The Reappraisal of the Reappraisal—CRAC Channels Are Activated by L-Type Ca2+ Channel Blockers, Reply to Bird et al

A Perspective on “A Reappraisal of the Effects of L-Type Ca 2 + Channel Blockers on Store-Oper a ted Ca 2 + Entry and Heart Failure”

data support the idea that Ca v 1.2 c hannel bloc kers, including amlodipine , nifedipine , ver apamil, and diltiazem acti v ate STIM proteins and CRAC channels inde pendentl y of stor e de pletion.4 We further showed that 0.5 μm amlodipine synergizes with suboptimal concentrations of the platelet-deri v ed gr owth factor (0.5 ng/mL PDGF) in a STIM1-dependent manner to induce arterial m y ocyte prolifer ation and migr ation to lev els appr oaching those produced by maximal PDGF stimulation (10 ng/mL).Bird et al. 5 challenged our study and contended that our findings r e pr esent an artifact of fluor escence Ca 2 + measur ements because "amlodipine is str ongl y fluor escent in the cytoplasm with an excitation spectrum that overlaps with that of fura-2."They r e ported Ca 2 + ima ging experiments using the singleexcitation/single-emission dye Cal520 but did not attempt to r e plicate our key biological observations, most nota b l y the synergistic effects of 0.5 μm amlodipine on PDGF-triggered arterial m y ocyte prolifer ation and migr ation, and their dependence on STIM1 (Figure 1H-K; Figure 7A-G; Figure S8). 4 We reject the contention of Bird et al. that our conclusions are incorrect due to an unrecognized contribution of amlodipine fluorescence to the Ca 2 + signal, based on the following evidence: (1) Contrary to the assertion of Bird et al., we hav e r ecognized the issue of extracellular amlodipine fluorescence, whic h w e ac knowledged, discussed, and corrected for in our Johnson et al. paper (first par agr aph in "Results" and Supplemental Material, Single Cell Ca 2 + Imaging ). 4 This was also recognized in our previous Liu et al. paper ("Methods," Ca 2 + measurements ). 6Amlodipine did not create a false fura-2 fluorescence-ratio signal that would be misinterpreted as a Ca 2 + increase.If it did, that signal would have been apparent in Figure 2I and J where amlodipine was added to STIM1/STIM2 double knockout cells or Or ai1/Or ai2/Or ai3 triple knockout cells, or in Figure 4A and B where amlodipine was applied to cells in 0 m m Ca 2 + extracellular solution, or in Figure 6K and L where amlodipine was applied to STIM1/STIM2 double knockout cells expressing STIM1 with N-terminal deletions that pr ev ent its r esponse to amlodipine.These negati v e r esults also rule out a detecta b le fluorescence-ratio contribution from amlodipine accumulated intracellularly.(2) Cruciall y, we pr ovided electr oph ysiological e vidence that amlodipine acti v ates both nati v e CRAC curr ents in HEK293 cells with endogenous levels of STIM and Orai and larger CRAC currents in cells co-expressing STIM1 and Orai1, with the expected biophysical properties (Figure 2C-H). 4The amlodipine-ev oked curr ents wer e of similar size to the currents elicited in the same HEK293 cells by a standard storede pletion pr otocol with BAPTA in the recording pipette, about 0.2-0.5 pA/pF for native CRAC current and 20-50 pA/pF when STIM1 and Orai1 are overexpressed.
(3) In this study (Figure 1F and G), 4 and in the earlier study wher e we originall y identified Ca v 1.2 channel b lockers as acti v ators of Ca 2 + signaling in glioblastoma cells using a small-molecule screen of a library of 1650 compounds (Figure S2A), 6 we showed that amlodipine at 0.5-20 μm significantl y and dose-de pendentl y elicited a Ca 2 + signal when the measurement was made with fluo-4, a dye similar to Cal520 and for which overlap with the amlodipine fluorescence spectrum is not an issue.(4) Unlike amlodipine, the drugs diltiazem, nifedipine, verapamil, mibefradil, and BayK8644 do not fluoresce at the w av elengths monitor ed for fura2 spectra.These drugs activated a similar Ca 2 + entry (Figure 2M-R; Figure S2; Figure S3A-D). 4(5) The fura-2 indi vidual-w av elength intensity plots in Bird et al. (Figures 1 and 2) 5 ar e irr elev ant, because the r elev ant measurement is the fluorescence ratio F 340 /F 380 .The fluorescence ratio F 340 /F 380 for amlodipine by itself is slightly less than 1.Hence, to the extent that uptake of amlodipine made any contribution to the ratiometric signal, uncorr ected amlodipine fluor escence would de pr ess the true fura-2 ratiometric signal and underestimate the actual increase in cytoplasmic Ca 2 + , as we have documented previously (Figure S2J and K).We also presented evidence that amlodipine and diltiazem r equir e an intermediate step or pathway to activate STIM (Figure 6M and N). 4 The Lines of Evidence Above Were Not Ac kno wledged by Bird et al.
The pharmacokinetics and pharmacodynamics properties of drugs ar e heavil y influenced by binding to plasma pr oteins, which affect drug distribution, absorption, and half-life. 7Drug concentrations in plasma of patients poorly correlate with clinical effects and can serve in monitoring only when protein binding is r elati v el y low (less than 50%). 8Concentrations of Ca v 1.2 c hannel bloc kers measur ed in the plasma of tr eated patients distribute over a wide range of ∼1-1 μm and 80%-99% of these drugs are in a protein-bound state. 9The high half-life and volume of distribution of amlodipine indicates a high absorption and distribution into tissues. 9While 0.5 μm amlodipine might be higher than the quoted range in serum of patients, it is not clear how either the concentration applied in our short-term in vitro experiments, or the plasma concentrations measured in patients during long-term treatment relate to amlodipine concentration at its apparent site of action on STIM in the ER lumen of arterial m y ocytes.Even if we could be sure that the ER concentrations of amlodipine in arterial m y ocytes of patients w ere low er than in our in vitr o experiments, gi v en that we showed that the cellular action of amlodipine and diltiazem on STIM is indirect, via an unknown intermediate step or steps, it would be pr ematur e to rule out a cum ulati v e effect of prolonged ER exposure to low concentrations of these drugs.As noted by Bird et al. (Figure S2), 5 amlodipine accumulates into intracellular compartments including the ER, its site of action on STIM.Ther efor e, our data are compelling.The Ca 2 + channel bloc kers-induced STIM-Or ai sensitization exists, and could limit the bloc kers' ther apeutic effecti v eness.We also acknowledge that the drugs concentration at which this pathway is robustly acti v ated is much higher than the clinically therapeutic plasma concentrations.Bird et al. provided "real-world" meta-analysis comparing incidence of heart failure between placebo patients and patients with no prior history of car dio vascular disease and newly prescribed a monotherapy of either Ca v 1.2 channel blockers or other anti-hypertensi v e medications.They assessed the risk of developing heart failure in the following year of follow-up after a minimum of 6 months of drug intake. 5This analysis of newly hypertensi v e patients, wher e Ca v 1.2 expr ession in m y ocytes w ould be high and CRAC c hannel activity w ould be absent, shows that Ca v 1.2 channel b lockers ar e effecti v e in these patients, as expected.However, this situation is not a reflection of advanced vascular remodeling, a progressive process that dev elops ov er years to decades wher e Ca v 1.2 is downr egulated, and STIM1/Orai1 increased.Our cross-sectional analysis (Figure 7T) 4 and that of Bird et al. (Figure 6) 5 a gr ee that angiotensin converting enzyme inhibitors, beta blockers, angiotensin receptor blockers, and diuretics are more protective against heart failure than Ca v 1.2 channel b lockers.Ther efor e, Bird et al. man ufactur e an argument they can win, by blurring the distinction between Ca v 1.2 channel blockers being effecti v e a gainst hypertension but with possible liabilities-our position-and these blockers being simpl y ineffecti v e .Contr ary to the assertion of Bird et al., we did not claim that Ca v 1.2 channel blockers are not protective, nor did we suggest they be r emov ed as first line anti-hypertensi v e drugs.
The study by Bird et al. was accompanied by a commentary and an editorial. 10 , 11Rajagopal and Rosenberg 10 commented: "One of the major differences from the present study and Johnson et al. was the choice of Ca 2 + -sensitive fluorescent dyes. ... [Bird et al.] found that amlodipine accumulated within the cytoplasm ov er sev eral min utes of exposur e, dominating the fluorescence signal in fura-2-loaded cells, and thereby mimicking a Ca 2 + transient. . .Thanks to the rigorous studies in this work, we now have a better understanding of the true effects of amlodipine and CCBs on SOCE, and the clinical utility and safety of amlodipine is no longer in question."This commentary missed the point that amlodipine fluorescence did not (and, mathematically, could not) register as a spurious increase in the fura2 ratiometric signal, and failed to critically and rigor ousl y discuss and contrast our findings with those of Bird et al.
The editorial by Verkhratsky and Petersen 11 titled " How do we clean up the scientific record " was interesting in that it concluded from the outset that " the results reported by Johnson et al. are wrong " and that Bird et al. showed that " [t]he finding of Johnson et al. is . . .an artifact ," thus failing to grasp the core experimental findings of our paper and to ev aluate criticall y the claims of Bird et al.Quite ir onicall y, the authors branded our study as a case of " serious scientific error ," and used it as a launchpad to offer an extended opinion on pr ob lems of scientific rigor and fraud and w ays to r emedy them.We think a good start would be to avoid omissions and misr e pr esentations by car efull y r eading the studies one wishes to comment on.
Verkhratsky and Petersen 11 went on to state: ". . .how to rectify straightforw ard err ors, whic h (as illustr ated by the case discussed in this editorial) carry dangerous implications?The only way is post-publication testing of the main findings and, in the case of error identification, making the academic community informed thr ough pub lication, which is of course exactl y what is happening in the case discussed here."This and other statements in this editorial raise a more profound question that goes far beyond the current case.Scientific discourse is healthy and welcomed as it advances our understanding of true biological mec hanisms and acceler ates the application of this knowledge in the clinic.However, an inherent premise is such discourse needs to be based on the scientific evidence and be judged by the scientific community.In this context, is it appropriate for tw o scientists, how ev er pr ominent they ar e, to self-appoint as the arbiters of the scientific record?A question for the community to address.
In summary, we stand by our conclusion that the current data "indicate caution against the use of L-type Ca 2 + channels blockers in elderly patients or patients with advanced hypertension and/or onset of car dio v ascular r emodeling, wher e levels of STIM and ORAI ar e elev ated." 4 A note of caution is pr ecisel y that: definiti v e guidelines for the clinical use of antihypertensi v e medications can only be esta b lished by further resear c h.We look forward to meaningful scientific discussions of our data and data coming from new resear c h in this area. 6

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In Figures3, 4C, and 4D, Bird et al.5  showed that 20 μm amlodipine and diltiazem acti v ate a significant Ca 2 + signal, though they dismissed it as small or tr ansient.5  Contr ary to what was implied by Bird et al., we did not claim that Ca v 1.2 c hannel bloc kers activate SOCE to the same extent as maximal acti v ation by thapsigargin; maximal SOCE is not a prerequisite for physiological significance.(7) SiRN A knoc kdown (F igure 2I and J) 6 and CRISPR/Cas9 knockout of STIM1/2 and Orai1/2/3 and cDNA rescue (Figure 2K-R; Figure S2A-L) 4 showed that members of all major classes of Ca v 1.2 channel blockers, namely dih ydrop yridines (amlodipine, nifedipine), verapamil (a phenylalkylamine), and diltiazem (a benzothiazepine) activate Ca 2 + entry but only when at least one STIM isoform and one Orai isoform ar e expr essed.(8) Diltiazem triggered STIM1 and Orai1 puncta formation and colocalization, induced F örster resonance energy transfer (FRET) between STIM1-eYFP and CFP-Or ai1 (F igure 3A-I; F igure 5A-C) and altered STIM1 intramolecular FRET (Figure 5D-H). 4(9) Bird et al. suggested that amlodipine depletes the endoplasmic reticulum (ER) stores based on measurements with the cytosolic Ca 2 + indicator Cal520 but this seemed to become evident only with amlodipine at 40 and 100 μm , 5 a concentr ation r ange higher than what w e used.We dir ectl y measur ed Ca 2 + stor es using a geneticall y encoded ER-targeted indicator and showed that 20 μm amlodipine and as high as 50 μm diltiazem failed to alter ER Ca 2 + content, whereas the subsequent addition of 1 μm ionomycin caused the same extent and rate of ER store depletion in Ca v 1.2 channel b lockers-tr eated and v ehicle-tr eated cells (Figur e 4C-I). 4At 20 μm amlodipine, Bird et al. found no Ca 2 + release from stores in Figures 4A-D, 5A-C, and 5D-E; and v er y minimal appar ent r elease fr om stor es ev en in the pr esence of 1 m m Gd 3 + to block extrusion of Ca 2 + by pumps (Figure 5F-G). 5Thus, Bird et al. arri v ed at exactl y the conclusion r eached in our study: Amlodipine (20 μm ) elicits Ca 2 + influx without de pleting stor es.A conserv ati v e statement of the differ ence betw een the tw o papers on this point is that the amlodipineevoked Ca 2 + signal is quantitatively less in Bird et al. under their conditions and with the Ca 2 + dye used, Cal520.(10) STIM1 dimerization assays on ER membranes isolated from amlodipine-and diltiazem-treated HeLa cells showed that, by comparison to membranes from vehicle-treated cells, amlodipine and diltiazem induced a significantly higher STIM1 dimerization at Ca 2 + concentrations ranging from 300 μm to 1 m m , demonstrating that treating cells with Ca v 1.2 c hannel bloc kers r enders STIM1 pr one to acti v ation at resting ER Ca 2 + concentrations (Figure 5I and J). 4 (11) Xenopus STIM1 is acti v ated by store depletion while being insensiti v e to acti v ation by amlodipine, and a truncated human STIM1 mimicking Xenopus STIM1 and lacking an Nterminal region (residues 31-40) loses activation by amlodipine but retains activation by store depletion (Figure 6A-L), 4 arguing that Ca v 1.2 channel blockers act, dir ectl y or indir ectl y, on the ER luminal N-terminal domain of STIM1.