Exposure to concentrated ambient particulate matter induces reversible increase of heart weight in spontaneously hypertensive rats

Whole-body ambient inhalational CAP exposures protocol

The protocol of animal experiments was approved by the Ohio State University Institutional
Animal Care and Use Committee, and all animals were treated humanely and with regard
for alleviation of suffering. Animal exposure and the monitoring of exposure atmosphere
and ambient aerosol were performed as previously described using a versatile aerosol
concentration enrichment system that was modified for long-term exposures 8]. Briefly, SHR (4-week-old males, n?=?6/group, 24 in total) were bought from Charles
River and were housed in standard cages in a mobile trailer with a 12-hr light/12-hr
dark cycle, temperatures of 65–75 °F, and relative humidity of 40–60 %. After 1 week
of acclimation, rats were exposed to CAP or filtered air (FA) in chambers of the Ohio
Air Pollution Exposure System for the Interrogation of Systemic Effects at The Ohio
State University. All rats, including both FA and CAP groups, were exposed at exactly
the same time. FA-exposed rats received an identical protocol with the addition of
a high-efficiency particulate air filter (Pall Life Sciences, East Hills, NY, USA)
positioned in the inlet valve to remove CAP in the filtered air stream, as previously
described 8]. The exposure protocol comprised exposures for 6 h/day, 5 days/week (no exposure
took place during weekends) for a total duration of 15 weeks.

Sampling and analyses of ambient PM
_2.5
and CAP in the exposure chamber

To calculate exposure mass concentrations of ambient PM
_2.5
and CAP in the exposure chambers, samples were collected weekly on Teflon filters
[Teflo, 37-mm, 2-?m pore (Pall Life Sciences, Ann Arbor, MI, USA)] and weighed before
and after sampling in a temperature- and humidity-controlled weighing room using a
Mettler Toledo no. 11106057 microbalance (Mettler Toledo, Columbus, OH, USA). The
filters were then wetted with ethanol and extracted in 1 % nitric acid solution. The
extraction solution was sonicated for 48 h in an ultrasonic bath and then allowed
to passively acid digest for a minimum of 2 weeks. We then analyzed sample extracts
for a suite of trace elements using inductively coupled plasma-mass spectrometry (ICP-MS)
(ELEMENT2; Thermo Finnigan, San Jose, CA, USA) 30].

Blood pressure assessment

The systolic blood pressure was obtained using a non-invasive tail-cuff monitor (MK2000;
Muromachi Kikai, Tokyo, Japan). The measurement was performed weekly (Saturday morning).
Before these analyses, all rats had been trained for 1 week. During assessment, the
rats were placed into a holder which was pre-warmed and kept at 30 °C during the whole
assessment and subjected to 20 measurements. The mean systolic blood pressure of these
20 measures for each animal were determined and presented.

Echocardiography

Cardiac function was analysed by echocardiography (40 MHz transducer, Vevo 121 2100;
Visualsonics, Toronto, Ontario), and the sonographer was blinded to the experimental
groups during both data collection and analysis. Internal temperature was maintained
at 37 °C as rats were continuously sedated with 1 % isoflurane (in 100 % O
₂
) during assessment, which was about 1 h. Pre-warmed ultrasound gel (Aquasonic, Parker
Labs, Fairfield, New Jersey) was used on the chest with a 15 MHz probe optimized for
rats placed in the parasternal, short axis orientation. Data were averaged from at
least three analyses per rat. LV dimensions (LVESd and LVEDd) and posterior wall thickness
(PWTs and PWTd) were assessed, using the leading-edge technique according to the American
Society for Echocardiography. Fractional shortening (FS) was calculated using the
equation: %?FS?=?[(LVEDd-LVESd)/LVEDd*100. Additional images were acquired to obtain
aortic and pulmonary dimension, and pulse-wave Doppler imaging was used to obtain
aortic and pulmonary velocities. Stroke volume (SV) was estimated utilizing the velocity
time integral trace multiplied by the cross sectional area of the vessel, and this
was used to calculate cardiac output (CO) as the product of SV and heart rate (HR).
To determine the effects of withdrawal from CAP exposure on cardiac function, half
rats were subjected to echocardiography shortly after CAP exposure, and the remaining
were analysed after 5 weeks of withdrawal from CAP exposure.

Tissue preparation

After anesthetized with pentobarbital sodium, rats were subjected to blood collection
from hearts using a 22 g needle, and rapidly headed. Hypothalamus was isolated using
incision sites as follows: rostral border of the optic chiasm, caudal border of the
mamillary body, ventral border of the anterior commissure and lateral borders of the
tuber cinereum and mamillary body complexes, and snap-frozen in liquid nitrogen. Thoracic
aortas were proximally isolated and cut at the root. The whole heart was then isolated
and placed on kimwipes to remove the remaining blood, and then snap-frozen in liquid
nitrogen. All tissues were stored at ?80 °C until processed.

Myography

Briefly, rats were anesthetized with pentobarbital sodium, and the thoracic aorta
was quickly removed and cleaned in physiological salt solution (PSS) containing (mM):
NaCl, 130; NaHCO
₃
, 14.9; KCl, 4.7; KH
₂
PO
₄
, 1.18; MgSO
₄
•7H
₂
O 1.18; CaCl
₂
•2H
₂
O, 1.56, EDTA, 0.026, glucose 5.5. The aorta was cut into 2-mm rings, and were then
mounted in a muscle bath containing PSS at 37 °C and bubbled with 95 % O
₂
-5 % CO
₂
. Isometric force generation was recorded with a Multi Myography System (Danish Myo
Technology A/S, Aarhus N, Denmark). A resting tension of 30 mN was imposed on each
ring, and the rings were allowed to equilibrate for 2 h. Arterial integrity was assessed
first by stimulation of vessels with 80 mM KCl. Endothelium-integrity was assessed
by measuring the dilatory response to ACh (10 ?M) in PE-contracted vessels (1 ?M).
To examine the contractility of those aortic rings, phenylephrine (PE) or U-46619
was added in a cumulative manner. To test relaxation responses, aortic rings were
pre-contracted by PE (0.3 ?M) that induced approximately 60 % of maximal contraction,
followed by addition of acetylcholine (ACh) in a cumulative manner.

Western blot analysis

About 1 g of left ventricle wall close to the apex of heart was cut on ice, and homogenized
in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA). 40 ?g/sample
proteins were resolved with 10 % SDS-PAGE and transferred to membrane. Immuno-staining
was performed by standard techniques with primary antibodies as follows: anti-GAPDH
from Cell Signaling Technology (Boston, MA, USA); anti-ACTA1 from ABGENT (San Diego,
CA, USA); anti-SERCA2 from Thermo Fisher Scientific (Rockford, IL, USA); and anti-MYH7
from GeneTex (Irvine, CA, USA). Signals were detected by supersignalâ„¢ chemiluminescence
(Pierce, Rockford, IL, USA) and analysed by densitometry.

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated from tissues with TRIzol reagent (Invitrogen, Carlsbad, CA,
USA). 4 ?g total RNA was reverse transcribed using random hexamers and the ThermoScript
RT-PCR System (Invitrogen). Quantitative RT-PCR was performed with the Stratagene
Mx3005 using SYBER Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). The
sequences of primers were previously described 31]: glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense, 5?-ATG ATT CTA CCC ACG
GCA AG-3?, antisense, 5?-CTG GAA GAT GGT GAT GGG TT-3?; tumor necrosis factor-? (TNF?):
sense, 5?-GAC CCT CAC ACT CA GAT CAT CTT CT-3?, antisense, 5?-TGC TAC GAC GTG GGC
TAC G-3?; interleukin-6 (IL-6): sense, 5?-CGA GCC CAC CAG GAA CGA AAG TC-3?, antisense,
5?-CTG GCT GGA AGT CTC TTG CGG AG-3?); IL-1?: sense, 5?-CCC TGC AGC TGG AGA GTG TGG-3?,
antisense, 5?-TGT GCT CTG CTT GAG AGG TGC T-3?; COX2: sense, 5?-GAT TGA CAG CCC ACC
AAC TT-3?, antisense, 5?-CGG GAT GAA CTC TCT CCT CA-3?. The relative expression level
was obtained as described previously with minor modification 8]. Briefly, Ct values were acquainted through analysis with software provided by the
manufacturer, and differences of Ct value between target gene and GAPDH (?Ct) and
then 2
^?Ct
were calculated. Results were expressed as ratio to the average of FA group (fold).

Statistics

If not specified, data are expressed as mean?±?SEM. Statistical comparisons of dose–response
curves were performed with two-way repeated-measures analysis of variance (ANOVA)
using GraphPad Prism version 4.0b (Graphpad Software Inc., La Jolla, CA, USA)]. Otherwise,
statistical comparisons were performed with one-way ANOVA or Student’s t-test. p??0.05 was considered to be statistically significant.