#M10117) and FITC antibody (#M10422) were both purchased from TransGen Biotech .Compound C was purchased from Calbiochem . Wortmannin was purchased from Selleckchem . Gö6983 was purchased from EMD Millipore . Fluo-4 AM was purchased from Invitrogen . Pertussis Toxin (PTX) and Gallein were purchased from Tocris Bioscience . Ryanodine was purchased from Cayman Chemical . 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid , Wuhan, China. A voucher specimens of S. alopecuroides L. was numbered (No. SC0859) and has been deposited in the Herbarium of School of Pharmaceutical Sciences, SCUN. Air dried aerial parts of S. alopecuroides L. (10°kg) were ground into powder and extracted using 85% EtOH . After evaporation of the collected solution, the residue was acidified with diluted HCl (5%) to a pH of two and partitioned between petroleum ether  and the acid water layer. The aqueous part was basified with aqueous NH3 to a pH of 10 and extracted with ethyl acetate to afford 135 g of crude alkaloid. A part of the crude alkaloid (125 g) was subjected to column chromatography over silica gel  and eluted with the isocratic gradient solvent system of CH2Cl2, methanol, and diethylamide (100: 10: 2) to yield six fractions (F1−F6) . After evaporation under vacuum, crude aloperine (33 g) was obtained from F3 and then dissolved in methanol (150 ml) at 50°C. The ALO solution was kept in refrigerator at 4°C to recrystallize and afford 19 g of pure ALO. 1H, 13C NMR and ESIMS data were obtained to support the identification of aloperine . L6 cells  were infected with freshly prepared viruses at the exponential growth phase. Polybrene (8 μg/ml) was used to improve the efficiency of virus infection. L6 Cells with fluorescence were isolated by fluorescence activated cell sorter (FACS) and seeded into 96-well plates. Finally, the clone with the highest fluorescence intensity was screened out after insulin (100 nM) treatment.pIRAP-mOrange cDNAs were inserted into the pQCXIP plasmid. The retrovirus was prepared by transfecting pQCXIP-IRAP-mOrange, vesicular stomatitis virus G (VSVG), and PHIT60  at a ratio of 2: 1: 1 through Lipofectamine 2000  into Plat E cells . Forty-e2. L6-IRAP-mOronge was inoculated on glass covered glass slides overnight, then starved in serum-free α-MEM for 2 h. The fluorescence imaging of L6-IRAP-mOronge was performed by laser scanning confocal microscope LSM 700 , and the dynamic displacement of irap-mrange was observed. The photos were taken after the addition of 30 μM ALO or other agents by 555 nm excitation laser every 5 min in 30 min. The fluorescence intensity of IRAP-mOrange at the PM was measured for reflecting the GLUT4 translocation as previously described (µM Compound C (AMPK inhibitor), 100 nM Wortmannin (PI3K inhibitor), or 10 μM Gö6983 (PKC inhibitor) for 30 min prior to treatment with ALO.GLUT4 and Insulin-regulated aminopeptidase (IRAP) co-localize in GLUT4 storage vesicles (GSVs) and previous studies indicated that they are highly co-localized . Therefoescribed . To clar4–5×104 cells/well in 100 μl α-MEM medium. After 12 h incubation, the L6 cells were treated with different doses of ALO  or 100 nM insulin or vehicle control dissolved in 100 μl glucose-free α-MEM medium that contained 150 μg/ml 2-NBDG. Plates were incubated at 37°C with 5% CO2 for 30 min. Following the treatment, the glucose uptake in the L6 cells was measured by the method described in the glucose uptake assay kit.Glucose uptake related experiments were carried out as previously described . Glucoseg for 10 min to remove unbroken cells and at 18,000 × g for 20 min to obtain the membrane fraction. All samples were subjected to SDS-PAGE analysis on 10% resolving gels. In a single experiment, each sample was loaded with the same amount of protein. The changes in expression of GLUT4 in separated proteins were detected by western blotting.The steps for purification of related components of L6 cell membrane are similar to our previous experiments . L6 cell2+ was analyzed and measured in a similar manner as previously described  solution containing 2 mM Ca2+ and the PSS solution containing 0 mM Ca2+ into the chamber. Or added the PSS solution containing 0 mM Ca2+ and BAPTA to the chamber, so that the cells were in such an external fluid. The images were taken after the addition of 30 μM ALO or other agents by 488 nm excitation laser every 5 min in 30 min. The fluorescence intensity of fluo-4 AM representing the intracellular Ca2+ concentration was measured and analyzed by the Zen 2010 software .Intracellular Caescribed . L6 IRAPg for 10 min. Supernatant protein denatured in SDS sample buffer. Equal amounts of protein samples were subjected to 8% SDS-PAGE. Separated proteins were electrophoretically transferred to Polyvinylidene fluoride (PVDF) membranes, and the membranes were incubated with primary antibodies and HRP-conjugated secondary antibodies. The protein bands were detected and quantified by a ChemiDoc XRS .Starve L6 cells for 2 h before experiment. The L6 cells were then washed with cold PBS three times, resuspended in HES buffer supplemented with protease inhibitor cocktail  and phosphatase inhibitor cocktail  at 4°C. The L6 cells lysing was performed as described previously . The lysn = 8) or a high-fat diet . After 4 weeks, the HFD rats were injected intraperitoneally with a low dose of streptozotocin . Rats in the NC group were injected with the citrate buffer vehicle. Over-night fasted rats with fasting blood glucose (FBG) level more than 11.1 mmol/L at 1 week after STZ injection were chosen for experiments. Thereafter, these T2DM rats were randomly divided into four groups with eight animals each: diabetic control (DC), diabetic with low dose ALO , diabetic with medium dose ALO , and diabetic with high dose ALO . ALO was made into a suspension with saline containing 0.5% CMC-Na. NC and DC groups were intragastric administered with the same solvent. All groups received intragastric administration once daily for four consecutive weeks.The care and use of animals and all procedures involving animals were performed in accordance with the Guidelines for Animal Experiments of South-Central University for Nationalities and were approved by the university animal ethics committee . Male Sprague-Dawley (SD) rats (8 weeks old) of Specific Pathogen Free (SPF) grade were purchased from the Beijing HFK Bioscience Co., Ltd. Each three rats were fed in one cage with enough food and water under standard conditions at 23 ± 2°C, 50–60% relative humidity, and with a constant 12 h light–dark cycle. After 7 days of acclimatization, the animals were randomly assigned to receive a regular chow diet , as the normal control group , triglycerides (TG), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) were evaluated by an automatic biochemical analyzer   and blood samples were collected from abdominal aorta. The serum was separated by centrifuging the blood samples at 3,000 × , Japan) . Serum fThe related experiments of Histology and immunohistochemistry are similar to the previous ones . Parts og, 4°C for 15 min. The concentration of protein contents was determined with BCA assay kit   . Eventuain vitro experiments were performed triplicate. Differences between groups were analyzed with one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test using Graphpad prime 5.0 software. A probability (p) value of less than 0.05 was considered as statistically significant.Data were shown as means ± standard error of the mean (SEM). All of the The effect of ALO on cellular glucose uptake was performed by 2-NBDG assay on different concentrations of ALO  along with both positive (insulin 100 nM) and vehicle control. Results indicated that glucose uptake were significantly enhanced with the increasing of concentration of ALO as compared to the vehicles . To eluc2+ plays an important role in insulin-induced glucose uptake . Moreover, when cytosolic Ca2+ was inhibited, ALO-induced increases of IRAP fluorescence at the PM also was restrained (upper). These data implied that intracellular Ca2+ involved in ALO-induced GLUT4 trafficking. Cytosolic Ca2+ is composed of intracellular store release and extracellular Ca2+ influx. ALO induced increases of intracellular Ca2+, which partly decreased following the removal of extracellular Ca2+ (bottom). However, ALO-induced increases of IRAP fluorescence at the PM remained unchanged (upper). These results suggested that releasing of intracellular Ca2+ is therefore played a key role in ALO-induced GLUT4 trafficking to the PM.It has been reported that the intracellular Cae uptake  and GLUTe uptake . Hence, BAPTA-AM , bottom.strained , upper. lar Ca2+ , bottom.nchanged , upper. 2+ and GLUT4 trafficking to the PM. Compound C is the primary reagent used as an AMPK inhibitor (µM) or PI3K inhibitor Wortmannin (100 nM) or PKC inhibitor Gö6983 (10 µM) for 30 min had no effect on ALO-induced increase of intracellular Ca2+ (bottom). However, Gö6983 strongly restrained ALO-induced increase of IRAP fluorescence intensity at the PM (upper) and Wortmannin partly inhibited ALO-induced increase of IRAP fluorescence intensity at the PM (upper). On the contrary, Compound C had no effect on ALO-induced increase of IRAP fluorescence intensity at the PM (upper). These data implied that PKC and PI3K/Akt might be involved in ALO-induced GLUT4 trafficking and expression. Thereafter, to validate this observation, we performed western blotting and observed that treating L6 cells with ALO (30 µM) for 5 and 30 min significantly enhanced PKC phosphorylation level (µM) for 30 min strongly restrained ALO-induced GLUT4 expression , a blocker of IP3Rs that regulates intracellular Ca2+ release to block the ALO-increased intracellular Ca2+. We found that 2-APB significantly inhibited ALO-induced increase of IRAP fluorescence intensity at the PM and intracellular Ca2+ under 2 mM  is also an important receptor that regulates intracellular calcium release  and p-Akt (phosphorylation of Akt) in skeletal muscle and WAT. The results showed that the treatment with ALO increased GLUT4 expression, phosphorylation of both PKC and Akt in skeletal muscle and WAT of T2DM rats. Especially, ALO was more effective in phosphorylating PKC than Akt . Moreover, the addition of ALO only induced an increase in Akt phosphorylation level at 5 min while the increase was reduced at 30 min . Therefore, we concluded that ALO enhanced GLUT4 translocation via G protein-PLC-PKC signaling pathway in L6 cells.AMPK , PI3K/Aklocation , sanggua pathway  and diacipocytes . Imagingolved in . Interesolved in , upper. t 30 min . We hypo pathway . Treatmelocation , upper. 2+, the translocation of GLUT4 caused by ALO stimulation was not affected, but only partially affected the increase of intracellular calcium ion concentration, indicating that Ca2+ release played a key role in ALO-induced GLUT4 translocation . Hence, ALO could increase the intracellular Ca2+via G protein-PLC-IP3R-Ca2+ pathway. In summary, in vitro studies demonstrated that ALO-induced enhancement of GLUT4 expression and translocation are mediated by G protein-PLC-PKC and PI3K/Akt pathways, thus increasing glucose uptake. Recent study showed that raising intracellular cytosolic Ca2+ concentration can activate PKCs which promote the gain of surface GLUT4 level .PZ and XY: contributed to the conception of the study; GS, YH, MX, and ZY: performed the experiments and analyzed the data; GS, YH, and MX: drafted the manuscript; QL and JS: assisted with the experiments; PZ and XY: revised the manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work.The present study was financially supported by National Natural Science Foundation of China grants , Fundamental Research Funds for the Central Universities, South-Central University for Nationalities (CZY20004), Wuhan Applied Basic Research Program of Science and Technology (2017060201010217) and Fund for Key Laboratory Construction of Hubei Province (2018BFC360).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the last three decades, timely myocardial reperfusion using either thrombolytic therapy or primary percutaneous intervention (pPCI) has allowed amazing improvements in outcomes with a more than halving in 1-year ST-elevation myocardial infarction (STEMI) mortality. However, mortality and left ventricle (LV) remodeling remain substantial in these patients. As such, novel therapeutic interventions are required to reduce myocardial infarction size, preserve LV systolic function, and improve survival in reperfused-STEMI patients. Myocardial ischemia-reperfusion injury (MIRI) prevention represents the main goal to reach in order to reduce STEMI mortality. There is currently no effective therapy for MIRI prevention in STEMI patients. A significant reason for the weak and inconsistent results obtained in this field may be the presence of multiple, partially redundant, mechanisms of cell death during ischemia-reperfusion, whose relative importance may depend on the conditions. Therefore, it is always more recognized that it is important to consider a “multi-targeted cardioprotective therapy”, defined as an additive or synergistic cardioprotective agents or interventions directed to distinct targets with different timing of application . Given that some neprilysin (NEP) substrates  exert a cardioprotective effect against ischemia-reperfusion injury, it is conceivable that antagonism of proteolytic activity by this enzyme may be considered in a multi-targeted strategy for MIRI prevention. In this review, by starting from main pathophysiological mechanisms promoting MIRI, we discuss cardioprotective effects of NEP substrates and the potential benefit of NEP pharmacological inhibition in MIRI prevention. The improvement of strategies for the protection of myocardium following acute ischemic insult represents, nowadays, the real challenge of the clinical research in cardiology. In fact, despite the widespread use of the best available therapies , up to 30% of patients with ST-elevated myocardial infarction (STEMI) still develop left ventricular remodeling (LVR) ,2,3,4,5 The reduction of the infarction area and the preservation of myocardial metabolism represent the pathophysiological basis of cardioprotection. The extension of the resulting infarction depends on (1) the size of the ischemic area at risk, (2) the duration and intermittency of coronary occlusion, (3) the magnitude of residual collateral blood flow, and (4) the extent of coronary microvascular dysfunction. In humans, 30–50% of the area at risk is still viable, and therefore, salvageable by reperfusion, after 4–6 h from the onset of anginal symptoms, as estimated from magnetic resonance imaging and biomarker analysis of myocardial salvage . Even afWhereas reperfusion is mandatory to salvage ischemic myocardium from impending infarction, it also inflicts a metabolic injury that is not only reversible, such as stunning ,11,12, bTherefore, by starting from MIRI pathophysiology, this review focuses on cardioprotective pathways activated by NEP substrates and the clinical role of NEP inhibition in the prevention of MIRI.The etiology of MIRI is multifactorial, and several molecular mechanisms have been shown to be involved, including reactive oxygen species (ROS) production, defects in calcium handling, mitochondrial damage, and inflammation . Indeed,Oxidative stress and calcium overload can both induce the abrupt opening of the mitochondrial permeability transition pores (MPTPs) during MIRI ,18. The Activation of cellular autophagy and degradation of damaged mitochondria by lysosomes  is generally beneficial because they reduce MIRI . As an e2+ overload improves the recovery of heart function and prevents the heart from contracture during reperfusion [+/Ca2+ exchange and Na+/H+ exchange, is one of the reasons for cell edema leading to myocardial stunning [The common clinical characteristics of MIRI are reperfusion arrhythmias , myocarderfusion . Consiststunning .Inflammation exerts a key role in cardiovascular diseases, as extensively reported in the literature ,38. InflIn addition, there is a growing body of evidence raising the hypothesis that the genetically-driven interaction between inflammation and thrombosis plays a pivotal role not only in the etiology of myocardial infarction (MI) ,45, but The coronary circulation with atherosclerotic plaque rupture and superimposed thrombosis is not only the culprit of myocardial ischemia but, after the restoration of coronary blood flow, it is also the target of IRI ,46. IRI Two main cardioprotective molecular pathways have been described ,20,71. TSurvivor Activating Factor Enhancement (SAFE) has been indicated as a further cardioprotective signaling pathway. Key components of SAFE have been suggested to be cytokines of the immune system and transcription factors . The cruInterestingly, drugs activating both pathways (such as the sphingosine-1-phosphate receptor agonist fingolimod) ameliorate MIRI and reduce MI size in a porcine model .NEP, an endothelial cell surface zinc metallopeptidase, is widely expressed in endothelial cells, smooth muscle cells, cardiac myocytes, renal epithelial cells, and fibroblasts. It is found in the lung, gut, adrenal glands, brain, and heart. NEP catalyzes the degradation of cardioprotective peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), Ang II, Bk, SP, and ADM at different degrees . The catFew data explored the effects of NEP inhibition using endogenous substrates at various concentrations, thereby limiting our knowledge about NEP activity in specific stress conditions. However, it could be hypothesized that NEP enzymatic affinity may change based on specific substrate concentrations. Accordingly, it has been demonstrated that elevated plasma BNP is an endogenous NEP inhibitor, and it works as an important self-regulation mechanism of NPs concentration . FurtherAn early event in STEMI is the acute and marked increase of ventricular expression and release of ANP and BNP . The timNeprilysin catalyzes the degradation of several substrates, which are promoters of peripheral vasodilation  or vasoconstrictors  . Here, wThe NPs include ANP, BNP, and CNP. ANP is derived from pre-proANP, a 151 amino acid precursor, and is primarily expressed and stored in the atria, although lower concentrations can be found in the ventricles and in the kidneys. Atrial wall stress stimulates ANP secretion. Human BNP is similarly synthesized as a pre-hormone, pre-proBNP, a 134 amino acid peptide, and cleaved into proBNP, which is 108 amino acids in length. ProBNP is subsequently cleaved into the biologically active 32 amino acid BNP, plus the 76 amino acid NT-proBNP. Although low levels of BNP are found in the atria, a greater concentration of BNP is stored in the ventricles, and it is released in response to cardiac stress, such as volume overload. CNP is the most expressed NP in the brain, but it is also produced in chondrocytes and endothelial cells. After a vascular insult or endothelial injury, CNP neo-intimal expression increases .NPs possess several physiological effects within the body ,95,96,97In vitro studies showed that synthetic ANP, via cGMP-forming trans-membrane guanylyl cyclase (GC)-receptor A (GC-A or NPR-A), reduces TNF-α-induced endothelial hyperpermeability  and limi+-ATP-channel opening [2+-induced hypercontracture caused by an increase in sarcoplasmic reticulum (SR) ATPase (SERCA) activity [It has been reported that cardioprotective action of NPs is mediated through mitochondrial, but not sarcolemmal, K opening ,105. Uro opening . ANP inf opening . Consist opening . It has activity .A reduced release of myocardial creatinine kinase isoform MB by ~15%, an increase in left ventricular ejection fraction by ~5%, and a decrease in reperfusion injuries  have been reported in a clinical placebo-controlled trial on ANP infusion during MI . FinallyCardiac RAS (cRAS) plays an important role in MIRI. Ang I converts into vasoconstrictor Ang II peptide, formed of eight amino acids, via the angiotensin-converting enzyme (ACE), whereas Ang I and Ang II are converted into vasodilator peptide Ang 1–7 by ACE2 and NEP .Ang II has been shown to reduce IS by affecting mitochondrial respiration and cardiac functions ,114. In Ang 1–7 regulates cardiac functions, blood pressure, cardiac hypertrophy, HF, and the growth of cells. In particular, Ang 1–7 has anti-proliferative action on the vascular smooth muscles, cardiac muscle cells, and improves endothelial functions by releasing Bk and nitric oxide (NO) ,117. TheThe kallikrein-kinin system has well-established cardioprotective actions. Although both plasma and tissue kallikrein (KLK1) produce kinin peptides, KLK1 plays a major role in cardioprotection. Studies of KLK1 gene knockout mice reveal an essential role for KLK1 in the maintenance of Bk levels in the heart . WhereasBk is a peptide made up of nine amino acids that increase vascular permeability and acts as a potent vasodilator through the stimulation of specific endothelial BKR2. BKR2 couples to Gi proteins and activates the downstream endothelial nitric oxide synthase (eNOS)/protein kinase G (PKG) and RISK pathways, including the two parallel cascades, PI3K-Akt, and MEK1-ERK1/2, against MIRI . FurtherHowever, Bk does not only protect the heart against MIRI, but also prevents apoptotic death induced by prolonged hypoxia in arterial endothelial cells. In particular, PC induces Bk synthesis by increasing KLK1 activity in endothelial cells . Releasem (Michaelis constant) of each peptidase [cat (turnover number), an enzyme with a low Km, such as ACE, may have a predominant role in Bk metabolism when Bk levels are low, whereas an enzyme with a higher Km  may play a more dominant role when Bk levels are high [Bk has a very short half-life because many different enzymes cleave it and may participate in its metabolism, and peptidase activity  plays a major role in the tissue-specific regulation of Bk levels . Therefoeptidase . Thus, dare high , such asare high ,132.Furthermore, the effect of an enzyme inhibition on Bk levels depends not only on the specific enzyme’s contribution to Bk metabolism relative to other enzymes, but also on the baseline degradation rate for Bk in each district . It has The pre-pro-apelin consists of 77 amino acid residues and is cleaved into shorter biologically active C-terminal fragments, including apelin-12, -13, -17, and -36 . A largeMoreover, the apelin/APJ system inhibits the generation of mitochondrial ROS, which leads to the abrupt opening of MPTPs and cell death in response to MIRI . Apelin-Apelin/APJ system increases NO formation and improves MIRI-induced cardiac dysfunction . The apeThe apelin/APJ system plays an important role in angiogenesis . Apelin-Apelin/APJ system may also attenuate endoplasmic reticulum (ER) stress response-induced cell death by promoting NO formation in MIRI ,155. The+/H+ exchange can reverse the effects of apelin-12 on LDH leakage and promote membrane damage in MIRI [+/H+ exchange.Apelin-12 and -13, as well as structural apelin-12 analogues, decrease cell membrane damage through better control of cell membrane integrity and ion homeostasis. Indeed, exogenous apelin-12 significantly decreases LDH leakage and reduces myocardial cell membrane damage by phospholipase-C (PLC), protein kinase C (PKC), PI3K, and MEK1/2 signaling . Inhibit in MIRI . Therefo2+-modulators and maintains Ca2+ transient in MIRI [2+ transient can inhibit Ca2+ transient overload and induce cardioprotection in MIRI. Furthermore, apelin-13 and apelin-36 inhibit reperfusion-induced hypercontracture of heart by delaying the abrupt opening of MPTPs [Finally, the apelin/APJ system significantly affects calcium handling. In isolated rat hearts, apelin-13 improves the redox states of SR Ca in MIRI . Maintaiof MPTPs .Despite their fundamental potency, apelin peptides are prone to fast proteolytic degradation ,162. So SP is an undecapeptide member of the tachykinin neuropeptide family, that acts as a neurotransmitter and as a neuromodulator . SP is rSP has two distinct effects in response to myocardial insult or overload. Short-term, SP vasodilatory effects appear to be protective by increasing myocardial perfusion, as demonstrated by ischemia-reperfusion studies. Indeed, SP induces bone marrow stem cell mobilization that has been experimentally and clinically investigated to regenerate damaged hearts and suppresses inflammation in ischemic injuries ,170,171.On the other hand, long-term up-regulation of SP appears to be associated with inflammation, apoptosis, matrix metalloproteinase activation, and alterations of the extracellular matrix, which may collectively lead to adverse remodeling and worsening HF .Human ADM is a 52 amino acid peptide, that is produced from its precursor molecule pre-pro-ADM. Since the discovery that the ADM gene is expressed to a greater extent in endothelial cells than in the adrenal medulla, it has been recognized that this peptide should be considered as a secretory product of the vascular endothelium, together with NO and endothelin .The beneficial effects of ADM on MIRI are well-known . It has Consistently, intermedin (IMD or ADM-2), an ADM similar peptide, plays an important role in MIRI prevention. Administration of IMD attenuates acute IRI in the isolated perfused rodent heart ,181 and IMD has been detected in patients without evident cardiac disease post-mortem in left ventricular cardiomyocytes, pericardial adipocytes, pericardial vein endothelial cells, and coronary artery vascular smooth muscle . IMD is ADM levels in the plasma and tissues are regulated by the proteolysis of NEP, the enzyme mainly involved in ADM degradation . TherefoDuring the last three decades, timely myocardial reperfusion using either thrombolytic therapy or pPCI has allowed amazing improvements in outcomes with a more than halving in 1-year STEMI mortality ,190,191.Actually, MIRI prevention represents the main goal to reach in order to reduce STEMI mortality ,193. NevAn important reason for the weak and inconsistent results obtained in this field may be the presence of multiple, partially redundant, mechanisms of cell death during ischemia-reperfusion, whose relative importance may depend on the conditions. Therefore, it is always more recognized that it is important to consider a “multi-targeted cardioprotective therapy”, defined as an additive or synergistic cardioprotective agents or interventions directed to distinct targets with different timing of application  .Given that NEP substrates exert a cardioprotective effect against IRI, it is conceivable that antagonism of proteolytic activity by this enzyme may be considered in a multi-targeted strategy for MIRI prevention. Sacubitril/valsartan  is a first-in-class approved angiotensin receptor NEP inhibitor (ARNI), that simultaneously provides NEP inhibition and blockade of the Ang II type I receptors (AT1Rs). This concomitant blocking is required because the single NEP inhibition increases Bk levels leading to clinically relevant episodes of angioedema . SAC/VALFirstly, concomitant NEP and AT1Rs antagonism increase levels of molecular compounds leading to activation of different pro-survival pathways, such as PI3K-Akt, PKC, PKG, AMPK, and ERK . In partAs proof of its molecular effects, NEP/AT1Rs inhibition gave good results in different animal experimental models of MI ,212,213.p < 0.001 for SAC/VAL versus enalapril at 1 week through 8 weeks) [The timing of SAC/VAL therapy is also important. Following oral administration, SAC/VAL dissociates into individual components with plasma concentrations of SAC, its active metabolite sacubitrilat (LBQ657), and VAL achieving peaks in 0.5 h, 2 h, and 1.5 h, respectively. In clinical study PIONEER-HF, the time-averaged reduction of the N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentration was significantly greater in the SAC/VAL group than in the enalapril group. Interestingly, the greater reduction in the NT-proBNP concentration with SAC/VAL than with enalapril was evident as early as week 1  . Further8 weeks) . Thus, SGiven these findings, in a “multi-targeted” strategy aimed at MIRI prevention, we hypothesize that optimal timing for SAC/VAL may be immediately at the end of pPCI, after RIC protocol administration and instead of Post-C. In fact, RIC has been shown to reduce the incidence of major adverse cardiac events, in particular of HF, whereas the role of Post-C has been not well defined . In thisInterestingly, SAC/VAL is not in contrast with other drugs commonly used in STEMI treatment. Dedicated drug interaction studies demonstrated that co-administration of furosemide, carvedilol, amlodipine, omeprazole, hydrochlorothiazide, metformin, and atorvastatin, did not alter the systemic exposure to SAC, sacubitrilat, or VAL . NeverthIt is hoped that most doubts about SAC/VAL utilization in the setting of acute MI may be clarified by the results of the ongoing PARADISE MI study (NCT02924727). Nevertheless, it is noteworthy to mention that the benefits of SAC/VAL on post-MI LV remodeling may be mitigated on the background of optimal medical therapy . Consistently, sodium-glucose transport protein 2 (SGLT2) inhibitors have been shown to ameliorate post-MI LV remodeling in a porcine model of MI . Thus, iIn STEMI, coronary reperfusion itself induces myocardial injury that can be prevented by endogenous compounds targeting different pro-survival pathways. These molecules are the substrate of various peptidases that regulate their plasmatic levels. NEP plays a pivotal role in enzymatic degradation of most of them , especially in the setting of acute MI. Thus, it is conceivable that NEP inhibition could represent an interesting target to prevent MIRI. SAC/VAL is a well-known ARNI yet successfully used in the treatment of chronic and acute decompensated HF. Because of its concomitant selective inhibitory action on NEP and RAS, SAC/VAL has a wide protective effect on myocardium in experimental models of MI. This is currently under investigation for cardiovascular protection against MIRI in humans (PARADISE MI study).


Insights from these published papers are as follows: (1) Chondrocyte preparations express a number of different ion channels that can regulate their ER. (2) Understanding the basis for ER requires knowledge of (a) the presence or absence of ligand (ATP/histamine) stimulation and (b) the extraordinary ionic composition and ionic strength of synovial fluid. (3) In our chondrocyte preparations, at least two types of Ca2+-activated K+ channels are expressed and can significantly hyperpolarize ER. (4) Accounting for changes in ER can provide insights into the functional roles of the ligand-dependent Ca2+ influx through store-operated Ca2+ channels. Some of the findings are illustrated in this review. Our summary diagram suggests that, in chondrocytes, the K+ and Ca2+ channels are linked in a positive feedback loop that can augment Ca2+ influx and therefore regulate lubricant and cytokine secretion and gene transcription.An improved understanding of fundamental physiological principles and progressive pathophysiological processes in human articular joints  requires detailed investigations of two principal cell types: synovial fibroblasts and chondrocytes. Our studies, done in the past 8–10 years, have used electrophysiological, Ca Advancing present understanding of mechanisms that underlie physiological responses in musculoskeletal systems requires detailed measurements and analyses made in both the active or excitable cell types  and the closely associated non-excitable cells  that make up these tissues and organs . In arti2+ levels ([Ca2+]i) is required for normal matrix metabolic and secretory functions, as well as cartilage differentiation [2+]i homeostasis is very likely to be an important factor in the initiation and rate of progression of chronic articular joint diseases such as arthritis i monitoring and spatial imaging, as well as immunocytochemical and molecular biology methods to identify and define some of the dynamic regulatory mechanisms for [Ca2+]i in chondrocytes. In this summary, our main goal was to define functional relationships between K+ channel regulation of the resting membrane potential (ER); and [Ca2+]i levels, both at baseline and following the application of physiological ligands. In addition, selected (previously published) data sets, based on total internal reflection fluorescence (TIRF) microscopy, provide a basis for relating changes in cellular or macroscopic properties to ligand-induced intracellular trafficking of defined components of ion channel complexes such as those for Ca2+-activated K+ (KCa) channels i [2+ or translocation of intracellular protein substituents using TIRF microscopy can be found in [+ channels in the generation of the chondrocyte ER [The methods and the data sets that we have used to generate 2+]i ,18 have found in . We haveocyte ER ,21.R therefore is of strong interest. Our groups o, in fact, produce only very small changes in transmembrane current in the region of membrane potentials close to the ER. We have explored this in detail i signaling in lymphocytes and T-cells i homeostatic response involves voltage-independent Ca2+ channel-mediated communication between the Ca2+ influx across the surface membrane of the cell and signaling that is dependent upon the Ca2+ content in intracellular organelles (mainly the ER) i in sub-compartments of the ER. Very shortly after its activation, the functional phenotype of the parent cell  changes significantly: i.Ca channels that are expressed.There is a marked hyperpolarization of plasma membrane due to activation of one or more of the subtypes of Kii.2+]i, by sometimes generating [Ca2+]i waves or oscillations i,  a,54 a2+]i matrix  contribute significantly to the regulation of [Ca2+]i levels both in the resting and histamine-activated state. In addition, in the OUMS-27 chondrocyte-like cell line i homeostasis and definition of cell phenotype can be described as a feedback loop involving: i.2+ from one or more intracellular stores .Ligand (ATP or histamine) triggered release of Caii.Targeted translocation of a specific intracellular protein (STIM1) from the ER to discrete spatial locations near the surface membrane (the ER-plasma membrane junction).iii.2+ to enter the chondrocyte over quite extended (s) time periods. This is referred to as SOCE, and takes place through CRAC channels.STIM1-induced conformational changes in a second partner protein (Orai1) and resulting formation of ion channels that readily allow Caiv.R triggered by increases in [Ca2+]i and resulting augmentation of Ca2+ influx then initiates or promotes functionally important enzymatic cascades or intracellular regulatory pathways (Ca2+-dependent phosphorylation/dephosphorylation).Alterations (hyperpolarization) in chondrocyte EOne of the main insights from this illustrated review is that, in the adult human chondrocyte , a significant regulatory paradigm for ) clearly demonstrated functional expression of CRAC channels in chondrocytes. A key component in this scheme is the ligand-triggered, dynamic formation of CRAC channels and subsequent maintained influx of Ca2+]i and gene expression, have consistently revealed expression and function of a small number of TRP channel transcripts in chondrocytes i oscillations or intracellular waves [2+]i-driven signaling can markedly enhance gene expression i oscillations can also coordinate chondrocyte migration with the level of repetitive mechanical activity of the articular joint i can activate KCa channels in chondrocytes the ATP flux through connexin and pannexin-based hemi channels produces a significant hyperpolarization of the ER. This results in a marked change in chondrocyte functionality  and phenotype  in both healthy and diseased conditions. Related multidisciplinary research that has also significantly advanced present understanding of chondrocyte cell physiology, mechanobiology, and pathology is based upon the discovery of expression and functional activity of additional connexin-like proteins. This is the pannexin family, , specific members of which have been identified in chondrocyte surface membranes . Recent + channel family members in chondrocytes, there is no doubt that Cl− expression can strongly regulate and even modify the ER in both developing and mature chondrocytes. We have provided detailed accounts of an important role for Cl− conductance in regulating ER in the chondrocyte-like cell line OUMS-27 i and the resulting significant, relatively long-lasting hyperpolarization of the ER. Published findings mainly concerning the electrophysiological regulation of the innate immune response ,43,44, hR is substantial, , and is often also relatively long lasting (1–10 s). This change in ER can either activate or significantly increase Ca2+ influx through both conventional Ca2+ channels and CRAC channels. The hyperpolarization also increases the electrochemical driving force for ion fluxes through other channels that are permeable to Ca2+ and Na+, as well as Cl− channels that are expressed in human chondrocytes [2+]i levels and membrane potential i.To evaluate and then implement a novel approach for regulating chondrocyte membrane potential by incorporating optogenetic tools , such aschannels  into choiii.R in populations of isolated cells. It may be possible to monitor and calibrate a signal obtained during flow cytometry assays [To improve throughput of data acquisition using methods that allow ligand-induced changes in chondrocyte Ey assays  to proviy assays . iv.2+-dependent steps in the diagram shown in 2+]i levels at baseline together with parameters describing ligand-induced transients and/or oscillations are much needed. These can be obtained by using recently published analytical software [2+]i transient waveforms has yielded interesting insights into some aspects of [Ca2+]i homeostasis i release and buffering as well as Ca2+ extrusion. The Hille group i levels and (ii) transmembrane voltage [Ca channels can also be strongly modulated by ‘endogenous ligands’ such as reactive oxygen species (ROS) or estrogen [2O2, ONOO−, NO, CO and H2S is generated. Each ROS probably influences wide range of physiological responses of chondrocytes and gets involved in OA pathogenesis. However, the effects of ROS appear to be very complicated: they can show both cytotoxic and protective effects on chondrocytes. It has been reported that ROS modulates BKCa channel activity by direct or indirect action on BKα or BKβ1 subunits [Ca channel may partially play roles in ROS-induced effects on chondrocytes during OA progression, but further studies are needed to clarify this point. In addition, the function of BKCa channels in chondrocytes is almost certainly strongly regulated by the ambient hypoxia of the articular joint. A very important downstream signaling pathway mediated by the hypoxia inducible factor (HIF)-1 family of transcription factors has been shown to significantly alter BKCa function [Ca channel function in this progressive disease may be altered. Interestingly it has recently been shown that BKCa channel activity can be regulated by circadian activity of the host cell [It is now well known that, even at baseline, BK voltage . Rather,estrogen . It is westrogen ,98. Whensubunits . Therefofunction ,100. Hypfunction  and hencost cell . Ca channel has long been known to consist of a signaling complex consisting of the pore or α subunit and an accessory β subunit. Recently, a γ subunit of the BKCa channel has been cloned and shown to have very strong, indeed profound effects on the voltage-dependence of BKCa channel and macroscopic current activation [The BKtivation ,104. Det2+ channel needs to be considered in the setting of known transcriptional activators [2+-sensitive K+ channels are likely to act in a stand-alone fashion, it has been shown that when they are co-expressed the level of activity of the intermediate conductance channel can strongly modulate that of the BKCa channel [Somewhat similarly, the functional expression of the intermediate conductance Cativators  and bothtivators . Finally channel . Within mature mammalian articular joints, the chondrocyte exists and functions as a single, relatively isolated cell. At present most research groups obtain these preparations (single chondrocytes) using (i) immortalized chondrocyte cell lines such as TC28a2, OUMS-27, SW-1353, (ii) chondrogenic progenitor cell line (ATDC5) and (iii) primary cultured chondrocytes that enzymatically dispersed from cartilage tissues. Such immortalized cell lines are easy to handle and maintain chondrocytic phenotype, e.g., Col2a1 and So×9 expression, but at the same time they lose some chondrocytic features such as NO production, expression of genes involved in matrix synthesis and turnover ,109,110.However, there is a limitation on using primary chondrocytes cultured in 2-D conditions. The environment of chondrocytes in 2-D culture is quite different from that within cartilage tissues. This difference is a reason why chondrocytes dedifferentiated after enzymic dissociation. It is important to recall and acknowledge that although this experimental paradigm yields useful results, for translational applications this approach may be limiting. 2+ imaging methods where chondrocytes in cartilage tissue were loaded with Ca2+ indicators and dynamic Ca2+ signaling were observed using a confocal microscope. In the articular joint, the functional unit is the chondrocyte together with its immediate pericellular coating. This combination has been denoted ‘the chondron’ . Both clAlthough our focus in this review, and in our experimental programs, is on ion channels in single isolated chondrocytes; we recognize the need to study the physiology of the chondrocyte in a scalable fashion by also taking account of research that relates the composition and dynamics of the extracellular matrix to both the chondron and the chondrocyte. This integrative approach  is neces


Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5′ splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5′ splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca2+ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca2+-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.Ca Tb) by metabolic means  during vertebrate evolution and by fossil records that were indicative of an increased locomotion and easier ventilation, such as maxilloturbinates  and free cytosolic (Ca2+c) calcium (2+m levels are elevated by plasma membrane depolarization of muscle fibres (2+ channels (IP3R and RYR1) in the sarcoplasmic reticulum  in the mitochondrial matrix, either indirectly by dephosphorylation (pyruvate DH) or directly by Ca2+-binding (isocitrate DH and 2-oxoglutarate DH)  . These e2+-dependent matrix DHs, OGDHC is most sensitive to low ATP/ADP ratios  to succinyl-CoA by OGDHC, generating NADH and electrons for the respiratory chain and increasing the cycle flux and the rate of ATP synthesis ,17,21–23P ratios . OGDHC bd humans  but is ad humans . OGDHC id humans , in lines of 2OG ,22,27,28oxoglutarate dehydrogenase; OGDH; EC 1.2.4.2). E1  has the lowest catalytic activity among OGDHC components and is rate-limiting in multiple organisms  4a and 4b  that was shown to be essential for Ca2+ binding and OGDHC activation  in the intervening intron. Human dBP adenines are located only 25–41 nucleotides (nt) downstream of the 5′ splice site (5′ss) of exon 4a, below a proposed steric threshold for simultaneous assembly of spliceosomal complexes recruited to 5′ss and BPs  downstream of mammalian dBPs. We propose that this enrichment contributed to the observed sensitivity of the MXE pair to RNA-binding proteins (RBPs) with uridine preferences that ensure reciprocal regulation of each exon. The dBP-driven and Ca2+-responsive isoform 4b+ led us to investigate the capacity of exonic auxiliary splicing elements to include/exclude binding sites for Ca2+ and other common metals in/from coding sequences. We further show that evolution of exonic splicing enhancers and silencers has been shaped by codons that are critical for formation of protein binding sites for metals at the opposite ends of the Irving-Williams affinity order: codons for amino acids required for binding weak metals, such as Ca2+, generally increase exon inclusion in mature transcripts whereas codons for amino acids required for strong binders, such as copper, promote exon exclusion. Finally, we hypothesize that the Ogdh alternative splicing facilitated evolution of endothermy by maximizing the aerobic scope of striated muscles while protecting other tissues from NADH oversupply.In this study, we first show that splicing of  and BPs . The sep and BPs ,38, inseOGDH reporter  by transformation. Plasmid DNA was extracted with the GeneJET Plasmid Miniprep kit (ThermoFisher) and correctly sized inserts were confirmed by gel electrophoresis following digestions with restriction enzymes. Sanger sequencing of 30 plasmids confirmed 9 (PstI) and 16 (EcoRV) constructs with unique MIR inserts  was amplified by PCR using degenerate primers  that tarP motifs . The PCR inserts . Plasmidmyc-tagged mammalian expression constructs, pcDNA3.1-GFP as transfection/loading control and jetPRIME (Polyplus) as a transfection reagent. Cells were depleted using small interfering RNAs (siRNAs) shown in 20 according to the manufacturers’ recommendations. RT-PCR reactions were performed using minigene- (35E1+PL4 or 35m-amplF) and vector-specific (PL4) primer combinations , washed with a buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10% glycerol (w/v), 3.6 mM β-mercaptoethanol and 20 mM imidazole and eluted using the same buffer upon addition of imidazole to 500 mM. Recombinant WT PUF60 was prepared using a construct cloned by inserting BamHI/XhoI amplicons into pET-28 with a 2xHis-lipoyl-TEV site, as described  pLysS Competent Cells (Promega). The cells were cultivated to OD 0.6 at 37°C and protein expression was induced with 1 mM IPTG at 37°C for 2 h. Bacterial pellets were dissolved in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10% glycerol (w/v) and 3.6 mM β-mercaptoethanol containing cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche) and were sonicated using SONOPLUS GM Mini20 (Bandelin Electronic). The recombinant protein was purified with Niescribed . For lon32P] ATP and T4 polynucleotide kinase, as described  at room temperature for 20 min. RNA-protein complexes were separated on native 6% polyacrylamide gels run in 0.5× TBE at 4°C. Their signal was measured using a Typhoon 9210 PhosphorImager and ImageQuant 8.2 . The data were fitted to the Hill equation (B = Bmax*[Ln/(nL + Kdn)], where Bmax is a bound fraction of RNA (B) at the saturating protein concentration L and n is the Hill coefficient) to determine dissociation constants (Kd).For gel shift assays, purified WT proteins were incubated with the indicated oligoribonucleotides (Eurofins) that were end-labeled with  < Cu2+/Zn2+), known as the Irving-Williams series (2+ and Ca2+) preferentially interact with residues encoded by splicing-enhancing codons whereas codons for residues interacting with tight binders (Cu2+ and Zn2+) act as splicing repressors, with moderate binders (Mn2+ and Fe2+) exhibiting intermediate values of ESE/ESS ratios and splicing activities  was flipped . Ultimately, the overall ESE/ESS gradient across the Irving-Williams series mirrored concentrations of these metal ions in the primordial (sulphid) ocean  favour exon inclusion whereas mutations generating codons for residues that interact with highly competitive binders (Cu/Zn) generally promote exon skipping Figure -C. Loss a/Mg favo Figures , E, acti Figures , allele  Figures . After e Figures , thereby2+m. Remarkably, the OGDH exon duplication arose about 2,000 Myrs after the earliest living organisms on the Earth had selected the inseparable tandem of ATP and low Ca2+c to control cellular signalling, apparently as a compromise between ATP-based energetics and poor Ca-ATP solubility , but no upstream dBPs, were found among a ‘match-only’ set of 77 668 BPs, but no intron 4a dBPs were reported in the high-confidence set of 59 359 BPs  and Amphimedon queenslandica (sponge), probably the oldest surviving metazoan. This suggests that Ca2+ sensitivity may have been selected for without duplication and this feature may have been lost in multiple lineages. This scenario is supported by evidence for the independent origin of Ca-binding DxDxDG motifs  and by L in Daphia pulex (water flea), however, rapid paralogue divergence and incomplete annotations leave us with uncertainties about their exact identities.Although ing site , genomicing site ,177 reveG motifs , which aG motifs . The firsitivity . Several2+. These metals have high coordination numbers and affinity for ligands of low polarizability . R. R183). OGDH MXE usage facilitated early evolution of endothermy by favouring organisms capable of maximizing sustained NADH and ATP supply in response to Ca2+ signals generated by striated muscles. The hypothesis is supported by the (i) crucial position of E1 in Ca2+-responsive energy supply pathways (Introduction and Discussion S1); (ii) regulation of E1 by Ca2+m in the TCA cycle, ensuring activity-dependent NADH and ATP provision ; (vi) OGDHC role in hypometabolic states such as torpor and hibernation (Discussion S3); (vii) self-promoting activity of exon 4b coding for the DADLD motif, contributing to the ubiquitous expression of dBP-led isoform 4b+  ev Figures ; (v) cri+ Figure  and  acted as drivers of the central metabolic conversion in animal history should be evaluated in more detail in future studies.First shown for muscle contraction ,188, Ca2cellence , Ca2+c hd so far , includid so far , parentad so far ,191 and d so far  (DiscussdBP RNA-seq data are available from the Array Express (accession number E-MTAB-9412).gkab046_Supplemental_FilesClick here for additional data file.