A Reappraisal of the Effects of L-type Ca2+ Channel Blockers on Store-Operated Ca2+ Entry and Heart Failure

Abstract

Dihydropyridines such as amlodipine are widely used as antihypertensive agents, being prescribed to ∼70 million Americans and >0.4 billion adults worldwide. Dihydropyridines block voltage-gated Ca²⁺ channels in resistance vessels, leading to vasodilation and a reduction in blood pressure. Various meta-analyses show that dihydropyridines are relatively safe and effective in reducing hypertension. The use of dihydropyridines has recently been called into question as these drugs appear to activate store-operated Ca²⁺ entry in fura-2-loaded nonexcitable cells, trigger vascular remodeling, and increase heart failure, leading to the questioning of their clinical use. Given that hypertension is the dominant "silent killer" across the globe affecting ∼1.13 billion people, removal of Ca²⁺ channel blockers as antihypertensive agents has major health implications. Here, we show that amlodipine has marked intrinsic fluorescence, which further increases considerably inside cells over an identical excitation spectrum as fura-2, confounding the ability to measure cytosolic Ca²⁺. Using longer wavelength Ca²⁺ indicators, we find that concentrations of Ca²⁺ channel blockers that match therapeutic levels in serum of patients do not activate store-operated Ca²⁺ entry. Antihypertensive Ca²⁺ channel blockers at pharmacological concentrations either have no effect on store-operated channels, activate them indirectly through store depletion or inhibit the channels. Importantly, a meta-analysis of published clinical trials and a prospective real-world analysis of patients prescribed single antihypertensive agents for 6 mo and followed up 1 yr later both show that dihydropyridines are not associated with increased heart failure or other cardiovascular disorders. Removal of dihydropyridines for treatment of hypertension cannot therefore be recommended.

Graphical Abstract

Figure 1.

Effects of amlodipine on cytosolic Ca²⁺-signals in fura-5F-loaded HEK 293 cells. (A) Acute application of 0.5 μm amlodipine slightly decreased the ratio of emitted signal for cells excited at 340 and 380 nm. Addition of 20 μm amlodipine resulted in a marked step decrease in the ratio signal. (B) The component 340 and 380 wavelength signal intensities that were used to derive the ratio changes in (A) are shown. While 0.5 μm amlodipine did not clearly change the 340 or 380 nm signal, 20 μm amlodipine caused an initial step increase in background fluorescence followed by a prolonged rise in cellular fluorescence that reached a steady state after 15 min. (C) The fluorescence intensity change observed in background bathing solution was subtracted from the fluorescence signals measured within fura-5F loaded cells. (D) Ratio (F340/F380) signal was derived from ratioing the “background corrected” 340 and 380 signals observed in (C). After “background” correction, 20 μm amlodipine did not raise cytosolic Ca²⁺. The data in (A) and (D) are mean ± SEM for n = 5 experiments.

Figure 2.

Effects of CCBs on cellular fluorescence signals in non-dye loaded HEK 293 cells. To dissect the effects of amlodipine besylate on HEK 293 cells, experiments were conducted as described in Figure 1, but now on cells that had not been loaded with fura-5F. (A) A coverslip containing HEK 293 cells was exposed to 1 μm amlodipine. Whereas cell-free regions did not show a detectable increase in background fluorescence, a small fluorescent rise occurred over tens of seconds within cells. (B) With 20 μm amlodipine, an initial step increase in background (cell-free) fluorescence occurred and this was followed by a prolonged rise in cellular fluorescence that reached a steady state after ∼15 min, and with a t_1/2 of 5.75 min. (C) Cells that already been incubated with 20 μm amlodipine for 15 min were bathed continuously with amlodipine-free external solution. Amlodipine was removed from the bathing solution at t =  60 sec. The accumulated intracellular fluorescence signal decreased very slowly (estimated t_1/2 of 42 min). The residual amlopidine fluorescence could not be quenched by the addition of ionomycin/Mn²⁺ (10 μm/20 mm, respectively). In panels (A-C), excitation wavelength was 380 nm. (D) Background (buffer) and cellular fluorescence are shown for different amlodipine concentrations at 340 nm excitation. (E) As in panel (D), but intensity of emitted light at 380 nm excitation is shown. The dashed lines in panels (D) and (E) indicate the typical fluorescent signals measured in fura-5F loaded HEK 293 cells at rest (indicated by “fura5F-cell” in F). (F) Intensity of signal following excitation at the wavelengths indicated are compared. (G) Fluorescence to 100 μm of either nifedipine or diltiazem are shown for the conditions indicated. Nifedipine fluorescence increased slightly inside cells compared with cells alone. Unpaired t-test was performed for the comparisons indicated (ns = not significant, ** = P ≤ 0.01). (H) Fluorescence signals were measured in non-dye loaded HEK 293 cells using the excitation and emission settings for Cal520 (ex 488 nm, em 520 nm). Amlodipine besylate (100 μm) did not increase the fluorescence intensity measured in buffer solution (background) or in cells . All data are mean ± SEM for n = 3 experiments.

Figure 3.

Effects of thapsigargin and amlodipine on Ca²⁺-signals in Cal520-loaded HEK 293 cells. (A) Stimulation with 0.5 μm amlodipine in the presence of external Ca²⁺ failed to raise cytosolic Ca²⁺ and only a very small, transient rise was seen in 20 μm amlodipine. By contrast, thapsigargin (TG; 2 μm) caused a prolonged rise in cytosolic Ca²⁺. (B) Aggregate data of “peak” response are compared. All data are mean ± SEM for n = 3 experiments. Unpaired t-test was performed for each condition against DMSO (ns = not significant, **** = P ≤ 0.0001). Changes in the Ca²⁺signal were monitored using a fluorescence imaging plate reader (FLIPR^TETRA).

Figure 4.

Effects of CCBs on Ca²⁺ release and Ca²⁺ entry. (A) Different concentrations of amlodipine were applied in Ca²⁺-free solution and then external Ca²⁺ readmitted as indicated. Upper panel shows thapsigargin (2 μm), amlodipine (0.5 μm), and solvent (DMSO) control. Middle and lower panels show 20 and 100 μm amlodipine. (B) As in panel (A), but nifedipine was used instead. (C) As in panel (A), but diltiazem was applied. (D-E), Effects of different concentrations of amlodipine on Ca²⁺ release and Ca²⁺ entry are shown. Changes in the Ca²⁺signal were monitored using a fluorescence imaging plate reader (FLIPR^TETRA) and reported as changes in F/Fo. All data are mean ± SEM for n = 4 experiments.

Figure 5.

Effects of amlodipine on intracellular Ca²⁺ pools. (A) Different concentrations of amlodipine were applied in Ca²⁺-free solution (first addition) and then ionomycin was applied (second addition), using a protocol based on data in Supplementary Figure S3. The second addition of ionomycin was used to estimate the extent of Ca²⁺ store depletion. As the concentration of amlodipine increased, the size of the ionomycin-accessible Ca²⁺ store decreased. (B) Aggregate peak responses for the first additions are summarized. (C) Aggregate data showing the size of the ionomycin response (second addition) after different amlodipine concentrations are compared. Included in (A-C) are the responses to thapsigargin (2 μm). All data are mean ± SEM for n = 3 experiments. (D) Ca²⁺ release to the various stimuli indicated, applied in Ca²⁺-free solution, was measured. (E) Aggregate peak responses are compared, and unpaired t-test was performed for each condition against DMSO (ns = not significant, ** = P ≤ 0.01, **** = P ≤ 0.0001). (F) As in panel (D), but 1 mm Gd³⁺ was present to prevent Ca²⁺ extrusion. (G) Aggregate data from experiments as in panel (F) are compared, and unpaired t-test was performed for each condition against DMSO (ns = not significant, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001). Ca²⁺ measurements were performed on Cal520-loaded HEK 293 cells using a FLIPR^TETRA.

Figure 6.

Results of network meta-analysis. Treatments with direct comparison are linked with a line, the thickness of which is proportional to the number of included trials (single number adjacent to the line). The odds ratio and 95% confidence interval for each pair-wise comparison are presented adjacent to the lines with those in bold representing the network meta-analysis estimate with DHP-CCB as the referrant treatment. ACEI: Angiotensin converting enzyme inhibitors; ARB: Angiotensin receptor blockers; BB: Beta blockers; CCB: Dihydropyridine calcium channel blockers; DI: Thiazide; P: Placebo.