Atazanavir and lopinavir also inhibited OATP1B3‐ or NTCP‐mediated transport with a mean IC50 of 1.86 ± 0.500 μM or 65.6 ± 10.7 μM and 5.04 ± 0.0950 μM or 20.3 ± 2.13 μM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug–drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration–time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors  were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP‐mediated estrone 3‐sulfate transport or OATP1B1‐mediated estradiol 17β‐D‐glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC i values into mechanistic static equations, enabled successful quantitative prediction of clinical AUCRs. Predictions suggest the mechanisms driving DDI are principally inhibition of intestinal BCRP and hepatic OATP1B1, with minimal contribution from OATP1B3, NTCP or OAT3.This work sought to clarify the mechanisms underlying clinical DDIs between rosuvastatin and protease inhibitors. Investigating in vitro inhibition of the critical transporter disposition pathways of rosuvastatin, and subsequent incorporation of cell test system derived determined K AUCarea under the plasma concentration–time curveAUCRarea under the plasma concentration–time curve ratioBCRPbreast cancer resistance proteinmaxCmaximum plasma concentrationDDIdrug–drug interactionDMSOdimethyl sulfoxideDPMdisintegrations per minuteƒefractio n excreted valueHEK293human embryonic kidney 29350ICconcentration that produces 50% inhibitionOATorganic anion transporterOATPorganic anion transporting polypeptideNTCPsodium taurocholate cotransporting polypeptide1Statins are widely prescribed for the effective treatment of hypercholesterolemia and as a drug class are generally well tolerated in humans.rosuvastatin, which has been shown to be more effective in managing dyslipidemia in HIV patients than other statins and has one of the largest prescription frequencies amongst the statins.atazanavir, darunavir or lopinavir/ritonavir (Crestor® drug label)ƒe] values) have been determined previously.BCRP) efflux as the barrier to absorption , hepatic organic anion transporting polypeptide 1B1  uptake for hepatic elimination, and active renal secretion of rosuvastatin mediated by organic anion transporter 3  uptake. Other minor pathways that contribute toward the overall hepatic elimination of rosuvastatin (ƒe = 0.7) include OATP1B3 (ƒe = 0.11) and sodium taurocholate cotransporting polypeptide .ƒe = 0.7) was considered using a literature OATP1B1 Ki as presumed surrogate for inhibition of “overall active hepatic uptake”, did the mechanistic approach predict (AUCR = 3.25) the clinical observation.i values similar to the surrogate OATP1B1 value, and that inhibition of renal OAT3 does not contribute to DDI.A particular concern is the potential for DDI between protease inhibitors and the hydrophilic statin 50) of atazanavir versus BCRP and OATP1B1 and to investigate atazanavir as an inhibitor of the minor rosuvastatin disposition pathway transporters OATP1B3, NTCP and OAT3 in vitro. Using these data in the mechanistic static rosuvastatin model described by Elsby et al.Therefore, in order to test this hypothesis, the aims of study were to determine the inhibitory parameters . Hanks balanced salt solution , Dulbecco's modified eagle medium (Gibco™ DMEM: high glucose with GlutaMax and pyruvate for human embryonic kidney (HEK)293 transfected cells; Gibco™ DMEM: high glucose and pyruvate for Caco‐2 cells), fetal bovine serum , mammalian protein extraction reagent (M‐PER), bicinchoninic acid protein assay kit, and 45% glucose solution were purchased from Fisher Scientific . All other chemicals, solvents and reagents were purchased from Fisher Scientific.Estrone 3‐sulfate sodium salt, estradiol 17β‐D‐glucuronide sodium salt, sodium taurocholate hydrate, novobiocin, rifamycin SV sodium salt, cyclosporin A, pioglitazone hydrochloride, probenecid, lucifer yellow dilithium salt, sodium butyrate, non‐essential amino acids and HEPES were purchased from Sigma‐Aldrich . 2 surface area) and Millicell 96‐well transport analysis plates were purchased from Millipore . Caco‐2 cells were obtained from American Type Culture Collection. 24‐Well tissue culture treated microplates and various 96‐well v‐bottom or round‐bottomed polypropylene microplates were supplied by Fisher Scientific.Biocoat™ Poly‐D‐lysine 24‐well multiwell plates, human organic anion transporting polypeptide (OATP) 1B1 (SLCO1B1)‐, human OATP1B3 (SLCO1B3)‐, human sodium taurocholate cotransporting polypeptide ‐, and human organic anion transporter (OAT) 3 (SLC22A8)‐expressing HEK293 TransportoCells™ and vector control cells were supplied by Corning BV Life Sciences . Millicell‐96 multiwell cell culture insert plates  fetal bovine serum, 2 mM L‐glutamine, 1% (v/v) nonessential amino acids and 50 U mL−1 penicillin and 50 μg mL−1 streptomycin) which was changed every two or three days.The methodology used was adapted from the previously validated Caco‐2 unidirectional  BCRP inhibition assay.3H]‐estrone 3‐sulfate 3H]‐estrone 3‐sulfate appearing in the receiver compartment was determined by sampling (50 μL) into a white walled clear bottomed multiwell plate, to which scintillation fluid (200 μL) was added, and then samples counted on a Microbeta2 liquid scintillation counter (PerkinElmer). The quantified amount  of [3H]‐estrone 3‐sulfate was subsequently converted to concentration (pmol/mL) and used to calculate apparent permeability (Papp), as described previously.app value in the absence or presence of test inhibitor, to give a corrected BCRP‐mediated B‐A Papp, which was subsequently converted to percentage (vehicle) control transport activity. For each test inhibitor, determined percentage control transport activity was plotted against nominal inhibitor concentration and fitted using SigmaPlot 12.5  to determine the concentration that produces half‐maximal inhibition of probe substrate transport . The Papp of co‐incubated lucifer yellow across the cell monolayer was determined and cell monolayer integrity was deemed acceptable if Papp < 1.0 × 10−6 cm/s.The BCRP inhibition experiments were performed between day 18 to 22 post‐seeding. The cell monolayers were washed twice with pre‐warmed (37°C) transport buffer  before being pre‐incubated with transport buffer containing protease inhibitor drug or positive control inhibitor (novobiocin) on both sides of the cell monolayer for 30 min at 37°C. After pre‐incubation, transport buffer was removed and the appropriate donor solutions were added to the basolateral donor compartment. Donor solutions  in transport buffer contained the probe substrate ‐estradiol 17β‐D‐glucuronide, 0.02 μM; NTCP: [3H]‐taurocholic acid, 1 μM; OAT3: [3H]‐estrone 3‐sulfate, 1 μM) was determined  over a specified linear incubation time  at 37°C in SLC transporter‐expressing cells and vector control cells, in the absence and presence of protease inhibitor drug at a single tested concentration, or positive control inhibitor . The final dimethyl sulfoxide (DMSO) concentration was 0.1% (v/v). The chosen concentration of each drug represented ≥10x its unbound maximum hepatic inlet concentration for hepatic OATP1B3 and NTCP, or ≥10x its unbound maximum plasma concentration for renal OAT3. This translated to tested concentrations of 50 and 15 μM for atazanavir, 15 and 10 μM for darunavir, 5 and 3 μM for lopinavir, or 0.3 and 0.1 μM for ritonavir against OATP1B3/NTCP and OAT3, respectively. Following the incubation period, active transport processes were stopped by removing (via aspiration) the incubation solutions and immediately washing the cells twice with ice cold uptake buffer before then placing plates on ice. Following the wash steps, M‐PER (400 μL) was added to each well and cells were lysed for at least 5 min at 250 rpm on an orbital shaker. An aliquot (300 μL) of cell lysate was added to a white walled, clear bottomed 24‐well visiplate, liquid scintillation cocktail (2 mL) was added, and samples were counted on a Microbeta2 scintillation counter in order to determine the total radioactivity (DPM) taken up in cells. In parallel, the protein content of cell lysates (25 μL) was determined using a Bicinchoninic acid (BCA) protein assay kit according to the manufacturer's instructions.Uptake of probe substrate  was normalized to the protein (mg) content of each well to calculate the uptake activity (pmol/mg). Uptake activity of probe substrate into vector control cells was subtracted from that determined into transporter‐expressing cells to calculate the corrected transporter‐mediated uptake. Corrected uptake activity (pmol/mg) was subsequently converted to percentage (vehicle) control transport activity.2.43H]‐estradiol 17β‐D‐glucuronide (0.02 μM) incubated for 2 min, with rifamycin SV (100 μM) as positive control inhibitor. For IC50 determinations, the only difference to the method performed above was that the 15‐min pre‐incubation step contained a range of six concentrations of protease inhibitor drug, and subsequent incubations were conducted with the same six concentration levels of drug rather than with a single concentration. All four protease inhibitor drugs were studied against OATP1B1 using pre‐incubation/incubation concentrations of either 0.1, 0.3, 1, 3, 10 and 30 μM for atazanavir and darunavir, or 0.03, 0.1, 0.3, 1, 3 and 10 μM for lopinavir and ritonavir. Based upon the results determined from the inhibition screen, only atazanavir and lopinavir were studied against OATP1B3 or NTCP using concentrations of 0.1, 0.3, 1, 3, 10 and 30 μM and 0.03, 0.1, 0.3, 1, 3 and 10 μM, or 0.3, 1, 3, 10, 30 and 50 μM and 0.1, 0.3, 1, 3, 10 and 20 μM, respectively.OATP1B1, OATP1B3, NTCP and vector control cell lines were cultured and subsequent transporter assays conducted as described above using the same transporter‐specific probe substrates and incubation times. For OATP1B1, the probe substrate was  = maximum enterocyte concentration (Ig = (FaFg × ka × dose (mol))/Qent) for intestinal transporters,max u = fu × Cmax) for renal transporters.u = unbound fraction in plasma, Cmax = maximum total plasma concentration of inhibitor at steady state, FaFg = fraction of the dose absorbed after oral administration, ka = absorption rate constant (min−1), Qh = hepatic blood flow (1617 mL/min), RB is the blood‐to‐plasma concentration ratio (default = 1.0) and Qent = enterocyte blood flow (300 mL/min).The mean ICEquation  previousEquation , and OAT2.6http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY,Key protein targets and ligands in this article are hyperlinked to corresponding entries in 33.13H]‐estrone 3‐sulfate (1 μM) with a determined mean (±SD) IC50 value of 42.2 ± 10.2 μM (Table gut] (dose (mol)/250 mL)/Ki value of 40 (1702/42.2), 31 (4382/143), 164 (2545/15.5) or 22 (555/25.4), respectively, indicating the potential for interaction in vivo (≥10) perpetrated by all drugs. The positive control inhibitor, novobiocin (0.1–100 μM) gave acceptable inhibition (mean IC50 of 1.81 ± 0.276 μM) in the test system.Atazanavir, darunavir, lopinavir or ritonavir demonstrated reproducible inter‐assay concentration‐dependent inhibition of BCRP‐mediated transport of [‐sulfate  μM with ‐sulfate  μM with ‐sulfate  μM with ‐sulfate  μM with ‐sulfate  μM with 3.23H]‐estradiol 17β‐D‐glucuronide (0.02 μM) with a determined mean (±SD) IC50 value of 0.734 ± 0.177 μM  transport when tested at concentrations greater than or equal to ten times their unbound [Cmax] , or NTCP‐mediated transport of [3H]‐taurocholic acid (1 μM), with mean (±SD) IC50 values of 1.86 ± 0.500 μM, or 65.6 ± 10.7 μM, respectively  and 20.3 ± 2.13 μM (R = 1.02), respectively  was also considered. For rosuvastatin, the critical pathways that culminate in its hepatic elimination in order of importance  are OATP1B1, NTCP and OATP1B3. Consequently, it was necessary to assess atazanavir as an inhibitor of BCRP, OATP1B1, NTCP, OATP1B3 and OAT3  to establish whether these additional pathways need to be incorporated into the mechanistic model to more accurately predict observed AUCR, aiding confirmation of the mechanism underpinning the DDI. To understand whether a similar mechanistic scenario might exist for other rosuvastatin‐protease inhibitor DDIs, darunavir, lopinavir and ritonavir's inhibitory properties were also assessed and used in predictions for confirming their mechanisms of DDI.When considering the victim rosuvastatin, a mechanistic static model incorporating inhibition of critical disposition pathways intestinal BCRP and hepatic OATP1B1, correctly predicted 91% of clinical DDIs.50 value for prediction of risk. However, the reality is that this is often not the case for practical reasons , but predominantly because the pharmaceutical industry (through regulatory guidance) generally favors the use of a single established in vitro “surrogate” probe substrate to evaluate routine inhibitory liability against specific transporters and metabolizing enzymes that can be subsequently applied to holistically risk assess perpetrators against multiple victim drugs. With respect to each specific transporter, in line with regulatory recommendations (since inhibition can be substrate‐dependent), the chosen in vitro probe substrate should either be clinically relevant to observed DDIs, a good surrogate of a clinically relevant in vivo substrate, or one that generates a lower IC50 for known inhibitors to avoid underestimating interaction potential.50 variation when assessing inhibition potential.m value at the transporter so that the calculated IC50 values equated to Ki values. Consequently, as such inhibition constants are widely accepted to be independent of substrate, then this ensures the right inhibitory parameter value is being determined for accurate DDI prediction. In addition, inclusion of a pre‐incubation step with a range of concentrations of inhibitor  acts to ensure that any adsorption of inhibitor to plasticware during the assay (were it to occur) is mitigated due to the masking of non‐specific binding sites prior to performing the co‐incubation with fresh inhibitor solution and probe substrate. Therefore, the final concentrations of inhibitor would be anticipated to remain as nominal further ensuring the accuracy of the determined Ki value.In an ideal world, assessment of inhibition potential would utilize the victim drug of the DDI under investigation  as the in vitro probe substrate in order to determine the clinically relevant ICi values to those obtained with rosuvastatin.i values for the known BCRP inhibitors novobiocin, Ko143, cyclosporin A, pantoprazole, sulfasalazine, atorvastatin, diclofenac, fluvastatin and nifedipine obtained in the previous validation with corresponding literature values derived from inside‐out membrane vesicles expressing BCRP,i is the correct one for assessing DDI risk at BCRP.For assessment of inhibition of BCRP function, the present study has utilized the regulatory industry gold standard methodology involving the polarized Caco‐2 cell monolayer test system, which has previously been extensively validated to correctly identify inhibitors of BCRP using rosuvastatin as probe substrate.50 (Ki) values were within 1.3‐ to 2‐fold of published literature values for atazanavir, darunavir, lopinavir or ritonavir (Tables 50 (Ki) values were also within 1.2‐ to 2‐fold of previous studies that utilized either the same recommended surrogate OATP1B1 in vitro probe substrate estradiol 17β‐D‐glucuronideThe present study confirmed that all the protease inhibitor drugs investigated inhibited BCRP and OATP1B1 with the same rank order of inhibitory potency of lopinavir > ritonavir > atazanavir >> darunavir at both transporters. The determined mean BCRP ICr Tables ,13, 1max u]/Ki ≥0.1 or R=1+[Iin max u]/Ki ≥1.1),50 (4.7 μM) was reported for ritonavir, this value is 47‐times higher than the clinically relevant concentration (0.1 μM) tested in this study. This confirms that OAT3 perturbation is not involved as a mechanism in the observed DDIs between rosuvastatin and these protease inhibitors. With respect to NTCP and OATP1B3, neither darunavir nor ritonavir inhibited these transporters at clinically relevant hepatic concentrations confirming that these pathways are also not involved in the mechanism of their respective rosuvastatin DDIs. Conversely, both atazanavir and lopinavir inhibited OATP1B3 and NTCP in the current study. The determined mean OATP1B3 IC50 (Ki) value for atazanavir was 3.9‐fold lower than previously published versus atorvastatin as probe substrate.trans‐inhibition alongside cis‐inhibition, then the 2014 study would only reflect cis‐inhibition, whereas the inclusion of the pre‐incubation step in this study would also account for any additional effect of trans‐inhibition on determined IC50 value. This same explanation likely explains the disconnect between the present study and that of Vildhede et al.For investigations involving OAT3, NTCP and OATP1B3, initial inhibition experiments were performed using a single inhibitor concentration screening format in which each of the protease inhibitor drugs were assessed at a concentration that was ≥10‐times either their systemic unbound maximum plasma concentration  or their unbound maximum hepatic inlet concentration (for hepatic NTCP and OATP1B3). These drug‐specific concentrations were chosen so that in the absence of any observed inhibition in vitro, based on regulatory basic static equation cut‐offs , despite the fold increase due to OATP1B1 (1.49) being close to its theoretical maximum (1.6‐fold) and indicating that there is around 80% inhibition of the transporter. This is likely the result of not accounting for the modest effect on AUCR of inhibition of OATP1B3 and NTCP by atazanavir. Whilst small, the predicted AUC increase of 1.09‐fold due to OATP1B3 inhibition actually represents 75% inhibition of this transporter's function , whereas the predicted AUCR due to NTCP inhibition (1.01) only reflects a 4% reduction in function. For these hepatic transporters, the magnitude of inhibition by atazanavir directly correlates with the calculated R‐value. To calculate a predicted AUCR due to combined inhibition of OATP1B1, OATP1B3 and NTCP by atazanavir it is not appropriate mathematically to simply multiply the individual AUCR values together because conceptually they are not isolated clearance pathways, rather they contribute to the overall hepatic elimination pathway of rosuvastatin. Therefore, it is this combined Whilst a similar effect with regard to inhibition of OATP1B3 and NTCP was observed with lopinavir, the individual predicted AUCR values are minimal at 1.01 and 1.00, respectively, which is consistent with the R‐values being below threshold. Nevertheless, despite the suggested lack of interaction, inhibition of both transporters still contributed to overall DDI via all hepatic uptake, as evidenced by the small increase in cumulative AUCR compared with that from OATP1B1 alone. When this cumulative prediction was combined with that due to BCRP, the overall predicted AUCR (2.16) matched the clinical AUCR. However, predictions based solely on BCRP and OATP1B1 (AUCR = 2.12) also match the clinical observation, suggesting that for lopinavir, the driving mechanism of DDI is simply inhibition of intestinal BCRP and OATP1B1 as previously reported.i determined here (reducing predictions from 1.33 to 1.20). Finally, whilst ritonavir is a potent inhibitor of both BCRP and OATP1B1 in vitro, it only flags for the potential to cause DDI in vivo through inhibition of intestinal BCRP, whereas due to a combination of low dose and low unbound exposure, it is not predicted to cause DDI through OATP1B1. The DDI risk due to BCRP inhibition is diminished further when enterocyte, rather than theoretical lumen (used in guidance), concentration is considered in the mechanistic model, thereby resulting in a predicted AUCR of only 1.03. When combined, the overall AUCR is predicted to be 1.04 indicating that ritonavir is unlikely to cause a clinical DDI with rosuvastatin if administered in isolation. In clinical practice however, ritonavir is coadministered to boost the absorption of a second protease inhibitor through inhibition of intestinal enzymes.In the case of darunavir, since it is not an inhibitor of OATP1B3 or NTCP, the mechanism for perpetrating its DDI is principally inhibition of BCRP and, to a lesser extent, inhibition of OATP1B1. The AUCR predictions in this study are marginally lower than that of Elsby et al.In conclusion, determined transporter inhibitory parameters and use of mechanistic static model predictions have successfully indicated that the clinical DDIs perpetrated by atazanavir, darunavir and lopinavir with rosuvastatin are driven by inhibition of intestinal BCRP and hepatic OATP1B1 with minimal contribution from OATP1B3, NTCP and OAT3. Furthermore, the use of mechanistic models for quantitative DDI prediction may help illuminate the involvement of additional pathways in DDI where predictions do not reconcile the clinical observation.Participated in research design: Atkinson, Coghlan, Edgerton, Elsby, Hodgson, Outteridge. Conducted experiments: Coghlan, Edgerton, Hodgson. Performed data analysis: Atkinson, Coghlan, Edgerton, Elsby, Hodgson, Outteridge. Wrote or contributed to the writing of the manuscript: Atkinson, Coghlan, Edgerton, Elsby, Hodgson, Outteridge.The authors are all employees, or a former employee (HC), of Cyprotex Discovery Ltd (an Evotec Company) which is a contract research organization.


Here, we investigated whether prolonged β-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that ‘dispersion’ of RyR clusters disrupts Ca This process of Ca2+-induced Ca2+ release (CICR) depends on the precise organization of LTCCs and RyRs within dyadic junctions of the two membranes, with LTCCs located in nanoscale apposition to RyRs across the dyadic cleft (2+ release units (CRUs) to generate Ca2+ sparks , and considerable data have linked these phenomena to t-tubule disruption in this condition . Recentlhronized . Similarhronized  and in mhronized . Thus, a2+/calmodulin-dependent protein kinase II (CaMKII)- and protein kinase A (PKA)-dependent phosphorylation of RyR disrupt its function during HF  for varying lengths of time . For the control group, cells were kept for 60 min following isolation prior to fixation for imaging. 3D dSTORM reconstructions revealed progressive RyR cluster dispersion as the duration of isoproterenol application increased . Indeed,We next performed 3D correlative imaging of RyRs and the t-tubular network to distinguish between dyadic and non-dyadic (‘orphaned’) RyRs . Based o2+ release from the SR  animals after isoproterenol treatment  or genetically ablated (S2814A). We found that the RyR arrangement in the phosphomimetic S2814D mutant cells reatment . Indeed,reatment , CRU sizreatment , and clureatment  were allreatment . In contreatment . Adminisreatment  and incrreatment . These freatment . Taken tFollowing β-AR stimulation, increased cyclic AMP levels lead to PKA-dependent phosphorylation of the RyR at ser-2808 . Based oTo further validate an involvement of PKA, we quantified RyR arrangement following application of the selective PKA activator 6MB-cAMP  . As illu2+ sparks since these events occur almost exclusively at dyads  and t-tubules , we generated four computational CRU geometries that were representative of the range of RyR dispersion observed in cells . For eac2+ release event was classified as being an observable spark if its ΔF/F0 was ≥ 0.3. However, we also noted the occurrence of smaller modeled release events not observable in experiments. These sub-spark events were characterized as Ca2+ quarks (0 < 0.3) or failed sparks if their Ca2+ release was negligible . These results suggest that when CRUs become markedly dispersed during chronic β-AR activation, both spark-mediated and silent leak contribute to declining SR content . Here, the antiarrhythmic actions of RyR dispersion appear to be linked to a reduction in SR content resulting from an increase in both spark-mediated and silent leak. We postulate that local regions of the cell with dispersed CRUs and lowered SR content may act as a physical barrier or ‘fire break’ to impede Ca2+ wave propagation.Taken together, the modeling results critically link changes in CRU configuration and spark morphology; phenomena that were observed separately in experiments. However, the modeling data also support the contention from experimental data that RyR cluster fragmentation can reduce arrhythmogenic spontaneous CaWe next examined whether dispersion of RyRs reported during HF  similarl3D dSTORM imaging of Sham and HF cardiomyocytes  revealed2+ release during HF could be linked to RyR phosphorylation and dispersion. Representative recordings of Ca2+ sparks are presented in 2+ transients were of lower magnitude in failing cardiomyocytes . Once the heart had been cleared of blood, perfusion was switched to the same solution containing collagenase type II  for 10–12 min at 37°C. Following digestion, left ventricular tissue was dissected and finely cut into 3–4 mm3 pieces. A secondary digestion was performed to liberate additional cells from tissue by transferring approximately 8 mL of tissue and collagenase solution to a 10 mL Falcon tube containing 0.2 mg DNase  in 500 µL bovine serum albumin (BSA). Cells were subsequently filtered and allowed to pellet in 0.2 mmol/L Ca2+.Isolation of cardiomyocytes was based on the protocol previously described by Isolated cardiomyocytes were washed with Dulbecco’s phosphate-buffered saline (PBS) , fixed with 4% paraformaldehyde for 10 min, quenched with 100 µmol/L glycine for 10 min, and permeabilized with 1% Triton X-100 for 10 min. The cells were then plated on glass-bottom dishes  that had been coated with laminin  and blocked using Image-iT FX Signal Enhancer (Thermo Fisher Scientific) prior to immunolabeling.3 in PBS.For RyR visualization, cells were incubated with mouse anti-RyR  overnight at 4°C. For t-tubule imaging, cells were incubated overnight at 4°C in a mixture of rabbit anti-Cav-3  antibody and a custom rabbit anti-NCX1 antibody as previously described   diluted in Tris-glycerol . This composition has been previously shown to produce comparable, if not superior, quality of dSTORM images compared with conventional oxygen scavenging-dependent systems .Cardiomyocytes were imaged using the Zeiss ELYRA/LSM 710 system . A diode laser  illuminated the sample via a plan-apo 63 × 1.4 NA oil objective, configured in a highly inclined and laminated optical sheet (HiLo). 3D imaging was achieved utilizing Phase Ramp Imaging Localization Microscopy (PRILM) technology . FluoresReconstruction of dSTORM data was carried out using the ‘PALM Processing’ module in the ZEN Black software (Zeiss). In short, an experimental 3D PSF with an axial range of 4 µm was acquired using 100 nm TetraSpecks . Individual single-molecule events were detected by employing an 11 pixel circular mask, with a signal-to-background noise ratio of 6. Drift correction was performed using a five-segment piecewise linear function, and a text-based points table was then generated containing the x, y, and z coordinates of each localization event. In order to minimize the inclusion of clusters with larger localization error, events from only the central 600 nm of the 4 µm stack were included . Lastly,3D quantification of RyR organization was performed as previously described . In shorThe Zeiss ELYRA system was also used for confocal imaging of RyRs  and t-tubules  for 3D correlative reconstruction of dyads. Prior to dSTORM imaging of RyRs, a 3-µm-thick confocal stack centered on the same region was acquired . Deconvolution of confocal images was performed using Huygens Essential software , with a signal-to-noise ratio of 5. Using the confocally imaged RyR channel as a reference, confocally measured t-tubule distributions were correlated to dSTORM-derived RyR positions. This was done with a custom-written Python script that corrected for both lateral, axial, translational, and scaling differences between the two imaging modalities to optimally align the dSTORM-derived RyR data with the confocal RyR data .To examine the arrangement of RyR clusters in relation to t-tubules, 3D volumetric geometries were constructed from the correlated images of these structures using the same approach as described in our earlier work . GeometrA 3D reconstruction of the t-tubule network was created from confocal NCX1/Cav-3 imaging by first fitting the data to 10 nm × 10 nm × 10 nm voxels, binarizing using an adaptive Gaussian threshold, and then pruning small non-contiguous components (<0.03 µm³). The resulting thresholded structures were skeletonized using a 3D skeletonization algorithm , and re-To render the 3D geometric reconstructions and produce geometric meshes for use in mathematical modeling, specific locations of individual RyRs were assigned by projecting the thresholded dyadic cluster data onto a cylindrical shell surrounding the t-tubules, leaving a 1-voxel-wide (10 nm) dyadic cleft. These overlap interfaces were then randomly filled with RyRs such that no channels were closer than a center-to-center distance of 30 nm. As shown in our earlier work, this approach yields RyR numbers that are in good agreement with our experimentally obtained estimates .To visualize the reconstructed geometry of t-tubules and RyRs, iso-surfaces were generated from the full geometries using the Lewiner Marching-Cubes algorithm , smoothe−12 µL, which is in good agreement with the values reported by electron microscopy tomography  terminals for each cluster were defined by iterative morphological binary dilation of the RyRs constrained to a 20-nm-wide cylindrical shell surrounding the t-tubules. Dilation was performed with a structuring element with a square connectivity of one for nine iterations. This method was chosen because it produces a jSR that evenly surrounds the RyR channels in the cluster, defines a well-constrained dyadic cleft space, and has a jSR volume on the order of 3–4 × 10mography . The nonmography  and serv2+ release. Roughly 1000 such CRU geometries were extracted and sorted based on the number of contained RyRs, their spatial spread (measured as the root mean square of their distance to the cluster center), and the number of individual clusters within the CRU. Four representative geometries were selected containing roughly the same number of RyRs, but with different degrees of compactness. These ranged from a completely solid CRU to a majorly dispersed CRU consisting of numerous, sprawled clusters  and superfused with HEPES-Tyrode solution containing (in mmol/L): 140 NaCl, 0.5 MgCl2, 5.0 HEPES, 5.5 glucose, 0.4 NaH2PO4, 5.4 KCl, and 1.8 CaCl2 . Cardiomyocytes were scanned along a 1024 pixel line drawn across the cell’s longitudinal axis at a temporal resolution of 1.5 ms for a total duration of 6 s. Ca2+ sparks were analyzed using a custom program  as previously described (0 ≥ 0.3). Spark characteristics outputted by the program included amplitude (ΔF/F0), TTP, full duration at half maximum (FDHM), and full width at half maximum (FWHM). Spark frequency was determined by normalizing spark count to the cell length and recording duration. Spark-mediated leak was calculated as the product of spark amplitude, FDHM, and FWHM.Using an LSM 7Live confocal microscope (Zeiss), spontaneous Caescribed , with sp2+ transients were similarly recorded by confocal line-scans in fluo-4-loaded cells, but during field stimulation via a pair of platinum electrodes (3 ms supra-threshold current pulses at 1 Hz). As previously described  across the cell and measuring the standard deviation of the values. We have termed this measure the ‘dyssynchrony index.’ Spontaneous Ca2+ waves were measured during pauses in the electrical excitation, and wave frequency was normalized to the recording duration. SR Ca2+ content was estimated by measuring the increase in cytosolic Ca2+ fluorescence after superfusing cardiomyocytes with 10 mM caffeine (Sigma-Aldrich). SERCA activity was estimated as the difference between the rate constants (1/τ) of the decline of steady-state Ca2+ transient (1 Hz) and the caffeine-induced Ca2+ transient (1/τ 1 Hz − 1/τ caffeine) acquired in the same cell  with the addition of a cOmplete EDTA-free protease inhibitor cocktail tablet  and a PhosSTOP tablet (Roche). SDS was then added to the homogenates to a final concentration of 1%, and protein concentrations were quantified using a micro BCA protein assay kit . BSA was used as standard protein.Protein homogenates (5 or 15 µg/lane) were size-fractionated on 4–15% or 15% Criterion TGX gels  and transferred to 0.45 μM PVDF membranes . The membranes were blocked in 5% non-fat milk or 5% Casein (Roche Diagnostics) in Tris-buffered saline with 0.1% Tween (TBS-T) for 1 hr at room temperature, and then incubated with primary antibody overnight at 4°C. The following primary antibodies were employed for immunoblotting: RyR2 (1:1000) , pSer2808 RyR2 (1:2500) , pSer2814 RyR2 (1:2500) , and GAPDH . Secondary antibodies were anti-rabbit  or anti-mouse . These were incubated for 1 hr at room temperature, and blots were developed using Enhanced Chemiluminescence . Chemiluminescence signals were detected with a LAS 4000 , and protein levels were quantified using ImageQuant software . Results were normalized to GAPDH and then to sham values.In order to examine the relative phosphorylation levels of isoproterenol-treated cardiomyocytes, we employed fluorescence-activated cell sorting. This technique has previously been shown to enable protein quantification in close correlation with results from Western blotting . Fixed cFollowing sorting, the secondary Alexa Fluor 647 antibody  in the single cardiomyocyte fraction was excited with the 638 nm laser and emission detected by a photomultiplier tube with a 665/30 band-pass filter. Data are presented as detected fluorescence intensity as a product of cell size. Confirmation of cell viability was performed using LIVE/DEAD Fixable Violet Dead Cell Stain (Thermo Fisher), excited with the 405 laser and emission detected via a 450/50 band-pass filter.2+ spark characteristics were interrogated using a mathematical model of Ca2+ release in the dyad. This previously described reaction-diffusion model includes independent, stochastic RyR channels coupled with deterministic Ca2+ diffusion and buffering . For simulations where the peak amplitude was below this threshold, we defined sub-spark events as ‘quarks’ (0 < 0.3) or ‘failed sparks’ if Ca2+ release was negligible (ΔF/F0 ≤ 0.1).For each simulation, we measured the peak amplitude of the Fluo signal and the rise time (TTP). Based on the measured amplitude, we then determined which simulations to consider sparks using the same threshold as in the experimentally measured Ca‘quarks’  , with Fisher’s least significant difference post-hoc comparison where appropriate. p-Values<0.05 were considered to be significant.Cardiomyocytes from rats were randomly selected for analysis of RyR localization and Cahttps://gitlab.com/louch-group/ryr-tt-correlative-analsyis  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Prolonged β-Adrenergic Stimulation Disperses Ryanodine Receptor Clusters in Cardiomyocytes: Implications for Heart Failure" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Although a significant number of suggestions are made, these are meant to be constructive towards the enhancement of your paper.Essential revisions:The reviewers make the following broad points:Reviewer 1 would like you to consider the following suggestions:First, in the absence of a control with RyR constitutively active at the S2808 (PKA) phosphorylation site, it would be interesting to see if direct activation of PKA using 6MB-cAMP is sufficient to induce disruption of the RyR-S2814A. Conversely, the ability of PKA inhibition to reverse the ISO-induced RyR disruption of RyR-S2814D could also be tested.Along similar lines, after prolonged stimulation of β-AR, or in failing cardiomyocytes inhibition of either CaMKII or PKA partially reverses RyR disruption and CICR disruption. Have you tested whether the disruption is completely reversed by the simultaneous inhibition of both?The link between β-AR stimulation and phosphorylation-dependent RyR dispersion in heart failure is assumed rather than tested (line 393). I note that care has been taken not to assert this too strongly and that phosphorylation-dependent RyR dispersion is the key claim, but I am curious to know if considered any ways to test it (perhaps by running experiments in post-infarction cells where β-AR has been inhibited)? I leave this as an open question as it isn't clear to me how best to do this and it is perhaps not trivial, possible, or necessary!Reviewer 2 makes the comments that:– A critical control is missing to demonstrate the effects of 2 hrs ISO treatment on RyR2 arrangements. This will strengthen the conclusions from the CaMKII and PKA inhibitor RYR2 dispersion reversal experiments which utilize a protocol that includes a 1 hr ISO stimulation followed by a 1Hr ISO + inhibitor treatment.– The mathematical modeling results would also benefit from more explanation of how they complement, align, and expand on the experimental studies.Reviewer #1 (Recommendations for the authors):The reviewers also make the following major points:1. A brief explanation of how CRU fragmentation would be reflected by changes to the reported dSTORM parameters  at the start of the Results section would help to orient the reader.2. Reconstructions of representative CRUs are presented in Figure 1, 2, and 3, with selected CRUs inset as compelling visual illustrations of CRU fragmentation or reformation. It would be beneficial to allow the reader to decide for themselves if they are truly representative by stating their dSTORM parameter values in the figure or legend. This is especially true where single representative CRUs are presented as in Figure 2a-c and Figure 3a and b.3. On line 192 the authors state 'We found that the RyR arrangement in the phosphomimetic S2814D 193 mutant cells at baseline resembled the fragmented RyR organization observed in wild type (WT) 194 animals after isoproterenol treatment . Indeed, cluster size , CRU size , and 195 cluster density  were all similar in WT + ISO and S2814D cardiomyocytes, and significantly 196 different from untreated WT cells. Furthermore, application of isoproterenol to S2814D cardiomyocytes 197 did not lead to significant additional RyR dispersion, and only tendencies toward smaller cluster and 198 CRU sizes .'The representative CRU reconstructions in Figure 2g and h seem to contradict the quantified dSTORM parameters in Figure 2h, j-l and the description of the results in the text (starting at Line 192). This indicates a possible error in the production of the figure or that the choice of representative CRUs could be improved. In the images, the S214D CRUs appear more similar to wt (aside from the CRU selected for the insert) rather than wt + ISO as expected from the plotted data. There also appears to be significantly greater disruption visible in the S2814D + ISO than S2814D as a result, again counter to the observation made in the text about the plotted data.4. There is a minor discrepancy between the naming of CaMKII mutants in Figure 2. panels (2814D and 2814A) and the text (S2814D and S2814A).5. A more precise description of the source of the S2814D and S2814A cardiomyocytes is needed for the reader to fully understand the strength of these control experiments. The authors currently state in the text and Materials and methods section that they are isolated from transgenic RyR2-S214D and RyR2-S214A mice provided by the Wehrens lab. The paper reference provided is only partially helpful as it describes the generation of homozygous knock-in mice RyR2-S214A but makes no mention of RyR2-S214D. I would assume that both are isolated from homozygous knock-in mice but please confirm if this is indeed the case and state it explicitly in the manuscript.2+ spark and wave properties as 'Mean data' and it is not clear what this means. It might be less confusing simply to use the term 'Experimental data' instead.6. Figure 2, 3, 4, and 6 legends refer to various bar charts of quantified dSTORM parameters and ca7. Connected to the previous point, the bar charts in Figure 1, 2, 3, 4, and 6 are nicely presented so that the mean, SEM, and variation in the datapoints of a given variable are clear. However, it is not always obvious whether each datapoint represents individual measurements or a cell average and this information should be stated clearly in either the legends or methods section.2+ leak is calculated from spark mass and frequency is missing from the Materials and methods section.8. A brief description of how spark-mediated ca9. Figure 5c-f: These panels seem to have shared keys for the data represented by unshaded and shaded bars that only appear in the final panel . It would be clearer if the difference between unshaded and shaded was described in the figure legend with reference to the applicable panels. The same applies to Figure 6f-g.10. The Ethical approval section of Materials and methods needs to be checked for references to other journals.Reviewer #2 (Recommendations for the authors):11. Supplementary Tables 1-3 would benefit from brief explanations of their contents.1) The authors report that a 1 hr ISO stimulation followed by a 1 hr ISO stimulation in the presence of AIP led to a reversal of the ISO-induced RyR2 dispersal and concluded that CaMKII thus mediated RyR2 cluster dispersion downstream of B-AR stimulation. However, in this protocol, the cells are fixed after a 2 hr total exposure to ISO and it is unknown what this duration of ISO treatment does to RyR2 clustering and distribution. To increase confidence in these findings, an important control must be added. RyR2 clusters should be examined after 2 hrs ISO treatment (without any inhibitors on board). Likewise, a similar protocol is employed to test the effects of PKA on cluster dispersal, with the authors reporting that H89 reversed the dispersal. Again, this finding would be strengthened by knowing what 2 hrs of ISO does to RyR2 clustering and distribution. The same 2 hr total ISO stimulation protocol is used when the authors assess whether H89 or AIP can reverse the slowing in spark kinetics. To strengthen confidence in this finding, spark kinetics should also be examined after 2hrs of ISO treatment. The results may be quite different from those seen after a 1 hr treatment especially given the finding that RyR phosphorylation at ser-2814 and ser-2808 both exhibited bell-shaped responses during the 1 h ISO stimulation. If one extends that treatment to 2 hrs, phosphorylation of S2814 and S2808 may continue to fall and this complicates the findings with the inhibitors.2) At each ISO-treated time point, the study would benefit from the inclusion of an accompanying no-ISO control. Does the RyR dispersal occur because the cells are deteriorating with more time post-isolation, or is it specifically and exclusively because of the acute ISO-induced stress? These controls would help address that question and strengthen the conclusions of the paper.3) There is no indication of statistical significance on any of the summary data plots or mention of P values. This should be addressed.2+ leak shifts from spark-mediated to silent leak when CRUs are dispersed, reducing the fidelity of spark generation" but in figure 4h, spark-mediated (non-silent) leak is strikingly increased after 60 mins of ISO treatment which leads to the most profound dispersal. Please clarify how these results align. Perhaps there is more of both spark-mediated leak and non-spark leak? What experimental data supports non-spark mediated leak? The idea that there could be localized regions of SR ca2+ depletion that act as "fire-breaks" to prevent the generation of ca2+ waves even though there are more ca2+ sparks is logical and provides a satisfying theory that unifies some of the seemingly paradoxical findings but this is only raised in the second to last paragraph of the discussion. Raising this idea earlier may help the reader connect the dots more easily.4) The modeling results need to be put into context and compared with what is going on in the experimental data. This section is somewhat confusing and is a bit of a jarring shift away from the experimental section. It should be better integrated. The authors state "the modeling results indicate that RyR ca Essential Revisions (for the authors):The reviewers make the following broad points:Reviewer 1 would like you to consider the following suggestions:First, in the absence of a control with RyR constitutively active at the S2808 (PKA) phosphorylation site, it would be interesting to see if direct activation of PKA using 6MB-cAMP is sufficient to induce disruption of the RyR-S2814A. Conversely, the ability of PKA inhibition to reverse the ISO-induced RyR disruption of RyR-S2814D could also be tested.Thank you for these suggestions for these additional control experiments, which we have now performed. As expected, 6MB-cAMP remained effective in dispersing RyR clusters in RyR-S2814A cells, supporting that 6MB exerts its actions independent from the CaMKII phosphorylation site on RyR. For the second experiment, we believe you meant to suggest applying PKA inhibition (H89) together with ISO treatment in the S2814A cells, not S2814D cells. This experiment would then show that H89-induced inhibition of RyR dispersion is mediated via PKA inhibition without contribution of the 2814 site. Performing these experiments confirmed this hypothesis. We have added these new data to the bottom of Figure 3, and included the following addition to the main text:Page 4, line 228:“Importantly, 6MB-induced RyR dispersion in S2814A cardiomyocytes  which was comparable to that observed in WT. Similarly, dispersion observed in S2814A cardiomyocytes treated with isoproterenol was reversed by the addition of H89 . These observations support that 6MB and H89 treatments exerted their effects on RyR configuration via modulation of PKA activity, without contribution from the CaMKII phosphorylation site on the RyR. Taken together, these data indicate that prolonged PKA-dependent phosphorylation of RyRs is sufficient to drive RyR cluster fragmentation, but that both PKA and CaMKII concertedly drive this process during β-AR stimulation.”Along similar lines, after prolonged stimulation of β-AR, or in failing cardiomyocytes inhibition of either CaMKII or PKA partially reverses RyR disruption and CICR disruption. Have you tested whether the disruption is completely reversed by the simultaneous inhibition of both?2+ sparks. We have now tested the combined actions on AIP and H89 in ISO-treated cells. As expected, we observed a significant further reversal of the RyR dispersion induced from ISO treatment . Similarly, we observed a tendency for further reversal of spark characteristics when AIP and H89 were combined .The reviewer is correct that inhibition of CaMKII or PKA individually did not fully reverse the effects of ISO treatment on RyR configuration and caWe have added text accordingly to two parts of the manuscript:Page 3, Line 216:“As in experiments investigating effects of CaMKII inhibition , it should be noted that PKA inhibition did not fully restore the RyR configuration at the CRU level. Indeed, simultaneous treatment of cardiomyocytes with AIP and H89 resulted in further reversal of the effects of isoproterenol on CRU size, beyond those observed with either agent alone . These observations are consistent with summative roles of PKA and CaMKII activation in promoting RyR dispersion”Page 4, Line 252:2+ spark properties beyond values observed at the 1 h time point . Taken together, these data indicate that, during prolonged β-AR stimulation, RyR dispersion induced by PKA and CaMKII activity is linked to slowing of ca2+ spark kinetics and increased ca2+ leak”“A tendency toward additive effects was noted when AIP and H89 treatments were combined . In control experiments, a 2 h treatment period with isoproterenol alone  did not alter caThe link between β-AR stimulation and phosphorylation-dependent RyR dispersion in heart failure is assumed rather than tested (line 393). I note that care has been taken not to assert this too strongly and that phosphorylation-dependent RyR dispersion is the key claim, but I am curious to know if considered any ways to test it (perhaps by running experiments in post-infarction cells where β-AR has been inhibited)? I leave this as an open question as it isn't clear to me how best to do this and it is perhaps not trivial, possible, or necessary!The Reviewer is correct in stating that the link between β-AR stimulation and phosphorylation-dependent RyR dispersion in heart failure is only assumed in our study. Although considerable previous work has supported the presence of a hyper-sympathetic state in heart failure, including in post-infarction rats , we have not tested effects of reducing sympathetic tone on RyR configuration. We have now clarified this point, and edited the following sentence in the Results section :“The striking similarity of these findings to experiments employing prolonged isoproterenol exposure  supports that fragmentation of RyR clusters during HF results from increased activation of both CaMKII and PKA, likely as a consequence of the chronic β-AR stimulation that is well established in this condition .”We have also raised this point in the Discussion, where we mention the importance of examining effects of β-blockers in future heart failure studies :2+ signaling impairments in experimental HF, and establish the reversibility of this relationship by the application of β-blockers.”“Future work should be aimed at directly linking β-adrenergic tone, RyR dispersion, and caReviewer 2 makes the comments that:– A critical control is missing to demonstrate the effects of 2 hrs ISO treatment on RyR2 arrangements. This will strengthen the conclusions from the CaMKII and PKA inhibitor RYR2 dispersion reversal experiments which utilize a protocol that includes a 1 hr ISO stimulation followed by a 1Hr ISO + inhibitor treatment.Your point is well taken that the total treatment time for ISO + AIP  or ISO + H89  is 2 hours. Thus, we have now included a 2 h ISO control, and observed no further RyR cluster dispersion beyond levels present in the 1 h ISO group. These data have been presented in Figure 2 —figure supplement 2a, b rather than the main figures which were becoming quite cumbersome. The follow edit has been made to the text on page 3, line 179:“In control experiments, the longer 2 h treatment period with isoproterenol alone did not alter RyR configuration beyond values observed at the 1 h time point .”2+ spark characteristics, and again observed no further effects beyond changes observed at 1 h . Text has been added on page 4, line 253 stating:Similarly, we have included 2 hr ISO control experiments for ca2+ spark properties beyond values observed at the 1 h time point .”“In control experiments, a 2 h treatment period with isoproterenol alone  did not alter ca– The mathematical modeling results would also benefit from more explanation of how they complement, align, and expand on the experimental studies.2+ content, and thereby inhibition of ca2+ waves following RyR dispersal. Finally, we have included a new paragraph at the end of the modeling Results section summing up our key findings :Thank you for this comment. We have now edited the description of the mathematical modeling data, and emphasized how these results complement and expand upon experimental findings. Importantly, we have noted how calculations of spark-mediated and silent leak observed in modeling contribute to reduction in SR ca2+ release (ca2+ waves). Here, the antiarrhythmic actions of RyR dispersion appear to be linked to a reduction in SR content resulting from an increase in both spark-mediated and silent leak. We postulate that local regions of the cell with dispersed CRUs and lowered SR content may act as a physical barrier or “fire break” to impede ca2+ wave propagation.”“Taken together, the modeling results critically link changes in CRU configuration and spark morphology; phenomena that were observed separately in experiments. However, the modeling data also support the contention from experimental data that RyR cluster fragmentation can reduce arrhythmogenic spontaneous caThe reviewers also make the following major points:Reviewer #1 (Recommendations for the authors):1. A brief explanation of how CRU fragmentation would be reflected by changes to the reported dSTORM parameters  at the start of the Results section would help to orient the reader.Your point is well taken, as these changes may be confusing to readers not familiar with previous work describing CRU dispersion. We have now included a brief explanation of how this phenomenon is expected to influence the number of RyRs per cluster and CRU, and the overall cluster and RyR densities, as well as an additional schematic  to illustrate hallmarks of RyR cluster dispersion. The following text has been added to the beginning of the Results section on page 2, line 129:“Using 3D dSTORM imaging, we investigated the effects of long-term phosphorylation on RyR organization in cardiomyocytes. We specifically hypothesized that phosphorylation would fragment or “disperse” RyR clusters in a time-dependent manner. Numerically, such breaking apart of RyR groupings is expected to be reflected by a reduction in the average number of RyRs contained in each cluster, as illustrated in Figure 1 —figure supplement 1. This change would be accompanied by a concomitant increase in the total number of clusters as there now more numerous, smaller clusters in a given 3D space. If marked RyR cluster dispersion occurs, the total number of RyRs contained in a CRU may also be reduced, as clusters are no longer located in close enough proximity to be grouped in functional release units . Notably, if RyR dispersion occurs without the addition or loss of channels, then the total number of RyRs detected by dSTORM imaging should remain unchanged.”2. Reconstructions of representative CRUs are presented in Figure 1, 2, and 3, with selected CRUs inset as compelling visual illustrations of CRU fragmentation or reformation. It would be beneficial to allow the reader to decide for themselves if they are truly representative by stating their dSTORM parameter values in the figure or legend. This is especially true where single representative CRUs are presented as in Figure 2a-c and Figure 3a and b.We have now added parameter values below each of the representative CRUs in Figures 1, 2 and 3. We hope that this gives the reader a feeling for how the example images compare with mean observed changes in RyR configuration.3. On line 192 the authors state 'We found that the RyR arrangement in the phosphomimetic S2814D 193 mutant cells at baseline resembled the fragmented RyR organization observed in wild type (WT) 194 animals after isoproterenol treatment . Indeed, cluster size , CRU size , and 195 cluster density  were all similar in WT + ISO and S2814D cardiomyocytes, and significantly 196 different from untreated WT cells. Furthermore, application of isoproterenol to S2814D cardiomyocytes 197 did not lead to significant additional RyR dispersion, and only tendencies toward smaller cluster and 198 CRU sizes .'The representative CRU reconstructions in Figure 2g and h seem to contradict the quantified dSTORM parameters in Figure 2h, j-l and the description of the results in the text (starting at Line 192). This indicates a possible error in the production of the figure or that the choice of representative CRUs could be improved. In the images, the S214D CRUs appear more similar to wt (aside from the CRU selected for the insert) rather than wt + ISO as expected from the plotted data. There also appears to be significantly greater disruption visible in the S2814D + ISO than S2814D as a result, again counter to the observation made in the text about the plotted data.We agree with the Reviewer that our previous choice of representative image for Figure 2h did not accurately reflect the mean characteristics of RyR configurations in the 2814D cells. We have now selected a more suitable example. The appropriateness of each representative example is now documented by the presentation of the RyR cluster and CRU measurements below each image (see previous comment).4. There is a minor discrepancy between the naming of CaMKII mutants in Figure 2. panels (2814D and 2814A) and the text (S2814D and S2814A).We have corrected the figure legend to read S2814D and S2814A.5. A more precise description of the source of the S2814D and S2814A cardiomyocytes is needed for the reader to fully understand the strength of these control experiments. The authors currently state in the text and Materials and methods section that they are isolated from transgenic RyR2-S214D and RyR2-S214A mice provided by the Wehrens lab. The paper reference provided is only partially helpful as it describes the generation of homozygous knock-in mice RyR2-S214A but makes no mention of RyR2-S214D. I would assume that both are isolated from homozygous knock-in mice but please confirm if this is indeed the case and state it explicitly in the manuscript.We have now added the reference for the generation of S2814D mice, and clarified that we employed homozygous S2814A and S2814D animals and their wildtype littermates in our study. The following text has been added to the Methods section on page 10, line 521:45) or genetically ablated (RyR2-S2814A46). Cells isolated from homozygous mutant mice were used for this study, with comparison made to their respective wild-type littermates which served as controls.”“We examined effects of the CaMKII phosphorylation site S2814 on RyR configuration by employing knock-in transgenic mouse models, where the site was rendered constitutively active  unless otherwise stated. For RyR localisation and caWe have also included a sentence in the figure legends which reads:“The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte.”2+ leak is calculated from spark mass and frequency is missing from the Materials and methods section.8. A brief description of how spark-mediated caSpark-mediated leak was calculated as the product of spark amplitude, FDHM and FWHM. This has now been mentioned on page 13, line 672 of the Methods section.9. Figure 5c-f: These panels seem to have shared keys for the data represented by unshaded and shaded bars that only appear in the final panel . It would be clearer if the difference between unshaded and shaded was described in the figure legend with reference to the applicable panels. The same applies to Figure 6f-g.9. Figure 5c-f: These panels seem to have shared keys for the data represented by unshaded and shaded bars that only appear in the final panel . It would be clearer if the difference between unshaded and shaded was described in the figure legend with reference to the applicable panels. The same applies to Figure 6f-g.This has now been clarified in the legend.10. The Ethical approval section of Materials and methods needs to be checked for references to other journals.Corrected .11. Supplementary Tables 1-3 would benefit from brief explanations of their contents.Thank you for requesting this clarification, we have now elaborated on the descriptions for these supplementary tables (now named supplementary files 1-3).Reviewer #2 (Recommendations for the authors):1) The authors report that a 1 hr ISO stimulation followed by a 1 hr ISO stimulation in the presence of AIP led to a reversal of the ISO-induced RyR2 dispersal and concluded that CaMKII thus mediated RyR2 cluster dispersion downstream of B-AR stimulation. However, in this protocol, the cells are fixed after a 2 hr total exposure to ISO and it is unknown what this duration of ISO treatment does to RyR2 clustering and distribution. To increase confidence in these findings, an important control must be added. RyR2 clusters should be examined after 2 hrs ISO treatment (without any inhibitors on board). Likewise, a similar protocol is employed to test the effects of PKA on cluster dispersal, with the authors reporting that H89 reversed the dispersal. Again, this finding would be strengthened by knowing what 2 hrs of ISO does to RyR2 clustering and distribution. The same 2 hr total ISO stimulation protocol is used when the authors assess whether H89 or AIP can reverse the slowing in spark kinetics. To strengthen confidence in this finding, spark kinetics should also be examined after 2hrs of ISO treatment. The results may be quite different from those seen after a 1 hr treatment especially given the finding that RyR phosphorylation at ser-2814 and ser-2808 both exhibited bell-shaped responses during the 1 h ISO stimulation. If one extends that treatment to 2 hrs, phosphorylation of S2814 and S2808 may continue to fall and this complicates the findings with the inhibitors.As we have noted above, your point is well taken that the total treatment time for ISO + AIP  or ISO + H89  is 2 hours. Thus, we have now included a 2 h ISO control, and observed no further RyR cluster dispersion beyond levels present in the 1 h ISO group. These data have been presented in Figure 2 —figure supplement 2a, b rather than the main figures which were becoming quite cumbersome. The following edit has been made to the text on page 3, line 179:“In control experiments, the longer 2 h treatment period with isoproterenol alone did not alter RyR configuration beyond values observed at the 1 h time point ”2+ spark characteristics, and again observed no further effects beyond changes observed at 1 h . Text has been added on page 4, line 253 stating:Similarly, we have included 2 hr ISO control experiments for ca2+ spark properties beyond values observed at the 1 h time point .”“In control experiments, a 2 h treatment period with isoproterenol alone  did not alter ca2) At each ISO-treated time point, the study would benefit from the inclusion of an accompanying no-ISO control. Does the RyR dispersal occur because the cells are deteriorating with more time post-isolation, or is it specifically and exclusively because of the acute ISO-induced stress? These controls would help address that question and strengthen the conclusions of the paper.For the control group, cells were kept for 60 min following isolation prior to fixation for imaging. This 60 min time point then matched the duration of the longest ISO exposure, ensuring that there was not time-dependent run down of cardiomyocyte quality that contributed to observed changes in RyR configuration. This has now been mentioned in the Results section on page2, line 142.3) There is no indication of statistical significance on any of the summary data plots or mention of P values. This should be addressed.Significant differences are now indicated in each plot by the use of comparison bars. Significance was assigned for P < 0.05, as noted in each figure legend, and the Methods section.2+ leak shifts from spark-mediated to silent leak when CRUs are dispersed, reducing the fidelity of spark generation" but in figure 4h, spark-mediated (non-silent) leak is strikingly increased after 60 mins of ISO treatment which leads to the most profound dispersal. Please clarify how these results align. Perhaps there is more of both spark-mediated leak and non-spark leak? What experimental data supports non-spark mediated leak? The idea that there could be localized regions of SR ca2+ depletion that act as "fire-breaks" to prevent the generation of ca2+ waves even though there are more ca2+ sparks is logical and provides a satisfying theory that unifies some of the seemingly paradoxical findings but this is only raised in the second to last paragraph of the discussion. Raising this idea earlier may help the reader connect the dots more easily.4) The modeling results need to be put into context and compared with what is going on in the experimental data. This section is somewhat confusing and is a bit of a jarring shift away from the experimental section. It should be better integrated. The authors state "the modeling results indicate that RyR caThank you for this important critique. We agree that in the original version of the article, it was difficult to understand the link between experimental and modeling data. We have added a paragraph to address this on page 3, line 282:“To link observed RyR cluster morphologies with measured spark properties we constructed spark models employing experimentally-observed CRU morphologies. We then ran the computational spark model with these geometries to examine the resulting sparks. We also examined the likelihood of “undetectable” local release events whose amplitude is too small to be detected in confocal images. While difficult to measure experimentally, this non-spark mediated leak can importantly affect local SR load.”2+ waves. We do not have experimental measurements of silent leak, so this insight from modeling is quite important. As suggested, we have now included mention of the “fire breaks” concept in the Results section where these data are first presented on page 6, line 340:In addition, we have reorganized this section considerably, changing the order of data presentation, and have repeatedly referred back to previous experimental findings to explain how they are paralleled by modeling results. The Reviewer correctly points out that data concerning spark-mediated and silent leak were not clearly presented. In the revised text, we have clarified that during β-AR, the modeling predicts that both types of leak are increased, which would promote reduction in SR content, and inhibition of ca2+ release (ca2+ waves). Here, the antiarrhythmic actions of RyR dispersion appear to be linked to a reduction in SR content resulting from an increase in both spark-mediated and silent leak. We postulate that local regions of the cell with dispersed CRUs and lowered SR content may act as a physical barrier or “fire break” to impede ca2+ wave propagation.”“Taken together, the modeling results critically link changes in CRU configuration and spark morphology; phenomena that were observed separately in experiments. However, the modeling data also support the contention from experimental data that RyR cluster fragmentation can reduce arrhythmogenic spontaneous ca


Stem cells maintain adult tissue homeostasis under physiological conditions. Uncontrolled stem cell proliferation will lead to tumorigenesis. How stem cell proliferation is precisely controlled is still not fully understood. Phosphorylation of Yun is essential for ISC proliferation. Yun is essential for the proliferation of normal and transformed intestinal stem cells. Our mass spectrometry and biochemical data suggest that Yun can be phosphorylated at multiple residues in vivo. Interestingly, we show that the phosphorylation among these residues is likely interdependent. Furthermore, phosphorylation of each residue in Yun is important for its function in ISC proliferation regulation. Thus, our study unveils the important role of post‐translational modification of Yun in stem cell proliferation. Yun (Luck in Chinese) is an intrinsic factor identified for intestinal stem cell (ISC) proliferation control. Yun is post‐translational modified by phosphorylation at five serine residues at its C‐terminus in vivo. Phosphorylation among these five residues is likely interdependent. Importantly, phosphorylation of each residue in Yun is important for Yun's function in ISC proliferation regulation. Thus, the proliferation  and differentiation of adult stem cells must be tightly balanced under physiological conditions. Disruption of this balance will result in excessive stem cell or precocious stem cell differentiation and finally stem cell depletion, eventually leading to various diseases, such as cancer and precocious aging.Drosophila intestine is an excellent model to study the regulation of stem cell proliferation/differentiation and tumorigenesis, which shares marked similarities in terms of development, cellular makeup, and genetic control with its mammalian counterparts.Drosophila intestinal stem cells (ISCs) are distributed along the basement membrane of the adult intestinal epitheliumThe adult diamond (dind).In our recent study, we identified a novel intrinsic factor Yun (Luck in Chinese) which sustains the proliferation of normal and transformed ISCs.In this study, we investigate whether PTMs exist in Yun and whether these PTMs are required for Yun's function in ISC proliferation. We find that Yun is phosphorylated in vivo. Several phosphorylation residues are identified by mass spectrometry. Site‐directed mutagenesis analyses indicate that the phosphorylation of these residues may be interdependent and is important for the proper function of Yun in ISC proliferation regulation.22.1Drosophila adult posterior midgut using an esgGal4, UAS‐GFP, tubGal80ts (esgts) driver, which is expressed in the progenitors .CG7705).Yun encodes a novel protein without any known domains or motifs. We examined its expression pattern by immunostaining using a Yun‐specific antibody and found that in adult intestines, Yun was mainly expressed in progenitors and EEs  showed a significant reduction in the number of esg+ cells  in progenitors causes rapid ISC proliferation and eventually leads to tumorigenesis , we used MARCM technique to examine the roles of Yun in tumorigenesis. The size of Rafgof MARCM clones is significantly increased compared to that of control, whereas the proliferation of Rafgof MARCM clones was completely suppressed in the absence of yun ‐ and phosphorylated serine (pS)‐specific antibodies, respectively. The results showed that Yun could be detected by both antibodies, supporting the notion that Yun is phosphorylated at serine residue(s) in vivo Figure . ConsistWe further examined the contribution of phosphorylation status of Yun by these candidate phosphorylation residues and whether the phosphorylation status of individual residue can affect phosphorylation of the other residues. We performed site‐directed mutagenesis of these residues, changing each of them to alanine to make phosphorylation dead mutants, and generated transgenic flies carrying these mutants individually Figure . We then2.5yun[5SA]), while the other expressed a phosphorylation mimic form of all five serine residues (yun[5SD]), and examined their function. Compared with control MARCM ISC clones, the ISC MARCM clones of yun null mutant failed to proliferate  failed to restore the proliferation defects observed in yun null mutant, suggesting that the phosphorylation of these five serine residues collectively is essential for Yun function in ISC proliferation control  only partially restored the proliferation defects observed in yun null mutant, suggesting that the proper phosphorylation status of each single residue in Yun is also important for its function in ISC proliferation control  failed to restore the proliferation defects observed in yun null mutant, suggesting that the phosphorylation of this single serine residue is critical for Yun's function in ISC proliferation control  significantly restored the proliferation defects observed in yun null mutant  or yun(S530D) was ectopically expressed, indicating that the phosphorylation of serine 530 residue is also important for Yun's function in ISC proliferation control , yun(S534A), and yun(S536A) failed to restore the proliferation defects observed in yun null mutant, while ectopic expression of phosphorylation mimic yun(S532D), yun(S534D), and yun(S536D) did not statistically restore the proliferation defects observed in yun null mutant, although ISC clones containing 2–3 cells could be occasionally observed, indicating that that the phosphorylation of serine 532, 534, and 536 residues is also necessary for Yun's function in ISC proliferation control  or phosphorylation mimic form (SD) of a single residue, respectively. Consistently, ectopic expression of wildtype  Figures  and 5T. l Figure . Whereast Figure . These dl Figure . Further Figures . Collect3The proliferation and differentiation of adult stem cells must be tightly balanced in order to maintain tissue homeostasis and prevent tumorigenesis. However, how stem cell proliferation is properly controlled, in particular how post‐translational modifications of intrinsic regulators affect their functions in stem cell proliferation control, remains not fully understood. Here, we find that novel intrinsic Yun is phosphorylated at multiple serine residues in vivo and that the phosphorylation of these serine residues in Yun protein is critical for its function in ISC proliferation control. Interestingly, the phosphorylation of these serine residues may be interdependent and phosphorylation of Yun on the function of Yun in sustaining ISC proliferation seems to be complicated and the role(s) played by individual serine residue may be different.yun and phosphorylation mimic yun(5SD) could not restore the proliferation defects in the absence of either casein kinase, suggesting that multiple downstream substrates are required for ISC proliferation control and expression of single substrate is not sufficient to restore ISC proliferation blockage upon their depletion  of phosphorylation status exists among these residues, and how the phosphorylation of these residues is coordinated to regulate the function of Yun in ISC proliferation control using site‐specific phospho‐Yun antibodies and corresponding knock‐in flies carrying single and/or combined phospho‐dead and phospho‐mimic 44.1esgGal4, UAS‐GFP, tubGal80ts , tubGal80ts,tubGal4, UAS‐Rafgof (BL2033), yunshRNA, yunΔ9, UAS‐flag‐yun, and hsFlp, ActGal4, UAS‐GFP, FRT82B‐tubGal80 , hsFlp, ActGal4, UAS‐RFP, FRT82B‐tubGal80 ,DlGal4, tubGal80ts , esgGal4, Gal80ts, UAS‐CC3Ai,prosGal4, UAS‐GFP, tubGal80ts , CkIαv20 (THU5314 and HMS02276), FRT19A‐CkIα8B12 (BL63802), CkIIβv20 , FRT19A‐Ck2βA (BL67713), UAS‐CkIα‐HA (BL55067 and BL55068), and UAS‐CkIIβ (F001372).Flies were maintained on standard media at 25°C. Two–three–days‐old flies were selected and transferred to 29°C, unless otherwise specified. Flies were transferred to new vials with fresh food every day and dissected at specific time points as indicated. In all experiments, only female posterior midguts were analysed. Information about alleles and transgenes used in this study can be found either in FlyBase or as noted: 4.2ts before dissection and immunostaining. The flies were transferred to new vials with fresh food every day.Crosses (unless stated otherwise) were maintained at 18°C to bypass potential requirements during early developmental stages. Two–three–days‐old progeny with the desired genotypes were collected after eclosion and maintained at 29°C to inactivate Gal804.3pUAST‐flag‐yun (CG7705) was constructed by cloning yun ORF  into pUAST‐nflag. attB‐pUAST‐CkIIβ‐myc was constructed by cloning the ORF of UAS‐CkIIβ (F001372) into the EcoRI and XbaI site of attB‐pUAST‐myc. All point mutation constructs were made by Hieff Mut™ Multi Site‐Directed Mutagenesis Kit . attB‐pUAST‐yun(5SA:S450/530/532/534/536A)‐flag‐GFP and attB‐pUAST‐yun(5SD:S450/530/532/534/536D)‐flag‐GFP vectors were constructed by cloning the mutated yun(5SA) and yun(5SD) into the EcoRI site of attB‐pUAST‐flag‐GFP respectively. attB‐pUAST‐flag‐yun and attB‐pUAST‐flag‐yun vectors were constructed by cloning the mutated yun(1SA) and yun(1SD) into the KpnI site of attB‐pUAST‐nflag. Transgenic flies were obtained by standard P‐element‐mediated germline transformation carrying attP site at 36B, 75B, or 86F.4.4The clonal analyses were achieved using the MARCM system.4.52PO4/Na2HPO4, 175 mM NaCl, pH 7.4), and fixed in 4% paraformaldehyde for 25 min at room temperature. Samples were washed with 1 x PBT (0.1% Triton X‐100 in 1 × PBS) and blocked in 3% BSA in 1 × PBT for 45 min. Primary antibodies were added to the samples and incubated at 4°C overnight. The following primary antibodies were used: mouse mAb anti‐Dl , mouse mAb anti‐Prospero , rabbit anti‐pH 3 , rabbit anti‐active caspase 3 , and rabbit anti‐Yun (1:1000).For standard immunostainings, intestines were dissected in 1 × PBS ] on ice for 30 min. After centrifugation, lysates were then diluted tenfold with RIPA buffer and subjected to immunoprecipitation using anti‐FLAG M2 affinity gel . The immunocomplexes were collected by centrifugation and washed with 1 ml of RIPA buffer three times. For western blotting, immunoprecipitated proteins were separated in SDS‐PAGE and then blotted onto PVDF membranes. The membranes were stained with primary antibody overnight at 4°C. Followed by washing, PVDF membranes were incubated with secondary antibodies conjugated with HRP, then the membranes were scanned using Luminescent Image Analyser . Rabbit anti‐Yun (1:1000),4.7Proteins were precipitated with 25% trichloroacetic acid (TCA) for at least 30 min on ice. The protein pellets were washed twice with 500 μl ice‐cold acetone, air dried, and then resuspended in 8 M urea, 100 mM Tris, and pH 8.5. After reduction  and alkylation , the samples were diluted to 2 M urea with 100 mM Tris, pH 8.5 and digested with trypsin at 1/50 (w/w) enzyme/substrate ratio at 37°C for 16–18 h. The digestion was then stopped by addition of formic acid to 5% .4.8All the samples were analysed using an EASY‐nLC 1000 system (Thermo Fisher Scientific) interfaced with a Q‐Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were loaded on a trap column  and separated on an analytical column  with a 60 min linear gradient at a flow rate of 200 μl/min as follows: 0–5% B in 2 min, 5–30% B in 43 min, 30–80% B in 5 min, and 80% B for 10 min . Spectra were acquired in data‐dependent mode: the top ten most intense precursor ions from each full scan  were isolated for HCD MS2  with a dynamic exclusion time of 30 s. The AGC targets for the MS1 and MS2 scans were 3e6 and 1e5, respectively, and the maximum injection times for MS1 and MS2 were both 60 ms. Precursors with 1+, more than 7+ or unassigned charge states were excluded.4.9Drosophila melanogaster protein database (database ID number of UP000000803) using ProLuCID with the following parameters: precursor mass tolerance, 3 Da; fragment mass tolerance 20 ppm; peptide length, minimum 6 amino acids and maximum 100 amino acids; enzyme, Trypsin, with up to three missed cleavage sites.The MS data were searched against a Uniprot 4.10esg+ cells, confocal images of 40× lens/1.0 zoom from a defined posterior midgut region of different genotypes indicated were acquired. The number of esg+ cells from each confocal image was determined using Image‐Pro Plus software, manually selecting the “filter” depending on the respective cell size to filter out background signals (referred to as the relative number of esg+ cells). The clone sizes were scored manually under Zeiss Imager Z2/LSM780 microscope for indicated genotypes. At least 10 different guts were analysed for each set. Statistical analysis was done using the Student's t‐test. PEMS 3.1 software was used for SEM analyses and Sigma plot and GraphPad prism software for graph generation. The graphs were further modified using Adobe Photoshop and Illustrator. nsp >0.05; *p < 0.05; **p < 0.01; *** p < 0.001; and **** p < 0.0001.The number of intestines scored is indicated in the text. To determine the relative number of The authors declare no conflicts of interest.Conceptualization, Zhouhua Li and Hang Zhao; investigation, Xuejing Ren, Hang Zhao, Lin Shi, Zhengran Li, Ruiyan Kong, Rui Ma, Lemei Jia, Shan Lu, Jian‐Hua Wang, Meng‐qiuDong, Yingchun Wang, Zhouhua Li; formal analysis, Xuejing Ren, Zhouhua Li, Hang Zhao; methodology, Rui Ma; validation, Xuejing Ren, Hang Zhao, and Zhouhua Li; writing—original draft preparation, Xuejing Ren, Hang Zhao, and Zhouhua Li; writing—review and editing, Hang Zhao and Zhouhua Li; supervision, Zhouhua Li; project administration, Zhouhua Li; funding acquisition, Zhouhua Li. All authors have read and agreed to the published version of the manuscript.Appendix S1Supporting InformationClick here for additional data file.


Lysobacter capsici VKM B−2533T β-lytic protease (Blp), a medicinally promising antimicrobial enzyme, was first solved. Blp was established to possess a folding characteristic of the M23 protease family. The groove of the Blp active site, as compared with that of the LasA structural homologue from Pseudomonas aeruginosa, was found to have amino acid differences. Biochemical analysis revealed no differences in the optimal reaction conditions for manifesting Blp and LasA bacteriolytic activities. At the same time, Blp had a broader range of action against living and autoclaved target cells. The results suggest that the distinction in the geometry of the active site and the charge of amino acid residues that form the active site groove can be important for the hydrolysis of different peptidoglycan types in target cells.The crystal structure of the  Lysobacter bacteria. The most lytically active but least studied among these three enzymes is Blp. The specificity of its action against living Gram−positive pathogens evokes much interest in this enzyme as an active basis for the creation of antimicrobial drugs.β−Lytic protease (Blp), together with α−lytic protease, was first described in 1965 . LysozymMyxobacter 495  × 1000 µL }/[5 min (time of reaction) × 5–25 µL (volume of sample) × 0.01 (correction coefficient for OD reduction per min)]. A unit of bacteriolytic activity (LU) was taken as an amount of the enzyme, which led to a decrease in absorption of the cell suspension by 0.01 o.u. per 1 min. The value of LU/mL was calculated by the following formula: {.The structures were determined by molecular replacement with Phaser  using th REFMAC5 . Manual  REFMAC5 . The fin REFMAC5 . Data anStatistical analysis was performed using GraphPad Prism version 8.0.1 . All experiments were conducted with 4–8 repeats.p < 0.05. The data are presented as means ± standard deviations, as well as in the form of boxplots (medians ± interquartile spans). The data were considered to be significant at t−test. An unpaired two−tailed Student’s t−test with Welch’s correction was used for unequal variances.The normal distribution of the data was verified using the D’Agostino–Pearson complex test. To determine the equality of the variances of two independent groups, the F−test was used for the normally distributed data of two groups, the unpaired two−sided Student’s