Figure 3.

Validation of HF in human heart tissue and doxorubicin‐treated cells and demonstration of intracellular‐free Zn²⁺ increase under HF. (A) Expression level of α‐actinin, ACTN and PUMA in both human heart tissue with heart failure (HF) (A) and doxorubicin (DOXO)‐treated H9c2 cells (B) with respect to their controls (CON). Representative Western blotting bands for protein expression levels are given upper parts of figures in (A) and (B). (C) Representing of measurement protocol of intracellular‐free Zn²⁺, [Zn²⁺]_i in doxorubicin‐treated (failing heart modelled, DOXO group) cells, loaded with Zn²⁺ selective fluorescent dye FluoZin‐3. Zn²⁺ ionophore pyrithione, Zn²⁺/Pyr (1‐μM) exposure (3 min.) and high‐affinity heavy‐metal Zn²⁺‐chelator, N,N,N',N'‐tetrakis(2‐pyridylmethyl)ethane‐1,2‐diamine, TPEN (50 μM). The mean (±S.E.M.) intracellular Zn²⁺level, as FluoZin‐3 intensity, respectively. (D) Bar graphs representing the [Zn²⁺]_i levels calculated as nM (see Methods and materials) in the treated cells comparison with the untreated cells (CON group). Bars represent mean (±S.E.M.) for each group. Number of human samples, n = 3 for HF group and n = 2 for CON group. All measurements with double assays in each sample from each group for each type of measurement. Significance level accepted at *P < 0.05 versus. CON.