Molecular and Cellular Determinants of Human Iron Overload Cardiomyopathy

Figure 1:. Viability and amount of iron accumulation within cardiomyocytes (CMs) and cardiac fibroblasts (CFs) in monolayer culture. (a.) Representative images of CMs survival analyzed by cell staining with Calcein-AM (green), Hoechst 33343 (blue) and EthD1 (red) with different concentrations of iron (0, 100, 250, 500 μM) fed through media for 5 days. (b. and c.) Quantification of the percent dead CMs and CFs after 5 days of treatment with iron. No significant differences were seen between control (CMs: n=13 fields of view, CFs: n=9) and 100 μM iron treated cells (CMs: n=12, CFs: n=8) while cells treated with 250 μM iron (CMs: n=12, CFs: n=9) or 500 μM iron (CMs: n=14, CFs: n=9) showed decreased viability. Data analyzed by one-way ANOVA, uncorrected Dunn’s multiple comparisons test. (d.) Colorimetric measurement of the relative levels of intracellular iron accumulation within CMs and CFs after exposure to 100 μM iron for 5 days. The amount of iron in CMs is three-fold higher than CFs when treated with the same concentration of iron (Mann-Whitney tests). Each data set represents at least two distinct differentiations or biological replicates, and there are at least three sample replicates…

Figure 2:. EHTs capture key contractile aspects of iron overload cardiomyopathy. (a.) Representative data trace showing the force produced by the EHT (orange) as a function of time in response to a length step (blue). Traces were used to calculate the active and passive forces. (b.) Active force and (c.) passive force exerted by the tissue as a function of length. n = 24 control tissues (blue) and n = 25 iron-treated tissues (red). Statistical testing was done by two-way ANOVA. Each data set represents three or more distinct differentiations or biological replicates. Data are median ± 95% CI, *p-value≤0.05, **p-value≤0.01, ***p-value≤0.01, and ****p-value≤0.01. Representative traces showing the fluorescence of the calcium sensitive dye as a function of time. EHTs were stimulated at 1 Hz. Traces for (d.) control and (e.) iron-treated EHTs are shown. Irregular waveforms are highlighted by purple boxes. Additional traces and quantification can be found in the supplementary materials.

Figure 3:. Effect of excess iron on the action potentials of engineered heart tissues. (a.) Image of a representative micro–heart muscle (μHM) engineered heart tissue formed in a dog bone shaped PDMS stencil. (b.) Representative normalized voltage waveform for a μHM. μHMs were treated with 100 μM iron for 5 days and loaded with a voltage sensitive dye to measuring action potentials by high optical resolution microscopy. Waveforms for control (blue) and iron-treated (red) μHMs. (c-f) Analysis of fluorescence waveforms to measure voltage. Shown are the action potential durations (APD) for the signal to return to 30, 50, 80, and 90% of the peak signal. Statistical testing was done using a Mann-Whitney test. Each point shows an individual tissue, the central bar shows the median, and the error bars show 95% confidence intervals. Data was drawn from three or more distinct differentiations. n = 33 for control tissues (blue) and n = 18 for iron-treated tissues (red). *p-value≤0.05, **p-value≤0.01, ***p-value≤0.01, and ****p-value≤0.01.

Figure 4:. Effect of excess iron on the calcium transients of engineered heart tissues. Calcium transients were measured using calcium-sensitive dye. (a.) Representative normalized calcium transient showing the fluorescence as a function of time for a single beat of control (blue) and iron treated (red) μHMs (b.) Representative calcium transient for control (blue) and iron treated (red) μHMs. (c-f) Quantification of key parameters from calcium transients. n = 44 control (blue) and n=22 iron-fed (red) μHMs. Statistical testing was done using a Mann-Whitney test. Shown are (c.) integrated calcium flux, (d.) amplitude, (e.) Time to relax by 30% (τ30) and (f.) 50% (τ50). (g.) The amount of reactive oxygen species (ROS) was measured for both CMs and CFs using a bioluminescence assay. Wells treated with iron for 5 days (CMs: n=8, CFs: n=6) have three-fold higher ROS compared to control (CMs: n=8, CFs: n=6) as seen by Dunnett’s T multiple comparisons test. Each data point represents an individual well of a plate. Data are drawn from three or more distinct differentiations. Symbols show median and error bars show 95% confidence intervals. *p-value≤0.05, **p-value≤0.01, ***p-value≤0.01, and…

Figure 5:. In vitro motility assays reveal iron effects on the actomyosin contractile apparatus. Motile rate of actin filaments moving over a bed of cardiac myosin. Proteins were treated with 1 mM iron (a.) overnight or (b.) 3 days. Iron was included in the buffer for some assays, and some assays contained the iron chelator deferoxamine (DFO). Each point represents an individual actin filament speed recorded over 30 seconds. Statistical testing was done using an ANOVA followed by post-hoc t-tests. *p-value≤0.05, **p-value≤0.01, ***p-value≤0.01, and ****p-value≤0.01.