L-arginine mitigates radiation-induced early changes in cardiac dysfunction: the role of inflammatory pathways.

Abstract

Our earlier studies demonstrated the ability of L-arginine (L-Arg) to reverse radiation-induced immune dysfunction. The aim of the present study was to investigate cardiac dysfunction up to 24 h after 2 Gy of total-body irradiation (TBI) and its mitigation by L-Arg. The current studies also explore the association of radiation-induced inflammation and electrocardiographic (ECG) abnormalities. TBI-induced cardiac iNOS and kinin B1 R, changes in the ECG profile like bradycardia, increased RR interval, ST elevation and increased QRS duration at 4 h and 24 h after TBI. TBI with 2 Gy induced inflammatory responses in spleen and cardiac tissue. L-Arg administered 2 h after TBI (TBI+L-Arg) mitigated the entire inflammatory response and ECG profile toward normalcy. L-Arg administered just before TBI (L-Arg +TBI) could not reverse the above-mentioned changes. Radiation-induced inflammatory responses at +4 h and +24 h after TBI in spleen and cardiac tissue correlated with the changes in ECG profile at the corresponding time. The results suggest the ability of L-Arg administered at the correct therapeutic window to mitigate radiation-induced cardiac dysfunction at 4 and 24 h after TBI.

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L-Arginine Mitigates Radiation-Induced Early Changes in Cardiac
Dysfunction: The Role of Inflammatory Pathways
Jyoti Shukla, Nazir M. Khan, Vikas S. Thakur and T. Balakrishna Poduval1
Immunology and Hyperthermia Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay,
Mumbai – 400 085, India
Shukla, J., Khan, N. M., Thakur, V. S. and Poduval, T. B.
L-Arginine Mitigates Radiation-Induced Early Changes in
Cardiac Dysfunction: The Role of Inflammatory Pathways.
Radiat. Res. 176, 000–000 (2011).
Our earlier studies demonstrated the ability of L-arginine (L-
Arg) to reverse radiation-induced immune dysfunction. The aim
of the present study was to investigate cardiac dysfunction up to
24 h after 2 Gy of total-body irradiation (TBI) and its mitigation
by L-Arg. The current studies also explore the association of
radiation-induced inflammation and electrocardiographic (ECG)
abnormalities. TBI-induced cardiac iNOS and kinin B1 R,
changes in the ECG profile like bradycardia, increased RR
interval, ST elevation and increased QRS duration at 4 h and
24 h after TBI. TBI with 2 Gy induced inflammatory responses
in spleen and cardiac tissue. L-Arg administered 2 h after TBI
(TBI+L-Arg) mitigated the entire inflammatory response and
ECG profile toward normalcy. L-Arg administered just before
TBI (L-Arg +TBI) could not reverse the above-mentioned
changes. Radiation-induced inflammatory responses at +4 h
and +24 h after TBI in spleen and cardiac tissue correlated with
the changes in ECG profile at the corresponding time. The
results suggest the ability of L-Arg administered at the correct
therapeutic window to mitigate radiation -induced cardiac
dysfunction at 4 and 24 h after TBI.
g2011 by Radiation Research Society
INTRODUCTION
Exposures to doses of radiation of 1–10 Gy may occur
during the course of radiation therapy or as the result of
radiation accidents or nuclear/radiological terrorism (1).
An inflammatory response and immune dysfunction is a
classical feature of radiation exposure and appears to be
a key event in the development of radiation injury (2).
Radiation-induced heart disease has been established as
indicating the clinical and pathological conditions
resulting from injury to the heart during therapeutic
irradiation of adjacent neoplasm (3–5). Inflammation
caused by proinflammatory cytokines such as IL-1b,
TNF-a and IL-6 have been implicated in critical injuries
caused by acute radiation injury, heatstroke, sepsis and
clinical pathological conditions such as SIRS, inflamma-
tory myocarditis, cardiac allograft rejection and heart
failure (2, 6–9). Nitric oxide (NO) produced by iNOS has
alsobeensuggested toplayaroleinthepro-inflammatory
cytokine-induced cardiac contractile failure.
Early inflammatory changes in the heart after total-
body irradiation (TBI) have not been studied so far. In
addition, no one has reported the early cardiac changes
induced by radiation and its association with the
inflammatory response. Our initial work had indicated
that TBI of mice caused changes in the ECG pattern
(10). Considering the above, studies were designed to see
the effect of a dose of 2 Gy on heart electrical activity at
4 and 24 h after TBI and its association with cardiac and
splenic inflammatory markers in the irradiated host. The
earlier studies had suggested the importance of the time
of administration of L-Arg and the L-Arg pathway in
mitigating the radiation-induced host immune dysfunc-
tion and inflammation caused by heatstroke, sepsis (2,
6–8). Based on the above, experiments were designed to
determine the ability of L-Arg to mitigate the inflam-
matory process and the early cardiac dysfunction when
administered at the correct therapeutic window. In this
study, we used a murine model, because it is the best
characterized animal model for initial assessment of
therapeutic agents to mitigate radiation injury (11).
MATERIALS AND METHODS
Animals
Swiss/Bh inbred male mice weighing 25–27 g and 7–8 weeks of age
were used for the studies; the animal care was as described previously
(2). All the experiments were carried out in accordance with the ethical
guidelines laid down by the Committee for the Purpose of Control and
Supervision of Experiments on Animals, Government of India.
Total-Body Irradiation and L-Arg Administration
Mice were divided into five groups consisting of four mice each.
Sham-irradiated mice were handled similarly but were not irradiated
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1Address for correspondence: Immunology & Hyperthermia
Section, Radiation Biology & Health Sciences Division, Bhabha
Atomic Research Centre, Trombay, Mumbai-400 085, India; e-mail:
tbpodu@.barc.gov.in.
RADIATION RESEARCH 176, 000–000 (2011)
0033-7587/11 $15.00
g2011 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR2523.1
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(Sham group). Mice were exposed to 2 Gy
Energy Canada, Model 220) at a dose rate of 10.79 cGy/s (TBI
group). L-Arg (Sigma Chemical Co., St. Louis, MO) in normal
pyrogen-free physiological saline (120 mg/kg) was administered
intraperitoneally (i.p.). The L-ArgzTBI group received the L-Arg
10 min before TBI and those in the TBIzL-Arg group received L-
Arg 2 h after TBI. A group of normal mice received only L-Arg at 0 h
(L-Arg group). After treatment, mice were given sterile food and
acidified water ad libitum. Care was taken to ensure that the bedding
of the cages was changed twice a day. The time of TBI was con-
sidered as 0 h, and various parameters were monitored with
reference to this time.
60Co c rays (Atomic
Semi-quantitative Reverse Transcriptase-Polymerase Chain
Reaction (RT-PCR)
Since RT-PCR has been used to monitor the possible biomarkers
for ionizing radiation exposure in human blood lymphocytes, we
used this method to monitor the upregulation of inflammatory
molecules in the irradiated host (12). Expression of various
cytokines, iNOS, kinin B1 R and transcription factor T-bet and
GATA-3 was monitored by one-step RT-PCR in spleen and heart
as reported earlier (2). For RNA preparation, splenic and cardiac
tissue were pooled from four mice per group per experiment The
RNA was reverse-transcribed by one-step RT-PCR by using the
Masterscript
described earlier (2). The program used for one-step RT-PCR was
as reported earlier with the exception of the annealing temperatures
for 45 s for various primers (Table 1). The sequences of
oligonucleotide primers of b-actin, iNOS, kinin B1 R, various
cytokines, T-bet and GATA-3 are presented in Table 1. Semi-
quantitative RT-PCR was performed using b-actin as an internal
control to normalize gene expression for the PCR templates. The
absence of PCR product signal from genomic DNA contamination
was confirmed by performing PCR with representative RNA
samples without reverse transcription amplification of mRNA.
Equal amounts of each PCR reaction product (10 ml) were run on
2% agarose gels containing ethidium bromide in Tris borate EDTA
TMRT-PCR System (5 Prime GmbH, Germany) as
buffer at 60 V. The intensity of the bands in the gels was visualized
under a UV lamp, and relative intensities were quantified using
GeneSnap Software (Syngene).
Animal Preparation for ECG
ECGs were performed with a Bio Amp device PowerLab System
(PowerLab 2/20; ADInstruments, Australia), which recorded bipolar
lead I. For the ECG recording, all animals were anaesthetized by i.p.
injection of urethane (1 g/kg body wt), and the recordings were taken
in a supine position. All the recording were performed between 10:00
a.m. and 2:00 p.m. to exclude the influence of diurnal variations or to
minimize circadian variations in a constant environment. Once the
corneal and withdrawal reflexes were absent in the mice, we inserted
MLA1204 needle electrodes subcutaneously into the right forelimb
and into each hind limb. The needle electrodes and pulse transducer
(ADInstruments) were connected to a PowerLab/4SP (ADInstru-
ments) through an ML136 Animal Bio Amp. Mice were allowed a 45-
min stabilization period before obtaining baseline ECG and HR
recordings. The procedure was done to avoid the inclusion of any
artifact due to anesthesia. Single-channel (lead II) ECG was recorded
for 20 min. For ECG recordings, four mice per group per experiment
were used.
Data Acquisition and Analysis
The ECG signal was amplified using a PowerLab/4SP system and
was digitized at a sampling rate of 1 kHz. All data were acquired on a
computer for further analysis using Chart 5 for Windows software.
Wave durations (in milliseconds) were calculated automatically by the
software after placement of the cursors. Measurements were average
values determined from 20-min consecutive ECG records. Records
were filtered (1 to 100 Hz) through a band-pass filter to minimize
environmental signal disturbances. ECG signal output was recorded
with Chart 5 for Windows. Data were analyzed using raw data for the
ECG signals acquired at a sampling rate of 1 K/s, lower pass 0.3 Hz
and upper pass 1 kHz. RR interval, QRS duration and the peak
height of T wave were analyzed using Chart 5 Pro (Version 5.5 for
Windows). The ECG analysis included the following measurements:
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TABLE 1
Primer Sequences for Amplification of cDNA during PCR with their Respective Annealing Temperatures
GenePrimer sequenceAnnealing temperature
b-ActinForward 59-CATCACTATTGGCAACGAGC-39
Reverse 59-GGACTGTTACTGAGCTGCGT-39
Forward 59-CTGCAGCACTTGATCAGGAACCT-39
Reverse 59-GGGAGTAGCCTGTGTGCACCTGGAA-39
Forward 59-AAGGATGGTGGAGTTGAACG -39
Reverse 59-CAGGTCTGTGAGCTCCTTCC -39
Forward 59-TGGAGTCACAGAAGGAGTGGCTAAG-39
Reverse 59-TCTGACCACAGTGAGGAATGTCCAC-39
Forward 59-CATCGGCATTTTGAACGAGGTCA-39
Reverse 59-CTTATCGATGAATCCAGGCATCG-39
Forward 59-ATGCAGGACTTTAAGGGTTACTTGGGTT-39
Reverse 59-TTTGGAGCAAACATGGAGAGAGGCTTTA-39
Forward59-TGGACCGCAACAACGCCATCTATGAGAAA-39
Reverse 59-GGAGCTGAAGCAATAGTTGGTATCCAGGG-39
Forward 59-ATGGCAACTGTTCCTGAACTCAACT-39
Reverse 59-CAGGACAGGTATAGATTCTTTCCTTT-39
Forward 59-GTTCTATGGCCCAGACCCTCACA-39
Reverse 59-TCCCAGGTATATGGGCTCATACC-39
Forward 59-CTTATCAAGCCCAAGCGAAG-39
Reverse 59-TAGAAGGGGTCGGACGAACT-39
Forward 59-GCCAGGGAACCGCTTATATG -39
Reverse 59-GACGATCATCTGGGTCACATTGT -39
47uC
iNOS 54uC
Kinin B1 R50uC
IL-657uC
IL-453uC
IL-1056uC
TGF-b
59uC
IL-1b
53uC
TNF-a
55uC
GATA-3 50uC
T-bet52uC
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FIG. 1. Splenic expression of (panel A) TNF-a, (panel B) T-bet, (panel C) TGF-b and (panel D) GATA-3 mRNA after TBI as determined by
RT-PCR as described in the Materials and Methods. Gene expression data were normalized to b-actin expression. Band intensity is shown as
means ± SEM of data from three independent animal experiments (three biological replicates) with similar results. In each experiment, spleens
from four mice were pooled for RNA isolation per group. *P , 0.05 compared to Sham group,$P , 0.05 compared to TBI group, **P , 0.05
compared to L-ArgzTBI group. Panel E: The splenic expression of GATA-3, TGF-b, TNF-a, T-bet and b-actin mRNA 4 and 24 h after TBI.
Lanes 1 to 5 are Sham, TBI, L-ArgzTBI, TBIzL-Arg and L-Arg, respectively. When there was not a basal level of expression, the bands are not
shown. The PCR band shown is representative of the three independent animal experiments with similar results.
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Radiation Researchrare-176-02-08.3d3/6/11 21:43:554Cust # RR2523
FIG. 2. Cardiac expression of (panel A) TNF-a, (panel B) IL-1b, (panel C) IL-6, (panel D) T-bet, (panel E) iNOS and (panel F) kinin B1 R
mRNA after TBI by RT-PCR as described in the Materials and Methods. Gene expression data were normalized to b-actin expression. Band
intensity is shown as means ± SEM of data from three independent animal experiments (three biological replicates) with similar results. In each
experiment, hearts from four mice were pooled for RNA isolation per group. *P , 0.05 compared to Sham group,$P , 0.05 compared to TBI
group, **P , 0.05 compared to L-ArgzTBI groups. Panel G: Cardiac expression of kinin B1 R, iNOS, IL-6, IL-1b, TNF-a, T-bet and b-actin
mRNA at 4 and 24 h. Lanes 1 to 5 are Sham, TBI, L-ArgzTBI, TBIzL-Arg and L-Arg, respectively. When there was not a basal level of
expression, the bands are not shown. The PCR band shown is representative of the three independent animal experiments with similar results.
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heart rate in beats per min, RR interval in ms, ST amplitude in mV
and QRS duration in ms. The software used a derivative-based QRS
detection algorithm to calculate the heart rate by detecting the peaks
of the R waves automatically.
Preparation for Heart Histology
Light microscopy analysis of the left ventricle of heart tissue was
performed on tissue slices fixed in neutral buffered formalin,
embedded in paraffin, sectioned at 5
hematoxylin and eosin. Stained tissue sections were examined
microscopically at 10003 magnification and recorded by a Carl-
Zeiss microscope attached to a Nikon Camera.
mm and stained with
Assay for Serum Markers for Cardiac Injury
Activity of creatine kinase (CK) and its isoenzyme CK-MB was
assayed in serum using a commercially available colorimetric kit
(Agappe Diagnostics, Mumbai) following the manufacturer’s proto-
col. The serum LDH levels were assayed using commercially available
colorimetric kits (Ecoline R; Merck Limited, Mumbai) following the
manufacturer’s protocol. Briefly, 50 ml of serum was added to 1000 ml
of reaction solution (4:1). The resultant reaction mixture was
incubated at 37uC for 5 min and the absorbance of the solution was
read at 340 nm.
Immunohistochemical Staining for T Cells and Macrophages in
Cardiac Tissue
For immunostaining, hearts from treated animals were fixed in
neutral buffered formalin, embedded in paraffin, sectioned at 5 mm
and stained for CD3 T cells or CD14 macrophages as described
previously (13). Briefly, sections were deparaffinized, rehydrated
using xylene and graded series of ethanol and distilled water,
permeabilized and stained with PE-labeled mouse anti-CD3 (BD
Biosciences) for the presence of T cells or with FITC-labeled mouse
anti-CD14 (BD Biosciences) for the presence of macrophages. Section
were then analyzed using an LSM510 confocal microscope (Carl
Zeiss, Jena GmbH, Germany) with a krypton-argon and He-Ne laser
coupled to an Orthoplan Zeiss photomicroscope.
Statistical Analyses
Values are expressed as means ± SEM unless otherwise stated.
Data from all the experiments were analyzed using one-way ANOVA
followed by post-hoc analyses using the Scheffe test. P , 0.05 was
considered to be statistically significant.
RESULTS
L-Arg Mitigates Radiation-Induced Splenic Inflammatory
Cytokine Response
Mouse spleens in the TBI and L-ArgzTBI groups had
significantly increased expression of TNF-a and T-bet
compared to the Sham group at 4 and 24 h (Fig. 1A, B,
E). In contrast, mouse spleens in the TBIzL-Arg group
had significantly reduced expression of both TNF-a and
T-bet compared to the TBI and L-ArgzTBI groups.
Mice in the TBIzL-Arg group had significantly
increased expression of TGF-b and GATA-3 compared
to the TBI and L-ArgzTBI groups at 24 h (Fig. 1C, D,
E). Mice in the L-Arg group had significantly increased
expression of T-bet, TGF-b and GATA-3 in their
spleens. Expression of these genes at the mRNA level
are shown in Fig. 1E. The expression of IL-4, IL-10 and
IL-6 was found to be absent in all five groups at 4 and
24 h (data not shown).
L-Arg Mitigates Radiation-Induced Heart Inflammatory
Cytokine Response
Mouse hearts in the TBI and L-ArgzTBI groups had
significantly increased expression of TNF-a, IL-6 and T-
bet compared to the Sham group at 4 and 24 h (Fig. 2A,
C, D, G). Mice in the TBIzL-Arg group had significantly
decreasedexpressionofTNF-a,IL-6andT-betcompared
to the TBI and L-ArgzTBI groups. Expression of IL-1b
inmousehearts was foundtobeelevatedat4h onlyinthe
TBI and L-Arg zTBI groups compared to the Sham
group and was significantly reduced in the TBIzL-Arg
group compared to the TBI and L-ArgzTBI groups
(Fig. 2B, G). IL-4, IL-10, GATA-3 and TGF-b were not
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FIG. 2. Continued
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expressed in any of the groups at 4 and 24 h (data not
shown).
L-Arg Mitigates Radiation-Induced Cardiac iNOS and
Kinin B1 R Expression
Mouse hearts in the TBI and L-ArgzTBI groups had
significantlyincreasedexpression ofiNOSandkininB1R
compared to the Sham group (Fig. 2E, F, G). Mice in the
TBIzL-Arg group had significantly decreased expression
of iNOS and kinin B1 R compared to the TBI and
L-ArgzTBI groups in their hearts. The expression of
these genes at the mRNA level is shown in Fig. 2G.
L-Arg Mitigates Radiation-Induced Bradycardia
The anesthetized mice were assessed at 4 and 24 h
after TBI for ECG patterns (Fig. 3). The mice exposed
to radiation showed significant bradycardia and an
increased RR interval (Fig. 4A, B). Bradycardia in mice
in the L-ArgzTBI group increased significantly com-
pared to mice from either the Sham and TBI groups.
Mice in the TBIzL-Arg group had significantly reduced
bradycardia and RR interval compared to the TBI and
L-ArgzTBI groups.
L-Arg Mitigates Radiation-Induced ST Elevation
The mice exposed to radiation showed ST elevation
(Fig. 4C). The ST elevation in L-ArgzTBI mice was
significantly higher compared to mice in the Sham
and TBI groups. Mice in the TBIzL-Arg group had
significantly decreased ST amplitude compared to the
TBI and L-ArgzTBI groups.
L-Arg Mitigates Radiation-Induced Increase in
QRS Duration
The TBI group showed increased QRS duration
(Fig. 4D). The QRS duration in L-ArgzTBI mice was
significantly higher compared to the Sham group. Mice
in the TBIzL-Arg group had a significantly reduced
QRS duration compared to either TBI and L-ArgzTBI
groups.
Heart Histology, Immunostaining and Serum Markers for
Cardiac Injury
The heart histology did not show any significant
myocardial structure disorder or necrosis (Fig. 5). To
assess the presence of inflammatory cells in the cardiac
tissue of treated animals, we used double immunofluo-
rescence staining and confocal analysis. It was found
that compared to the control (Sham) group, the
irradiated groups did not show fluorescence of either
CD3 (red) or CD14 (green), demonstrating the absence
of T cells or macrophages in the cardiac tissue at both 4
and 24 h (Supplementary Figs. 1 and 2, respectively;
http:dx.doi.org/10.1667/RR2523.1.S1). The serum levels
of LDH, CK and CK-MB were studied as markers for
cardiac injury and were not significantly different any of
the four groups compared to the Sham group (data not
shown).
DISCUSSION
The aim of these studies was to look at the changes in
cardiac electrical activity and the gene expression of
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r
FIG. 3. The ECG profile of mice 4 and 24 h after TBI. The profile is representative of four independent animal experiments with similar results.
In each experiment, the ECG profile of four mice per group was recorded. The ECG profile shown is the averaged recording obtained during the
analysis with Chart 5 Pro software for 20 min of data acquisition after 60 min of normalization. Panels A and E: Sham; panels B and F: TBI;
panels C and G: L-ArgzTBI; panels D and H: TBIzL-Arg; panels I and J: L-Arg.
FIG. 3. Continued
RADIATION-INDUCED INFLAMMATION AND CARDIAC DYSFUNCTION
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inflammatory mediators 4 and 24 h after TBI. The
results from the current experiments indicated that
mice in the TBI and L-ArgzTBI groups had marked
bradycardia, increased RR interval, ST elevation,
increased QRS duration and increased expression of
inflammatory markers in cardiac and splenic tissue.
L-Arg administered 2 h after TBI (TBIzL-Arg group)
shifted the entire cardiac and inflammatory parameters
toward normal. These studies correlate with our earlier
studies that suggested the ability of L-Arg to reverse the
radiation-induced immune dysfunction when adminis-
tered after TBI (2). Our results also suggested that
there was no structural damage in the heart tissue of
irradiated mice, as indicated by histology, immunohis-
tochemical staining for T cells and macrophages, and
markers for cardiac injury in serum. Alterations in the
ECG profile suggest that there are functional alterations
in the heart tissue. Since the ECG evaluates the electrical
activity of the heart our results suggest that TBI causes
significant changes in the electrical activity of the
heart. These new data helped us in designing an anti-
inflammatory approach to mitigate radiation-induced
cardiac dysfunction, based on the earlier studies, which
demonstrated the ability of L-Arg administered postir-
radiation in reversing radiation-induced inflammation
and immune dysfunction (2). Supplemental dietary
arginine given after abdominal irradiation accelerated
intestinal mucosal regeneration and enhanced bacterial
clearance after radiation enteritis in rats (14). However,
when L-Arg was fed to rats before exposing the abdomen
to radiation, significantly increased damage to various
segments of intestine was seen compared to rats on other
feeding regimens (15). These observations are line with
our findings and suggest the importance of the correct
time window for L-Arg administration (2). Our studies
indicated that L-Arg administration prior to radiation
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FIG. 4. Means ± SEM of (panel A) heart rate (beats/min), (panel B) RR interval (ms), (panel C) ST height (mV), and (panel D) QRS duration
(ms) in mice 24 h after TBI. Data are from one of the four independent animal experiments with similar results. In each experiment, data from
four mice per group were assessed. Analysis was done using Chart 5 Pro software for 20 min of data acquisition after 60 min of normalization.
*P , 0.05 compared to Sham group,$P , 0.05 compared to TBI group, **P , 0.05 compared to L-ArgzTBI group.
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exposure accelerated the production iNOS, which
contributes to increased NO production and worsens
the response of radiation injury. L-Arg administered
after radiation exposure significantly reduced the levels
of iNOS with a concomitant increase in the levels of
arginase, indicating the importance of the time of
administration of L-Arg in shifting the arginine pathway
away from production of excess NO. The studies offer a
mechanistic explanation by demonstrating the impor-
tance of the time of L-Arg administration in modulating
the L-Arg pathway away from excess production of NO
in the therapy of ARS. In the current studies, L-Arg
administered just before TBI (L-ArgzTBI group)
caused further ECG changes compared to TBI alone,
again supporting the earlier work on the importance of
the time of administration of L-Arg and the role of
arginine pathways in altering the course of radiation
injury (2).
Results from cancer patients treated with radiation
indicate that radiation-induced heart disease is a long-
term complication of radiation exposure (3–5). Mani-
festations of radiation-induced heart disease include
accelerated atherosclerosis, pericardial and myocardial
fibrosis, conduction abnormalities and injury to cardiac
valves (16). Release of proinflammatory cytokines like
TNF-a, IL-1b from macrophages and monocytes
aggravate the radiation-induced inflammatory cascade.
Activation of these cytokine cascades play a role in the
development of late radiation effects (17). Our studies
demonstrate that changes occurred in ECG and gene
expression of inflammatory mediators within 24 h after
TBI, which has not been reported before. Hence halting
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FIG. 5. Hematoxylin and eosin staining of left ventricle of heart. Data shown are representative of one of the two independent experiments.
No morphological changes in heart histology were found among the groups. All the images were taken at 10003 magnification.
RADIATION-INDUCED INFLAMMATION AND CARDIAC DYSFUNCTION
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the inflammatory cascade at an early stage after
irradiation would be an approach to treat the develop-
ment of radiation-induced heart disease. Therefore,
treatment of mice with L-Arg after TBI, which prevents
the expression of early inflammatory mediators induced
by radiation, could contribute to preventing the
development of long-term complications of radiation.
Radiation-induced inflammatory responses and their
potential side effects on the host have been demonstrat-
ed (2, 18). The differentiation of naı ¨ve T-helper (Th)
cells to Th1 or Th2 cells is regulated by the transcription
factors T-bet and GATA-3, respectively. T-bet/GATA-3
ratio has been used as a measure of the Th1/Th2
cytokine profile in mixed cell populations (19). The T-
bet/GATA-3 ratio in the spleen at 24 after TBI increased
to 5.1 in the TBI group compared to the Sham group,
increased further to 6.25 in the L-Arg z TBI group, and
decreased to 0.25 in the TBIzL-Arg group. The
decreased T-bet/GATA-3 ratio and Th1/Th2 ratio in
the spleens of mice in the TBIzL-Arg group suggest the
reversal of the inflammation. Increased TNF-a and T-
bet in the spleen and heart in the L-ArgzTBI group
correlated with depressed cardiac function. These
decreased inflammatory markers in the spleen and
cardiac tissue from the TBIzL-Arg group correlated
with the reversal of the cardiac dysfunction. The cascade
of cytokines and the corresponding transcription factors
produced immediately after irradiation contributed to
the course of radiation injury. Our studies suggest that
therapy to treat the cardiac dysfunction should aim to
lower the levels of TNF-a and T-bet. These results
suggest that for immediate reversal of the injury, the
Th1/Th2 ratio should be in favor of Th2 cytokines. Our
findings are in agreement with our previous studies of
acute injuries like acute radiation syndrome, heatstroke
and sepsis, which suggest the need to shift the L-Arg
pathway away from excess NO production (2, 6–8) to
initiate host recovery from the injury. The work
demonstrated that exogenous administration of L-Arg
at 2 h after TBI induces host homeostasis, which may be
critical in establishing conditions suitable for an
organism to respond to radiation injury. The ability of
L-Arg to upregulate both the transcription factors T-bet
and GATA-3 and the cytokine TGF-b in the spleen
(Fig. 1) correlates with our earlier studies of the
increased splenic proliferation with the administration
of L-Arg (2). In this way, immunomodulator like L-Arg
administered at the right dose and time may mitigate the
radiation-induced immune dysfunction.
Kinin B1 R is one of the most profoundly regulated G-
protein-coupled receptors and is not expressed in normal
physiology but is induced during inflammation (6, 20, 21).
Normal functioning of kinin B1 R is important for
mammalian heart physiology (21). Kinin B1 R activation
plays a significant role in the progression of inflammatory
cardiovascular disease, and kinin B1 R-antagonists has
beneficial therapeutic effects in halting the progression of
inflammatory cardiovascular disease (20, 23). Ours is the
first report of the role of kinin B1 R in radiation-induced
cardiac dysfunction. Since L-Arg has been shown to
downregulate the expression of kinin B1 R in heatstroke
(6), we tested the ability of L-Arg to suppress the radiation-
induced expression of cardiac B1 kinin R. The results
convincingly demonstrate the ability of L-Arg to suppress
radiation-induced expression of kinin B1 R and bring the
cardiac function of the irradiated host toward normalcy.
Excessive production of NO induced by radiation is
involvedinradiationinjury(2,24).NOplaysacentralrole
in cardiovascular regulation (21). Excessive NO produced
by iNOS contributes to profound cellular disturbances
leading to heart failure (25, 26). NO produced by iNOS is
sympathoinhibitory and is known to induce hypotension
andbradycardia(27).TheincreasediNOSgeneexpression
in cardiac tissue and bradycardia in mice in the TBI group
supports this observation. In the current work, mice given
L-Arg before TBI had pronounced bradycardia with a
concomitant increase in iNOS gene expression. The
decrease in iNOS gene expression in the group of mice
given L-Arg at 2 h after TBI correlates with the reversal
of bradycardia. Induction of Th1 cytokines TNF-a and
IL-1b is associated with an increase in iNOS activity (28,
29). Enhanced NO generation from iNOS was thought to
be responsible for marked cardiac dysfunction as seen in
most inflammatory heart diseases (30, 31). Increased
cardiac iNOS contributes to depressed myocardial con-
tractility and beta-adrenergic responsiveness in heart
failure rats (30). Selective iNOS blockage improved the
cardiac function. In the current work, L-Arg administered
at 2 h after TBI significantly reduced cardiac iNOS, kinin
B1 R and inflammatory cytokines. These results suggest
that regulation of pro-inflammatory molecules and
control of NO release by myocardial iNOS by the
administration of L-Arg at the correct therapeutic window
may be a good therapeutic strategy for the treatment of
cardiac diseases.
In the present study, radiation-induced expression of
inflammatory molecules, kinin B1 R and iNOS in the
heart was accompanied by altered electrical activity of
the heart. Earlier studies in rats did not find statistically
significant differences in the ECGs monitored 24 h after
15 Gy irradiation of the heart (32). However, in our
studies we observed significant ECG changes at 4 h after
exposure to 2 Gy that persisted until 24 h after TBI. This
suggests that alterations in ECGs caused by radiation
are due to complex interactions of central nervous
system, the immune system and other systemic respons-
es. In general, ST segment elevation reflects myocardial
injury (33). There was no significant increase in the levels
of CK, CK-MB and LDH in serum (data not shown)
and structural alterations in the heart histology. The
results suggest that immediate alteration of the electric
property of the heart and not the functional expression
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of the damage is responsible for the changed ECG
profile. Changes observed in QRS duration indicate
either nonspecific intraventricular conduction delays or
ectopic rhythms originating in the ventricles due to the
disturbances in pacemaker rhythm, which is a clear
indication of altered cardiac excitability (34, 35).
Our current studies along with earlier studies demon-
strate that L-Arg when administered at the correct time
can reverse both cardiac and immune dysfunction (2).
However, it is not always necessary that gene expression
reflect the changes at protein or enzyme levels. Further
confirmation of these data is required at the protein or
enzyme level. These results could form the basis of
detailed studies in mice regarding the long-term effects
of radiation on cardiac function, inflammation of the
host and its modulation by L-Arg. Exogenous adminis-
tration of L-Arg after TBI induces host homeostasis,
which may be critical in establishing conditions in which
an organism can respond to radiation injury. The
pathophysiological mechanisms of these associations
remain to be elucidated. This may have implications
for military and civilian triage where L-Arg can be
considered to mitigate the performance decrement
caused by exposure to sublethal doses of ionizing
radiation, (36, 37). Radiation-induced ECG changes
can potentially be used as a noninvasive and inexpensive
diagnostic and prognostic indicator of radiation injury
along with other established biomarkers to assess
radiation exposure and to identify potential biological
response modifiers that are capable of reversing
radiation injury. Because L-Arg is a well-characterized
least toxic amino acid whose pharmacodynamics is well
characterized, it can easily enter clinical trials.
ACKNOWLEDGMENTS
The project was completely funded by Bhabha Atomic Research
Centre, Government of India. The authors sincerely acknowledge Mr.
Mr. Kashinath Munankar and Narendra S. Sidnalkar for their useful
technical assistance.
Received: December 7, 2011; accepted: March 28, 2011; published
online:
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