rVλ6Wil fibrils bind cultured cardiomyocytes.

<p>(A) Fluorescent synthetic fibrils composed of rVλ6Wil (Alexa Fluor-488, green; arrow) bound specifically to cultured AC10 cardiomyocytes (blue, nuclei; red, f-actin). (B) Confocal micrographs of cultured cardiomyocytes (blue, nuclei; purple, f-actin) after incubation with 1 μM fluorescent rVλ6Wil fibrils (green). Optical sections at the surface (Z-slice 20), center (Z-slice 45), and bottom (Z-slice 55) demonstrated the presence of fibrils on the cell surface (arrow) and rare intracellular material (arrowhead). All images were enhanced equivalently by increasing the brightness.</p

Metabolic dysfunction of cardiomyocytes is dependent on the presence of rVλ6Wil fibrils.

<p>(A) Supernatants from a suspension of rVλ6Wil fibrils generated by centrifugation of a fibril suspension at either 435,000 x g or 16,000 x g do not decrease MTT reduction by cardiomyocytes (supernatants were used at a 1x or 10x volume equivalent [vol. eq] to the unfractionated fibril suspension). Filtrate from a suspension of rVλ6Wil fibrils passed through a 100,000 (100K) MWCO filter did not contain material that affected MTT reduction (n = 3, per assay). (B) HPLC analysis of supernatant samples from the 435,000 x g (435K) and 16, 000 x g (16K) centrifugation as compared to the same volume of starting rVλ6Wil monomer (M) material. (C) Over the course of rVλ6Wil fibrillogenesis, only samples of the reaction mixture containing ThT-positive material significantly decreased MTT reduction when added for 24 h to AC10 cardiomyocytes. Gray circles, thioflavin T fluorescence (excitation = 450 nm; emission = 490 nm); black circles, MTT reduction (n = 3 sample per time point).</p

rVλ6 amyloid fibrils cause metabolic dysfunction in cultured cardiomyocytes.

<p>Synthetic fibrils composed of rVλ6Wil (A), Jto (B), JtoR68S (C), and non-amyloid fibrils elastin (D) were shown to be fibrillar by using electron microscopy (left panels; bar = 100 nm; arrows indicate the region of fibrils shown inset). MTT reduction was measured in cultured AC10 human cardiomyocytes in the presence of fibrils (light) and monomeric proteins (dark) from 1 nM– 1 μM. *<i>p</i><0.05, for an unpaired t-test (right panel, n = 3). EM images were modified equivalently by increasing contrast and applying a shadowing effect, using Image J.</p

rVλ6Wil fibrils cause a decrease in cardiomyocytes MTT reduction without inducing cell death.

<p>(A) Fluorescence microscopy demonstrating the increase in binding of Alexa Fluor-488 labeled rVλ6Wil fibrils (1 μM) over 24 h of incubation (blue, nuclei; red, f-actin). (B) Quantitation of rVλ6Wil fibril binding to cardiomyocytes (μm²/cell; closed circles) correlated inversely, over 24 h, with MTT reduction (open circles). (C) Cell number, quantified using crystal violet (black) did not decrease over 72 h of incubation with 1 μM rVλ6Wil fibrils despite a decrease in MMT reduction (grey; n = 3 samples per time point). (D) Fluorescence micrographs of AC10 cardiomyocytes incubated with or without non-fluorescent 1 μM rVλ6Wil fibrils (blue, nuclei; green, f-actin; numbers represent cell count in 10x objective field of view). (E) Cell viability of AC10 grown in culture for 4 days in the presence or absence of 1 μM rVλ6Wil fibrils for 24, 48, or 72 h, was assessed by CMFDA fluorescence (original objective, 20x. Images were cropped and digitally magnified 4x. Numbers represent the mean live/dead cells, <i>n = 4</i> independent fields of view).</p

rVλ6Wil fibrils cause oxidative stress in cardiomyocytes without a decrease in cellular ATP.

<p>(A) After a 2 h incubation with rVλ6Wil fibrils or monomer, cellular ATP levels, as detected by luminescence (black bars), were not significantly reduced relative to the PBS-treated control. A decrease in MTT reduction (gray bars) was observed in the presence of fibrils (n = 3). (B) Oxygen consumption rate was significantly higher in AC10 cells (<i>p</i> < 0.05) treated with 1μM rVλ6Wil fibrils as compared to treatment with PBS or rVλ6Wil monomer protein (n = 6 wells). (C) Cardiomyocytes labeled with DCFH-DA exhibited a dose-dependent increase in ROS production after 24 h of exposure to rVλ6 Wil fibrils (n = 3).</p