Iron blockade effects of LTCC and TTCC blockers on iron-overloaded cardiomyocytes.

<p>In this CALG-AM fluorescent assay, HL-1 cells were pretreated with LTCC blockers, amlodipine (AML) and verapamil (VER), and TTCC blocker, efonidipine (EFO), at logarithmic scale from 0.1 to 100 µM. 3 indicated doses of Fe(III) (<b>A</b>) and Fe(II) (<b>B</b>) were loaded 30 min after blocker treatment. Fluorescence readings were at 70 min after iron loading. Fluorescence signal changes were normalized to respective negative controls of each treatment arm. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. The results represented as mean ± SEM of four independent triplicate experiments.</p

Cellular toxicity of LTCC blockers and the comparison with deferiprone.

<p>(A) Apoptotic effects of 10 or 100 µM AML, VER and deferiprone on HL-1 cells for 72 hr were assessed by annexin V/PI flow cytometry assay. (B) HL-1 cells were challenged with 300 µM Fe(III) or Fe(II), followed by treatments of 10 or 100 µM deferiprone 20 min after iron loading. Apoptosis was determined after 72 hr incubation by annexin V/PI assay. Data were shown as total annexin V positive cell portion with normalization to respective negative controls. * <i>p</i><0.05; ** <i>p</i><0.01. The results represented as mean ± SEM of three independent experiments, except 100 µM AML and VER (n = 1).</p

Calcium channel blockers could not salvage HL-1 cells from iron overload induced apoptosis.

<p>HL-1 cells were pretreated with LTCC blockers AML or VER, and TTCC blocker EFO for 1 hr, followed by Fe(III) (<b>A</b>) and Fe(II) (<b>B</b>) loading for 72 hr. Controls were defined as treatments without blockers. Apoptosis was determined by annexin V/PI flow cytometry assay. Total annexin V positive cell portion was counted. The results represented as mean ± SEM of three independent experiments.</p

Exogenous iron entered cardiomyocytes in a time- and dose- dependent manner.

<p>(<b>A</b>) Fe(III) uptake by live HL-1 cells treated at 3 indicated doses, detected by CALG-AM fluorescent assay. Fluorescence intensity was carried out by fluorescent plate reader Fusion. Local average reading at 10 min was set as initial fluorescence level. Each reading at any given time was normalized to the local initial fluorescence level. FeCl₃ was load at 30 min. Impermeant chelator DTPA was loaded at 115 min; permeant iron chelator ICL 670 was loaded at 136 min. Control was defined as treatment without addition of iron, DTPA and ICL670. (<b>B</b>) Fe(III) and Fe(II) uptake at 100 min of the assessment time point indicated in (A), i.e. 70 min after iron loading. FeCl₃ loaded with ascorbate represented Fe(II) treatment. Both controls with and without ascorbate were shown. *, †, ‡, <i>p</i><0.05; **, ††, ‡‡, <i>p</i><0.01; ***, †††, ‡‡‡, <i>p</i><0.001; * versus respective controls. The results represented as mean ± SEM of five independent triplicate experiments.</p