Subchronic exposure to titanium dioxide nanoparticles modifies cardiac structure and performance in spontaneously hypertensive rats

Abstract

Background: Non-communicable diseases, intended as the results of a combination of inherited, environmental and biological factors, kill 40 million people each year, equivalent to roughly 70% of all premature deaths globally. The possibility that manufactured nanoparticles (NPs) may affect cardiac performance, has led to recognize NPs-exposure not only as a major Public Health concern, but also as an occupational hazard. In volunteers, NPs-exposure is problematic to quantify. We recently found that inhaled titanium dioxide NPs, one of the most produced engineered nanomaterials, acutely increased cardiac excitability and promoted arrhythmogenesis in normotensive rats by a direct interaction with cardiac cells. We hypothesized that such scenario can be exacerbated by latent cardiovascular disorders such as hypertension.

Results: We monitored cardiac electromechanical performance in spontaneously hypertensive rats (SHRs) exposed to titanium dioxide NPs for 6 weeks using a combination of cardiac functional measurements associated with toxicological, immunological, physical and genetic assays. Longitudinal radio-telemetry ECG recordings and multiple-lead epicardial potential mapping revealed that atrial activation times significantly increased as well as proneness to arrhythmia. At the third week of nanoparticles administration, the lung and cardiac tissue encountered a maladaptive irreversible structural remodelling starting with increased pro-inflammatory cytokines levels and lipid peroxidation, resulting in upregulation of the main pro-fibrotic cardiac genes. At the end of the exposure, the majority of spontaneous arrhythmic events terminated, while cardiac hemodynamic deteriorated and a significant accumulation of fibrotic tissue occurred as compared to control untreated SHRs. Titanium dioxide nanoparticles were quantified in the heart tissue although without definite accumulation as revealed by particle-induced X-ray emission and ultrastructural analysis.

Conclusions: The co-morbidity of hypertension and inhaled nanoparticles induces irreversible hemodynamic impairment associated with cardiac structural damage potentially leading to heart failure. The time-dependence of exposure indicates a non-return point that needs to be taken into account in hypertensive subjects daily exposed to nanoparticles.

Keywords: Arrhythmias; Cardiac electrophysiology; Cardiac fibrosis; Nanotoxicology; Titanium dioxide nanoparticles.

Fig. 1

Study population and experimental protocol. Flowchart describing the number of animals subjected to the different experimental procedures before and after intra-tracheal instillation of saline solution (CTRL group) or saline solution added with TiO₂, at a final concentration of 2 mg/kg (TiO₂-NP group). * timepoints for oxidative stress and inflammation; § timepoints for real-time PCR analysis; # timepoints for PIXE. For more details see “Outline of the experimental protocols” section

Fig. 2

Electrocardiographic waveform and interval durations. Basic electrophysiological parameters evaluated in CTRL (triangles) and TiO₂-NPs treated (square) animals. a P wave duration (ms). b PQ segment duration (ms). c QRS complex duration (ms). d RR interval duration (ms). e QTc duration (ms). Kruskal-Wallis (post hoc analyses: Dunn’s multiple comparison) was performed and statistical significance was set at p < 0.05. * vs corresponding 1° week; # vs corresponding 3° week. Data are represented as median and interquartile range (IQR)

Fig. 3

Heart rate variability indexes and arrhythmia evaluation. Heart rate variability parameters and spontaneous arrhythmic events in CTRL (triangles) and TiO₂-NP (square) groups. a SDRR duration (ms). b r-MSSD duration (ms). c Sinus pauses (SP, number of events). d Atrio-ventricular blocks (AV block, number of events). Kruskal-Wallis (post hoc analyses: Dunn’s multiple comparison’s) was performed and statistical significance was set at p < 0.05. ● vs CTRL; * vs corresponding 1° week; # vs corresponding 3° week; § vs corresponding 6° week. Data are represented as median and IQR

Fig. 4

Cardiac refractoriness, excitability, anisotropic conduction velocities and arrhythmia induction. Electrophysiological parameters and the index of arrhythmia inducibility in CTRL (triangles) and TiO₂-NPs treated (squares) animals. a Effective refractory period (ERP, ms). b Rheobase (μA). c Chronaxie (ms). d Threshold intensity for a 1 ms duration impulse (μA). e ventricular conduction velocities along fiber (CVl, m/s). f ventricular conduction velocities across fiber (CVt, m/s). g ventricular conduction velocities ratio (CVl/CVt). Kruskal-Wallis (post hoc analyses: Dunn’s multiple comparison) was performed and statistical significance was set at p < 0.05. ● vs CTRL; * vs corresponding 1° week; # vs corresponding 3° week; § vs corresponding 6° week. Data are represented as median and interquartile range (IQR)

Fig. 5

Cardiac tissue fibrosis evaluation. Masson’s trichrome staining sections were analyzed by optical microscopy to evaluate perivascular and interstitial fibrosis in the LV myocardium (greenish). Representative images of heart sections from control (a and b) and TiO₂-NP treated animals (c and d). a CTRL LV myocardium; black rectangle area is shown at higher magnification in panel (b). c TiO₂-NPs treated LV myocardium; black rectangle area is shown at higher magnification in panel (d). Scale bars: a and c = 200 μm; b and d = 100 μm

Fig. 6

Ultrastructural features of the alveolar lung parenchyma from a representative untreated CTRL and TiO₂-NPs treated SHR. a a type I pneumocyte (PI) and three capillaries (*) are present in the alveolar septum separating air spaces (As) in CTRL lung. b several TiO₂-NPs are present in the cytoplasm of a large alveolar macrophage showing, in addition to vacuoles and microvescicles, onionskin-like multimembrane ultrastructures suggestive of autophagy. The area inscribed by white rectangle is shown at higher magnification in the inset. c an aggregate of electrondense TiO₂-NPs is located nearby an endothelial cell lining the lumen of a capillary recognizable by the red blood cell (RBC). The white rectangle inscribes an area shown at higher magnification in d to document that TiO₂-NPs are not yet internalized. e internalization of TiO₂-NPs in a capillary endothelial cell that is better appreciable at higher magnification in (f). Scale Bars: a 5 μm, b, c, e: 2 μm, d, f: 500 nm

Fig. 7

TEM analysis of the heart from a representative untreated CTRL and TiO₂-NP treated SHR. a low magnification image of a CTRL heart to illustrate the sarcolemma (arrowheads) lining the surface and gap junctions (arrows) delimiting cardiomyocytes filled of mitochondria and myofibrils. On the left, collagen bundles (Col) are present in the interstitial space between an endothelial cell (Ec) lining a capillary lumen (L) and cardiomyocytes. N: cardiomyocyte nucleus. b aggregates of TiO₂-NPs within the cardiomyocyte cytoplasm showing effaced myofibrils and swollen mitochondria. c the white rectangle inscribes an area shown at higher magnification in (d) in which the arrowhead points to NP located in the interstitial space between the vessel wall inscribing a lumen (L) and cardiomyocytes (CM). e low magnification image of a treated SHR myocardium to illustrate widening of the interstitial space by abundant fibrotic deposition (Col) surrounding cardiomyocytes (CM) and an endothelial cell (Ec) lining a capillary. Arrowheads point to gap junctions delimiting two CMs. The white rectangle inscribes an area shown at higher magnification in f to document the internalization of TiO₂-NP located in proximity of the CM sarcolemma bordered by collagen bundles (Col). Scale Bars: A, C, E: 5 μm, B: 2 μm, D: 1 μm, F: 500 nm

Fig. 8

Inflammation and toxicological markers in the heart tissue. Lipid peroxidation products and different inflammatory and toxicological markers evaluated in CTRL (white bars) and TiO₂-NPs treated (black bars) animals. a TBARS evaluation in the heart tissue. b IL-6 evaluation in the heart tissue r. c MCP-1 evaluation in the heart tissue. d TIMP-1 evaluation in the heart tissue. Two-way ANOVA (post hoc analyses: Bonferroni test) was performed and statistical significance was set at p < 0.05. ● vs CTRL; * vs corresponding 1° week; # vs corresponding 3° week. Data are represented as mean ± SEM

Fig. 9

Molecular analysis displaying different gene expression linked to fibrosis deposition. Graph of different gene expression in CRTL (triangles) and TiO₂-NPs treated (square) animals. a ACTA2 gene expression; b COL1A2 gene expression; c COL3A1 gene expression; d COL4A1 gene expression; e TGF-β1 gene expression; f CTGF gene expression. Kruskal-Wallis (post hoc analyses: Dunn’s multiple comparison) was performed and statistical significance was set at p < 0.05. ● vs CTRL; * vs corresponding 1° week; # vs corresponding 3° week. Data are represented as median and IQR