Titanium dioxide nanoparticles promote arrhythmias via a direct interaction with rat cardiac tissue

16 Pages·2015·3.37 MB·English

Titanium dioxide nanoparticles promote
arrhythmias via a direct interaction with rat
cardiac tissue
Savi et al.
Savietal.ParticleandFibreToxicology (2014) 11:63
DOI10.1186/s12989-014-0063-3
Savietal.ParticleandFibreToxicology (2014) 11:63
DOI10.1186/s12989-014-0063-3
RESEARCH Open Access
Titanium dioxide nanoparticles promote
arrhythmias via a direct interaction with rat
cardiac tissue
Monia Savi1,3†, Stefano Rossi1,3†, Leonardo Bocchi1†, Laura Gennaccaro1, Francesca Cacciani1, Alessio Perotti1,
Davide Amidani1, Rossella Alinovi2,3, Matteo Goldoni2,3, Irene Aliatis4, Pier Paolo Lottici4, Danilo Bersani4,
Marco Campanini5, Silvana Pinelli2,3, Marta Petyx6, Caterina Frati2, Andrea Gervasi7, Konrad Urbanek8,
Federico Quaini2, Annamaria Buschini1, Donatella Stilli1, Claudio Rivetti1, Emilio Macchi1,3, Antonio Mutti2,3,
Michele Miragoli3,9* and Massimiliano Zaniboni1,3*
Abstract
Background: Inlight of recent developments innanotechnologies, interest is growing to better comprehend the
interaction ofnanoparticleswith body tissues, in particular within thecardiovascular system.Attention has recently
focused on thelink between environmental pollution and cardiovascular diseases. Nanoparticles<50 nm in size are
known to passthealveolar–pulmonary barrier, enterinto bloodstream and induce inflammation, butthedirect
pathogenic mechanismsstill need to be evaluated. We thus focused our attentionon titanium dioxide (TiO )
2
nanoparticles, themost diffuse nanomaterial in polluted environments and one generally considered inertfor the
human body.
Methods: WeconductedfunctionalstudiesonisolatedadultratcardiomyocytesexposedacutelyinvitrotoTiO and
2
onhealthyratsadministeredasingledoseof2mg/KgTiO NPsviathetrachea.Transmissionelectronmicroscopywas
2
usedtoverifytheactualpresenceofTiO nanoparticleswithincardiactissue,toxicologicalassayswereusedtoassess
2
lipidperoxidationandDNAtissuedamage,andaninsilicomethodwasusedtomodeltheeffectonactionpotential.
Results:VentricularmyocytesexposedinvitrotoTiO hadsignificantlyreducedactionpotentialduration,impairmentof
2
sarcomereshorteninganddecreasedstabilityofrestingmembranepotential.Invivo,asingleintra-trachealadministration
ofsalinesolutioncontainingTiO nanoparticlesincreasedcardiacconductionvelocityandtissueexcitability,resultingin
2
anenhancedpropensityforinduciblearrhythmias.Computationalmodelingofventricularactionpotentialindicatedthat
amembraneleakagecouldaccountforthenanoparticle-inducedeffectsmeasuredonrealcardiomyocytes.
Conclusions:AcuteexposuretoTiO nanoparticlesacutelyalterscardiacexcitabilityandincreasesthelikelihoodof
2
arrhythmicevents.
Keywords:Pollution,Cardiacarrhythmia,Experimentalmodel,Titaniumdioxidenanoparticles,Supernormalconduction,
Membraneleakage
*Correspondence:[email protected];massimiliano.
[email protected]
†Equalcontributors
3CERT,CenterofExcellenceforToxicologicalResearch,DepartmentofClinical
andExperimentalMedicine,ViaGramsci14,Parma43126,Italy
1DepartmentofLifeSciences,UniversityofParma,Parma,Italy
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Savietal.;licenseeBioMedCentral.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain
Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
unlessotherwisestated.
Savietal.ParticleandFibreToxicology (2014) 11:63 Page2of15
Background Mapping (EPM) and cellular motion detection – that
The explosive growth in nanotechnology – i.e., the design acute exposure (<5 hours) to TiO -NPs (diameter range:
2
anddevelopmentofsystemsattheatomicornanoscale– 30–100 nm) is detrimental for cardiac performance and
andbioengineeringnanotechniqueshasledtothedevelop- increases the propensity for arrhythmia. Biophysical
ment of a large number of new nanomaterials with novel characterizationoftheNPswasconductedwithanumber
biological, physical and chemical properties. Nanoparticles of techniques – i.e., Atomic Force Microscopy (AFM),
(NPs) have been manufactured for several decades on an Dynamic Light Scattering (DLS), Raman spectroscopy,
industrial scale. Several metal oxide NPs possess photo- and Transmission Electron Microscopy (TEM). TiO toxi-
2
catalytic ability, high electrical conductivity, ultraviolet cologywasalsocharacterizedwithROSandThioBarbituric
absorption and photo-oxidizing capacity against chemical AcidReactiveSubstance(TBARS)analyses.
and biological species [1]. Some commercial products,
suchascosmetics,arealsolikelysourceofNPsthatcould
Results
become uncontrollably delivered to the human environ-
Particlessize,typeandaggregation
ment[1].Because ofthewidespreadpresenceofpotential
AFMimagingrevealedthatarelevantfractionofTiO -NPs
sources of NPs, environmental release of manufactured 2
had a diameter <100 nm (Figure 1A): specifically, single
NPs is expected to increase for the near future. Thus,
NPshadadiameterinthe25–35nmrange(Figure1B);the
understandingthemolecularandcellularbasisofnanotoxi-
overall size distribution frequency of the NPs is given in
cityisanessentialchallengetoday.
Figure 1C. In addition, NP aggregates of variable size and
We focused the present study on one of the most-
morphology were also present, composed of up to tens of
produced NPs species, namely titanium dioxide (TiO ),
2 single particles (Figure 1D). By measuring volume, we
1.45 million metric tons of which were produced in the
estimatedthat~40%ofNPshadadiameter<100nm,with
United States in 2007 alone [2], and which was considered
theremainingparticulatesmadeupofaggregates.
biologically inert up to recently [3]. Most studies concern-
TheRamanspectrumoftheTiO -NPs(Additionalfile1:
ingTiO -NPtoxicologyhavefocusedtodateonpulmonary 2
2 FigureS1)hadpeakscorrespondingtoamixtureofanatase
inflammation [4-7] and have elucidated its behavior inside
(tetragonal polymorph, space group I4 /amd, characterized
cells. Unfortunately, it has been very difficult to detect, byRamanpeaksat~143,196,396,5161and638cm−1)and
track and precisely quantify TiO -NPs at the cellular level.
2 rutile (tetragonal polymorph, P4 /mnm, with characteristic
Thus,theeffectsoncellularandnuclearmembraneprotein Raman frequencies at ~143, 2382, 445 and 609 cm−1) TiO
domains, as well as on the interference with metabolic 2
minerals.AllpeaksforTiO -NPswerelargerthanthoseof
pathways,remainpoorlyinvestigated. 2
thepurepolymorphs,confirmingthepresenceofnanosized
The National Institute of Occupation Safety and
(<100 nm) TiO particles [13,14]. The amount of anatase
Health (NIOSH) recommends airborne exposure limits 2
was determined with a calibration procedure using the
of 2.4 mg/m3 for fine TiO and 0.3 mg/m3 for ultrafine
2 intensities of the Raman peaks of the two polymorphs
TiO , as time-weighted average concentrations for up to
2 present in the mixture (see Additional file 1: Methods
10hoursperdayduringa40-hourworkweek,butNIOSH
section). The results of this procedure on different Raman
alsoadmitsthatthereisinsufficientdatatoclassifyTiO as
2 peakscoherently indicated 93 wt% anatasein theTiO -NP
ahazardforhumanhealth[8,9]. 2
powder, with an estimated uncertainty of about ±1%.
Recently, attention has moved to the study of how
Finally, in order to better characterized charge and size of
TiO translocates from lungs to other systemic organs
2 the adopted NPs, DLS was employed: Z-potential and
[1,10], including the gastrointestinal tract, kidney and
hydrodynamicdiametervaluesarereportedinTable1.
heart [11].Indeed,theheart isthefirstorganthatinhaled
TiO -NPs may reach, after passing the alveolar epithelial
2
fenestrated barrier and entering the pulmonary circulation. TiO nanoparticlesimpaircardiomyocytecontractility
2
Thus, we hypothesized that acute exposure to TiO -NPs The effect of TiO on contractility was investigated on
2 2
might cause detrimental effects on cardiac electromech- cardiomyocytes isolated from adult rats: n=104 non-
anical function. In fact, we have previously described exposed control cardiomyocytes (CTRL), and n=102
in cultured engineered neonatal cardiac tissue that TiO -NP-exposedcells(NP ).Figure2A–Cgiverepresenta-
2 C
TiO -NPs may influence cardiac action potential (AP) tive recordings of shortening under different pacing fre-
2
by enhancing conduction and upstroke velocities and quencies(0.5Hz,1Hzand2Hz)inCTRL(blackline)and
disrupting myofibrils via production of reactive oxygen NP (red line). The average diastolic sarcomere length
C
species (ROS) [12]. However, comprehensive in vivo (Figure 2D) was nearly identical in CTRL and NP at all
C
assessment of cardiac risk is lacking. pacingrates(1.73±0.003μmand1.74±0.003μm,respect-
Here, we demonstrate with conventional electrophysio- ively). In contrast, fractional shortening (FS) and maximal
logical techniques – i.e., patch-clamp, Epicardial Potential rates of shortening and re-lengthening were significantly
Savietal.ParticleandFibreToxicology (2014) 11:63 Page3of15
Figure1AtomicForceMicroscopyanalysisoftitaniumdioxidenanoparticles(TiO -NPs)depositedonpoly-ornithine-treatedmica.
2
A.ImageofdeposedTiO-NPs.B.HeightprofilealongthewhitelineshowninA.C.HeightdistributionofTiO-NPs.D.ImagesofTiO-NP
2 2 2
aggregates(scalebars=100nm).
reduced in NP at all frequencies (~ −50%, p<0.01; recording periods in the absence of electrical stimulation;
C
Figure 2E–G). similarly to motion measurements, cells were condition-
Spontaneous contractions (SCs) were then assessed in ally trained (40 beats at 5 Hz) before V recording. We
r
cardiomyocytes that had been conditionally trained for found three types of V fluctuation (Figure 3A): in one
r
60 s with field stimulation set at 0.5 Hz. The percentage group of cardiomyocytes (type 1), V was stable over
r
of cells exhibiting SCs in the 60 s after stopping electrical time – considering the expected signal-to-noise ratio
pacing was significantly higher in NP than in CTRL forthistypeofvoltagerecording–withasharpfrequency
C
(Figure 2H). Furthermore, in cardiomyocytes exhibiting distribution(Figure3B,leftpanel);inasecondgroup(type
SCs, the number of events per cell within the 60 s period 2),V underwent small (~1 mV), continuous fluctuations,
r
was two-fold higher in NP (Figure 2I, Additional file 1: resultinginacontinuouslydistributedfrequencyarounda
C
FigureS2). peak value of −70 mV (Figure 3B, middle panel); finally,
type 3 cardiomyocytes displayed dispersed transitions to
TiO nanoparticlesslightlydepolarizerestingmembrane discrete V levels (ΔV up to 3–4 mV), resulting in
2 r r
potentialbutdramaticallyalterotheractionpotential well-separated peaks within the frequency distribution
parameters (Figure 3B, right panel). The fraction of type 2 and 3
In order to assess whether SCs were associated with cardiomyocytes increased after exposure to TiO -NPs
2
sub- or supra-threshold fluctuations of resting membrane (64% in NP vs. 36% in CTRL). Overall, ΔV and the
C r
potential (V), we analyzed variability (ΔV) over 60 s coefficient of variability of V (CV ) were signifi-
r r r Vr
cantly higher in NP (ΔV : 2.8±0.24 mV in CTRL vs.
C r
4.0±0.35 mV in NP ; CV 0.53±0.04 in CTRL vs.
Table1BiophysicalpropertiesonTiO NPsindifferent C Vr:
2 0.70±0.07inNP ).TiO -NP-exposedcardiomyocytesalso
solutions C 2
had, on average, a remarkable reduction in AP duration
TiO -NPs Water Saline Tyrode PBS-BSA
2 (APD) with respect to both the early (APD ) and late
20
Z-Potential(mV) −31.74±1.02 −18.36±2.30 −24.60±1.61 −13.05±2.13
(APD ) phases of repolarization (Figure 4A,B). A dose-
60
Hydrodynamic 154±3.00 498±0.55 538±0.37 449±0.85 dependent effect was found at concentrations from 5 to
Diameter(nm) 50μg/ml(Additionalfile1:FigureS3).Ofnote,despiteAPD
Savietal.ParticleandFibreToxicology (2014) 11:63 Page4of15
Figure2RepresentativetracesofsarcomereshorteningrecordedinCTRL(black)andNP (red)cardiomyocytesfield-stimulatedat0.5
C
(A),1(B)and2(C)Hz.Graphsofrestingsarcomerelength(D),sarcomerefractionalshortening(FS)(E),maximalrateofshortening(−dl/dt )
max
(F),andmaximalrateofre-lengthening(+dl/dt )(G).H.Piechartsofthepercentagesofcardiomyocytesexhibitingspontaneouscontractions
max
(SCs,stippledareas)inCTRL(white)andNP (red)cellsafter60sofconditionaltrainingat0.5Hz.I.GraphofnumberofSCs/cardiomyocytein
C
the60smeasurementperiod.*,p<0.01vs.CTRL.
shortening – which is expected to decrease beat-to-beat significant changes in V (compare Figures 4A and F;
r
variability of APD in this cell type [15] – CV Additional file 1: Figure S4). Moreover, we ran simulations
APD60
increased significantly by 28% (Figure 4C). ofPandit-modelledAPswithandwithouttheadditionofa
APupstroke(UPS)andelectricalmembranecapacitance K+ leakage current, and setting extracellular [K+] at values
(C ) values were also significantly reduced after acute ranging from 3.0 to 23.2 mmol/l. We found that the
m
exposure to TiO -NPs (Figure 4D–E). Mean values of V, simulated leakage current led to an increase in dV/dt
2 r max
resting membrane resistance (R ), AP amplitude (APA), from 180 to 183 V/s (Additional file 1: Figure S5, left
m
and rheobase and chronaxie values were comparable in panel), with a maximum peak corresponding to a [K]
o
NP andCTRL(Additionalfile1:TableS1). of about 6 mmol/l, which is known to characterize
C
In order to provide a mechanistic explanation for the supernormal conduction of sodium current in engineered
cellular findings, we ran simulations with a modified neonatalratcardiactissue[17](Additionalfile1:FigureS5,
versionofthePanditetal.ratventricleAPmodel[16].The rightpanel).
above-presented in vitro findings could be reproduced in
silico by introducing into the Pandit model’s pool of ion ECGandepicardialelectrogramsindicatefasterelectrical
currentsa1.5nSleakageconductanceselectivelypermeable activationafterexposuretoTiO
2
to potassium ions (for the sake of comparison, amounting Rats were anesthetized as described below and in-
only to ~5% of maximum I conductance). Of note, stilled tracheally with either physiological solution
K1
the experimental and simulated APs were similar, with a (Vehicle) or 2 mg/Kg TiO -NPs (NP ). Rats were left
2 R
quasi-superimposable reduction of APD without any to recover for 4 hours before undergoing anesthesia
Savietal.ParticleandFibreToxicology (2014) 11:63 Page5of15
A
Type1
1 s
Type 2
V
m
1
Type 3
B
210x4 Type1 210x4 Type2 210x4 Type3
1.6 1.6 1.6
yneuqerFc 10..28 Frequency 10..28 Frequency 10..28
0.4 0.4 0.4
0-72 -71 -70 -69 -68 -67 -66 -65 -64 0-72 -71 -70 -69 -68 -67 -66 -65 -64 0-72 -71 -70 -69 -68 -67 -66 -65 -64
V (mV) V (mV) V (mV)
r r r
Figure3VariabilityinrestingmembranepotentialincardiomyocytesexposedtoTiO NPs.A.Tracesrepresentativeofthethreetypesof
2
V behaviorfoundovera60srecordingperiodsubsequenttoconditioningtrainingat5Hzfor40beats.B.FrequencydistributionofV forthe
r r
threeΔV types.
r
(see Figure 5A). NP had a normal QRS pattern of NP :0.64±0.04ms;p<0.05)inNP ,pointingtoanoverall
R R R
activation, as evidenced by 3-lead ECG (data not increaseincardiacexcitability(Table2).
shown). Electrograms (EGs) derived from the electrode From the isochrone maps, we evaluated the average
arraypositionedontheepicardialsurface(Additionalfile1: transverse and longitudinal conduction velocity (CVt and
Figure S6) did not indicate any qualitative difference CVl, respectively) (Figure 5C). While CVt was unchanged,
in the two experimental groups. Indeed, EGs (Figure 5B) CVlincreasedsignificantly by 11% in NP (Table 2, **,
R
had the expected biphasic form, with positive electro- p<0.005). The anisotropy ratio CVl/CVt – the augmenta-
cardiographic R and negative S waves whose ampli- tion of which is recognized asa key arrhythmogenic factor
tudes were correlated to the area explored by the [18]–was19%higherinNP (**,p<0.005).
R
electrode array. In detail, EGs had an Rs shape (see
Additional file 1: Methods section) when positioned near TrachealinstillationofTiO nanoparticlesexacerbates
2
the heel of the pulmonary artery, and an rS shape near arrhythmogenesis
breakthroughpoints. Refractoriness was measured as the effective refrac-
Since EG waves were not qualitatively different, we tory period (ERP) using a dedicated protocol [19,20]
focused our attention on their duration (right panel (Figure 6) – see Additional file 1: Methods section - for
ofFigure5B,Table2).WefoundthatPwavedurationand parameters definition. An increase in ERP duration and
thePQsegmentweresignificantlyreduced(−12%),aswas ERP spatial dispersion – commonly recognized as an
QRS complex duration (−5%), in NP compared with index of vulnerability to arrhythmia – was also found in
R
Vehicle. There was a non-significant increase in heart NP (Figure6B–C).
R
rate (R-R reduction), while QT duration was reduced Similarly, the likelihood of establishing arrhythmic
22% in NP (Table 2). events during the ERP was assessed from 60 electrode
R
positions in NP and 44 in Vehicle. We observed a
R
ExposuretoTiO nanoparticlesincreasescardiac roughly two-fold increase in induced ventricular ectopic
2
excitabilityandconductionvelocity activity, mainly extra-systole couplets (Figure 6A, top)
In vivo, we observed a non-significant decrement of and ventricular fibrillation (Figure 6A, bottom), when
rheobase(Vehicle:21.4±1.68μA;NP :20.6±2.73μA)and pacing neartheERP (33% ofthe total electrodes for NP
R R
significantly decreased chronaxie (Vehicle: 0.75±0.04 ms; vs.18%Vehicle; Figure 6C).
Savietal.ParticleandFibreToxicology (2014) 11:63 Page6of15
AA
CTRL NP
C
0mV
-20mV APD
20
-60mV APD
60
10ms
B C D E
120 2 12 250
*
100 10
200
1.5
* *
80 8 *
150
60 % 1 mV 6 F
p
100
40 4
* 0.5
50
20 2
0 0 0
APD APD CV UPS C
20 60 APD60 m
CTRL NP
C
F
60
G = 5% I leakage
K1 CTRL K leak
V)
m
(
m
V
-80
10ms
Figure4TiO NPs-inducedchangesincellularelectrophysiology.A.Representativeactionpotential(AP)waveformsrecordedfromcontrol
2
(CTRL,blackline)andTiO-NP(NP ,redline)cardiomyocytesatthephysiologicaldrivingrateofratheart(5Hz).B–E.Graphsofactionpotential
2 C
duration(APD)measuredat−20mV(APD )and−60mV(APD ),beat-to-beatvariabilityofAPD (CV ),APupstroke(UPS)andmembrane
20 60 60 APD60
capacitance(C ).Inallgraphs,CTRLisgivenbywhitecolumns,andNP bytheredcolumns(n=37NP andn=49CTRL).*,p<0.05vs.CTRL.
m C C
F.APssimulatedwiththePanditmodel,without(blacktrace,CTRL)andwith(redtrace,NP )asimulated1.5nSconstantpotassiumleakage.
C
TiO nanoparticlesacutelyreachlungsandventricles,and in vivo into left and right ventricular cardiomyocytes of
2
areinternalizedwithincells TiO -instilled rats, suggesting that contamination of
2
A key aspect for the interpretation of the above findings cardiac tissue can occur via the lungs. In particular,
was to identify the presence of TiO -NPs not only in the morphologic evidence provide by TEM indicates that
2
lungs but also in exposed cardiomyocytes. We found NPs leave the capillary lumen, cross the endothelial layer,
that TiO -NPs entered not only directly into cultured penetrate the sarcolemma and reach the myoplasm
2
cardiomyocytes (Additional file 1: Figure S7A,B), but also by establishing intimate contact with myofibrils and
Savietal.ParticleandFibreToxicology (2014) 11:63 Page7of15
A
0 1 2 3 4 5 8
B RMS
P PQ QT
40 mV
QRS
30 ms
Vehicle NP
C R
10
11 15 11 14
10 8 9 13 14 7 8 12 15
6
7 10 12 9 16
6 8 5
5 13
5
9 10
5 7
6 14
7 9 9 11
17
8 11 11 7 6 8
Figure5InstillationofTiO -NPsandinvivorecordingsofcardiacelectricalperformance.A.Time-scale(hours)oftheexperimental
2
protocol.B.RepresentativeEGsrecordedfroman8×8epicardialelectrodearray.Eachwaveformofthegridrepresentsthetime-courseofextracellular
potentialatthecorrespondingposition.TheschemeontherighthandsideexplainstheEGparameters,asmeasuredfromtheirrootmeansquare
(RMS)-derivedsignals.Magentathintracerepresentsthefirsttimederivative,whoseminimumvalueistakenasamarkeroftheendoftheQTinterval
C.Representativeactivationtimemaps(isochrones,ms)fromVehicle(left)andNP (right),showingdifferencesinlongitudinal(redarrows)and
R
transverse(bluearrows)propagation.
mitochondria (Figure 7A–C). Ultrastructural analysis heart. While only tracheal tissue showed positive TBARS
alsoshowedthepresenceofTiO -NPsinlungtissue,with values in Vehicle –probably owing to the instillation
2
a tendency to agglomerate in the cytoplasm of alveo- maneuver – NP had lipid peroxidation in the lungs
R
lar cells and macrophages (Figure 7D–H). as well as in the heart (Figure 8C), indicating membrane
damageofcardiomyocytesduetoROSproduction.
TiO Nanoparticlesarecardiotoxicandgenotoxic
2
The interplay between TiO -NPs and cardiomyocytes Discussion
2
denoted signs of intracellular damage after acute adminis- Our findings indicate that TiO -NPs can: i) reach the
2
tration. Indeed, we observed a 25% increase in damaged heart via the respiratory system; ii) enter ventricular
nuclear DNA already after one hour of exposure, accom- cardiomyocytes; and iii) enhance the susceptibility to
paniedbya16%increaseinROSformation(Figure8A,B). cardiac arrhythmias via shortening of repolarization time,
We also analyzed TBARS ex vivo, from trachea, lungs and and by increasing cardiac excitability. To the best of our
Savietal.ParticleandFibreToxicology (2014) 11:63 Page8of15
Table2In-vivoelectrophysiologicalparameters andslightdepolarizationofV.Inordertoprovideamech-
r
Vehicle NP anistic explanation of the increment of CVl found in vivo,
R
we simulated electrically paced AP trains by means of
Pwave(ms) 33.0±0.22 28.9±0.21**
the Pandit model endowed or not with the leakage cur-
PQsegment(ms) 24.2±0.18 21.3±0.21**
rent. Simulated APs exhibited the highest ΔdV/dt for
max
QRScomplex(ms) 16.0±0.09 15.2±0.09**
[K] =5.8mmol/l,whichcorrespondstothepointofsuper-
o
QTinterval(ms) 40.0±0.28 31.1±0.54** normal sodium conduction [17] in cardiac tissue, suggest-
RRinterval(ms) 237.6±0.90 231.3±2.47 ing that the small TiO -induced depolarization may: i)
2
Rheobase(μA) 21.4±1.68 20.6±2.73 speed up the kinetics for reaching the activating threshold,
and ii) lead to the observed increment in CVl, which can
Chronaxie(ms) 0.75±0.04 0.64±0.04*
thus be ascribed to supernormal conduction [12]. Notably,
CVI(m/s) 0.63±0.004 0.70±0.004
conductionblockandincreasedlikelihoodofalternans[28]
CVt(m/s) 0.33±0.002 0.32±0.002
consequenttosupernormalconductionareknowntomake
Anisotropyratio 1.96±0.01 2.33±0.02** cardiac tissue prone to structural and functional reentrant
*p<0.05vsVehicle**p<0.005vsVehicle. arrhythmias [17,29]. Although arrhythmogenesis is fre-
quently associated with prolongation of repolarization,
knowledge,thisisthefirsttimethatadirecteffectofTiO measured at the cellular and organ level (APD and QT),
2
on cardiac electrical performance has been reported and the role of QTand APD shortening in favoring transition
explainedinvivo. toarrhythmiceventshasbeenalsoextensivelydocumented
It was described recently that, depending on size and [30]. This is particularly relevant for our findings, where
bioavailability, NPs may reach the bloodstream after TIO -NPs–incontrastwithdiesel-exhaustparticulates,for
2
passing the pulmonary barrier [10]. Furthermore, we instance[31]–inducedAPDandQTshortening.
previously reported that electrically charged NPs modulate Also,physiologicalsarcomerecontractionisundoubtedly
excitability by producing membrane disruption (positively affected by the presence of TiO . In fact, not only were
2
charged NPs) or life-compatible nanopores (negatively NPs detected among the myofibrils by TEM, but dynamic
chargedNPs)[21].Wealsoreportedthatnegativelycharged sarcomere measurements on the same cells indicated that
nanoparticles can be internalized via clathrin-mediated their excitation–contraction coupling machinery was sig-
membrane protrusions [22]. Such findings are in line with nificantly impaired. Besides the reported arrhythmogenic
our leakage hypothesis and explain our observation of behavior due to dyssynchronous sarcomere contractions
a significant decrease in membrane capacitance due to mediatedbythemechano-electricfeedback[32,33],wealso
membranelosscausedbytheinternalizationprocess. found an increased incidence of spontaneous contractions
TiO is widely used as a nanomaterial because it is (cf.Figure3)followinghigh-frequencyconditioningpacing,
2
considered inert [23,24]: its toxicity, documented in which may be synergic with an increment of inducible
cellular and animal models, remains controversial [25,26] arrhythmicevents.
since surface charge may differ depending on the vehicle Although our experiments do not reveal any evidence
[26]. Z-potential characterization demonstrated that NPs for NP-induced changes in rate-dependent shortening of
were negatively charged in our experimental setting and, the sarcomere, their lengths, FS and maximum rates of
therefore, able to transiently produce nanopores that, in shortening and re-lengthening (±dl/dt ) were all affected
max
turn, resulted in the development of leakage currents, bythepresenceofNPs.Ourmulti-levelapproachgenerated
asreportedpreviously[21].Itisknownthat,upontracheal parallel in vitro and in vivo results (reduction in the APD,
instillation,TiO generatesadetrimentalsequenceofdys- increase in the APD temporal dispersion, reduction in
2
functions, which might ultimately lead to atherosclerosis the QT interval and increase in its dispersion), which
and,thus,affectthefunctionoftheheartpump[27].Here, substantiallyruleoutcardiacinflammatoryprocesscaused
we show how quickly TiO -NPs may directly affect by TiO -NPs as the main mechanism responsible for
2 2
cardiacelectricalactivityviathereductionofrepolarization excitation–contraction coupling unbalance. Nonetheless,
velocity, enhancing temporal dispersion and increasing chronic effects, including inflammatory progression,
excitability. certainly need further evaluation.
In order to test the hypothesis that an NP-induced A recent study on Langendorff-perfused isolated heart
leakage current might provide, per se, a mechanistic elegantly demonstrated that TiO -NPs produced abnormal
2
explanation of our electrical findings, we introduced a electrical activity accompanied by a dose-dependent
1.5 nS potassium leakage into the membrane equations of increase in heart rate [34]. Our study confirms those
the Pandit rat ventricular AP model [16]. Of note, simula- findings in in vivo anesthetized rats (ECGs and EGs) and
tions were consistent with the results obtained in patch- focuses for the first time on the underlying mechanism.
clamped cardiomyocytes, i.e., reduction of APD and UPS, Indeed, the evaluation of excitability parameters in vivo
Savietal.ParticleandFibreToxicology (2014) 11:63 Page9of15
A
S1S2
Vehicle
B
C
NP
R
100 35
90
** 30
80
a
70 hmi25
yt
ms) 60 arrh20
PRE( 4500 ucible 15
d
n
30 of i10
%
20
5
10
0 0
Figure6SusceptibilitytoarrhythmiasinVehicleandNP rats.A.Ventricularectopiccouplet(top)andventricularfibrillation(bottom)
R
recordedduringevaluationoftheeffectiverefractoryperiod(ERP).Scalebar=500ms.B.EvaluationofERPinVehicleandNP.**,p<0.005.
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C.Percentageofinduciblearrhythmcevents.
indicated that cardiac tissue becomes more excitable, in in inducible arrhythmias occurred after perfusion with
line with previously described results [12]. Moreover, we TiO -NPs, which were rapidly internalized within cardio-
2
adopted a single NP dose (2 mg/Kg) in solution in order myocytes.ROS-inducedmembranedamageandNP-induced
to be aligned not only with NIOSH recommendations on leakages are expected to locally depolarize cardiac tissue
the time-evaluation of human exposure to TiO -emitting and promote arrhythmogenesis [37] via source-sink
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sources, but also because such a concentration in ~50 μl electrical mismatch. Occurrence of arrhythmias was
of instilled solution is a good compromise between the estimated in healthy rat hearts in vivo by the well-known
volume of vehicle needed and the optimal disaggregation S1-S2 protocol (see Additional file 1: Methods section).
of NPs (cf. Figure 1). A half-dose instillation of TiO -NPs Through this protocol, we found a significant increase in
2
(1 mg/Kg) evaluated in three rats produced the same the incidence of ectopic events (duplets or triplets)
effects observed with the full dose (data not shown). and of ventricular fibrillation in rats administered TiO .
2
Although the pathological effects on the cardiovascular Therefore, these malignant events may be exaggerated by
system are gaining more attention than they have had in acuteandsub-acuteexposuretoanenvironmentpolluted
the past [35], the effect of NPs on cardiovascular tissue is withTiO .
2
usually viewed as secondary to inflammatory processes
initiated at the lungs [36]. Despite the fact that the latter Studylimitations
mechanism can be present chronically, we observed While this study proposes supernormal sodium-based
in the acute setting that ROS production and an increase conduction at the single-cell level as a physiological