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. 2022 Jan 22;11(3):373.
doi: 10.3390/cells11030373.

Beta-Amyloid Instigates Dysfunction of Mitochondria in Cardiac Cells

Sehwan Jang  1 , Xavier R Chapa-Dubocq  1 , Rebecca M Parodi-Rullán  2 , Silvia Fossati  2 , Sabzali Javadov  1
Affiliations

Beta-Amyloid Instigates Dysfunction of Mitochondria in Cardiac Cells

Sehwan Jang et al. Cells. .

Abstract

Alzheimer's disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ1-40 and Aβ1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ1-40 and Aβ1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ1-42 had more adverse effects than Aβ1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ1-40 and Aβ1-42 significantly increased Ca2+ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells.

Keywords: Alzheimer’s disease; beta-amyloid; cardiomyocytes; coronary artery endothelial cells; mitochondria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Aβ decreased cell viability and impaired cell morphology (A,B): Primary HCAEC grown for 20 days in the presence and absence of 10 µM Aβ1-40 or Aβ1-42. (A): Cell viability. n = 4 per group. * p < 0.05 vs. control. (B): Representative phase-contrast images of control (vehicle: DMSO) cells and cells grown with 10 μM Aβ1-40 or Aβ1-42. Bar = 100 µm (C,D): H9c2 cardiomyocytes grown with Aβ1-40 or Aβ1-42 for 96 h. (C): Cell viability. n = 4 per group. * p < 0.05 vs. control. (D): Representative images of control (vehicle: DMSO) cells and cells grown with 10 μM Aβ1-40 or Aβ1-42. Bar = 100 µm.
Figure 2
Figure 2
Accumulation of aggregated Aβ. (A): Immunostaining of Aβ in primary HCAEC grown with 10 µM Aβ1-40 or Aβ1-42 for 20 days. Bar= 100 µm (B): Immunostaining of Aβ in H9c2 cardiomyocytes grown with 10 µM Aβ1-40 or Aβ1-42 for 96 h. Bar = 100 µm.
Figure 3
Figure 3
Aβ-induced mitochondrial and cellular dysfunction. (A–E): Primary HCAEC cultured for 20 days in the presence and absence of 10 µM Aβ1-40 or Aβ1-42. (A): mtROS levels measured by MitoSOX Red (Thermo Fisher). (B): Cellular ROS levels measured using Ampiflu Red (Sigma) in permeabilized cells. (C): ΔΨm measured by JC-1 (Thermo Fisher). (D): Activated caspase 3/7 levels measured using the CellEvent™ Caspase-3/7 Green Detection Reagent (Thermo Fisher). (E): ATP levels measured by ATP-Red (Sigma). (F–J): H9c2 cardiomyocytes cultured for 96 h in the presence or absence of 10 µM Aβ1-40/Aβ1-42. All parameters were measured by the same methods used for primary HCAEC (A–E). (F): mtROS levels. G: Cellular ROS levels. (H): ΔΨm. (I): Activated caspase 3/7 levels. (J): ATP levels. n = 4 per group for all parameters of primary HCAEC and H9c2 cardiomyocytes. * p < 0.05, ** p < 0.01 vs. control (C).
Figure 4
Figure 4
Cells and mitochondria demonstrated early response to Aβ. (A–G): Primary HCAEC grown for 48 h in the presence and absence of 10 µM Aβ1-40 or Aβ1-42. (A): Cell viability after 48 h of treatment. n = 4 per group. (B): Representative phase-contrast images of control (vehicle: DMSO) cells and cells treated with 10 μM Aβ1-40 or Aβ1-42 for 48 h. Bar = 100 µm (C): mtROS levels (D): Cellular ROS levels. (E): ΔΨm. (F): Activated caspase 3/7 levels. (G): ATP levels. (H–N): H9c2 cardiomyocytes grown for 48 h in the presence and absence of 10 µM Aβ1-40 or Aβ1-42. (H): Cell viability after 48 h of treatment. (I): Representative phase-contrast images of cells. Bar = 100 µm (J): mtROS levels (K): Cellular ROS levels. (L): ΔΨm. (M): Activated caspase 3/7 levels. (N): ATP levels. n = 4 per group for all parameters of primary HCAEC and H9c2 cardiomyocytes. * p < 0.05 vs. control (C).
Figure 5
Figure 5
Aβ1-42 decreased CRC. (A,B): Primary HCAEC grown for 48 h in the presence and absence of 10 µM Aβ1-40 or Aβ1-42. (A): Representative traces of the fluorescence intensity of Calcium Green 5N. (B): Quantification of CRC calculated from the burst cycles (cycles that showed the highest fluorescence signal increase). (C,D): H9c2 cardiomyocytes were grown for 48 h in the presence and absence of 10 µM Aβ1-40 or Aβ1-42. (C): Representative traces of the fluorescence intensity of Calcium Green 5N. (D): CRC was calculated from the burst cycles. The addition of 1 nmol calcium was done every 3 min (one cycle) at 37 °C. Assays without cells were used as a baseline. n = 6 per group. * p < 0.05 vs. control.
Figure 6
Figure 6
Visualization of Aβ aggregates and mitochondria by immunostaining. (A): Representative images of primary HCAEC. (B): Representative images of H9c2 cardiomyocytes. Primary HCAEC and H9c2 cardiomyocytes grown for 48 h in the presence and absence of 10 µM Aβ1-40 or Aβ1-42 were fixed and stained with anti-amyloid beta (green) and anti-ATP5A antibody (red). Nuclei were stained by DAPI. Bar = 10 µm.
Figure 7
Figure 7
The effect of Aβ on isolated cardiac mitochondria. (A,B): Isolated rat heart mitochondria were used to analyze mitochondria swelling. (A): Mitochondrial swelling measured as decrement of A525 during the first 2 min of the calcium overload. Sanglifehrin A (SfA), a cyclophilin D (an mPTP regulator) inhibitor, was used to demonstrate that the swelling was mediated by mPTP. (B): Quantification of mitochondrial swelling rate presented as a percentile of control. Aβ1-40 or Aβ1-42 were added 10 min before the experiment to 10 µM. 0.1% DMSO was used as a vehicle. n = 3–6 per group. (C): mtROS levels (D): ΔΨm. (E): ATP levels. n = 4 per group. * p < 0.05 vs. control.
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