Silica nanoparticles induce cardiotoxicity interfering with energetic status and Ca2+ handling in adult rat cardiomyocytes

Abstract

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO₂) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO₂-induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO₂ particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO₂ cell membrane association and internalization were analyzed. SiO₂ showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca²⁺ handling, which showed impaired contractility and intracellular Ca²⁺ transient amplitude during β-adrenergic stimulation in SiO₂ treatment. The time to 50% Ca²⁺ decay increased 39%, and the Ca²⁺ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity. Moreover, SiO₂ treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H₂O₂ generation were also observed. These data indicate that SiO₂ increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca²⁺ release, and reduced cell shortening. This mechanism of SiO₂ cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death.NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca²⁺ handling.

Keywords: Ca2+; cardiomyocyte; nanoparticle; silicon dioxide; toxicity.

Fig. 1.

Characterization of silica particles by scanning electron microscope (SEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and ζ-potential. A: representative micro-SiO₂ micrograph showing size and morphology. Insert shows size distribution of micro-SiO₂ obtained by field emission gun (FEG)-SEM (n = 100 particles). B: XRD analysis with the composition of the talline domains of nano- and micro-SiO₂. All samples show the characteristic diffraction pattern of amorphous silica presenting a broad peak (10–30°) with a maximum around 22°. C: the hydrodynamic sizes of nano- and micro-SiO₂ particles in ultrapure water. D: the ζ-potentials of nano- and micro-SiO₂ particles in ultrapure water. Black and gray curves correspond to micro-SiO₂ and nano-SiO₂, respectively, in each graphic.

Fig. 2.

Viability of adult cardiomyocytes after 24-h exposure of SiO₂ particles and mechanism of cell death. A: nano-SiO₂ and micro-SiO₂ viability evaluated by Alamar blue viability test. B: representative dot plots from flow cytometry with AnnexinV-PE-Cy7/Ghost Red 780 cell death analysis. A positive control of apoptosis cell death (56°C for 10 min) is shown in comparison to control (CT), nano-SiO₂, and micro-SiO₂ treated groups at 99.5 µg/ml. C: the percentage of necrotic, live, and apoptotic cells was quantified and is shown in the bar graph. D: time-dependent cytotoxicity by lactate dehydrogenase leakage at 0–24 h, in cultured cardiomyocytes with 24-h incubation of nano-SiO₂ and micro-SiO₂ at its half-maximal inhibitory concentration (IC₅₀) (99.5 µg/ml for nano-SiO₂ and 1,500 µg/ml for micro-SiO₂ particles). Values are means ± SE, n = 3–10; *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

Fig. 3.

SiO₂ cellular association and internalization in adult rat cardiomyocytes. Representative SEM micrographs showing nano-SiO₂ (A) and micro-SiO₂ (B) individual and agglomerates/aggregates association to the cellular membrane. Representative SEM–energy-dispersive X-ray spectroscopy (EDS) transversal cut images of untreated (C), Nano-SiO₂ (D), and Micro-SiO₂ (E) particle internalization after 24 h of incubation (99.5 μg/ml). F: quantification of internalized SiO₂ particles by particle-induced X-ray emission, as a function of time (for nano-SiO₂ 99.5 μg/ml and micro-SiO₂ 1,500 μg/ml). G: representative confocal microscopy image of the F-micro-SiO₂ shown as x, y and z-stacks. The cytoskeleton is shown in red and the brighter dots correspond to F-micro-SiO₂ particles. The arrows indicate the SiO₂ particles in the cell. The symbol N (in G) marks the nucleus location in the cell. Values are means ± SD; a t-test was used as statistical analysis between nano- and micro-SiO_2. Values are means ± SE, n = 3–5. *P < 0.05 vs. nano; ‡P < 0.05 vs. basal (time 0).

Fig. 4.

Cardiomyocytes surface showing silica particles by energy-dispersive X-ray spectroscopy (EDS). Right: FEG-SEM images of micro-SiO₂ (A) and nano-SiO₂ (B) individual and agglomerates/aggregates association to the cellular membrane and an adult rat cardiomyocyte as a control sample (C). Left: the respective four EDS images of elemental maps for carbon (red), sodium (cyan), silicon (magenta/yellow), and oxygen (green).

Fig. 5.

Micro-SiO₂ internalization in adult myocytes. A: semiquantitative increase in fluorescence in a dose-dependent manner of F-micro-SiO₂ particles. B: representative images from ventricular myocytes at the control (CT) condition and 150 µg/ml F-micro-SiO₂ concentration. The cytoskeleton is shown in red and F-micro-SiO₂ in green. Values are means ± SE, n = 4–6. *P < 0.05 vs. CT.

Fig. 6.

Cell shortening and Ca²⁺ handling in cardiomyocytes treated with nano- and micro-SiO₂ particles. The percentage of cell shortening before (CT) and after a 24-h treatment of nano-SiO₂ and micro-SiO₂ at its IC_50. Values are means ± SE, n = 15. **P < 0.001 vs. CT.

Fig. 7.

Calcium transient characterization at basal condition and after β-adrenergic stimulation. A and B: representative images from field-stimulated control and nano-SiO₂-treated myocytes under basal conditions and after isoproterenol (ISO) perfusion, the arrow indicates the analyzed transient for semiquantitative results. C: peak Ca²⁺ transient amplitude. D: time to 50% of decay (T_50%). Values are means ± SE. CT: n = 22–33 cells/3 animals; nano-SiO₂: n = 19–34 cells/3 animals. *P < 0.05 vs. CT; **P < 0.001 vs. CT; ‡‡P < 0.001 vs. basal; ‡‡‡P < 0.0001 vs. basal.

Fig. 8.

Calcium sparks characterization in isolated myocytes. A: line scan images from control conditions (left) and after 24-h incubation with Nano-SiO₂ (right). Surface plots can be seen above line scan images. Line profiles from selected 2-µm regions (black marks in line scan images) can be seen on images. Pooled data describing spark frequency (B) and amplitude (C). Values are means ± SE, n = 39 cells/3 animals/CT; 33 cells/3 animals/nano-SiO₂). *P < 0.05 vs. CT; **P < 0.001 vs. CT.

Fig. 9.

Calcium content in the sarcoplasmic reticulum. Representative images from caffeine-induced Ca²⁺ transients from control (CT) (A) and nano-SiO₂-treated cardiomyocytes (B). Black arrow indicates caffeine application (10 mM). C: pooled data for peak Ca²⁺ transient amplitude [sarcoplasmic reticulum (SR) load; n = 25 cells/3 animals/CT; 24 cells/3 animals/Nano-SiO₂].

Fig. 10.

Mitochondrial membrane potential, ATP content, reactive oxygen species (ROS) production, and glutathione depletion. Representative images from control (CT) (A) and nano-SiO₂-treated cardiomyocytes (B) loaded with TMRE by confocal microscopy. ATP content (luminescence relative units, LRU) (C) and relative fluorescence from mitochondrial membrane potential (%) (D) from CT and nano-SiO₂-treated cardiomyocytes. H₂O₂ production (E) and glutathione content (F) in cultured cardiomyocytes with 24-h incubation of nano-SiO₂ at its IC₅₀. Values are means ± SE, n = 10–11 cells (a–D); n = 3 (E and F); *P < 0.05 vs. CT; ***P < 0.0001 vs. CT.

Fig. 11.

Proposed mechanism of SiO₂-induced cardiotoxicity. Scheme of the suggested mechanism by which nano-SiO₂ particles induces cardiotoxicity interfering with energetic status and Ca²⁺ handling in cardiomyocytes. GSH/GSSG, reduced/oxidized glutathione; NCLX, Na⁺/Ca²⁺ exchanger; O₂^·−, superoxide radical; LTCC, L-type calcium channels; SR, sarcoplasmic reticulum; RyR2, ryanodine receptors; TnC, troponin C.