Mouse GMCs cultured under normal glucose conditions proliferated and synthesized ECM proteins in response to stimulation by mouse UII. UII-induced GMC proliferation and ECM protein synthesis were dependent on TRPC4 channel-mediated store-operated Ca2+ entry (SOCE) and sequential activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Ca2+/cAMP response element-binding protein (CREB) transcription factor. Under high glucose conditions, GMCs synthesized UII. Moreover, proliferation and ECM production in high glucose-challenged GMCs were attenuated by selective UTR antagonist, TRPC4 channel blocker, and CaMKII and CREB-binding protein/p300 inhibitors. These findings indicate that UII-induced SOCE via TRPC4 channels stimulates CaMKII/CREB-dependent GMC proliferation and ECM protein production. Our data also suggest that UII synthesis contributes to GMC proliferation and ECM accumulation under high glucose conditions.Glomerular mesangial cell (GMC) proliferation and matrix expansion are pathological hallmarks of a wide range of kidney diseases, including diabetic nephropathy. Although the circulating level of peptide hormone urotensin II (UII) and kidney tissue expression of UII and UII receptors (UTR) are increased in diabetic nephropathy, it remains unclear whether UII regulates GMC growth and extracellular matrix (ECM) accumulation. In this study, we tested the hypothesis that UII-induced Ca Alterations in UII and UII receptor (UTR) tissue expression and circulating and urinary levels of UII have been reported in human and experimental animals with cardiovascular and renal diseases, including hypertension, renal failure, congestive heart failure, atherosclerosis, renal fibrosis, glomerulonephritis, and diabetes8. Plasma and urinary concentrations of UII are elevated in proteinuric and non-proteinuric diabetic patients with progressive loss of renal functions7. Increased expressions of UII and UTR in kidney specimens from human and animals with diabetic nephropathy have also been reported8. However, the association between UII signaling and cellular events that underpin diabetic nephropathy is poorly understood.Experimental data from a variety of animal models suggest that peptide hormone urotensin II (UII) regulates renal functions, including vascular bed perfusion, glomerular filtration, and electrolyte homeostasis10. Increased mesangial expansion leads to encroachment of the Bowman’s space, obstruction of the glomerular capillaries, and progressive impairment of glomerular hemodynamics10. Exposure of cultured glomerular mesangial cell (GMCs) to high glucose concentrations induces proliferation, ECM protein synthesis, and hypertrophy, thereby mimicking the effect of hyperglycemia in diabetic nephropathy11. Mechanisms that underlie GMC responses to high glucose conditions are not fully resolved, but may include modulation of intracellular Ca2+ ([Ca2+]i), a major regulator of signaling pathways associated with cell cycle control12.The pathological hallmarks of diabetic nephropathy include glomerular ultrastructural changes, such as basement membrane thickening, extracellular matrix (ECM) accumulation, and mesangial expansion2+]i concentration can be triggered by an influx of extracellular Ca2+ into the cells via plasma membrane-localized Ca2+-permeable channels or Ca2+ release from the intracellular stores or both. Changes in [Ca2+]i is turned into biological responses by regulatory proteins that propagate Ca2+-sensitive signal transduction mechanisms such as protein phosphorylation and de-phosphorylation to the nucleus to influence gene transcription13. Like in many other cell types, Ca2+-sensitive transcription factors, such as the nuclear factor kappa-light-chain-enhancer of activated B cells, nuclear factor of activated T-cells, and Ca2+/cAMP response element-binding protein (CREB) control GMC survival16. High glucose stimulates CREB phosphorylation in GMCs17. Inhibition of [Ca2+]i elevation by Ca2+ channel blockers, inhibited proliferation, ECM protein synthesis, and CREB activity in GMCs14. Hence, CREB target genes are downstream effectors of Ca2+-dependent cellular events that promote GMC proliferation and ECM protein accumulation.An increase in i elevation, we first examined whether ML204, a selective TRPC4 channel blocker26 alters UII-induced SOCE in the cells. The effect of Pyr3, a selective TRPC3 channel blocker27 on UII-induced SOCE was also examined. In the absence of extracellular Ca2+, UII-induced [Ca2+]i elevation was unaffected by ML204 and Pyr3  was attenuated by ML204, but not, Pyr3  receptor antagonist araguspongin B, and [Ca2+]i chelator BAPTA, all attenuated UII-induced GMC proliferation  immunostaining was detected in cells treated with UII, an effect abolished by KN-93, a CaMKII inhibitor  is a substrate for CaMKII-induced phosphorylation30, we investigated the hypothesis that UII-induced [Ca2+]i elevation and CaMKII activation stimulate nuclear pCREB activity in GMCs. Immunofluorescence staining showed that endogenous CREB was unchanged in control and cells treated with UII  glucose DMEM to mimic hyperglycemic conditions typical of diabetic mice36. As an osmotic control, normal (5.5 mM) glucose-containing DMEM was supplemented with 19.5 mM mannitol. As shown in Fig. Since UII levels and UTR expression are increased in diabetic nephropathy2+]i elevation and CaMKII/CREB activation are involved in high glucose-induced GMC proliferation, we studied GMC growth in cells pretreated with a TRPC4 channel blocker and inhibitors of CaMKII and CREB coactivators. High glucose-induced GMC proliferation was attenuated by ML204, KN-93, SGC-CBP30, and BAPTA  protein complex38. Similarly, STIM1, Orai1, and TRPC1 contribute to UII-induced SOCE in aortic smooth muscle cells24. In human GMCs, SOCE is dependent on protein complexes, including TRPC1, 4, and STIM122. Whether UII-induced TRPC4 activation in mouse GMCs involve its association with TRPC1, STIM1, or Orai1 is not addressed in this study and requires further investigation.A previous study reported that TRPC4 channel knockdown attenuated SOCE in mouse GMCs by ~83%, indicating that TRPC4 forms store-operated Ca2+]i elevation stimulated by UII in mouse GMCs. Activation of CaMKII and CREB phosphorylation is required for UII-induced vascular smooth muscle cell proliferation24. Since UII stimulates CaMKII and CREB phosphorylation via TRPC4-mediated SOCE in mouse GMCs, we investigated the concept that UII-induced GMC proliferation involves CaMKII/CREB activation. We show that at the concentration that prevented UII-induced CaMKII phosphorylation, KN-93 attenuated UII-induced nuclear pCREB binding activity and proliferation in the cells. CREB, a ubiquitous transcription factor controls a wide variety of cellular processes, including GMC survival and ECM protein synthesis39. Phosphorylation of CREB on Ser133 stimulates its interaction with coactivators CBP or its homolog p300, leading to target gene activation32. CBP/p300 induction promotes cellular apoptosis, proliferation, and development40. In rat GMCs, angiotensin II-induced fibronectin upregulation involves activation of CREB and p30041. Here, we show that pharmacological inhibition of the CBP/p300 bromodomain34 suppressed GMC growth in UII-treated cells. These data suggest the involvement of CBP/p300-dependent gene transcription in UII-induced GMC proliferation. Corresponding to its effect on cell number, UII increased type IV collagen and fibronectin secretion by GMCs. Immunofluorescence staining of type IV collagen and fibronectin also indicated that UII stimulates their production by individual cells. These data are consistent with studies that reported an increase in fibronectin and collagen mRNA expression in rat proximal tubular epithelial cells and cardiac fibroblasts treated with UII43. Thus, a combination of increased proliferation and synthesis by individual GMCs may account for UII-induced ECM accumulation. Our data suggest that UII-induced ECM protein production is dependent on TRPC4 channels, CaMKII, and CBP/p300. Together, our findings highlight the contribution of TRPC4-mediated SOCE and CaMKII/CREB activation to increased growth and ECM production in UII-treated GMCs. CBP/p300 can interact with other transcription factors, such as members of the activator protein 1 (AP-1) family, including c-Fos and c-Jun40. Decoy oligodeoxynucleotides targeting AP-1 suppressed high glucose- and angiotensin II-induced rat GMC proliferation and matrix gene expression44. Other studies have demonstrated the importance of c-Fos and c-Jun in GMC proliferation46. Given the molecular and functional crosstalk between CREB and AP-1 signaling pathways47, UII-induced CREB activation and downstream GMC proliferation and ECM accumulation may involve AP-1 components.Our data show that CaMKII and CREB phosphorylation are sequential downstream targets of TRPC4-mediated i elevation inhibited proliferation and triggered apoptosis in GMCs49. The intracellular effectors of thapsigargin-induced SOCE in GMCs are unclear. Thus, it is likely that the effects of SOCE-mediated signal transductions on GMC survival and ECM protein production are dependent on the stimulant, the level of [Ca2+]i increase, or downstream Ca2+ sensitive signal transduction cascades that are activated. Acute exposure of rat GMCs to high glucose increased [Ca2+]i concentration by a mechanism that partly involves the store-operated Ca2+ channels50. Cyclopiazonic acid- and IP3- induced SOCE and store-operated Ca2+ currents, respectively have also been shown to be amplified in human GMCs under chronic high glucose treatment, and associated with increased protein expression of STIM1 and Orai151. These studies suggest that additional mechanisms of high-glucose-induced modulation of GMC function may involve direct alterations in basal [Ca2+]i levels or expression of the molecular components of the store-operated Ca2+ channels.Blockade of TRPC4 channels inhibited UII-induced SOCE, CREB activation, proliferation, and ECM production in GMCs. Similarly, TRPC4 channel inhibition attenuated high glucose-induced GMC growth and type IV collagen synthesis. Together, these data indicate that SOCE stimulated by UII contributes to high glucose-induced GMC proliferation and ECM protein accumulation. A recent study reported that activation of SOCE by thapsigargin abolished fibronectin and type IV collagen protein expression in human GMCs cultured in a high glucose medium, suggesting that SOCE elicited by thapsigargin negates ECM accumulation in GMCs2+ signaling promotes CaMKII/CREB-dependent proliferation and ECM protein production in GMCs. Our data also suggest that high glucose elicits UII synthesis, which in turn induces CaMKII/CREB-dependent proliferation and ECM protein production in the cells. Conceivably, increased UII production in GMCs and the resultant proliferation and ECM protein accumulation are involved in the pathophysiological mechanisms that underlie mesangial expansion in diabetic nephropathy.In conclusion, we present new findings demonstrating that UII-induced CaThe use of GMC lines was approved, and experiments were performed in accordance with the guidelines and regulations of the Institutional Biosafety Committee of the University of Tennessee Health Science Center. Mouse GMC line (CRL-1927) was purchased from the American Type Culture Collection . The cells were subcultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin.2+]i concentration was measured in GMCs using the ratiometric Ca2+ indicator Fura-2 AM, as we have previously done53. Briefly, mouse GMCs cultured in glass-bottom dishes were loaded with Fura-2 AM (10 μM) and 0.5% pluronic F-127 for ~1 h at room temperature. Cells were then washed with modified Krebs’ solution  for ~30 min to de-esterify Fura-2 AM molecules. Changes in intracellular Ca2+ ([Ca2+]i) was measured at room temperature with a ratiometric Ca2+ imaging system . Fura-2 AM fluorescence was recorded by using a hyperswitch light source (Ionoptix) to excite at wavelengths of 340 and 380 nm. The fluorescence was collected simultaneously from cells located in one imaging field per dish. Fura-2 AM ratios were background-subtracted and collected at 510 nm using a MyoCam-S CCD digital camera (Ionoptix). Data were then analyzed with the IonWizard software (Ionoptix).[Ca6) in 100 µl of antibiotic-free DMEM and a pool of 3 target-specific TRPC4 siRNAs (~1 µg) or a non-targeting scrambled control siRNA  were electroporated in cuvettes for 100 milliseconds at 150 V. Following recovery (~10 min at room temperature), cells were transferred into flasks or microplates and cultured for 72 hours. Western immunoblotting was used to confirm knockdown efficiency.siRNA transfection was performed using the BTX ECM 830 square wave electroporator . Cells . Denatured proteins (LDS sample buffer + DTT and heated at 70 °C for 10 min) were separated on polyacrylamide gels in a Mini Trans Blot Cell (Bio-Rad). Proteins were transferred onto nitrocellulose membranes using a Pierce Fast Semi-Dry Blotter (Thermo Scientific). Nonspecific binding sites on the membranes were blocked for ~1 h using Tris Buffered Saline supplemented with 0.05% Tween (TBS-T) and 5% nonfat milk. The membranes were probed with respective primary antibodies overnight at 4 °C. After a wash in TBS-T, the membranes were incubated in HRP-conjugated secondary antibodies for 45 min at room temperature. Immunoreactive proteins were visualized on a Kodak gel imaging system using the femtoLUCENT PLUS-HRP chemiluminescent reagent (G-Biosciences).53. GMCs plated in flat-bottom tissue culture microplates (~1 × 104/well) were starved overnight by culturing in DMEM that contained 0.1% FBS. The cells were then placed in a chamber apparatus of the IncuCyte ZOOM live content microscopy system  in an incubator. GMC growth was monitored in real-time and quantified from cell confluence changes automatically acquired at two-hourly intervals by the IncuCyte cell density detection software and interface. Phase contrast images (segmentation mask illustrated in goldenrod) demonstrating cell density were also acquired with the IncuCyte software.GMC growth was studied using live content microscopy as we have previously describedFor immunofluorescence staining, cells that were sparsely grown on glass coverslips were fixed in 4% formaldehyde for ~20 min and permeabilized with 0.2% Triton X-100 for ~15 min at room temperature. Non-specific binding sites in the cells were blocked for 1 h by incubation in MAXblock blocking medium . The cells were then treated overnight at 4 °C with respective primary antibodies. Next day, cells were washed with PBS and incubated with cross-adsorbed Alexa Fluor 555 goat anti-rabbit  for 1 hour at room temperature. Following a wash and mount, fluorescence images were acquired using a Zeiss LSM 710 laser-scanning confocal microscope.To determine nuclear immunoreactivity of pCREB, the total number of pCREB positive nuclei were counted in randomly-selected microscopic fields and expressed as the percentage of the total number of DAPI-stained nuclei in the same fields. The pCREB transcription factor assay kit  was used to detect pCREB activity in GMC nuclear extracts. GMC nuclear proteins were extracted using a nuclear extraction kit . pCREB DNA binding activity in nuclear extracts (5 μg protein/well) was determined according to the manufacturer’s instructions.6/microplate well) were starved overnight. Thereafter, the cells were treated with respective test substances and cultured for 24–72 hours. Type IV collagen, fibronectin, and UII concentrations in cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits purchased from Exocell Inc. , ScienCell Research Laboratories , and BioTrend Chemicals, LLC , respectively.Cultured GMCs  and Antibodies Inc. , respectively. Rabbit polyclonal anti-CaMKII, -pCaMKII, and -pCREB were purchased from Cell Signaling Technology . Rabbit polyclonal anti-collagen IV and -fibronectin were obtained from Bioss Inc. . HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Thermo Scientific  and Abcam, respectively. Cross-adsorbed Alexa Fluor 555 goat anti-rabbit was purchased from Life Technologies . Fura-2 AM, Pluronic F-127, mouse UII, urantide, β-mercaptoethanol, SGC-CBP30, and Pyr3 were purchased from Life Technologies, AnaSpec , Phoenix Pharmaceuticals , Peptides International, Inc. , Bio-Rad, Tocris Bioscience (Bristol UK), and Cayman Chemical , respectively.P value < 0.05 was considered significant.All data are expressed as mean ± standard error of mean (SEM). Data were analyzed using the InStat software . Statistical significance was determined by Student’s t-tests for paired or unpaired data and analysis of variance with Bonferroni post-hoc test for multiple comparisons. A


The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses.We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the  Native-like SOSIP trimers are a platform for development of immunogens aimed at inducing broadly neutralizing antibodies and, hence, a possible vaccine against HIV-1 infection. No previous study has reported on immune responses to more than one such trimer. Here, we assess how rabbits respond to immunization with two or three different trimers, based on virus sequences from HIV-1 clades A, B and C, to gain insights into whether each is immunogenic under various regimens. We find that autologous Tier-2 neutralizing antibody responses can be raised against each trimer immunogen, whether they are delivered simultaneously or sequentially. We also observed some boosting of the neutralization response to the first trimer when a second trimer was administered later. Cross-reactive neutralizing antibodies were seen but only sporadically. We also found that the key immunogenic epitopes on the Env trimers involved holes in the glycan shield, which normally protects the virus from antibody binding. These various findings will guide the design of future experiments in animals and eventually in humans. ECTO subunits and stabilization of the metastable trimer by engineered sequence changes )] or reciprocal inhibitory dilution [1/(ID50 [ppV])] are henceforth referred to simply as “titers” or “IC50 values”. We sometimes observed that neutralization curves plateaued at values of <100% (sometimes < 50%), particularly with the B41 Env-pseudotyped virus; a mechanistic analysis of the variable plateaus will be described elsewhere. We recorded IC50 values only when the maximum percentage neutralization was > 50%; other values were recorded as IC50 < 20. The titers against the MLV control virus were consistently < 30. We therefore deemed titers >40 to represent robust neutralization.In addition, sera from a subset of time points were tested at DUMC against two Tier-1A Env-pseudotyped viruses, MN.3 (clade B) and MW965.27 (clade C); and against a panel of nine Tier-2 viruses: Ce703010217_B6 (clade A), 246-F3_C10_2 (clade AC), CNE55 (CRF01_AE), TRO.11 (clade B), X1632-S2-B10 (clade B), BJ0X002000.03.2 (CRF07_BC), CH119.10 (CRF07_BC), 25710–2.43 (clade C) and Ce1176_A3 (clade C) , 43. An env genes containing point substitutions, most of which introduced potential sites of N-linked glycosylation, were made as previously described . Env-pseudotyped virus mutants based on these genes, together with the corresponding wild-type viruses, were used in TZM-bl cell neutralization assays, as described above. Viruses based on various clones of the MG505 virus are derived from the mother of the BG505 HIV-1-infected infant [Mutant BG505 and B41 full-length d infant , 46. Thed infant . The infThe mutant Env-pseudotyped viruses were tested in TZM-bl cell neutralization assays as described above. At a low dilution of serum , the extent of neutralization was expressed as a percentage of that obtained with the wild type virus (defined as 100%). In addition, sera were titrated to > 40,000-fold dilution, and the inhibition curves for wild-type and mutant viruses were compared.3 structure. Modeling and visualization were performed by the UCSF Chimera method (PMID 15264254).The trimer model was derived from the PDB 5ACO structure . The gly50) were derived from the titration curves and serve as measurements of the trimer-binding antibody response to the immunogens [Anti-trimer capture ELISAs with the BG505-D7324, B41-D7324 and DU422-D7324 trimers were performed as described previously . Brieflymunogens , 15.The peptide APTKAKRRVVQREKR corresponding to the epitope-tag on the B41-D7324 trimers was obtained from GenScript, as was a scrambled control peptide, KKQARRARPVREVKT). The peptides were used to coat Maxi-Sorp (Nunc) ELISA plate wells at 0.6 μg/ml. After standard washing and blocking procedures, rabbit sera were added at a dilution of 1/20 in PBS with 2% milk and bound antibodies were detected with a goat anti-rabbit IgG-HRP conjugate diluted 1/3000 (BioRad). As positive controls, sheep Ab D7324 was titrated from 10 to 0.013 μg/ml, and a pool of IgG purified from HIV-1 infected humans  was titrated from 1000 to 1.3 μg/ml. The detection antibodies were rabbit anti-sheep IgG-HRP (Thermo) and goat anti-human IgG-HRP (BioRad), respectively.Groups were compared by two-tailed Mann-Whitney U tests. Two time points for the same rabbits were compared by Wilcoxon matched-pairs test; the matched pairs results are presented only when the pairing was efficient, according to correlation analyses within the test. Otherwise the results of Mann-Whitney U test are given. To determine whether titers were significantly above the cut-off value of a titer of 20, we applied the Wilcoxon signed rank test. Correlations were analyzed non-parametrically as Spearman rank correlations and the concomitant significances were calculated. All statistical analyses were performed in Prism (GraphPad).Group sizes of 5 for rabbit immunization studies limit the robustness of conclusions from group comparisons. Here, and in earlier papers , we showThe 8 groups of rabbits and the immunization regimens are summarized in The initial phases (weeks 0–24) of immunization groups 1 (B41) and 3 (BG505), before heterologous boosting, serve as comparators for other groups . One BG5The rest period from the fourth immunization (week 24) to the fifth at week 36 (see below) allowed us to look at how both autologous NAb responses diminished over time. We were unable to determine the half-lives of the titers because of the paucity of time points, and are aware that a second phase of titer decay might commence during the 10-week period from the assay at week 26. However, we note that the autologous responses to the BG505 and B41 trimers in groups 1 and 3 dropped ~10-fold during this time. Responses in these and other groups to later boosting with clade C trimers are discussed further below and are shown in We analyzed the effect of immunogen dose, and whether one co-administered trimer could interfere with the autologous NAb response to the other. These analyses are described in the SI see . In summGroups 5 and 6 were also affected by the BG505 contamination (~20%) of the initial B41-D7324 trimer preparations . NonetheThe B41 NAb response for group 5 at week 22 was unexpectedly strong in the three responding rabbits, considering that these animals had received only two doses of B41-D7324 trimer (at weeks 0 and 4) and then a heterologous boost by BG505 trimers at week 20 . Thus, tThe BG505.T332N NAb response in group-6 rabbits was consistently boosted by each immunization given during the 60-week duration of the experiment. This was the case even when B41-D7324 trimers were given at week 36, which is therefore another example of cross- boosting . Thus, aad hoc extension to the rabbit experiment did not include a study of clade C trimers in naïve rabbits (this experiment is now in progress). Hence, there is no comparator arm to judge whether the response to the DU422 and CZA97 trimers was influenced by prior exposure to the clade A and/or clade B trimers.We explored heterologous boosting with clade C trimers by immunizing group 1 with DU422 trimers and groups 2, 3, 4 and 8 with CZA97 trimers at weeks 36, 48 and 60, and selected animals also at week 73 . In eachvs. 12 weeks) could all have influenced the observed outcome.After the second immunization at week 48, autologous NAb responses developed in 2 of 5 rabbits immunized with DU422 trimers and were boosted by the third and fourth immunizations at weeks 60 and 73 ; the other three animals were non-responders but #5713, #5714 and the responder #5715 had cross-reactive titers of 60 at week 36 . The CZAvs. 620) and likewise they were higher after the second clade C trimer boost at week 50 than at week 48 (490 vs. 330) ; the same effect was seen at week 50 compared with week 48 (p = 0.013)  with clade C trimers  of rabbits previously administered clade A and/or B trimers cross-boosted the existing B41  and BG505.T332N  NAb titers  in most animals, although to varying degrees . For BG5vs. 330) . The cor = 0.048 . The cla= 0.013) . In thes= 0.013) . No cros= 0.013) . OverallWe also explored whether boosting with clade C trimers broadened the overall neutralization response. One notable observation was that NAbs against the BG505.T332N virus were detected in 5 of 5 rabbits from group 1 at week 38  Figs  and 5. TDuring the 60- or 73-week immunization period, the 15 rabbits in groups 2, 4 and 8 received three different trimers from clades A, B and C . All 15 In groups 5 and 6, after the sequential immunization with BG505 and B41 trimers, all 10 rabbits developed medium/strong NAb responses against BG505.T332N (peak titers 630–8200). Six of these rabbits also raised NAbs against B41 (peak titers 340–4700), whereas the other four  did not .We then analyzed the heterologous Tier-1  NAb titers in all rabbits . We compTo supplement the above analyses, we plotted the Tier-1 NAb titers (again MN.3 and MW965.26) for all groups against the various autologous Tier-2 NAb titers at weeks 22 and 62 . SpearmaWhy are the autologous Tier-2 NAb responses to multiple trimers strong and consistent in some rabbits, but weak or absent in others? As described above, the poor Tier-2 NAb responses in the latter subset of rabbits are not attributable to a global inability to respond to the trimers, as the Tier-1 NAb titers in these animals were not atypical. The explanation might be rooted in whether the antibody repertoires of individual animals can recognize the types of Tier-2 NAb epitopes that are described below. Thus, whereas all the rabbits may be capable of raising antibodies to Tier-1 NAb epitopes, some may be incapable of responding to Tier-2 NAb epitopes. Moreover, the different characteristics of the epitopes for Tier-1 and Tier-2 NAbs may explain why their titers are uncorrelated.We based our mapping strategy on the hypothesis that autologous Tier-2 NAbs recognize small holes in the glycan shield where a glycan is absent that is present in multiple HIV-1 strains . AccordiWe analyzed the autologous NAb response to the BG505 trimer in 30 different rabbits. The key results are based on the peak BG505.T332N responses recorded in each rabbit, and represent relative neutralization at a single serum dilution of 1/50 . One serOverall, the 30 rabbits fell into two major sub-groups based on whether the sera could neutralize BG505.T332N virus mutants containing glycan knock-ins at positions 241 and/or 289, or the S241K point substitution. Neutralization by 16 of the 30 sera was partially or completely eliminated when a glycan was present at both of these sites; the single glycan knock-in mutants and the double 241+289 mutant viruses yielded highly concordant data . In addiIn contrast, the other 13 sera efficiently neutralized the 241 and 289 single and double glycan knock-in mutant viruses, and also the S241K mutant . It is pAdditional clues, and also complexities, are provided by neutralization assays with clones of the MG505 maternal virus and mutants thereof. These clones differ from BG505.T332N in several positions, including at position-241 . The MG5The MG505 cl.H3 virus was resistant to all 30 of the rabbit sera , S4 Fig,As noted above, heterologous NAbs against BG505.T332N were induced in four group 1 rabbits immunized with B41 followed by DU422 trimers Figs  and 5. TSera from B41 trimer-immunized rabbits were tested against two virus mutants containing knocked-in glycans to occlude potential holes at positions 130 and 289 . The B41We tested two clones of the CZA97 virus and found that they were either highly sensitive  or strongly resistant  to sera from rabbits immunized with CZA97 trimers . A notab50 cutoff of > 20 was used, and 12 sera did so when a more rigorous cut-off of > 40 was used. There were four examples of heterologous NAb titers > 100 (of three different sera). Several of the heterologous responses were detected at the week-22 time point (2 weeks after the third immunization), but none at week 26 (2 weeks after the fourth). A few additional positive responses were seen at weeks 50 and 60, but none persisted until week 62; several more, but still sporadic, responses were recorded at weeks 73 and 75.To assess the breadth of neutralization, we tested sera from all 40 rabbits at weeks 22 and 26 and, for the appropriate groups also at weeks 50, 62 and 75, against a panel of 9 heterologous Tier-2 viruses . Sera frThe three most frequently neutralized viruses in the panel were: 25710–2.43 (clade C), TRO.11 (clade B), and Ce1176_A3 (clade C), while the least frequently hit was BJOX002000.03.2 (CRF07_BC). The glycan sites in the Env proteins of these isolates are shown in 50) to all four of the trimer immunogens . The strongest correlations between autologous binding and neutralizing antibody titers were seen after the BG505 and B41 monovalent trimer immunizations, and thus support the conclusions we previously drew about how the soluble SOSIP.664 immunogens are antigenic and structural mimics of virion-associated functional trimers , 10. The. FurtherNative-like soluble trimers, exemplified by SOSIP.664 and next-generation derivatives, are now being used to explore how bNAbs may eventually be induced by immunization , 26. StuRabbits respond to immunization with either BG505 or B41 trimers by inducing autologous NAbs against the corresponding Tier-2 viruses, as well as cross-reactive NAbs to Tier-1 viruses . Howeverde novo responses to these two trimers in rabbits, but it seems unlikely they would be very different from what was seen in the present experiment. The strong responses to the CZA97 SOSIP.664 trimers stand in marked contrast to the inability of uncleaved, non-native CZA97 gp140-Foldon proteins to induce autologous NAbs in immunized animals [When rabbits were immunized sequentially with the BG505 and B41 SOSIP.664 trimers they eventually generated autologous Tier-2 NAbs against both viruses. The consistency and high titers of these NAb responses were little different from when the same trimers were administered alone. Thus, rabbits can respond efficiently to more than one native-like trimer, when each is given three times over a prolonged period (~20 weeks). Fewer immunizations do not generate consistent and strong responses; we have not yet explored the minimum rest period required for the third immunization to be optimally effective. After the later clade C trimer boosts, several rabbits given three different trimers raised autologous NAbs to each one at various times. The response to the first trimer did not interfere markedly with the ability to raise autologous NAbs to the second one. In some cases, there was evidence for cross boosting, in that a second trimer modestly increased previously primed NAb titers to the first; this effect was observed most consistently when clade C trimers were given as heterologous boosts, particularly in respect to boosting the BG505.T332N NAb response. Both clade C trimers were immunogenic for autologous Tier-2 NAb responses, CZA97 being markedly more so than DU422. We have not yet obtained data on the  animals . Taken t animals , 26, 53.When BG505- and B41-based trimers were co-delivered at similar doses, autologous Tier-2 NAbs were raised against both viruses with no strong evidence for interference. Whether more substantial interference can arise in tri- and tetravalent immunizations will be determined from ongoing studies. The available evidence does not allow any strong conclusions about whether it is better to deliver two trimers each at the normal dose or at half of it . The best way to co-deliver multiple (3 or more) trimers as a cocktail is being assessed experimentally.We saw no sign, however, that sequential or simultaneous immunizations with two or three trimers was sufficient to generate consistent, high-level heterologous neutralization breadth at the Tier-2 level. Developing a bNAb response is unlikely to be as simple as mixing a cocktail, which is not to say that using more than one flavor of trimer has no value. When boosting previously primed responses, for example in germline antibody-targeting strategies, that approach may be exactly what is required. Nevertheless, we did detect NAbs against the BG505.T332N virus at substantial titers in several rabbits immunized with clade B and then clade C trimers. Thus, the NAbs against this Tier-2 virus arose in animals that had never seen BG505 SOSIP.664 trimers, and in some cases these NAbs appeared to target the 241/289 glycan hole of the BG505.T332N virus. However, the overall breadth of the Tier-2 response was limited to sporadic neutralization of heterologous viruses at titers that were generally <100.We mapped autologous NAb responses to the BG505, B41 and CZA97 trimers to holes in their glycan shields, which supports an earlier suggestion about the nature of neutralization vulnerabilities for Tier-2 viruses [We note that the lack of the 241-glycan creates a site of vulnerability on the BG505 virus and a corresponding immunogenic site on the BG505 trimer, and that no such exposed site is present on the maternal MG505 virus. It seems plausible that changes at position-241, perhaps in concert with others arising elsewhere , are involved in neutralization escape in this particular virus lineage. Because most circulating HIV-1 strains (~97%) resemble MG505 by bearing a glycan at the position-241 site they will be resistant to the induced antibodies, which is what we observed when the rabbit sera were tested against a Tier-2 virus panel. Moreover, even if a glycan hole were present at position-241 on a particular virus, the identity of the now-exposed residues at the base of the hole would also be important. Thus, the BG505.T332N S241K mutant, which lacks the glycan but contains a point substitution at position-241 compared with the trimer immunogen, was resistant to many rabbit sera. Once more, the sequence diversity of HIV-1 will be a formidable obstacle to vaccine development.The rabbit response to the B41 trimers was predominantly directed against a glycan hole at residue-289 in C2 (where 60% of HIV-1 isolates have a glycan), while that to the CZA97 trimers seems centered on the V4 region and is likely to involve a glycan hole at position-411. Studies with additional mutant viruses would be required to define these epitopes in greater detail. We have not yet mapped the autologous NAb response induced in 2 of the 5 rabbits given the DU422 trimers. The finding that the BG505.T332N and B41 viruses share a glycan hole that is impeded by the introduction of a glycan at position-289 may be relevant to understanding some observations of heterologous boosting and heterologous cross-neutralization. Thus, in the group-1 animals, we observed that immunization with B41-based trimers followed by boosting with DU422 trimers induced NAbs that neutralized the BG505.T332N virus. These heterologous NAbs targeted the same 241-glycan hole as the autologous NAbs raised against the BG505 trimers. Perhaps some antibodies induced by the immunogenic 289-glycan hole on the B41 trimers can recognize the similar hole on the BG505.T332N virus, and hence drive a degree of cross-neutralization. Cross boosting of the BG505.T332N and B41 NAb responses by the clade C trimers, again in a manner sensitive to the 289-glycan in both cases, may be rooted in the same mechanism. Among the three most frequently, but still sporadically, neutralized heterologous Tier-2 viruses we note that 25710–2.43 lacks the 241-glycan. Various observations in individual rabbits or sub-groups are not easily understood, including some of the boosting effects of the DU422 trimers that have glycans at both positions 241 and 289. Overall, these various ideas are tentative and would require additional experiments to confirm or refute.Although our overall emphasis is on suppressing rather than inducing Tier-1 NAb responses, we did quantify them . The kinThe trimer genotype is now emerging as an additional important variable, in this and in other experiments that we have been conducting. For example, we observed here that, in the heterologous boost context, the CZA97 trimer was more immunogenic than its DU422 counterpart. Moreover, a few rabbits were non-responders to CZA97, DU422 and B41 trimers but none to BG505, through influences that we are yet to understand. We are now generating and testing hypotheses about what specific features of individual trimers, and the corresponding viruses, most influence the induction of autologous Tier-2 NAb responses. Whether the same characteristics will also affect how different trimers cross boost existing NAb responses, or induce heterologous responses, will also need to be understood. Host factors are also important. Within a group of rabbits, there can be a wide range (2–3 orders of magnitude) of autologous NAb titers to the same trimer, and some animals do not respond at all. Whether an animal responds or not to a particular trimer immunogen by generating autologous Tier-2 NAbs, and the magnitude of any response, may be influenced by the genetics of both the antibody repertoire and of factors that affect the development of humoral immunity more generally. Whether different genetic factors affect the generation of Tier-1 and autologous Tier-2 NAb responses should also be considered in light of the different characteristics of the relevant epitopes. An influence of host genetic factors will not, of course, be unique to rabbits, although the identities of the factors involved may be species-dependent.This series of experiments was performed in rabbits, because the strong and consistent autologous NAb responses to the BG505 and B41 trimers in this species allow endpoints to be determined . Guinea In conclusion, the observations and inferences from this complex set of experiments may be a valuable guide on how best to design and use trimer-based immunogens in the quest to induce bNAb responses in humans. The limitations of the study, the restricted statistical power of small groups, and perhaps other issues inherent to the choice of model species, must be recognized. Nevertheless, the present results suggest that the induction of sufficient neutralization breadth will probably not be achieved through current regimens simply by combining a few native-like trimers into a cocktail without further advances in immunogen delivery methods. The combination approach may, however, be useful for boosting responses primed by germline bNAb-targeting trimers, particularly if our understanding of genotype-dependent influences on immunogenicity advances. Knowledge of the nature of the epitopes responsible for neutralization of Tier-2 viruses is likely to aid the design of immunogens intended to convert narrow-specificity NAb responses that, perhaps, evade glycans into broader ones that, in some cases, accommodate their presence. Thus, glycan holes may be the initial targets for several bNAbs in their ontogeny in the infected human –57 and tS1 Text(DOC)Click here for additional data file.S1 FigD, the glycan holes are defined in relation to the majority of the Env sequences in the Los Alamos Sequence Data Base (http://www.hiv.lanl.gov).Amino-acid differences are highlighted in gray and variable Env regions in yellow. Glycan sites are indicated in red, while the absence of a glycan (a glycan hole) in relation to comparator sequence or other isolates is highlighted in green. In panel (PDF)Click here for additional data file.S2 FigTop panels: NAb titers at week-22 are compared for the Tier-1 viruses MN.3 (left) and MW965.26 (right) and the autologous Tier-2 viruses BG505.T332N  and B41 , as indicated. Bottom panels: NAb titers at week-62 are compared for the Tier-1 viruses MN.3 (left) and MW965.26 (right) and the autologous Tier-2 viruses BG505.T332N, B41, DU422 (group 1) and CZA97 . The week-62 BG505.T3322N and B41 correlations with Tier-1 titers involve all the groups of rabbits bled at that time point, since some cross-neutralizing responses had arisen against BG505.T332N, albeit generally at low titers. No cross-neutralization of B41 was observed at week-62, however. Spearman-correlation analyses of these comparisons are recorded in (TIFF)Click here for additional data file.S3 Fig50, mean ± s.e.m. on a log-scale) are shown on the y-axis for the groups of five rabbits indicated on the category axis.NAb titers against the BG505.T332N (left) and B41 (right) viruses are compared at the peak (week-22) of the responses to single or dual immunizations as follows: group 1 (30 μg clade B trimer), group 3 (30 μg clade A trimer), group 4 , group 7 , group 8  and group-2 . The NAb titers (IC(TIFF)Click here for additional data file.S4 FigThe values shown represent the reductions in neutralization titer for (A) BG505.T332N virus mutants relative to wild-type ; or (B) CZA97 clones or mutants thereof relative to cl.12 (= 100%). The sera listed are the subset from the groups presented in (TIFF)Click here for additional data file.S5 FigThe scatterplots show NAb titers on the y-axes and the antibody binding titers to SOSIP.664-D7324 trimers on the x-axes. Within each plot the symbols corresponding to neutralization of the BG505.T332N, B41, DU422 and CZA97 viruses are color-coded as indicated on the figure panels. Spearman correlation coefficients  and the corresponding significances  are color-coded analogously. The scatter plots show the BG505, B41 and DU422 or CZA97 NAb and binding antibody titers for groups-1 and -3, during the period of DU422 or CZA97 trimer boosting immunizations from weeks 38–62 .(PPTX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.Efficient lysosomal Ca Lysosomes are the cell’s recycling centers in which lysosomal enzymes digest endocytosed or excess materials. Emerging evidence suggests that lysosomal membrane proteins including ion channels and transporters also play essential roles in material recycling by creating optimal environment for the enzymes or regulating lysosome membrane trafficking. Deficiencies in either the enzymes or membrane proteins results in a group of inherited metabolic disorders known as the lysosomal storage disorders (LSDs)134562+ release channel that is essential for the regulation of lysosomal membrane trafficking. TRPML1 deficiency results in enlarged lysosomes and abnormal lysosomal storage, as shown in cells from the patients with mucolipidosis type IV (ML4), a LSD caused by loss-of-function mutations of human TRPML182+ released via TRPML1. Activated BK provides K+ influx to facilitate TRPML1 opening by maintaining a lysosomal membrane potential beneficial for TRPML1. Therefore, BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosomal membrane trafficking and functionTransient Receptor Potential Mucolipin-1 (TRPML1) is a well-known lysosomal Ca2+ release, and subsequently alleviates the cellular phenotypes of these LSDs by promoting lysosomal exocytosis. Thus, promoting BK activity could be a common therapeutic approach to cure LSDs.Defective lysosomal membrane trafficking is commonly seen in LSDs4510111210111213342+ release is compromised in NPC1 human fibroblasts4781720NPC1 disease is caused by defects in the gene NPC1 that transports cholesterol across lysosomal membrane. NPC1 mutations result in impaired lysosomal membrane trafficking, leading to abnormal lipofuscin, cholesterol and sphingomyelin accumulation in lysosomes4Promoting lysosomal exocytosis (lysosome fusion with the plasma membrane) is known as a promising therapeutic approach to attenuate lysosomal storage and treat LSDs3456222324The rescue effect of NS1619 on NPC1 cellular phenotypes could be attributed to activating either BK or TRPML1. To tease apart these two possibilities, we directly studied NS1619’s effect on lysosomal BK and TRPML1 channel activity. Under whole-lysosome recording mode, TRPML1 displays strongly inward rectification while BK shows strongly outward rectification2+ release using GECO-TRPML1, a single-wavelength genetically encoded Ca2+ indicator72+ (free Ca2+ < 10 nM) solution, an increase in GECO-TRPML1 signal which is monitored by measuring GECO fluorescence (F470) represents TRPML1 activation. GECO-TRPML1 was expressed in HEK293T cells, and GECO-TRPML1 fluorescence in response to ML-SA1 was compared between NS1619 and control vehicle. As shown in 2+ content, because glycyl-phenylalanine 2-naphthylamide , a substrate of the lysosomal exopeptidase cathepsin C that induces lysosome osmolysis to deplete lysosomal Ca2+ pools2+ release through TRPML1.We have shown that BK activation facilitates TRPML1 function by maintaining a beneficial lysosomal membrane potential for TRPML1escence F70 repres2+ release in NPC1 cells. As shown previously42+ content was not affected Although all ML4 patients are characterized by motor impairment, mental retardation, and retinal degeneration, the severity of the phenotypes are different depending on the type of mutation33 content  or in GEDefective TRPML1 channels results in abnormal lysosomal storage, which often leads to the formation of autofluorescent lipofuscin, as seen in LSD human skin fibroblasts42+ release and lysosomal storage4410112+ release, we hypothesize that BK upregulation rescues NPA phenotypes. As expected, NPA cells displayed increased lipofuscin autofluoresence, as compared to wild type human fibroblasts4In Niemann-Pick type A (NPA) and type B (NPB) diseases, sphingomyelin accumulation in lysosomes due to insufficient activity of acid sphingomyelinase results in reduced TRPML1-mediated lysosomal Ca2+ release in NPA cells. As shown in 2+ content   . NotablyWT cells . Taken t2+ release channel that regulates lysosomal membrane trafficking74The lysosome acts as a garbage dump or recycling center inside of the cell. Dysfunction of the lysosome causes accumulation of waste products in the cell, eventually leading to cell death which is manifested by a large number of inherited metabolic disorders, called lysosomal storage diseases (LSDs). In general, LSDs are subdivided into two groups. One subgroup of LSDs is caused by deficiency of specific enzymes that are normally required for the breakdown of certain complex macromolecules including carbohydrates, lipids, proteins, and nucleic acids. The other subgroup of LSDs result from some defective lysosome membrane proteins which are essential for creating optimal environment for lysosomal enzymes, transporting metabolites, or lysosomal membrane trafficking125111213125111213153452+ release and membrane trafficking using a positive feedback mechanism. In this study we further strengthen this notion using a number of LSD models. We found that upregulating BK rescued the phenotypes of patient fibroblasts from NPC1, F408∆-ML4, NPA and Fabry disease by promoting lysosomal Ca2+ release and subsequent lysosomal exocytosis. Therefore, in certain types of LSDs, activating BK channels strengthens the positive feedback loop between TRPML1 and BK, and subsequently alleviates the diseases342+ release and to clear lysosomal storage in skin fibroblasts from a number of LSDs by improving lysosomal exocytosis. Given that TRPML1 is a nonselective cation channel that permeates both Ca2+ and Na+ + efflux could also carry some important functions, such as membrane potential regulation and Na+-dependent solute transport, to correct the cellular phenotypes of these LSDs. The demonstration that BK helps clear lysosome storage in these LSD models would argue that Ca2+ but not Na+ efflux represents the main cellular function of the TRPML1 channel in rescuing LSDs. This is further supported by the finding that the rescue effect of upregulating either TRPML12+ sensor using Syt VII DN. Collectively, our data demonstrate that small molecules targeting on BK channels can be used to restore lysosomal function and rescue disease associated abnormalities in patient cells with lysosomal storage. This may lead to a new clinical intervention strategy for treating LSDs in the future.We have previously demonstrated that BK forms a macromolecular complex with TRPML1 in lysosomes to ensure sufficient lysosomal Ca2+ release and membrane trafficking14323334353637In addition to LSDs3456811122 atmosphere. For some experiments, cells were seeded on 0.01% Poly-D-Lysine coated glass coverslips and cultured for 24 hrs before further experimentation. Cos1 and HEK293T cells were transiently transfected with indicated DNA using Lipofectamine 3000® following manufacturer’s manual. Fibroblasts were transiently transfected with indicated DNA by Neon® electroporation  and cultured for 24 hrs before use.Cos1 cells and HEK293T cells were obtained from ATCC  and maintained in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum . Human skin fibroblasts from NPC1 patient , NPA (GM00112) ML4 patient (GM02629), Fabry disease patient (GM00107) and non-disease control cells (GM00969) were obtained from Coriell Institute for Medical Research . Human fibroblasts carrying ML4 mutations  were from Baylor Institute of Metabolic Disease. All the fibroblasts were maintained in DMEM supplemented with 15% non-heat-inactivated FBS. Cells were cultured at 37˚C in a 5% COThe following chemicals were used: Texas Red 10 kD dextran ; NS1619 (Sigma); NS11021 (Sigma); GPN (Santa Cruz Biotechnology); Paxilline ; ML-SA1 (Tocris); Ionomycin (Cayman), ML-SI1 (Enzo Life Sciences Inc).Plasmids cMyc-BK-GFP constructs were prepared as before8http://crispr.mit.edu/), and two sequences were chosen: CATCTTGGGCTCGTGGACCG (target 1) and CTAGGCTGAGATGGTTCGCG (target 2). After being annealed with their reverse complementary sequences, the short double strand DNA’s (hereafter named gDNA) were ligated with Bbs I digested pX330-2A-GFP and the new constructs were named BK-CRISPR1 and 2, respectively. Human skin fibroblasts were transfected with individual pX330-2A-GFP/BK gDNAs using Neon® system following optimized protocol and cultured for 72 hours before use. The transfection efficiency for human fibroblasts was >70%.The operation followed detailed instructions of the published protocol2, 0.39 mM CaCl2, 20 mM HEPES . The pipette  solution was a standard extracellular solution contained 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose (the pH was adjusted with HCl to 4.6). Whole-lysosome currents were digitized at 10 kHz and filtered at 2 kHz. Liquid junction potential was corrected. Data were collected using an Axopatch 2A patch-clamp amplifier, Digidata 1440, and pClamp 10.2 software (Axon Instruments). All experiments were conducted at room temperature (21 °C–23 °C).Lysosomal electrophysiology was performed in isolated enlarged late endosome/lysosome vacuoles using whole-lysosome patch clamp method as described previously2+ release was measured under a ‘low’ external Ca2+ solution that contained 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA and 20 mM HEPES , and repeated three times independently.HEK293T cells or human fibroblasts were transfected with GECO-TRPML1 for 24–36 hrs and then plated onto glass coverslips. Imaging was carried out within 0.5–2 hrs after plating when cells still exhibited a round morphology. The fluorescence intensity at 470 nm (F470) was recorded using the EasyRatioPro system (PTI). Lysosomal Ca340/F380) were used to monitor changes in intracellular [Ca2+] upon stimulation. Lysosomal Ca2+ release was measured under a ‘low’ external Ca2+ solution. ML-SA1 (50 μM) and GPN (400 μM) were used to induce Ca2+ release from lysosomes. Ionomycin (1 μM) was added at the conclusion of all experiments to induce a maximal response for comparison. Ca2+ imaging was carried out within 0.5–2 h after plating and when cells still exhibited a round morphology.Cells were loaded with 5 μM Fura-2 AM (Invitrogen). Fluorescence was recorded at different excitation wavelengths using an EasyRatioPro system (PTI). Fura-2 ratios  for 2 hrs at room temperature. Cells were rinsed three times with PBS and viewed with Zeiss LSM710 confocal microscope (40 × or 63× oil-immersion objective lens) using 340–380 nm excitation and 510 nm emission.Confocal fluorescent images were taken using an inverted Zeiss LSM510 Axiovert 200M confocal microscope with a 63× oil-immersion objective. Sequential excitation wavelengths at 488 nm and 543 nm were provided by argon and helium-neon gas lasers, respectively. Emission filters BP500-550 and LP560 were used for collecting green and red images in channels one and two, respectively. After sequential excitation, green and red fluorescent images of the same cell were saved with ZEN2012 software. In some experiments, autofluorescence (green) detectable within the excitation wavelength of 480 nm is an indicator of lipofuscin accumulation. Images were analyzed by Zeiss software. Intensity of fluorescent was analyzed using ImageJ, The image size was set at 1,024 × 1,024 pixels.p-nitrophenyl N-acetyl-β-D-glucosaminide in 0.1 M citrate buffer  for 1 hour at 37 °C. Reactions were quenched by addition of 200 μl 0.1 M sodium carbonate buffer. Absorbance was read at 405 nm. Absorbance values were background subtracted using medium only values. LDH activity was determined by measuring the NADH oxidation with pyruvate as substrate. For each assay, a 50 μl sample was incubated with 50 μl reaction buffer  for 10 min at 22 °C and the fluorescence at 340 nm was measured using a spectrophotometer. Results were scaled to the complete cell death induced by exposure to 1% TritonX100 at room temperature for 20 min. Percent of cell death was defined as = 100 × (sample’s OD - background medium OD)/(Triton-X100 OD - medium background OD).NPC1 cells were cultured in 24-well plates. β-hexosaminidase activity in 100 μl supernatant samples was measured by incubation with 100 μl of 1 mM P < 0.05; **P < 0.01.Experiments were repeated at least three times independently. Data are presented as mean ± SEM. Analysis of variance (ANOVA) or Student’s t test was applied for statistical comparisons. P values of <0.05 were considered statistically significant. NS: not significant; *How to cite this article: Zhong, X. Z. et al. BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases. Sci. Rep.6, 33684; doi: 10.1038/srep33684 (2016).


Better understanding of the mechanisms behind basic ER Ca2+ content regulation is important, since current hypotheses on the possible ultimate causes of ER stress point to deterioration of the Ca2+ transport mechanism to/from ER itself. We measured [Ca2+]i temporal changes by Fura-2 fluorescence under experimental protocols that inhibit a host of transporters  channels, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), Na+/ K+ ATPase (NKA)) involved in the Ca2+ communication between the extracellular space and the ER. Following histamine-stimulated ER Ca2+ release, blockade of NCX Ca2+-influx mode (by 10 μM KB-R7943) diminished the ER refilling capacity by about 40%, while in Orai1 dominant negative-transfected cells NCX blockade attenuated ER refilling by about 60%. Conversely, inhibiting the ouabain sensitive NKA (10 nM ouabain), which may be localized in PM-ER junctions, increased the ER Ca2+ releasable fraction by about 20%, thereby supporting the hypothesis that this process of privileged ER refilling is junction-mediated. Junctions were observed in the cell ultrastructure and their main parameters of membrane separation and linear extension were (9.6 ± 3.8) nm and (128 ± 63) nm, respectively. Our findings point to a process of privileged refilling of the ER, in which NCX and store-operated Ca2+ entry via the stromal interaction molecule (STIM)-Orai system are the sole protagonists. These results shed light on the molecular machinery involved in the function of a previously hypothesized subplasmalemmal Ca2+ control unit during ER refilling with extracellular Ca2+.We investigated the role of Na The online version of this article (doi:10.1007/s00424-017-1989-8) contains supplementary material, which is available to authorized users. The subsequent [Ca2+]i elevation is a preamble underpinning basic EC function like nitric oxide (NO) synthesis by the endothelial NO synthase and endothelin-1 production o EB was composed of (in mM) 73 NaCl, 70 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with KOH. The Ca2+-free EB contained 0.1 mM EGTA instead of CaCl2.The 70 mM i were monitored using the Fura-2 technique as previously described  changes. Therefore, after calculating the mean Ca2+ signal traces from each type of experiment and referring to the protocol description in “nd histamine transient to the 1st and of the n Ca2+ phase transient to the 2nd histamine one. A third interesting parameter for our analysis is the rate of Ca2+ re-entry in the cytosol during the n Ca2+ phase. This is calculated as the slope of the roughly linear portion of the Fura-2 signal at the onset of the n Ca2+ phase.Given the slightly different behavior observed in different sub-populations of cells, our approach to determining significance in the effects recorded under the stimuli and inhibitors used in our experiments was to analyze the ratio between the amplitudes of the Cahttp://www.libreoffice.org).The Fura-2 340/380 fluorescence ratios were recorded as text files and then analyzed by a combination of in-house C programs, Linux shell scripts and LibreOffice Calc Spreadsheet functions (http://www.xfig.org) to measure PM-ER junctional separations and extensions, as well as “pillar” features. Plots were generated with gnuplot (http://gnuplot.info).The electron micrographs were recorded as TIFF image files and analyzed with xfig  on the PM, SERCA, and IP3R on the ER. Indirectly involved with Ca2+ handling are the NKA pumps on the PM i rise  has been shown to be rather variable [2+]i signal during the n Ca2+ phase, while the ER is refilled to the same level observed in cells with resting Vm entry in the cytoplasm and the ER Ca2+ release peak is significantly lower than when extracellular Ca2+ is present in the superfusate. We also observed that the remnant ER Ca2+ in this case is largely due to re-uptake by SERCA pumps during the first ER Ca2+ release, since, by blocking SERCA with 2,5-Di-tert-butyl-1,4-benzoquinone (BHQ) during the first histamine stimulation, we found that the Ca2+ left after the second stimulation under 0 Ca2+ conditions throughout the protocol was even further reduced  in the superfusate during the n CaEA.hy926 , reside EA.hy926 , 21, thi2+ is still releasable from the ER even after inhibition of NCX Ca2+ entry. This is likely only due to SOCE, since under membrane depolarization conditions and in absence of cell stimulation (note that histamine washout begins 3 min before the n Ca2+ phase), any contribution to Ca2+ entry by the TRPC should be negligible. Therefore, we performed a series of NCX Ca2+ entry mode inhibition experiments on a population of cells, which were deprived of functional Orai1 channels by means of transfection with Orai1dn. This experimental condition should ensure inhibition and elimination of NCX and SOCE extracellular Ca2+ entry pathways, respectively. Results obtained with this protocol are reported in Fig. 2+ phase of the mean trace in Fig. 2+ was omitted , still 2+ influx mode by blocking the ouabain-sensitive NKAα2/3 pumps with 10 nM ouabain . If, liy NKAα1 , 21, thi ER Fig. a. This iset Fig. c right, per Fig. d. Previons in EC . It is tle cells , may be 2+ influx mode inhibition and amplification experiments are consistent with a picture suggesting that the extracellular Ca2+ entering through the NCX is for the most part directly sequestered in the ER, with perhaps a minor amount contributing to the cytosolic [Ca2+]. Moreover, the ouabain-sensitive-NKA inhibition experiments also support the conjecture that the NCX Ca2+ transport takes place at PM-ER junctions. In this view, the fraction of bulk [Ca2+]i contributed by the NCX appears marginal and due to spill-over from the junctions, which would agree with the observed decreased or increased Ca2+ re-entry rate upon NCX Ca2+ entry mode inhibition or amplification, respectively. In the former case, diminished spill-over would hinder Ca2+ accumulation rate in the bulk signal, in the latter, somewhat “over-driving” NCX-dependent Ca2+ influx would also increase Ca2+ escape from the junctions, which can manifest itself as a higher rate of entry in the cytosolic signal during the initial n Ca2+ phase of the protocol.Taken together, the findings from the NCX Ca2+ entry in a population of cells transfected with Orai1dn, we clearly observed that SOCE via Orai1 channels contributes almost all of the releasable fraction of ER Ca2+ remaining after NCX inhibition in wild type cells. This can be seen in Fig. nd and 1st histamine-stimulated transient amplitudes is further reduced to about 30% of the control experiments in (depolarized) wild type cells. Since Orai1 Ca2+ transport is “by definition” junctional, Orai1 SOCE must contribute to the bulk [Ca2+]i via spill-over from PM-ER junctions, besides partially refilling ER Ca2+.When we inhibited NCX Ca2+]i signal, Orai1 channels are responsible for the near totality of the Ca2+ re-entry signal, which can be inferred from the complete elimination of the n Ca2+-phase signal in Orai1dn experiments  contribution to the i and [Na+]i lets us infer that the ER refilling mechanism may be assisted by PM-ER junctions also in non-depolarized cells. In Fig. ENCX = 3 ENa − 2 ECa, where ENa and ECa are the Nernst potentials for Na+ and Ca2+, respectively, as a function of [Ca2+]i at 3 different possible values of [Na+]i. Two values of the membrane potential of our cells are also plotted: Vm ≈−2 mV (dashed blue line) is the one we expect in depolarized conditions (as we determined in i = 10 mM and [Ca2+]o = 2 mM, the NCX can be expected always to function in Ca2+ entry mode at bulk [Ca2+]i = 100 nM .Since the NCX is in CaVm being much more negative implies that the NCX reversal condition is barely respected at bulk [Ca2+]i = 100 nM and [Na+]i = 10 mM (the [Na+]i = 10 mM curve crosses the Vm ≈−50 mV line in the neighborhood of [Ca2+]i = 100 nM). As the plots suggest, assuming that [Ca2+]i equals its bulk value of 100 nm, the condition would be more safely obeyed at higher values of [Na+]i . However, according to presently available data, we can expect to observe such high Na+ transients only in subplasmalemmal regions of cells, which are hypothesized to correspond to S/ER junctions [2+]i in such junctions is highly variable due to their sheer dimension and can easily reach micro-molar values [+]i closer to 20 mM would be necessary to achieve NCX reversal, which further confirms the conjecture arrived at by our functional measurements, in the depolarized system, that the NCX-mediated ER Ca2+ refilling mechanism must occur via PM-ER junctions in non-depolarized EC as well. On the other hand, under physiological conditions of bulk cytoplasmic and of extracellular ionic concentrations normally observed in EC environment, the unctions , 44. Butr values . In this2+ refilling may occur via PM-ER junctions in a privileged manner partly segregated from bulk cytosolic Ca2+ prompted us to study the ultrastructure of EA.hy926 cells in search for occurrences of such junctions. A survey of 29 electron micrographs suggests that peripheral ER appositions to the PM occur regularly in these cells and possess features also observed in S/ER junctions of other cells systems, which are involved in privileged Ca2+ signaling, such as the cardiac dyads [2+ refilling. The architectural features of the PM-ER junctions we report herein would enable them to provide the tightly confined environment necessary to generate spatially and temporally localized high [Ca2+]i and [Na+]i transients putatively responsible for Ca2+ signal segregation leading to the focal ER refilling we infer from our data. Confirmation of this hypothesized role of the PM-ER junctions in EC will only be possible by a combination of specific labeling of the NCX1, Orai1, and TRPC and realistic quantitative modeling of the junctional transport dynamics.The strong indication from our Fura-2 measurements, as well as from our analysis of the electrochemical potential, that ER Caac dyads , the mitac dyads , the PM-ac dyads , 43. We 2+ signal measurements and ultrastructural observations in the human umbilical vein-derived cell line EA.hy926 elucidate important steps in the mechanism of ER Ca2+ refilling and indicate that most of the ER Ca2+ releasable fraction is transported from the extra-cellular space to the ER lumen by the NCX and the Orai1 channels in similar proportions. In light of a hypothesized link between Na+/ Ca2+ exchange-mediated Ca2+ entry and eNOS activation which had been observed upon Na+ loading in ECs [2+ transport much like the hypothesized “subplasmalemmal Ca2+ control unit (SCCU)” [In conclusion, our findings based on previously unavailable specific Cag in ECs , our con (SCCU)”  and the  (SCCU)” .(PDF 610 KB)Below is the link to the electronic supplementary material.


This process can be modulated by nitric oxide (NO), a gas molecule synthesized endogenously by (nitric oxide synthase) NOS isoforms. At nanomolar concentrations NO activates soluble guanylate cyclase (sGC), which in turn activates protein kinase G via conversion of GTP into cyclic GMP. Alternatively, NO post-translationally modifies proteins via S-nitrosylation of the thiol group of cysteine. However, the mechanisms of action of NO on Ca2+ homeostasis during muscle contraction are not fully understood and we hypothesize that NO exerts its effects on Ca2+ homeostasis in skeletal muscles mainly through negative modulation of Ca2+ release and Ca2+ uptake via the NO-sGC-PKG pathway. To address this, we used 5–7 days-post fecundation-larvae of zebrafish, a well-established animal model for physiological and pathophysiological muscle activity. We evaluated the response of muscle contraction and Ca2+ transients in presence of SNAP, a NO-donor, or L-NAME, an unspecific NOS blocker in combination with specific blockers of key proteins of Ca2+ homeostasis. We also evaluate the expression of NOS in combination with dihydropteridine receptor, ryanodine receptor and sarco/endoplasmic reticulum Ca2+ ATPase. We concluded that endogenous NO reduced force production through negative modulation of Ca2+ transients via the NO-sGC pathway. This effect could be reversed using an unspecific NOS blocker or sGC blocker.Vertebrate skeletal muscle contraction and relaxation is a complex process that depends on Ca This dynamic and fast Ca2+ movement from the SR into the myoplasm and then back into the SR is denominated Ca2+ transient. The time course and the amplitude of the Ca2+ transients determine force production  is a small bioactive gas molecule, with complex arrays of message intermediation in vivo , whose activity is Ca2+ independent , increasing cGMP levels, which leads to activation of a myriad of physiological processes such the activation of PKG, cGMP-cation gated channels, cGMP-hydrolyzing phosphodiesterases (PDE), and cGMP-hydrolyzing PDEs , were used. All animal experiments have been performed in accordance with the guidelines of the state of Baden–Wuerttemberg and it has also been approved by the ethics committee of the University of Heidelberg Interfaculty Biomedical Research Facility (T54/16).3, 4.7 KCl, 1.2 MgCl2, 1.2 KH2PO4, 2.5 CaCl2, and 11.1 Glucose, pH 7.4) solution. The head was then removed with microsurgical scissors and forceps under the microscope . Two pieces of aluminum foil (both 2 * 8 mm) with cyanoacrylate glue were prepared and bent to a right angle. Anterior and caudal sections were set on the aluminum foil and mounted into the force transducer immersed in freshly prepared MKH and bubbled with 5% CO2/95% at room temperature. After fixation to the force transducer, the preparation was incubated in a glass chamber, which was perfused continuously (4 ml/min) with MKH. Two platinum electrodes flanking the preparation provided electrical field stimulations controlled by an isolated pulse stimulator  and a Grass SD9 stimulation unit. The mechanical signals from the force transducers were digitally converted by a PowerLab 15T AD converter (AD Instruments) and sampled (1 kHz sampling frequency) with LabChart 7 software (AD Instruments) and stored in a computer. The optimal length was established adjusting the length between the force transducer and the holder applying supramaximal stimulation in order to establish rheobase and chronobase for each experiment. Force-frequency curves were recorded with 200 ms stimulation trains at 2 ms pulse duration and supramaximal voltage, in 2 min interval. The stimulation frequency was increased in steps .The force measurement protocol using zebrafish larvae was described in detail elsewhere  5 mM, caffeine (250 μM), verapamil hydrochloride (20 μM), cyclopiazonic acid (CPA) 2.5 μM, NiCl2 2 mM, 1H-oxadiazoloquinoxalin-1-one (ODQ) 10 μM, and N-ethylmaleimide (NEM) 100 μM.All pharmacological agents were purchased from Sigma-Aldrich if not stated otherwise. NO donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP) 100 μM and Diethylamine NONOate diethylammonium salt  1 μM, unspecific NO blocker N2PO4, 4 MgSO4, 50 Taurin, 20 Glucose, 10 HEPES with 0.75 mg/ml Collagenase Type II; 4 mg/ml BSA, pH 6.9). A mix of transfer buffer A (TBA) and transfer buffer B (TBB) was used to slowly increase Ca2+ concentration. TBA (in mM): 117.2 NaCl, 25.2 NaHCO3, 4.7 KCl, 1.2 MgCl2, 1.2 KH2PO4, 11.1 Glucose, 10 HEPES, 10 BDM, and 5 BSA, pH 7.4. As control solution, TBB (in mM) was used: 117.2 NaCl, 25.2 NaHCO3, 4.7 KCl, 1.2 MgCl2, 1.2 KH2PO4, 2.5 CaCl2, 11.1 Glucose, 10 HEPES, and 5 BSA, pH7.4. The cell suspension was then centrifuged for 2 min at 500 rpm and the cell pellet was re-suspended in transfer buffer (TB)-1 (4.9 ml TBA in 0.1 ml TBB). This procedure was twice repeated in TB-2 (4.5 ml TBA in 0.5 ml TBB) and in TB-3 (2.5 ml TBA in 2.5 ml TBB). The cells were then seeded on 22 mm coverslip covered with 50 μl ECM placed on a 35 mm petri dish and incubated at 5% CO2 at 25 ± 1.5°C for 30 min prior use.Isolated skeletal muscle cells were obtained by adapting the procedure developed by Liao and Jain . A pool 2 was selected to record the Ca2+ transients from each cell. In order to introduce myocyte stimulation, a pair of platinum electrodes connected to a stimulator was placed to flank the cells. Three consecutive stimulations at 0.5 Hz 18 V for 10 ms were given and the change in fluorescence intensity was AD-converted  and sampled at 10 KHz in a PC using LabChart Pro7 software (AD Instruments). The analyses of calibrated Ca2+-transients were done using the Peak Analysis package from LabChart Pro 7. The traces were analyzed using 8 biophysical parameters: Peak , Duration , Duration50 , Peak Area , Time to Peak , Slope , Relaxation Time in force or Uptake Time for Ca2+ transients  and Tau . At the end of each experiment, the same cells were used to convert Fluo-4 fluorescence intensity into [Ca2+]i, as described previously  140 LiCl, 5 KCl, 1.2 KH2PO4, 1.2 MgCl2, 4 CaCl2, 20 HEPES, 0 EGTA, 0.005 Ionomycin, 0.01 CPA, 5 Caffeine and 1 Oubain and the resulting fluorescence was designated Fmax. Cells were then exposed to a similar solution, but now containing a Ca2+-free solution that contained (in mM) 140 LiCl, 5 KCl, 1.2 KH2PO4, 1.2 MgCl2, 0 CaCl2, 20 HEPES, 4 EGTA, 0.005 Ionomycin, 0.01 CPA, 5 Caffeine and 1 Oubain and the resulting fluorescence was designated Fmin. The following equation was used to convert Fluo-4 fluorescence readings F into [Ca2+]i:Isolated myocytes were incubated for 30 min with Fluo-4 AM  diluted to a final concentration of 10 μM in TBB. After incubation, the cells were washed once with TBB and the imaging dishes were mounted to a fluorescence microscope (Olympus Second Generation OSP-3 system) equipped with a photomultiplier unit and a Xenon light source system, filtered to provide an excitation wavelength of 488 nm. A photomultiplier was used to have a continuous recording of the fluorescence signal and thus a better time resolution necessary for the analyses of the biophysical parameters. An area of 7.5 μmd) of 345 nM was assumed according to the manufacturer's declaration.The Fluo-4 dissociation constant  perfused with TBB. Field electrical stimulation was performed as described above . The line scan trace was set at 1,024 pixels every 0.2 ms at the center of the myocyte's short axis and was moved down from 0 to −5 μm, in 1 μm steps in vertical direction. Continuous line scan was performed to monitor the change of the fluorescence intensity . The signal was recorded and processed using FLUOVIEW (Version 3.3.16) software.2HPO4, and 2 KH2PO4, adjust pH to 7.4), permeabilized with 0.25% Triton X-100 in PBS and rinsed three times with 1x PBS. To block unspecific binding sites, myocytes were incubated for 30 min with 1x PBS containing 0.01% Tween 20 and 1% BSA. Diluted first anti-bodies were incubated over night at 4°C, rinsed with 1x PBS three times for 5 min following the incubation of the secondary antibody for 1 h at room temperature and finally rinsed with 1x PBS three times for 5 min. The samples were mounted with Dako mounting media according to the manufacturer's instructions and analyzed in a fluorescence microscope. The immunofluorescence pictures were taken at the sub-sarcolemmal region of the isolated myocyte. The primary and secondary antibodies were purchased from Abcam and diluted as follows: anti-CACNA1S antibody [1A], cat no. ab2862, dilution 1: 100; anti-Ryanodine Receptor antibody [C3-33], cat no. ab2827, dilution 1: 200; anti-SERCA1 ATPase antibody [VE121G9], cat no. ab2819, dilution 1: 200; anti-nNOS  antibody, cat no. ab5586, dilution 1: 200; Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647), cat no. ab150107, dilution 1: 500; Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488), cat no. ab150077, dilution 1:500. The quantitative periodic distance analyses of the images were done using TTorg plugin form ImageJ . Normalized force production under the effect of L-NAME was not different from control at low and medium stimulation frequency (from 1 to 60 Hz). However, at high frequency of stimulation (90–200 Hz), the force was higher  in control solution only or with addition of an unspecific NOS blocker (L-NAME) or the NO donor (SNAP) compared to control solution Figure . Since tr Figure . In pres50 parameters were significantly reduced with SNAP. These results suggest that SNAP's mechanism of action to reduce force production is reducing the initial contraction; on the other hand, the effect of L-NAME was reducing the biophysical parameters related to uptake . These results suggest that NO modulates the force production based on initial contractions and the lack of NO alters relaxation.Further analyses of the biophysical parameters during single twitches 1 Hz) were done summarized in Table  Hz were Based on the modulatory effect observed with the force-frequency data described above, we mathematically evaluated the spatial expression of nNOS in zebrafish myocyte and correlated it with the expression of DHPR, RyR1, and SERCA1 using TTorg plugin of ImageJ Figures . Confoca2+ homeostasis (Ca2+ release and uptake) during each contraction. Therefore, the analyses of Ca2+ transients from isolated skeletal muscles provide evidence of the modulatory effect of NO. The size of a typical isolated skeletal myocyte from zebrafish larvae was ~15 × 100 μm, similar to a rectangular shape , two Ca2+ release (Time to Peak and Slope) and two Ca2+ uptake (Uptake Time and Tau). As shown in Figure 2+ transient, whereas the presence of L-NAME induces a slight enhancement. Compared to control, 100 μM SNAP significantly reduced Peak . First, we blocked the DHPR with verapamil (20 μM) in presence of SNAP or L-NAME. Compared to control conditions, verapamil radically suppressed the Peak of Ca2+ transients , we locked RyR1 at a half open stage with lower ryanodine concentration (100 nM) in presence of SNAP or L-NAME  as an activator of RyR1. At this concentration, the Peaks were significantly higher compared to control solution  completely blocked CaE Figure . We obseon Table . Both SNe Figure , except e Figure . To furton Table . This en2+ uptake in Ca2+ transients. In order to address this question, we evaluated two major Ca2+ uptake mechanisms: SERCA and NCX. We evaluated the role of NO on NCX function blocking SERCA with CPA (2.5 μM). The analysis of the Peak parameter did not show any substantial change attributable to NCX . As expected, release parameters of myocytes treated with NiCl2 were similar to control solution, showing that at this concentration NiCl2 did not interfere with DHPR or RyR activities. SNAP in combination with NiCl2 did not modify the Peak parameters  in order to evaluate the effect mediated by sGC and the S-nitrosylation blocker NEM (100 μM) to block the effect mediated by S-nitrosylation. Results are shown in Figure 2+ transient peak  we analyzed our data based on single twitch stimulation and not from submaximal tetanic stimulation, (3) we used L-NAME to evaluate the endogenous effect of NO on muscle contraction and on Ca2+ transients, and (4) we used zebrafish larvae as an animal model. These differences allowed us to quantify in detail each aspect of NO-dependent force and Ca2+ transient changes, measuring the effect of endogenous NO on force and Ca2+ transients in a well-established animal model for patho-physiological muscular approaches that have been extensively used due to easy and fast production of mutants, helpful model to test new therapeutic strategies and low-cost maintenance  Since it was not possible to calculate the cross sectional area for normalization of force production as performed in other muscle models, we found that in our model single twitch force at 1 Hz prior to the force-frequency protocol was suitable for normalization to standardize the results obtained from different sizes of zebrafish larvae. The increase of force production across the entire force-frequency protocol revealed that larvae did not produce a sigmoidal force increase in zebrafish  on the biophysical parameters of Ca2+ transients were absent once SNAP was simultaneously applied with 100 nM ryanodine. At high ryanodine concentrations, the RyRs were completely blocked and it was no possible to elicit Ca2+ transients even at higher or longer electrical stimulation. However, at 100 nM ryanodine, the RyRs are locked at a subconductance state and do not open to a full conductance state . Tau parameter was not different from NiCl2 solution with either SNAP or L-NAME. It is interesting to note that higher NiCl2 concentrations inhibit calcium transients, possibly because it blocked calcium release. We observed that only at 2 mM there was no such interference of NiCl2. These results suggest that SERCA is the main cytosolic Ca2+ removal protein in zebrafish myocytes, while NCX showed no measurable effect. Regarding the effect of NO on cytosolic Ca2+ removal, there were no significant differences in the tau parameter, suggesting that neither endogenous nor exogenous NO would modulate either NCX- or SERCA-dependent Ca2+ uptake.Based on the results obtained above Figure , we invel Figure . In mamml Figure  and it hl Figure  or via dl Figure ; however2+ transients described above could be mediated by NO-sGC pathway or via S-nitrosylation mechanism. In order to clarify this question, we analyzed the alterations of our proposed biophysical parameters under the effect of the sGC blocker, ODQ (10 μM). Moreover, we analyzed the effect of the S-nitrosylation blocker NEM (100 μM) on the same biophysical parameters. We observed that the sGC blockage with ODQ produced a similar effect as observed with L-NAME, suggesting that the effect of endogenous NO could be via the NO-sGC pathway. We wanted to exclude that this effect could also be related to NO-sGC-independent mechanisms evaluating whether the S-nitrosylation could contribute to the NO-mediated alterations of the biophysical parameters of the calcium transient. We observed that NEM, an S-nitrosylation blocker did not differ from control solution, except for Duration and Uptake time suggesting that S-nitrosylation via endogenous NO was not required for normal Ca2+ homeostasis. These two sets of data allowed us to propose that the main effect of NO on Ca2+ transients of 5–7 days old zebrafish skeletal muscle is via the NO-sGC pathway.The NO action on Ca2+ release from the SR via NO-sGC pathway, an effect probably mediated by protein kinase G (PKG) since this effect was antagonized by the action of NOS inhibitor or blocker of sGC. The next step would be to evaluate the targets and how PKG activation is able to decrease Ca2+ transient and thus force production in zebrafish skeletal muscle. Furthermore, in light of our results it would be of future interest to address the role of NO in other muscle pathways such as PKG activation as seen in skeletal muscle myofilaments (Beckendorf and Linke, In summary, our results support previous reports of the NO effect in mammalian skeletal muscle (Kobzik et al., ZX and MM: Contributed with design and performing all experiments, acquisition, analysis, and interpretation of data presented in the manuscript. He also drafted the manuscript and gave his final approval of the version to be published, agreeing with all aspects of the manuscript. RF: Contributed with interpretation of data for the work, drafting the manuscript and final approval of the version to be published and agreed with all aspects of the manuscript.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. The reviewer RLC and handling Editor declared their shared affiliation, and the handling Editor states that the process met the standards of a fair and objective review.