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

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.

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.

the technological advances of the 20th century have been the most powerful and fastest phenomena in human history. From the largest to the smallest artifacts, newly developed micro/nanomaterials have provided the main base for creating better and more versatile items in almost every area of knowledge. In this regard, nanotechnology, as we know it in the 21st century, is gaining enormous acceptance in the electronic, manufacturing, agriculture, consumer-product, and food additive industries as well as, more recently, the biotechnological and biomedical fields. Nanomaterials, such as titania (titanium dioxide) and silica (silicon dioxide, SiO₂), are used to improve and create new functional properties for common products (51). While nanomaterials based on biomaterials like polymers, such as poly (lactic-co-glycolic acid) (PGLA) or hydrogels, are considered of low toxicity, recent evidence has shown that some nanoparticles, such as metal, metal oxides (e.g., silica and titania), and carbon nanotubes, may pose potential risks to the environment, the food chain, and human health (35). Several studies have shown that nanoparticles can penetrate the body by different routes, including the skin, respiratory system, and gastrointestinal tract. In normal conditions, the inhalation of SiO₂ is likely the major route by which nanoparticles enter the body, and defective particle clearance in the airways increases the chance of particles translocating to different organs, including the heart, lungs, and kidneys (1, 45). Moreover, novel clinical diagnostic protocols and therapeutic drug treatments allow biological systems to undergo major and extended nano- and microparticle exposure, which can lead to an inflammatory response, fibrosis, oxidative stress, and cell death (15). Nano- and micro-SiO₂ particles have drawn a lot of attention in the biomedical field. Currently, different sizes of SiO₂ particles are being used in the clinical setting (23). Silica’s usefulness as a biomedical tool has led to focused use in relation to human diagnostic, imaging, and labeling and in new targeting systems, which allows these nano- and micromaterials to interact directly with the body, leading to increased concentrations in the bloodstream and higher organ accumulation. In recent years, SiO₂ nano- and microparticles have been used extensively because of their chemical properties and the capacity to manipulate their surface for different biomedical purposes, including for imaging and for therapeutic vehicles (5, 42, 50). However, while silicon-based particles have been considered to be of low toxicity and safe for use as a therapeutic vehicle (40), recent studies have shown evidence of toxicity, though this toxicity depends on a variety of factors, including the composition, differential toxicity among cell types, and—remarkably—particle size (13, 34, 38, 45). In particular, nanoscale particles (<100 nm) are a controversial topic of debate, and the related research conclusions lack consistency. For instance, in vitro and in vivo effects exerted by SiO₂ particles have recently been studied at the cardiovascular level, which showed cytotoxicity related to induced oxidative stress (10). Acute in vivo SiO₂ exposure also leads to an inflammatory response and endothelial dysfunction, which are strongly related to oxidative stress production and to impairments in myocardial antioxidant enzymes (8). Chronic exposure to SiO₂ particles has also been associated with alterations to different molecular mechanisms related to angiogenesis, heart formation and development, and pericardial edema and bradycardia, which can lead to cardiovascular diseases (10). More recently, microarray analysis showed that SiO₂ cardiotoxicity in a zebrafish model was related to oxidative stress and neutrophil-mediated cardiac inflammation (9). However, the mechanisms involved in SiO₂-induced toxicity in mammalian heart cells are still unknown.

In this study, we explored, for the first time, SiO₂-induced toxicity and impaired contractility and calcium handling in isolated adult cardiomyocytes exposed to 24 h incubation with two different sizes of SiO₂ particles: 7-nm AEROSIL 380 Fumed Silica, which will be referred to henceforth as nano-SiO₂, and 670-nm particles synthesized by our group, which will be referred to henceforth as micro-SiO₂. We explored the mechanism of cell death caused by nano- and micro-SiO₂ at their half-maximal inhibitory concentration (IC₅₀) and related these effects to the phenomenon of SiO₂ particle association with membrane and cellular uptake. Using confocal microscopy, we evaluated cardiomyocyte shortening and intracellular Ca²⁺ handling. In addition, we measured the mitochondrial membrane potential, oxidative stress, and ATP content to determine whether SiO₂ particles induced mitochondrial dysfunction and whether impairment of bioenergetics contributed to SiO₂-induced cardiac dysfunction.

MATERIALS AND METHODS

Chemicals.

All reagents were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO) unless otherwise stated.

Synthesis of micro-SiO₂.

The 670-nm SiO₂ particles were synthesized in accordance with the Stöber method (49). Briefly, a mix of 85% of ethanol, 3.6% of concentrated ammonium hydroxide solution, and 11.5% (vol/vol) of ultrapure water was stirred. Separately, a solution was prepared with 30.2% of tetraethyl orthosilicate, 13.2% of (3-aminopropyl) triethoxysilane, and 56.6% (vol/vol) of ethanol. This second solution was added slowly to the first one, and the mixture was magnetically stirred for 12 h. The resulting colloidal suspension was centrifuged, and the precipitate was rinsed with pure ethanol at least three times. The solid was then dried in a muffle at 70°C for 24 h. For the preparation of fluorescent silica spheres (F-micro-SiO₂), dried micro-SiO₂ spheres obtained of the aforementioned procedure were subsequently calcined at 400°C for 2 h. These calcined particles were then washed three times with ethanol in an ultrasonic bath. The fluorescence is attributed to the introduction of carbon and oxygen defects in the silica network after the calcination of (3-aminopropyl) triethoxysilane (20).

Nano-SiO₂ and micro-SiO₂ characterization.

Nano-SiO₂ (Degussa, Parsippany, NJ), micro-SiO₂, and F-micro-SiO₂ were characterized by a field emission gun–scanning electron microscope (FEG-SEM) (Model 200 Nova NanoSEM FEI Company). The measurements were performed under low vacuum conditions with a Helix detector and a 10–18 kV electron beam. Zeta potential and size measurements using dynamic light scattering (DLS) were performed on a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Briefly, nano- and micro-SiO₂ were suspended in ultrapure water, Tyrode, M-199, and M-199 + bovine serum albumin (BSA) (5%) solutions and were irradiated with a red laser (HeNe laser, wavelength λ = 632.8 nm), and the intensity fluctuations of the scattered light (detected at a backscattering angle of 173°) were analyzed to obtain an autocorrelation function. The software (DTS v5.03) provided both the mean hydrodynamic diameter and polydispersity index by using the method of cumulants (according to the international standard ISO 13321:19963) and a size distribution by using a regularization scheme by intensity, volume, and number. Crystalline structure was determined by X-ray diffraction (XRD) on a Panalytical Empyrean diffractometer (PANalytical, Westborough, MA).

Cardiomyocytes isolation.

All the studies were performed in accordance with the animal care guidelines of the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). All procedures were approved by the Institutional Animal Use and Care Committee (protocol number 2011-Re-017). Rat ventricular myocytes were isolated by collagenase II digestion of perfused hearts (14). Briefly, male Wistar rats weighing 250–300 g were used to isolate cardiac cells. Animals were heparinized and anesthetized with pentobarbital sodium (1,000 U/kg and 100 mg/kg, ip, respectively) before removal and hanging the heart. Hearts were mounted on a Langendorff apparatus and then perfused with Tyrode medium (TM) (in mM): 128 NaCl, 0.4 NaH₂PO₄, 6 glucose, 5.4 KCl, 0.5 MgCl-6H₂O, 5 creatinine, 5 taurine, and 25 HEPES, pH 7.4 at 37°C for 5 min and digested by 0.1% collagenase type II (Worthington Biochemical, Lakewood, NJ), dissolved in TM for 15 min. Ventricles were dissected and cells mechanically disaggregated. Cardiomyocytes were washed in crescent concentrations of calcium (0.25, 0.5, 1, and 1.5 mM) plus 0.1% albumin contained in the TM. Intact cardiomyocytes were cultured in M-199 medium supplemented with (in mM) 5 taurine, 5 creatine, 2 l-carnitine, 2.5 sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin on laminin-coated culture dishes at a density of 1 × 10⁴ viable cells/cm². Cells were incubated at 37°C in 95% air-5% CO₂ for 2 h before all experiments.

Determination of nano-SiO₂ and micro-SiO₂ cytotoxicity and cell death mechanisms.

To determine SiO₂ cytotoxicity, varying concentrations of nano- and micro-SiO₂ were added to culture media, and the incubation was continued for 24 h. At the end of the incubation period, cytotoxicity of SiO₂ was determined by the Alamar blue viability test (Life Technologies, Carslbad, CA). The IC₅₀ was calculated with Origin software (Northampton, MA). Necrosis and apoptosis were measured with Ghost Dye Red 780 and Annexin V-PE-Cy7-stained cells by using a BD FACSCantoII flow cytometer (BD Biosciences, Heidelberg, Germany). Cells were stained according to the manufacturer’s instructions. Briefly, 1 μl of Ghost Dye (Tonbo Bioscience, San Diego, CA) solution was added in 1 ml of TM and incubated for 30 min, washed, and resuspended in 300 μl with 5 μl of fluorochrome-conjugated Annexin-V-PE-Cy7 (eBioscience, San Diego, CA) for 10 min at room temperature. Finally, cells were washed and fixed using 4% paraformaldehyde for 20 min. After incubation, cell monolayers were washed twice with TM and detached from the culture plate by scrapping. In total, 10,000 cells were analyzed per measurement in TM solution, and data was analyzed with FlowJo 8.8.6 software (Treestar, Ashland, OR). The release of cytoplasmic lactate dehydrogenase (LDH), as a marker of cell integrity and necrosis, was determined enzymatically following the interconversion of pyruvate and lactate and with concomitant oxidation of NADH to NAD⁺, followed by using spectrophotometry at 340 nm. Values were adjusted to an LDH calibration curve. The activity of caspases-3 and -7, to assess SiO₂-induced apoptosis, was measured in cell pellets by using the Apo-ONE fluorescent substrate (Promega, Madison, WI).

Characterization of nano-SiO₂ and micro-SiO₂ in the cardiomyocyte.

To assess the interaction between cardiomyocytes membrane and SiO₂ particles, a 24-h cell incubation with nano- and micro-SiO₂ was analyzed by FEG-SEM. Samples were prepared by seeding cardiomyocytes on to 5- × 7-mm silicon chips specimen supports (Ted Pella, Redding, CA). Samples were fixed with 2.5% glutaraldehyde in PBS buffer (Electron Microscopy Sciences, Hatfield, PA) for 20 min and were then dehydrated in increasing concentrations of ethanol for 10 min. Specimens were mounted on SEM stubs (Ted Pella) with conductive adhesive tape (Ted Pella) and coated with a gold thin film. FEG-SEM surface images were acquired at different magnifications under high vacuum at 15.00 kV, spot size 3.0, with a Nova NanoSEM 200 (FEI, Hillsboro, OR). The energy-dispersive X-ray spectroscopy (EDS) analysis was performed with an Oxford Instruments analyzer at 15 kV. In addition to surface images, transversal cuts were done on random cardiomyocytes by focused ion beam, and EDS was performed to assess the intracellular location of the SiO₂ particles using a Zeiss Crossbeam 340, using 30 kV for the focused ion beam and 12 kV for the SEM. Ten individual cells from each treatment were selected randomly for imaging and EDS analysis.

Particle-induced X-ray emission (PIXE) was used to quantify the total amount of silica present in the cardiomyocytes. This technique has been used recently for nanomaterial quantification in complex matrices (25). A 2.5-MeV proton beam was used to irradiate the cells with currents < 1 nA to reduce sample damage. The cardiomyocytes were incubated with nano- and micro-SiO₂ at its IC₅₀ with different end time points (0, 1, 12, and 24 h). After each incubation time, cells were 1) washed with M-199 to remove free particles, 2) detached from the culture plate, and 3) centrifuged at 500 rpm through a 250-mM sucrose solution to separate noninternalized and nonadhered silica particles as well as dead cardiomyocytes from the intact cells. The pellet (alive cells) was resuspended in 200 µl of MilliQ H₂O, finally transported sequentially into truncated sample holders, and removing the aqueous media was removed as previously reported (26). Data analysis was done with GUPIXWIN software (7).

Intracellular localization of F-micro-SiO₂ by confocal microscopy examination.

To determine the intracellular localization of F-micro-SiO₂, cardiomyocytes were seeded into laminin-covered glass coverslips. Cells were incubated with F-micro-SiO₂ for 24 h, followed by fixation and staining of cellular structures. Briefly, after SiO₂ incubation, cells were fixed using 4% paraformaldehyde in PBS buffer for 20 min. Immunofluorescence staining was performed by cell permeabilization with 0.1% Triton X-100 (Thermo Fisher Scientific, Waltham, MA) in PBS for 3 min. Cells were rinsed three times in PBS, and a blocking step with 1% bovine serum in PBS was performed for 10 min. Actin filaments were stained with 0.5 U/μl of Alexa Fluor 568-conjugated phalloidin (Life Technologies, Grand Island, NY) for 20 min at room temperature. Glass coverslips were rinsed with PBS and mounted with Vectashield mounting media (Vector laboratories, Burlingame, CA). Samples were imaged with a Leica TCS SP5 confocal microscope equipped with a D-apochromatic 63X, 1.2 NA, oil objective (Leica Microsystems, Wetzlar, Germany). To assess cellular internalization of F-micro-SiO₂, a stack of two-dimensional (2-D) images, with a focal plane thickness of 1 µm, were collected every 1 µm along z-axis using an excitation (ex) and emission (em) of 488 nm and 500–700 nm, respectively. For the representative images to assess cellular internalization of F-micro-SiO₂, wavelengths of an ex of 488 and 543 nm and em of 500–600 and 620–720 nm, for F-micro-SiO₂ and cytoskeleton were used, respectively. The tridimensional reconstructions were made from z-stacks with a focal plane thickness of 2 µm using the 3-D viewer plugin from ImageJ (National Institutes of Health, Bethesda, MD).

Ca²⁺ handling and cell shortening in intact cardiomyocytes.

The Ca²⁺ transients, Ca²⁺ sparks, and cell shortening parameters were measured as previously described (29, 53). Twenty-four hour cultured cardiomyocytes on laminin-covered glass coverslips, with Nano- and Micro-SiO₂ at its IC₅₀, were incubated in TM with Fluo-4 AM or Fluo-3 AM (Life Technologies) to evaluate Ca²⁺ handling and cell shortening. Afterward, the cells were washed with a fluorophore-free solution. Loaded cells were mounted in a superfusion chamber. All fluorescence measurements were acquired with a Leica TCS SP5 confocal microscope equipped with a D-apochromatic 63X, 1.2 NA, oil objective (Leica Microsystems). Line scan images were recorded along the longitudinal axis of the cell at 400 Hz with an argon laser to excite the fluorophore at 488 nm, and its emission was collected at 500–600 nm. For cell shortening, a pinhole optimized for a 4-µm-thick section in the focal plane was used, while a 1-µm-thick section was used for Ca²⁺ signaling. Cell shortening was evaluated under field stimulation at 0.5, 1, and 2 Hz (MYP100 MyoPacer Field Stimulator; Ion-Optix, Milton, MA). For Ca²⁺ transient and Ca²⁺ sparks, the cells were field stimulated by 3 to 5 electric pulses at 1 Hz to attain steady-state sarcoplasmic reticulum (SR) Ca²⁺ content. The Ca²⁺ signaling under β-adrenergic stimulation was evaluated by perfusion of isoproterenol (ISO) at 100 nM, and the records were taken after 10 min of exposition. For SR Ca²⁺ content, the cells were field stimulated at 1 Hz before a rapid caffeine application at 10 mM. Fluorescence data were normalized as ΔF/F₀, where F is fluorescence intensity and F₀ is average fluorescence at rest.

Bioenergetic status measurements.

Mitochondrial membrane potential (Δψ) was evaluated in cultured cardiomyocytes by confocal microscopy using a Leica TCS SP5 confocal microscope equipped with a D-apochromatic 63X, 1.2 NA, oil objective (Leica Microsystems). Cardiomyocytes on laminin-coated coverslips were treated with nano-SiO₂ for 24 h, washed with TM to remove particles, and loaded with 300 nM tetramethylrhodamine ethyl ester perchlorate (TMRE) (Thermo Fisher Scientific) during 30 min at room temperature and darkness. TMRE accumulates in metabolically active mitochondria producing a fluorescence that is proportional to Δψ (44). Afterward, the cells were rinsed with fluorophore-free TM and placed in a recording chamber. TMRE signal was measured with an ex at 543 nm and an em to 560–700 nm using oil immersion 40X objective. To evaluate the Δψ, records were taken after field stimulation at 1 Hz. The 2-D images were acquired with Leica LAS AF version 2.7 format at 400 Hz with 512 × 512 image size and a focal plane thickness of 1 µm. The total fluorescence from the 2-D images was evaluated and plotted as a percentage of relative fluorescence, the control group being 100%. ATP content was measured in control group (CT) cardiomyocytes as well as those treated with nano-SiO₂ at its IC₅₀, after a 24-h exposure using the CellTiter-Glo Luminescent Assay (Promega).

Oxidative stress markers.

A change in the concentration of H₂O₂ in the medium was detected by fluorescence of the oxidized Amplex Red (Life Technologies) using wavelengths of ex at 550 nm and em at 585 nm. The response of Amplex Red to H₂O₂ was calibrated by sequential additions of known amounts of H₂O₂ from 100 to 1,000 pmol/min. Glutathione (GSH) was measured as previously described (39). Briefly, cells were dissolved in medium containing (in mM) the following: 50 KH₂PO₄, pH 7.5, with the addition of 0.2% Tx-100, 1 PMSF, and 0.6% sulfosalicylic acid for 10 min a 4°C and centrifuged at 8,000 rpm for 10 min. Protein concentration was measured by the Lowry method. Thirty micrograms of protein were incubated in a medium that contained (in mM) the following: 50 KH₂PO₄, 1 EDTA, pH 7.5 with the addition of GSH reductase 10 U/ml, 0.1 DTNB and 2 NADPH. The reduction of DTNB was followed at 412 nm and quantified with extinction coefficient (13.6 M/cm).

Statistical analyses.

Data were analyzed by ANOVA followed by Dunnett’s multiple comparisons tests using Graph Pad InStat (Graph Pad Software, San Diego, CA) or by Student’s t-test, as indicated. Data were expressed as means ± SE. A P value of <0.05 was considered statistically significant.

RESULTS

Characterization of SiO₂ particles.

The characterizations of the nano- and micro-SiO₂ particles are presented in Fig. 1. The field emission gun-scanning electron microscope (FEG-SEM) images show that the micro-SiO₂ had a spherical shape and exhibited dispersibility (Fig. 1A). The size distribution shown by FEG-SEM analysis had an average diameter of 670 ± 32 nm (Fig. 1A, insert), while the average diameter of nano-SiO₂ was 7 nm according to the manufacturer’s technical sheet. Analysis of the crystalline structure using XRD showed that the nano- and micro-SiO₂ particles presented an amorphous structure (Fig. 1B). The hydrodynamic diameters (Fig. 1C) of the nano- and micro-SiO₂ particles in ultrapure water were 91 ± 22 and 712 ± 212 nm, respectively. The ζ-potentials of the nano- and micro-SiO₂ particles (Fig. 1D) were −27.1 ± 4.4 and −14.4 ± 5.48 mV, respectively. Hydrodynamic diameters and ζ-potentials were also measured in Tyrode, M-199, and M-199 + BSA solutions (Table 1). In addition, EDS analysis showed that the purity of the silica particles was higher than 95%, and no traces of other metals were detected in the particle suspensions (data not shown).

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.

Table 1.

Characterization of silica particles by different techniques

Particle (SiO2)	Crystallinity (XRD)	Purity (EDS), %	Size Distribution		Surface Charge (ζ-potential), mV
			Feret diameter (FEG-SEM), nm	Hydrodynamic diameter (DLS), nm
Nano-SiO2	amorphous	>95	7*	91 ± 22 (water)	−24.1 (water)
				120 ± 25 (Tyrode)	−22.0 (Tyrode)
				220 ± 50 (M-199)	−21.6 (M-199)
				220 ± 106 (M-199 + BSA)	−20.8 (M-199 + BSA)
Micro-SiO2	amorphous	>95	666 ± 32	712 ± 212 (water)	−14.4 (water)
				1,106 ± 281 (Tyrode)	−19.5 (Tyrode)
				1,190 ± 272 (M-199)	−18.8 (M-199)
				1,281 ± 488 (M-199 + BSA)	−12.3 (M-199 + BSA)
F-micro-SiO2	amorphous	>95%	611 ± 22	712 ± 153 (water)	−33.6 (water)
				712 ± 152 (Tyrode)	−17.5 (Tyrode)
				1,484 ± 294 (M-199)	−13.4 (M-199)
				1,484 ± 812 (M-199 + BSA)	−12.9 (M-199 + BSA)

XRD, X-ray diffraction; EDS, energy-dispersive X-ray spectroscopy; FEG-SEM, field emission gun scanning electron microscope; DLS, dynamic light scattering; ELS, electrophoretic light scattering. BSA, 5 mg/ml

^*Size as referred by the provider’s data sheet.

Dose- and time-dependent cytotoxicity of nano- and micro-SiO₂ particles.

To assess the effects of SiO₂ on cardiomyocyte viability, adult rat ventricular myocytes were cultured with different concentrations of nano- and micro-SiO₂ particles, and cell death was examined after 24 h of treatment. As shown in Fig. 2A, nano- and micro-SiO₂ induced significant cytotoxic effects in a dose-dependent manner, with IC₅₀ values of 99.5 ± 12.4 µg/ml and >1,500 µg/ml, respectively, which indicates that nano-SiO₂ has a 15-fold higher toxicity than micro-SiO₂. Indeed, when the cardiomyocytes were cultured in the presence of nano- and micro-SiO₂ at the IC₅₀ of nano-SiO₂, only nano-SiO₂-induced cell death occurred as a result of the necrotic pathway. No evidence of apoptosis was observed in any case, as shown by the lack of single positive Annexin-V cells (Fig. 2, B and C) and the lack of caspase-3/7 activity (data not shown). In addition, necrosis induction also occurred faster with nano-SiO₂ than with micro-SiO₂ as shown in Fig. 2D by the LDH leakage. Nano-SiO₂ promoted cardiomyocyte necrosis in the first 3 h of incubation, while micro-SiO₂ only led to notable LDH leakage after 12 h at their corresponding IC₅₀ values.

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.

SiO₂ particles associate with the cellular membrane and internalize in the cell.

To relate cellular death to particle membrane association or to the potential internalization of the SiO₂ particles, we analyzed the association of single or agglomerates/aggregates of nano- and micro-SiO₂ to the cardiomyocytes’ cellular membranes, as shown in Fig. 3, A and B. The FEG-SEM images show the association of the SiO₂ particles with the cell membrane 24 h after administration in the cultured adult rat cardiomyocytes at 37°C (Fig. 3, A and B). Figure 4 depicts the surface of the cardiomyocytes using EDS, which shows that the nano- and micro-SiO₂ particles have individual and agglomerate/aggregate associations with the cellular membrane with respect to the four EDS images of the elemental maps for carbon, sodium, silicon, and oxygen. Transversal cuts in the cardiomyocytes with nano- or micro-SiO₂ (Fig. 3, C–E) showed that the particles could internalize in the cardiomyocytes after 24 h of incubation. Figure 3F shows the SiO₂ content quantified by PIXE in the cardiomyocytes as a function of time. A time-dependent increase was observed in SiO₂ in the cardiomyocytes treated with nano- and micro-SiO₂ particles, suggesting an association/internalization of the silica particles in the cardiomyocytes. Furthermore, EDS analysis in the transversal cut in the cardiomyocytes indicates that the SiO₂ signal is higher in nano-SiO₂ vs. micro-SiO₂ particles, which suggests that smaller SiO₂ particles are more prone to cellular internalization in cardiomyocytes. As shown in Fig. 3G, we explored, by confocal microscopy, the internalization of micro-SiO₂ after 24 h of incubation, and we analyzed the confocal z-stack acquisitions. For this purpose, we synthesized a micro-SiO₂ particle with fluorescent properties (F-micro-SiO₂) that had a similar size, morphology, dispensability, and ζ-potential as micro-SiO₂ (Table 1). Using this approach, we confirmed the presence of multiple single and agglomerate/aggregate F-micro-SiO₂ at different subcellular levels by using a tridimensional reconstruction captured from cardiomyocytes, thereby confirming the intracellular localization of SiO₂ particles (Fig. 3G and video in Supplemental Material; Supplemental Material for this article is available online at the Journal website). We also visualized the internalization of the F-micro-SiO₂ through confocal z-stack analysis, which confirmed the presence of particles (shown as bright spheres) near the nuclei area (shown as dark ovals) (Fig. 3G). Figure 5 shows a semiquantitative analysis wherein the total fluorescence in a z-stack was evaluated to determine the internalization of the F-micro-SiO₂ in intact cardiomyocytes exposed to different concentrations of particles during 24 h of incubation. F-micro-SiO₂ exhibited dose-dependence at low concentrations (Fig. 5A) and was able to reach a saturating concentration without promoting major cellular death (data not shown), which is similar to micro-SiO₂ at the same concentration (Fig. 2A), and we showed that cardiomyocytes treated with 150 µg/ml of F-micro-SiO₂ preserved their normal morphology (Fig. 5B). At the maximum evaluated concentration of 150 µg/ml, we observed a twofold increase in fluorescence; the 1 ± 0.17-fold fluorescence presented in the untreated (CT) group increased to 2.02 ± 0.41-fold in the F-micro-SiO₂-treated group (Fig. 5B).

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.

SiO₂ treatment induced a reduction in cell shortening, decreased SERCA activity, and impaired β-adrenergic stimulation.

Cardiomyocyte sarcomere shortening (Fig. 6) following nano- and micro-SiO₂ treatment reduced by 34 and 36%, respectively, compared with the CT group. This impairment in cell shortening was maintained at 0.5 and 2 Hz. However, no difference was evident between the nano- and micro-SiO₂ groups. For the purpose of exploring the mechanism of this impairment, we chose to work with nano-SiO₂ particles at 100 µg/ml to match the physiological conditions of airways and to avoid high range concentrations (IC₅₀ values of >1,500 µg/ml for micro-SiO₂) that could not be detected in vivo after a short exposure.

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.

Generally, the changes in cardiomyocyte Ca²⁺ handling followed the time course of the changes in contractility. In this sense, Fig. 7, A and B, shows a representative recording of Ca²⁺ transients evoked by field stimulation for the CT and nano-SiO₂-treated cells. Figure 7C shows the pooled data of the change in the Ca²⁺ transient peak amplitude upon physiological β-adrenergic stimulation ISO in both cell treatments. We found that under basal conditions, the peak Ca²⁺ transient amplitude was similar in the SiO₂-treated and CT cells (6.03 ± 0.27 ΔF/F₀ CT vs. 6.72 ± 0.37 ΔF/F₀ nano-SiO₂). However, after 10 min of continuous β-adrenergic stimulation, the Ca²⁺ transient amplitude increased to only 7.95 ± 0.72 in the nano-SiO₂-treated cells, while the CT group increased to 9.13 ± 0.59 (P < 0.05).

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.

For relaxation, the released Ca²⁺ must be removed from the cytosol; SERCA and the sarcolemmal Na⁺/Ca²⁺ exchanger perform this activity. In this context, the Ca²⁺ transient time to 50% decay (T_50%) provides an index of the combined activity of the Ca²⁺ removal systems; however, the SERCA activity underlies more than 90% of Ca²⁺ removal (4). Figure 7D shows the Ca²⁺ transient T_50% plotted under basal conditions and as a function of time during ISO perfusion. Under basal conditions, the T_50% of the nano-SiO₂-treated cells was 331 ± 23 ms, while that of the CT cells was 239 ± 11 ms (P < 0.05). In both cases, there was an increase in the cytosolic Ca²⁺ removal rate after adrenergic stimulation; however, there was still a difference between the CT and nano-SiO₂-treated cells (183 ± 7 and 233 ± 14 ms, respectively; P < 0.05). This result suggests impairment in SERCA activity.

Because alterations in Ca²⁺ handling in other pathological settings are also reflected at the level of diastolic Ca²⁺, we measured whether the spontaneous Ca²⁺ sparks were altered in the SiO₂-treated cardiomyocytes. Figure 8A shows the typical Ca²⁺ spark reconstruction in the CT (left) and nano-SiO₂-treated cardiomyocytes (right). Calcium spark analysis revealed a significant decrease in frequency in the nano-SiO₂ group (1.81 ± 0.28 sparks·100 µm⁻¹·sec⁻¹ nano-SiO₂) compared with the CT group (2.79 ± 0.26 sparks·100 µm⁻¹·sec⁻¹ CT) (P < 0.05) (Fig. 8B) and a significant decrease in amplitude (0.82 ± 0.02 F/F₀ CT vs. 0.65 ± 0.03 F/F₀ nano-SiO_2; P < 0.05) (Fig. 8C), which might indicate reduced SR calcium content. We estimated a steady-state SR Ca²⁺ content by the cytosolic peak of the caffeine-evoked Ca²⁺ release in the CT and SiO₂-treated cells. Figure 9, A and B, shows representative confocal images of caffeine-evoked SR Ca²⁺ release in the control and nano-SiO₂ treated cells, respectively. Pooled data of the peak caffeine-evoked Ca²⁺ release is shown in Fig. 9C. The nano-SiO₂ cells had a slightly lower SR Ca²⁺ content; the difference was not statistically significant under basal conditions (5.48 ± 0.35 and 4.59 ± 0.46 for CT and nano-SiO₂ treated cells, respectively; P < 0. 12).

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].

SiO₂ treatment induces a decrease in mitochondrial membrane potential and ATP content.

Efficient Ca²⁺ handling, which is necessary for proper contraction and relaxation, depends strongly on the ATP supply in the vicinity of SERCA (11). In this regard, we explored whether SiO₂ treatment affected the bioenergetics status in nano-SiO₂-treated cardiomyocytes (Fig. 10, A and B). Figure 10C shows a 29.5% decrease in the mitochondrial membrane potential (Δψ) with nano-SiO₂ treatment compared with the CT group after 24 h of incubation. This finding was strongly related to the ATP content in the cardiomyocytes, as nano-SiO₂ treatment reduced the ATP content by 60% compared with the CT cells (Fig. 10D). These findings suggest that the SiO₂ particles impaired mitochondrial function.

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.

SiO₂ induces ROS production and glutathione depletion.

Modification of the redox state by nano- and microparticles has been reported as one of the main mechanisms of cytotoxicity at the cellular level (35). In this regard, we incubated cardiomyocytes with nano-SiO₂ for 24 h and then measured H₂O₂ production and glutathione (GSH) content. Figure 10E shows that the SiO₂ particles significantly increased H₂O₂ production and markedly depleted GSH (Fig. 10F) compared with the CT group. Nano-SiO₂ induced a significant reduction in GSH (down to 6.5 nmol/mg) in comparison to the CT group (15.4 nmol/mg), which shows that silica particles exerted a prooxidative status on adult rat cardiomyocytes.

DISCUSSION

The purpose of this study was to evaluate, for the first time, the potential cardiotoxicity of SiO₂ particles in adult cardiomyocytes and their effects on excitation-contraction coupling. In addition, recent results have shown that silicon-based microparticles associate and accumulate in a failing myocardium (in contrast to a healthy myocardium) following intravenous administration, and the microparticles reach the cardiomyocytes. This finding points to a novel avenue for developing nanotechnology-based therapeutics and diagnostics for heart failure and other cardiomyopathies that involve endothelial dysfunction and a proinflammatory milieu (40). In addition, there is great interest in using these materials therapeutically as vehicles for carrying genes, siRNA, drugs, or peptides, and there are promising results (5, 42) because of their physicochemical properties, the capacity of biochemical functionality and surface modification to immobilize biomolecules, and the easy preparation of diverse particle sizes (nano and micro) with uniform shapes and structures (37).

SiO₂ nanoparticle internalization is associated with cardiotoxicity.

SiO₂ particles have displayed various cytotoxic effects at the cellular level, which has led to growing concern about their safety (15, 33, 35). Importantly, the toxicity of SiO₂ particles might depend on their crystalline nature and morphology, but this was not considered as a variable in this work because nano- and micro-SiO₂ have a spherical shape and the same crystalline nature. However, one important factor for consideration in this work is the particle size (45). It is generally accepted that the smaller the particles, the greater the toxicity exerted in biological systems. In our study, we found differential cardiotoxicity between nano- and micro-SiO₂. In agreement with many previous studies (34), nano-SiO₂ particles exerted potent cytotoxicity compared with micro-SiO₂ particles at the same concentration (e.g., 300 µg/ml). Nano-SiO₂ reduced viability nearly 80%, as compared with 30% for micro-SiO₂. Our results indicate that nanoparticles are more toxic to adult rat ventricular myocytes than microparticles. Consistent with our results, Duan et al. (9), using 70-nm SiO₂ nanoparticles, observed pericardial edema and cardiac toxicity in zebrafish embryos, which affected heart rhythm. These observations should be considered when designing new products for medical applications or for novel vehicles for gene/drug delivery.

We previously mentioned our decision to evaluate SiO₂ particle size and stability in ultrapure water and, more importantly, in culture media (Tyrode, M-199, and M-199 + BSA) to know SiO₂ particle properties where all experiments have been performed. One of the main characteristics of the DLS technique is that DLS “is a function of the relative refractive indices of the particle or molecule and the dispersant” (24). In our case, when measurements are made in a culture medium, the molecules within it scatter light, which causes interference; thus the data can probably not be gathered with 100% efficiency. However, the changes in the physicochemical properties (particle size distribution and surface charge) of the SiO₂ particles in the culture media were determined by using an incubation protocol adapted from Monopoli et al. (32) to assess the formation of the hard protein corona around the particles. The results, as shown in Table 1, effectively indicate that the more complex the media in which the SiO₂ particles are exposed, the greater the tendency to increase in size; nevertheless, we showed that their surface charge remained relatively unmodified, as it remained negative for all samples under all tested conditions. The ζ-potential of the nano-SiO₂ was −24.1 mV, while for the micro-SiO₂ and its fluorescence-modified counterpart it was −14.4 and −33.6 mV, respectively. Overall, these changes are not great enough to expect dramatic changes in their interaction with cells, such as in the internalization effects. It was not until relatively recently that the ζ-potential, which represents the surface charge of a given set of particles, became relevant as a key factor that influences processes such as opsonization and internalization. Examples of such dramatic changes were presented by Arvizo et al. (2), who tested these aforementioned key factors in gold nanoparticle-coated polymers that conferred surface charges that were positive, negative, or neutral. They found that particles with a neutral surface charge (i.e., a ζ-potential < |2| mV) had longer circulation times and consequently higher accumulation in tumors. Other studies with cancer cell lines, such as HeLa (18, 30), and endothelial cells (43) found in vivo that particles with a positive surface charge had higher internalization rates than negative or neutral particles. For cardiac cells specifically, Miragoli et al. (31) found that exposure in cultured neonatal rat myocytes to 50-nm-charged polystyrene particles showed contrasting effects based on the type of surface charge: positively charged particles showed higher cytotoxic effects, while negatively charged particles showed lower cytotoxicity but formed 50–250-nm nanopores that induced proarrhythmic events.

In this work, we observed differences in the cell release levels of LDH after 24-h incubation of SiO₂. As shown in Fig. 2C, nano-SiO₂ produced a twofold increase in activity compared with micro-SiO₂-treated cells. This difference was made more evident by the fact that SiO₂-induced necrosis occurred in a time-dependent manner, in accordance with previous reports, and nano-SiO₂ caused LDH leakage in endothelial cells at similar times and doses (10). Figure 2D shows cell membrane damage as early as in the first 3 h of incubation with nano-SiO₂; this phenomenon is probably due to the size of nano-SiO₂, which allows it to penetrate faster through the cell membrane, in turn causing an early permeabilization process that has important cytotoxic effects. This cytotoxic effect was also exerted by the micro-SiO₂ as early as the first 2 h, but notable LDH release was shown only after 12 h of incubation. In this scenario, membrane permeabilization and LDH release indicate the beginning of necrosis in SiO₂-treated cells. Apoptosis cell death was not observed, potentially because of the reduced energetic metabolites.

As observed, micro-SiO₂ exerted a lower cytotoxic effect compared with nano-SiO₂. To understand this difference, we developed a hypothesis with two scenarios: 1) micro-SiO₂ does not associate properly with cardiomyocyte membranes, and 2) the larger particle size of micro-SiO₂ prevents it from penetrating into cardiomyocytes, thus avoiding membrane disruption and cell damage. On the basis of this, we explored the association of SiO₂ particles with cellular membranes. The FEG-SEM micrographs show that nano- and micro-SiO₂ associated with cells as individuals and agglomerates/aggregates, and there was evidence of an internalization process and a strong interaction between SiO₂ and cell membranes. In accordance with this, the quantification by PIXE revealed that there was a concentration of SiO₂ in the cardiomyocytes, while no traces of SiO₂ were found in the unexposed cells (CT group). The concentration increased 3.3- and 4.5-fold for nano- and micro-SiO₂, respectively. These concentrations correspond to particles that were either internalized or that adhered to the cardiomyocytes.

To explore the second hypothesis, we incubated fluorescent micro-SiO₂ and analyzed its internalization using confocal microscopy. We confirmed that micro-SiO₂ was localized inside the cardiomyocytes, following a dose-dependent manner, with some association near the nucleus, an interesting phenomenon that is widely observed in different phagocytic cellular types (13, 27) but rarely explored in nondividing cells. Adult cardiomyocytes, as a nondividing cell type and with an apparent nonphagocytic capacity, represent an interesting opportunity to study the biological phenomena of nano- and microparticle internalization and intracellular trafficking. Our group recently studied the mechanisms for the internalization, trafficking, and perinuclear particle localization of these apparently not quiescent cardiomyocytes (40).

Calcium mishandling is associated with SiO₂ nanoparticle cardiotoxicity.

Aberrant Ca²⁺ handling is an important contributor to the electrical and contractile dysfunction associated with cell death. Recently, Gilardino et al. (16) found evidence that, in a neuronal cell line, SiO₂ nanoparticles induce oscillatory changes in intracellular Ca²⁺. Although Ca²⁺ returns to baseline in ~4 h, these authors suggest that several voltage-dependent Ca²⁺ channels on the plasmatic membrane are responsible for these effects. Our results do not agree with this, as they suggest that extracellular Ca²⁺ transport remains functional between the L-type Ca²⁺ channel and SR interaction because the time to peak of Ca²⁺ release remains intact (data not shown). On the other hand, we observed that the decreased contractility in SiO₂-treated cardiomyocytes could also be due to alterations in Ca²⁺ signaling. In this regard, we found that the electrically evoked Ca²⁺ transients were blunted with slow decay and were less sensitive to physiological β-adrenergic stimulation during ISO perfusion. The reduced Ca²⁺ transient amplitude could be due to either decreased SR Ca²⁺ content or a decrease in the L-type Ca²⁺ channel current density. The general consensus is that there is a modest change, or no change, in the Ca²⁺ current, even in the context of pathologies as profound as heart failure. However, there is much evidence associated with a state of SR Ca²⁺ depletion in pathologies such as doxorubicin-induced cardiomyopathy (36). Decreased SERCA activity and lower intra-SR Ca²⁺ content in SiO₂-treated cardiomyocytes might explain the smaller amplitude of Ca²⁺ transients and Ca²⁺ sparks. The lower SR Ca²⁺ content in SiO₂ nanoparticle-treated cells, as well as the slow rate of Ca²⁺ transient decay, could be explained by different events that can alter the SERCA function, such as low ATP content, as shown in this work (Fig. 10), or a decreased SERCA expression, which was recently observed in microarrays from zebrafish embryos treated with SiO₂ nanoparticles (9). However, several studies have shown that decreased SERCA activity is not necessarily accompanied by decreased protein expression (55), and it has been pointed out by our group that Ca²⁺ signaling alterations may precede the changes in protein expression (24). To explain diminished capacity of SERCA to reload the SR, we should consider the consequences of mitochondrial dysfunction due to the drop in membrane potential and the low ATP content (Fig. 10), particularly during high cardiac workloads. A reduced capacity for energy supply (22) and the ineffective removal of the end products of ATP hydrolysis have been observed in heart failure, which leads to a reduction in the phosphorylation potential, which can in turn affect the ATPases involved in Ca²⁺ handling (22). SERCA is one of the most energy-demanding, and therefore most sensitive, cardiac enzymes in ATP depletion. On the basis of this, dysfunctional mitochondria and a lower ATP content could undermine cytosolic Ca²⁺ removal by SERCA, decrease contractile strength, and slow muscle relaxation, which we observed in SiO₂-treated cardiomyocytes. However, while the SR Ca²⁺ content was slightly lower in the SiO₂-treated cells, under basal conditions we found a decrease in the spontaneous Ca²⁺ spark amplitude and frequency. Because a steady-state SR Ca²⁺ content results from the balance between SERCA activity and SR Ca²⁺ leakage, the latter reflected spontaneous Ca²⁺ sparks, and the decrease in SR Ca²⁺ leakage might explain the lower SR Ca²⁺ content observed in cells treated with SiO₂ nanoparticles; this phenomenon could also be exacerbated and deregulated under continuous β-adrenergic stimulation, as we showed in our electrically evoked Ca²⁺ transients under ISO stimulation (Fig. 7). Altogether, these findings are in agreement with reports on ventricular dysfunction and ischemic heart disease in which diastolic Ca²⁺ release is increased (12, 53).

Mitochondrial dysfunction as a trigger of SiO₂ toxicity.

As we mentioned before, in cardiomyocytes the excitation-contraction coupling and metabolic adaptations are based on the coordination between the mitochondria and SR, which facilitates the ATP supply for SERCA activity and ensures energy replenishment by Ca²⁺ and ADP exchange (14, 52). For this reason, mitochondrial function is recognized as an important player in regulatory Ca²⁺ signaling in the heart (52). However, the impact of SiO₂ nanoparticles on energy metabolism and mitochondrial function is largely unclear. Our findings demonstrate that exposing cardiomyocytes directly to SiO₂ nanoparticles for 24 h induced mitochondrial membrane depolarization and energy debacle (Fig. 10), and even more membrane depolarization and energy debacle after 10 min of continuous β-adrenergic stimulation under ISO treatment (data not shown).

Xue et al. (54) observed SiO₂ nanoparticle-induced suppression of mitochondrial dehydrogenase activity and ATP synthesis in hepatocytes. After in vitro exposure, the internalization of SiO₂ nanoparticles correlates with the disturbance of the mitochondrial structure and the release of reactive oxygen species (ROS) in hepatocellular carcinoma cells (47). In this context, cardiac mitochondria allow energy supply-demand matching and regulate the generation of antioxidant defenses, such as nicotinamide adenine dinucleotide phosphate and GSH (28). However, under the Ca²⁺ handling impairment—as observed during SiO₂ nanoparticle interaction—increased electron leakage from the electron transport chain, and the subsequent production of ROS and depletion of antioxidant reserves, has been observed (19). Similar to previous results, it was observed that nano-SiO₂ incubation induced a notable amount of H₂O₂ (a highly toxic type of oxygen-free radical) and a potent depletion in GSH (a first-line antioxidant defense), which indicates SiO₂-induced cytotoxicity that is at least partially due to an imbalance in the cellular redox status. In this regard, it is possible that SERCA and/or the ryanodine receptor might be oxidized, as has been described in other works (3), and this could, at least partially, underlie the slow rate of Ca²⁺ transient decay and the decrease in the amplitude and frequency of sparks. An enhanced diastolic Ca²⁺ leak may not only contribute to the decreased SR Ca²⁺ content but also increase the risk of delay after depolarization and Ca²⁺-triggered arrhythmias, as previously observed by Szebeni et al. (48) with lipid-based nanoparticles in a pig model.

A recent study also showed another possible mechanism of cardiotoxicity with metal-oxide nanoparticles. Savi et al. (41), using nano-TiO₂ particles that had a ζ-potential similar to that of our nano-SiO₂ particles but a different crystalline nature (a mixture of anatase and rutile, as compared with our amorphous SiO₂), observed that nano-TiO₂ particles promoted spontaneous contraction in cardiomyocytes (an arrhythmia trigger at the cellular level). This effect was related to the slight depolarization of the resting membrane potential, which consequently reduced the action potential duration. Then, using an in silico model, they suggested that nano-TiO₂ particles generate transient nanopores that lead to resting membrane potential instability, possibly due to K⁺ leakage (41).

In this context, our results with nano-SiO₂ particles suggest that there was no change in the action potential because the kinetics of Ca²⁺ release from the SR were not affected (time to peak; data not shown), and spontaneous contraction was not observed. However, our experiments were performed at 24 h, while the TiO₂ particles were analyzed 1 h after particle addition, so further experiments on patch-clamped cardiomyocytes with simultaneous measurement of Ca²⁺ handling might help to establish a potential connection between these mechanisms of cardiotoxicity. Interestingly, the nano-TiO₂ particles induced lipid peroxidation in the heart because of ROS production, which is similar to our findings regarding GSH and H₂O₂ levels.

Putting all of this information into context, as summarized in Fig. 11, the Krebs cycle generates the NADH required for oxidative phosphorylation through the electron transport chain (complex I–IV, in blue). This step-by-step transfer of electrons allows protons from the mitochondrial matrix to be transported uphill across the inner mitochondrial membrane, which forms a proton concentration gradient (ΔΨ_m). Thus free energy released during the oxidation of NADH is stored both as an electric potential and a proton concentration gradient across the inner membrane. The movement of protons back across the inner membrane, driven by this force, is coupled to the synthesis of ATP from ADP and P_i by the ATP synthase. During SiO₂ nanoparticle perfusion (indicated by gray arrows), defects in the mitochondrial homeostasis contribute to the energetic mismatch observed (ATP depletion). SiO₂ nanoparticles internalize and directly (dotted line) or indirectly produce ROS, such as the superoxide radical (O₂^·−), which is transformed to H₂O₂ by superoxide dismutase (SOD) and then converted to H₂O using glutathione peroxidase activity (GPx), depleting the (GSH) and increasing oxidative stress. When a cardiomyocyte is depolarized, Ca²⁺ enters through the L-type calcium channels (LTCC). This Ca²⁺ triggers a subsequent release of Ca²⁺ that is stored in the SR; through ryanodine receptors (RyR2), the Ca²⁺ released by the SR increases the intracellular Ca²⁺ (lower Ca²⁺ SR release), and then free Ca²⁺ binds to troponin C (TnC) and interacts with several proteins, which results in the sarcomere length being shortened (cell shortening decrease). In the relaxation phase, the SR sequesters Ca²⁺ using an ATP-dependent calcium pump (SERCA2a), which lowers the cytosolic Ca²⁺ concentration and removes calcium from the TnC, but because of the energy depletion, SERCA reduces its activity, which decreases the Ca²⁺ SR content.

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.

Clinical relevance.

The World Health Organization estimated that there would be ~20 million cardiovascular disease deaths in 2015, accounting for 30% of all deaths worldwide. In this context, the likelihood of cardiovascular diseases being associated with particulate air pollution is well documented. A series of scientific statements from the American Heart Association stressed that exposure to elevated levels of particulate matters is strongly linked with heart diseases, particularly when more than 75% of the total number of particles are nanoparticles (6, 17, 21). However, the correlation between heart disease and nanoscale particles is still being debated, and the related research conclusions lack consistency. Therefore, studying the cardiovascular toxicity of nanoscale particles is necessary and has profound scientific significance, particularly with regard to nanoparticle types, such as metal, metal-oxide, and carbon nanotubes, that have been reported to produce toxicity in other organs (35). To the best of our knowledge, we have demonstrated for the first time that SiO₂ nanoparticles impair the bioenergetics status and may consequently impact Ca²⁺ handling and contraction in rat cardiomyocytes. The mechanism of cardiotoxicity for SiO₂ particles mimics the pathological mechanisms of a failing heart. These findings will be helpful in managing risk and providing guidance to reduce the hazardous effects of nanoscale particles. In addition, a better understanding of the mechanism through which nanoparticles produce cardiotoxicity could uncover novel avenues for avoiding the side effects associated with the use of these particles.

In summary, the findings of this study support the notion that SiO₂-induced cardiomyocyte toxicity is strongly size dependent. First, we showed differential, dose-dependent toxicity after 24 h of incubation, with differential time-dependent cell death by necrosis (as shown by LDH leakage) and no activation of apoptosis. Second, we visualized, by confocal microscopy, an internalization phenomenon in these nondividing, nonphagocytic primary-culture cell types, thus opening up a new field of study. Third, our results showed the role of oxidative stress and the direct effect of SiO₂ particles on cell function: impaired Ca²⁺ handling and a reduction in cell shortening. These effects were all due to mitochondrial malfunction, which is shown as a drop in the membrane potential and ATP content (Fig. 11). These data enable us to explore and carefully design new medical devices and clinical therapeutic protocols that take into account the advantages and disadvantages of nanoparticles, in particular SiO₂.

GRANTS

This work was partially supported by Endowed Chair in Cardiology (Tecnológico de Monterrey, 0020CAT131) as well as the CONACYT Grants 151136, 133591, 269399, and Fronteras de la Ciencia Grant (0682) and Xignus Research Fund. C. Guerrero-Beltrán was supported by Postdoctoral Fellowship CONACYT, 290885. SEM-EDS work was supported by National Institutes of Health Grants 5 G12 RR-013646-12 and G12 MD-007591.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

C.E.G.-B., J.B.-R., O.L., Y.O.-A., E.C.C., J.R.G., N.G., J.V., A.G.-G., E.O., and N.O.-S. performed experiments; C.E.G.-B., J.B.-R., O.L., Y.O.-A., and E.C.C. analyzed data; C.E.G.-B., J.B.-R., O.L., E.C.C., G.T.-A., and G.G.-R. interpreted results of experiments; C.E.G.-B. and G.G.-R. drafted manuscript; C.E.G.-B., O.L., G.T.-A., and G.G.-R. edited and revised manuscript; J.B.-R. prepared figures; C.E., G.B., and G.G.-R. approved final version of manuscript; C.E.G.-B. and G.G.-R. conceived and designed research.