Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes

Abstract

Background: Silica nanoparticles (nanoSiO₂) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO₂ has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO₂.

Results: The cumulative administration of nanoSiO₂ reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC₅₀) of 93 μg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO₂ was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨ_m). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO₂ and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO₂ exposure, suggesting that nanoSiO₂ induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO₂, enhanced viability and reduction of H₂O₂ were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO₂-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO₂ in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 μg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO₂, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO₂ affects cardiac function via mitochondrial dysfunction through the opening of the mPTP.

Conclusion: The aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO₂ exposure.

Keywords: Calcium overload; Cardiotoxicity; Heart; Mitochondria; Oxidative stress; Permeability transition; Silica nanoparticles.

Fig. 1

nanoSiO₂ accumulates in heart tissue, diminishing contractility and affecting predominantly LVP and HR. a Silicon quantification in myocardial tissue by SEM-EDS after 100 μg/mL nanoSiO₂ perfusion in ex-vivo heart. b, c Heart rate pressure product (RPP = HR × LVP) during 60 min after time- and dose- dependent nanoSiO₂ administration. d The RRP dependence on nanoSiO₂ administration reduced the frequency, and in some cases the amplitude of LVP and dP/dt, in addition a reduced HR. Values are percentage of control and represent mean ± SEM, n = 4

Fig. 2

Exposure to nanoSiO₂ to rat and human cardiac mitochondria results in mitochondrial dysfunction, observed by a reduced oxygen consumption rate and mitochondrial membrane potential. For rat cardiomyocyte mitochondria (a-d), at incremental nanoSiO₂ concentrations: a Representative recordings of OCR. Addition of succinate and ADP are denoted by arrows. b Decrease of OCR evaluated in state 4 and state 3. c Representative recordings of ΔΨ_m. Addition of succinate and ADP are denoted by arrows. d Decrease of ΔΨ_m. For human cardiomyocyte mitochondria, at incremental nanoSiO₂ concentrations: e Representative recordings of OCR. Addition of oligomycin, FCCP, rotenone and Antimycin A are denoted by dashed lines. f Basal and maximum OCR, and spare reserve. The exposure of mitochondria to nanoSiO₂ was 5 min prior to measurements. Values represent mean ± SEM

Fig. 3

nanoSiO₂ promotes mitochondrial membrane permeability associated to mPTP opening. a-b Representative recordings of mitochondrial CRC at increasing concentrations: (a) as a function of nanoSiO₂, and (b) as a function of CsA. Arrows represent 10 μM Ca²⁺ bolus addition. c-d Semiquantitative analysis of CRC: (c) as a function of nanoSiO₂, and (d) as a function of CsA. e-f Representative recordings of: (e) mitochondrial depolarization, and (f) swelling in presence and absence of CsA. The exposure of mitochondria to nanoSiO₂ was 5 min prior to measurements. The concentration of nanoSiO2 in (b,d-f) was 100 μg/mL. CsA was applied at the same time of nanoSiO₂ administration. Values are percentage of control and represent mean ± SEM

Fig. 4

nanoSiO₂ disturbs mitochondrial enzymatic activity and promote the oxidation of mitochondrial proteins. MitoTEMPO, a mitochondrial antioxidant, partially prevents nanoSiO₂ oxidation effects. a Representative recordings of mitochondrial calcium transport in nanoSiO₂ (30 μg/mL) incubated in mitochondria after the addition of 10 μM Ca²⁺. MitoTEMPO improved mitochondrial calcium transport. b Aconitase enzyme activity, c free thiol content, and d mitochondrial thiols in the ANT interlinked by n-ethylmaleimide (NEM) binding in isolated heart mitochondria after nanoSiO₂ treatment (30 μg/mL) in presence or absence of MitoTEMPO (25 μM). The exposure of mitochondria to nanoSiO₂ was 5 min prior to measurements. MitoTEMPO was applied 30 min prior to nanoSiO₂ administration. Values are percentage of control and represent mean ± SEM. *p ≤ 0.05 vs control, #p ≤ 0.05 vs SNP. e Schematic interaction of NEM with SH groups in proteins

Fig. 5

The toxicity mechanism of nanoSiO₂ in cardiac cells is driven by reactive oxygen species and the opening of the mPTP. a MitoTEMPO dose-dependent cellular death prevention with 200 μg/mL of nanoSiO₂ administration in H9c2 cells. b H₂O₂ production after nanoSiO₂ administration (200 μg/mL) in presence or absence of MitoTEMPO (100 μM) in H9c2 cells. c Cellular viability in ventricular myocytes after nanoSiO₂ administration in the absence or presence of CsA (0.5 μM). d ATP production in cardiomyocytes after nanoSiO₂ administration (100 μg/mL) in absence or presence of CsA (0.5 μM). For human cardiomyocytes: (e) LDH release activity, (f) PI positive cells. MitoTEMPO or CsA were applied 30 min prior to nanoSiO₂ administration. nanoSiO₂ was incubated during 24 h. Values are percentage of control and represent mean ± SEM. *p ≤ 0.05 vs control, #p ≤ 0.05 vs CsA

Fig. 6

nanoSiO₂ induces an increase in mitochondrial ROS production, leading to dysfunction in cardiac contractility. Hearts perfused with nanoSiO₂ showed a compromised contractility, finding nanoSiO₂ accumulation (heart representation, left side). Once nanoSiO₂ internalizes into mitochondria, production of ROS is increased, compromising mitochondrial function. This leads to several oxidative damages, reducing the activity of the aconitase, and affecting the activity of key mitochondrial proteins such ANT through the oxidation of thiol groups. ANT oxidation promotes the mPTP formation, causing a decrease in mitochondrial membrane potential, which is the electrochemical force to synthetize ATP, compromising cellular viability. MitoTempo, a mitochondrial antioxidant agent, or CsA through delaying the formation of the mPTP, partially prevented these adverse effects of nanoSiO₂ exposure