Download Methods and results related to supplementary data

SEROTONIN 5-HT2B RECEPTOR BLOCKADE PREVENTS REACTIVE OXYGEN
SPECIES-INDUCED CARDIAC HYPERTROPHY IN MICE
SUPPLEMENTAL ONLINE MATERIAL
Short title: 5-HT2B, superoxide and cardiac hypertrophy
Laurent Monassier, MD, PhD*§; Marc-André Laplante, PhD**¶; Fabrice Jaffré, PhD#; Pascal
Bousquet, MD, PhD§; Luc Maroteaux, PhD#; Jacques de Champlain, MD, PhD¶.
§ Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, INSERM, U-715,
Faculté de Médecine, Strasbourg, France; (L. Mo, P.B.)
¶ Département de Physiologie, Faculté de Médecine, Université de Montréal and
Laboratoire de Recherche sur le Système Nerveux Autonome, Institut de Recherche Clinique,
Montréal, Canada; (M.A.L., J.DeC.).
# INSERM, U-389, Université Pierre et Marie Curie, Paris, Institut du Fer à Moulin,
Paris, F-75005, France; (F.J., L.Ma.)
* Corresponding author
**Both two first authors (L.M., M.A.L.) contributed equally to the work
Laurent Monassier, MD, PhD, LNPCV, INSERM U-715, Faculté de Médecine, 11 rue
Humann, Strasbourg, France. E-mail [email protected]
Tel: +333 90 24 33 92
Total word count: 1211
Fax: +333 90 24 33 88
Methods and results related to supplementary data
Induction of Cardiac Hypertrophy by ISO and AngII
In 9 week-old mice, vehicle (distilled water), ISO (30 mg.kg-1.d-1) or ISO associated with the
5-HT2B receptor antagonist, SB215505 (1 mg.kg-1.d-1) or with the antagonist of -adrenergic
receptors, propranolol (5 mg.kg-1.d-1) was delivered by subcutaneous osmotic minipumps
(1007D, Alzet Corporation) (all chemicals used in this study were from Sigma). In addition, a
group of mice was implanted with an ISO delivering minipump, 24h after having started a
treatment with the NAD(P)H oxidase inhibitor apocynin (1.5 mM, drinking water). Both
drugs were delivered until the end of ISO perfusion i.e. 5 days. Nox2-/- mice were also
perfused during 5 days either with vehicle or ISO. One group of mice received ISO +
apocynin in the drinking water. In 8 week-old mice, vehicle (distilled water), AngII (0.2
mg.kg-1.d-1) or AngII associated with the 5-HT2B receptor antagonist SB215505 (1 mg.kg-1.d1
) were delivered during 14 days by osmotic minipumps (1002, Alzet Corporation).
Similarly, 5-HT2B-/- mice were infused by either ISO (30 mg.kg-1.d-1) or AngII (0.2 mg.kg-1.d1
).
At the end of infusion periods, heart rate and systolic arterial pressure were recorded by the
tail-cuff method (Visitech). Transthoracic echocardiograms were then performed in 2%
isoflurane anaesthetized mice as described previously 1. Two hours later, mice were killed by
CO2 and weighed, the heart excised, weighed and quickly frozen after separation of right and
left ventricles and atria. In some experiments, the abdominal aorta was sampled for lucigenin
measurements. Tissues were stored at 80°C until use.
Involvement of NAD(P)H oxidase in chronic ISO induced cardiovascular alterations
To elucidate if NAD(P)H oxidase is involved in hypertrophy-induced by -adrenergic chronic
stimulation, we treated WT simultaneously with ISO and the selective NAD(P)H complex
inhibitor apocynin. This drug prevented the left ventricular hypertrophy measured by
echocardiography (LVM:BW 3.50.2 mg.g-1 vs. 6.30.3 mg.g-1 in ISO alone-infused mice,
P<0.05) and reduced the HW:BW ratio (Table 1). This prevention was associated with a
normalization of EDD which was similar to vehicle-treated animals (Table 1). This compound
did not significantly affect the cardiac contractility (Table 1) or the blood pressure (data not
shown). To confirm these results, Nox2-/- mice were treated with ISO.
ISO was still able to induce an increase in HW:BW ratio (+30% vs. controls Nox2-/- mice;
Supplementary data figure 1A), however no significant left-ventricular dilatation occurred
(EDD: 3.80.1 mm in controls vs. 4.30.2 mm in ISO, P>0.05). This constitutive inactivation
of the Nox2/gp91-phox subunit of the NAD(P)H oxidase did not prevent the ISO induced
tachycardia (56024 bpm in controls vs. 7716 bpm in ISO treated, P<0.05).
Other Nox isoforms are expressed in the heart that could contribute to a NAD(P)H oxidasemediated cardiac hypertrophy. To test this hypothesis, we simultaneously treated Nox2-/(gp91phox knockout) male mice (Jackson Laboratory) with ISO and apocynin. Apocynin
markedly reduced the ISO-induced cardiac hypertrophy (Table 1) attesting of a nonNox2/NAD(P)H oxidase mediated left-ventricular hypertrophy due to -adrenergic
stimulation. Nox1 expression was significantly increased by ISO in these animals while Nox4
isoform was not significantly affected (figure S1B and C). The Nox1 antibody revealed a
band of 60 KDa that was suppressed by incubating the Nox1 antibody with an excess of
antigenic peptide. The Nox4 antibody revealed bands at 65 and 80 KDa that were suppressed
by incubating the Nox4 antibody with excess antigenic peptide. The apocynin treatment
prevented the ISO-induced Nox1 overexpression indicating that SA is an inducer of Nox1
expression. Apocynin did not modify the Nox4 expression.
SB215505 is not a non-specific antioxidant
In this set of experiments, the spontaneous hematoxylin oxidation was followed with a UVvisible recording spectrophotometer (Shimadzu Corp. Kyoto, Japan) at 560 nm according to
the method of Martin et al. 2 modified by Chattopadhyay et al. 3. The enzymatic reaction was
performed with 50 M hematoxylin. Reagents were purchased at Sigma Chemical Co., StLouis.
In this assay, SB215505 was tested at 10-7, 10-6, 10-5 and 10-4 M and no antioxidant effect was
observed. However, the well known antioxidants glutathione (GSH) and ascorbic acid (AA)
reduced or suppressed the hematoxylin oxidation (figure S2). Therefore, this experiment rules
out any non-specific antioxidant property of SB215505.
Chronic infusion with SB215505 does not affect basal and NAD(P)H stimulated ventricular
superoxide anion concentration
Mice were subcutaneously treated with SB215505 (1 mg.kg-1, Alzet osmotic minipumps).
After 5 or 14 days of infusion, basal or NADPH stimulated production of superoxide anion
was measured in the left ventricles. As compared with controls (vehicle-infused), we did not
observe any effect of this compound attesting of the lack of NAD(P)H oxidase blockade
(figure S3).
References
1.
Nebigil CG, Hickel P, Messaddeq N, Vonesch JL, Douchet MP, Monassier L, Gyorgy
K, Matz R, Andriantsitohaina R, Manivet P, Launay JM, Maroteaux L. Ablation of
serotonin 5-HT(2B) receptors in mice leads to abnormal cardiac structure and
function. Circulation. 2001;103:2973-2979.
2.
Martin JP, Jr., Dailey M, Sugarman E. Negative and positive assays of superoxide
dismutase based on hematoxylin autoxidation. Arch Biochem Biophys. 1987;255:329336.
3.
Chattopadhyay A, Biswas S, Bandyopadhyay D, Sarkar C, Datta AG. Effect of
isoproterenol on lipid peroxidation and antioxidant enzymes of myocardial tissue of
mice and protection by quinidine. Mol Cell Biochem. 2003;245:43-49.
Legend to figure S1: Isoproterenol (ISO) induces an increase in heart weight:body weight
ratio (HW:BW) in Nox2-/- mice that was reduced by the NAD(P)H oxidase inhibitor,
apocynin (A) (n=6 per group). ISO induces a Nox1 overexpression that is prevented by
apocynin (APO) (n=5 per group) (B). The Nox4 expression is not affected by ISO in Nox2-/animals (n=5 per group) (C). The standardization was performed for each lane and the data
presented in the graph reflect the average values of 5 experiments. All the variations for actin
are taken into account in the values displayed in figure 1C. AU: arbitrary units. #: P<0.05 vs.
Nox2-/- control mice; *: P<0.05 vs. Nox2-/- ISO treated mice.
Legend to figure S2: Assessment of the direct chemical SA scavenging properties of
SB215505 compared to glutathione and ascorbic acid through its capacity to prevent
spontaneous hematoxylin oxydation. *P<0.05 vs. CT (normal rate of hematoxlin oxydation in
a physiological buffer). GSH: glutathione; AA: ascorbic acid.
Legend to figure S3: Basal and NAD(P)H oxidase stimulated superoxide anion production in
ventricles from mice treated with SB215505 alone. SA: superoxide anion
Figure S1
Figure S2
Figure S3