Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

Abstract

TTR (transthyretin) amyloidoses are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membrane protein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a KD near 650 nM. Only amyloidogenic V122I TTR compete with fluorescent V122I for cell-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase 3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.

Figure 1. Aggregation capacity and cytotoxic potential of fluorescent-labelled V122I TTR variants

(a) Percentage of aggregated (black bars) and soluble (white bars) protein for several V122I TTR variants after 3 days incubation at pH 4.4 and 37°C, with respect to total soluble protein at time zero. (b) Percentage of metabolic activity of the AC16 human cardiomyocyte cell line treated with V122I (black bars) or f-V122I (C10A/V122I/P125C-Alexa Fluor 488) TTRs, with respect to vehicle-treated cells.

Figure 2. The interaction of f-V122I TTR with AC16 cells is saturable

AC16 cells were incubated with increasing concentrations of f-V122I TTR or f-V122I TTR with 100× molar excess of unlabelled V122I to measure NSB. f-V122I TTR associated with the cells was measured by fluorescence intensity. These data were normalized for total protein content in the cell lysates measured by BCA assay. Total binding is denoted by open circles, NSB by open squares and specific binding (i.e. total-NSB) by black triangles. Specific binding data were fitted by a one site binding saturation curve (black line).

Figure 3. Native V122I TTR competes with f-V122I TTR for the same interaction sites in human cardiomyocytes

(a) AC16 cells were co-incubated with f-V122I TTR (320 nM) and increasing concentrations of amyloidogenic V122I TTR or non-amyloidogenic T119M TTR for 3 h at 4°C. The fluorescence associated with the cells was measured and normalized for total protein content. The data were normalized by the fluorescence from the samples incubated with f-V122I TTR only (100%). Filled triangles, V122I TTR; open circles, T119M TTR. (b) AC16 cells were co-incubated with f-V122I (600 nM) and equimolar amounts of unlabelled V122I TTR, T119M TTR, V122I stabilized with a small molecule (V122I-SM) or HBSS. Fluorescence associated with the cells was measured, normalized by total protein content and plotted as percentage with respect to the fluorescence of cells that had been incubated with f-V122I and HBSS only. The data show that similarly to T119M TTR, V122I-SM is unable to efficiently displace cell-associated f-V122I, whereas under the same conditions V122I TTR displaces more than 50% of f-V122I TTR.

Figure 4. Differential interaction of the amyloidogenic V122I TTR and the non-amyloidogenic T119M TTR with the human cardiac cell line AC16

AC16 cells were treated with trypsin (or HBSS as control) to remove membrane-associated proteins. V122I and T119M TTR were then incubated with the cells for 4 h at 4°C or 37°C. Cell lysates were prepared and analysed by SDS—PAGE (15% gel) and Western blot using an anti-TTR and anti-actin antibodies. The lower panel shows densitometry analysis (absorbance) of total cell-associated TTR normalized to actin. Error bars represent S.D. of the biological duplicates. The percentage of TTR/actin ratio in cells pre-treated with trypsin (trypsin +) with respect to control cells (trypsin −) is shown.

Figure 5. Aberrant interaction of V122I TTR with human cardiomyocytes

AC16 cells were incubated for 3 h with amyloidogenic V122I TTR or non-amyloidogenic T119M TTR (4 μM) at 4 or 37°C. Half of the experimental samples were then incubated with trypsin to remove the extracellular cell membrane-associated TTR. Cell lysates were analysed by SDS–PAGE and Western blot using an anti-TTR antibody and actin (upper panels). Band intensities were quantified by densitometry (absorbance) and plotted in the lower panels as TTR/actin ratio.

Figure 6. V122I TTR and T119M TTR accumulate in the presence of proteasomal (MG132) and lysosomal (NH₄Cl) activity inhibitors

AC16 cells were pre-incubated for 30 min with 0.5 μM MG132, 10 mM NH₄Cl or vehicle only (control). V122I TTR and T119M TTR were then added at a final concentration of 4 μM and the cells were incubated for 24 h at 37°C. Cell lysates were prepared after removing cell membrane-associated TTR with trypsin, and analysed by SDS–PAGE and Western blot developed with anti-TTR antibody and anti-actin antibody. Data are presented as TTR/actin ratio for each treatment and normalized to the average ratio values obtained in cells incubated with the vehicle only.

Figure 7. MBCD decreases V122I TTR-induced cytotoxicity in human cardiomyocytes in a dose-responsive manner

AC16 cells were incubated with several concentrations of MBCD (0 to 8 mM) for 15 min followed by incubation with amyloidogenic V122I TTR (grey bars) or non-amyloidogenic T119M TTR (white bars) at 8 μM. After 24 h incubation at 37°C, the percentage of cell viability with respect to vehicle-treated cells was measured by resazurin assay.

Figure 8. MBCD interferes with the interaction of TTR with human cardiac cells

AC16 cells were incubated with amyloidogenic (V122I) or non-amyloidogenic (T119M) TTR in the presence or absence of 8 mM MBCD for 4 h at 4 or 37°C. Afterwards, half of the samples were treated with trypsin to remove membrane-associated TTR. Cell lysates were prepared and analysed by SDS–PAGE and Western blot and developed with an anti-TTR antibody (a). TTR band density was measured and the percentage of TTR associated with the MBCD-treated cells was calculated with respect to vehicle (DMSO)-treated cells (b). The percentage of internalized V122I TTR (c) and T119M TTR (d) was calculated from the density values of cells treated with trypsin with respect to cells not treated with trypsin.

Figure 9. Amyloidogenic V122I but not non-amyloidogenic T119M TTR induces the generation of superoxide species and caspase 3/7 activation in human cardiomyocytes

(a) AC16 cells were treated with V122I TTR, T119M TTR, Antimycin A (positive control) or HBSS (vehicle control) for 24 h. The insults were then removed and the cells loaded with the superoxide-specific reagent DHE as detailed in the experimental section. Red fluorescence produced by DHE oxidation is shown. (b) AC16 cells were treated with V122I TTR, T119M TTR or, HBSS for 6 h at 37°C. Cell lysates were prepared as detailed in the experimental section and were added to Ac-DEVD-AFC caspase 3/7 substrate. Fluorescence intensity generated by cleavage of the substrate was measured. Asterisks in both panels indicate statistical significance with P<0.05 of experimental samples compared to HBSS (vehicle control) treated cells (unpaired t test).