Nutrition, Metabolism & Cardiovascular Diseases (2014) xx, 1e8

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Nutrition, Metabolism & Cardiovascular Diseases

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Maternal low-protein diet induces changes in the cardiovascular

autonomic modulation in male rat offspring
M.A.V. Barros a, J.L. De Brito Alves a, V.O. Nogueira a, A.G. Wanderley b,
J.H. Costa-Silva a,*
a
Department of Physical Education and Sport Sciences, Academic Center of Vitoria (CAV), Federal University of Pernambuco, 55608-680, Brazil
b
Department of Physiology and Pharmacology, Federal University of Pernambuco, Brazil

Received 4 June 2014; received in revised form 16 July 2014; accepted 24 July 2014
Available online - - -

KEYWORDS Abstract Background and aims: Maternal undernutrition induces development of the arterial
Protein hypertension. We investigated the effects of a maternal low-protein diet on cardiovascular auto-
undernutrition; nomic control in the offspring.
Developmental Methods and results: Male Wistar rats were divided into two groups according to the diets of
plasticity; their mothers during gestation and lactation: the control (normal protein, NP, 17% casein;
Autonomic n Z 14) and low-protein (LP, 8% casein; n Z 14) groups. Direct measurements of arterial pressure
cardiovascular (AP) were recorded from wakeful 90-day-old male offspring. The LP offspring presented higher
control; mean AP than did the NP rats (NP: 93
4 vs. LP: 113
2 mmHg; p < 0.05), whereas the heart
Hypertension rate (HR) was similar in the two groups. In the spectral analysis, the LP group showed higher po-
wer at low (NP: 1.98
0.25 vs. LP: 3.7
0.3 mmHg2; p < 0.05) and high (NP: 1.28
0.18 vs. LP:
2.13
0.42 mmHg2; p < 0.05) frequencies of systolic arterial pressure (SAP). In the pulse interval,
the LP group presented an increase in the LF/HF ratio (NP: 0.32 vs. LP: 0.56; p < 0.05). After pro-
pranolol (4 mg/kg, intravenous (iv)), the bradycardia was higher in the LP group (NP: 36
8 vs.
LP: 94
12 bpm; p < 0.05), after methylatropine (2 mg/kg, iv), the tachycardia was similar to
NP group. After administration of the ganglionic blocker (hexamethonium; 25 mg/kg, iv), the LP
animals showed larger delta variation in the AP (NP: 33.7
5 vs. LP: 53.6
4 mmHg;
p < 0.05).
Conclusion: The rats subjected to protein malnutrition presented an increase in the cardiovascu-
lar sympathetic tone, which contributed to the elevated AP observed in these animals.
2014 Elsevier B.V. All rights reserved.

Introduction development of non-communicable diseases of the

Epidemiological and experimental studies have shown
that malnutrition that occurs during gestation and lacta-
tion is an important predisposing factor for the
* Corresponding author. Ncleo de Educao Fsica e Cincias do
Esporte e CAV e UFPE, Rua Alta do Reservatrio, S/N, Bela Vista, Vitria
de Santo Anto, PE, CEP 55608-680, Brazil. Tel./fax: 55 81 35233351.
E-mail addresses: joao.hcsilva@ufpe.br, joaohenriq@hotmail.com (J.
H. Costa-Silva).
offspring in adulthood [1e4]. In rats, studies have shown
that the consumption of a low-protein (LP) diet during the
pre- or postnatal periods induces changes in cardiovas-
cular function and predisposes the offspring to the
development of hypertension [5,6].
Although there are several studies showing the rela-
tionship between malnutrition in early life and hyperten-
sion in adulthood, the mechanisms involved in this
process are not yet well understood. We recently demon-
strated that protein restriction during gestation and

http://dx.doi.org/10.1016/j.numecd.2014.07.011
0939-4753/ 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
2 M.A.V. Barros et al.
lactation leads to increased arterial pressure (AP) after 90
days of life in the offspring, and this increase might be due
to changes in the respiratory system during early life. [2]
The role of cardiovascular autonomic modulation was not
considered or studied in that work, but it is has been
described as an important factor in the development of
other hypertension models [7,8].
AP control is a complex mixture of hormonal, neural
and intrinsic factors, which all act on different time scales
and with different means of control. The action of these
factors on the cardiovascular system promotes sponta-
neous oscillations, named a high-frequency (HF) oscilla-
tion related to respiration and associated with cardiac
vagal modulation and a low-frequency (LF) oscillation
related to cardiac and vasomotor sympathetic modulation
of the heart and blood vessels [9,10]. Experimental studies
have shown that spontaneously hypertensive rats (SHR)
exhibited an increase in the LF band of the AP and a high
LF/HF ratio, which could contribute to sympathetic over-
activity and the development of hypertension [11].
In the present study, we investigated long-term effects
of the maternal low-protein diet on cardiovascular auto-
nomic modulation of the adult male offspring. To under-
stand the importance of the autonomic nervous system in
the maintenance of AP, we hypothesised that rats from
dams subjected to protein undernutrition during gestation
and lactation could exhibit sympathetic overactivity,
which could lead to the development of arterial hyper-
tension in this experimental model.
Methods
Animals and diets
Wistar rats were used and all experimental protocols
were approved by the Ethical Committee of the Federal
University of Pernambuco, Brazil. Two groups were
formed based on dietary manipulations: mothers fed a
17% casein diet (normal-protein (NP) group, n Z 6) and
mothers fed an 8% casein isoenergetic diet (LP group,
n Z 6) ad libitum. During the suckling period, the
mothers continued to be provided with the experimental
diet of either 8% or 17% of casein. The diets were made
according to the American Institute of Nutrition [2]. At
weaning, the male offspring received a standard diet
(Labina; Purina Agriband Sao Paulo, Brazil) and water ad
libitum until they were 90 days old. The experimental
groups were formed with two or three rats from each
mother (NP offspring, n Z 14; LP offspring, n Z 14).
Measurement of arterial pressure and heart rate
Male Wistar rats at 90 days were anaesthetised with ke-
tamine (80 mg/kg) and xylazine (10 mg/kg) to insert
femoral artery and vein catheters. After 18e24 h, the AP
and heart rate (HR) were recorded in conscious animals by
appropriate system (LabChart 7 Pro; ADInstruments, Bella
Vista, NSW, Australia). Each animal was placed in the
recording chamber for a period of acclimatisation
Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
(approximately 60 min). The pulsatile AP was recorded for
60 min under basal conditions, and the values of the mean
arterial pressure (MAP) and HR were calculated by selec-
tion of the 10 min of this period.
Spectral and symbolic analyses of cardiovascular
variability
The cardiovascular autonomic evaluation was performed
using the frequency domain analysis of the systolic arterial
pressure (SAP) and pulse interval (PI) by an appropriate
software program (CardioSeries-v.2.4; www.
danielpenteado.com). The spectra were integrated in the
LF (0.2e0.75 Hz) and the HF bands (0.75e3 Hz). To assess
the sympathovagal index, the LF/HF ratio of the variability
was calculated. Moreover, the symbolic analysis was used,
a non-linear method based on the conversion of the series
into a sequence of symbols [12].
Evaluation of sympathovagal tonus and intrinsic HR
Methylatropine (2 mg/kg, iv) and propranolol (4 mg/kg,
iv) were used to evaluate the parasympathetic and sym-
pathetic tonus on HR, respectively. First, methylatropine
was injected and the HR recorded during the next 15 min,
then, the propranolol was injected, and the HR was
recorded over 15 min. On the next day, this sequence was
reverse. Only the rst value of each sequence was
considered. After each sequence, the intrinsic HR (iHR)
was calculated [13].
Evaluation of sympathetic tonus on the vascular system
The sympathetic vascular tone was evaluated by an
intravenous injection of hexamethonium (25 mg/kg,
Sigma, St Louis, MO, USA) [14] and calculated by difference
between the MAP after the blocker and the baseline MAP.
Spontaneous baroreex sensitivity
In this study, the spontaneous baroreex sensitivity was
calculated through sequence method by computer soft-
ware CardioSeries (v.2.4) [15].
Statistical analysis
The results were expressed as the mean
the standard
error of the mean and compared using Students unpaired
t-test, except for LF/HF ratio that was expressed as median
and compared using the ManneWhitney test. These tests
were performed after analysis of data distribution (Kol-
mogoroveSmirnov and ShapiroeWilk test). The compari-
sons were performed using GraphPad Prism software
(GraphPad, v.5), and differences were considered signi-
cant at p < 0.05.
Maternal low-protein diet induces changes in the cardiovascular autonomic modulation 3

Results
Haemodynamics
At 90 days, the male rats born to the LP diet dams
exhibited an increase in the baseline MAP (NP: 93
4 vs.
LP: 113
2 mmHg; p < 0.05; Fig. 1B), but similar baseline
HR compared with the NP group (NP: 371
6 vs. LP:
390
9 bpm; Fig. 1C).
of the PI (NP: 0.32 vs. LP: 0.56; p < 0.05; Fig. 2D). Similar
to the observations in the PI variability, in the HR vari-
ability evaluated by the symbolic analysis, the percentage
of occurrences of the patterns of 0V sequences was
signicantly higher (NP: 41.3
4.3 vs. LP: 53.4
3.2%;
p < 0.05), and the patterns and sequences 2V were
signicantly lower in the LP group compared with the NP
group (NP: 21.4
2.6 vs. LP: 14.3
1.8%; p < 0.05;
Fig. 2E).

Cardiovascular variability
LF oscillations of the SAP (NP: 1.98
0.25 vs. LP:
3.7
0.3 mmHg2; p < 0.05) and of the PI (NP: 24.5
3.2
vs. LP: 35.2
2.9 nu; p < 0.05) were increased in the LP
animals. Whereas, the HF components of the SAP were
higher (NP: 1.28
0.18 vs. LP: 2.13
0.42 mmHg2;
p < 0.05) and of the PI were smaller in the LP animals
compared with the NP animals (NP: 75.5
3.2 vs. LP:
63.6
3.7 nu; p < 0.05) (Fig. 2). Protein restriction during
the perinatal period elicited an increase in the LF/HF ratio
Sympathovagal tonus and intrinsic HR
Figure 3 shows that the NP and LP animals had a similar
vagal tone (NP: 70
8 vs. LP: 70
10 bpm; Fig. 3D), but,
the LP group presented a signicantly increased sympa-
thetic tone (NP: 36
8 vs. LP: 94
12 bpm; p < 0.05;
Fig. 3C). After the double pharmacological blockade, it was
observed that the LP animals had a lower iHR than the NP
animals (NP: 379
9 vs. LP: 349
11 bpm; p < 0.05;
Fig. 3E).

Figure 1 Baseline cardiovascular parameters. Representative recordings of the baseline pulsatile arterial pressure (PAP), mean arterial pressure
(MAP), heart rate (HR) (panel A) and its mean values, of the normoproteic (NP, black bars; n Z 14) and low-protein (LP, grey bars; n Z 14) (panel B
and C) groups. The values were expressed as the mean
SEM. (*) p < 0.05 compared with the NP group (unpaired Students t-test).

Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
4 M.A.V. Barros et al.

Figure 2 Cardiovascular variability. Representative spectra of the systolic arterial pressure (SAP). The low- and high-frequency bands of the
spectra are highlighted in white and grey, respectively (panel A). Spectral analysis of the SAP (panel B), the pulse interval (PI) (panel C), the LF/HF
ratio (panel D) and nonlinear heart rate variability (panel E) of the normoproteic (NP, black bars; n Z 14) and low-protein (LP, grey bars; n Z 14)
groups. The values were expressed as the mean
SEM in the B and C panels (unpaired Students t-test). In the D panel, the values were expressed as
median and compared by ManneWhitney test. (*) p < 0.05 compared with the NP group.

Sympathetic tonus impact on the vascular system Spontaneous baroreex sensitivity

After the administration of a ganglionic blocker, the delta
AP variation was larger in the LP group (NP: 33.7
5 vs.
LP: 53.6
4 mmHg; p < 0.05; Fig. 4B). After this block, in
the spectral analysis, also it was observed that the LP an-
imals had higher delta variation of the LF band in the SAP
(baseline LF subtracted from LF after blocking) compared
with the NP animals (NP: 1.42
0.38 vs. LP:
2.92
0.49 mmHg2; p < 0.05; Fig. 4C); for the PI, the range
was similar between the groups in all frequency bands
(Fig. 4D). A similar pattern in the percentage of the vari-
ation of the symbolic analysis between the LP and NP
groups was observed (Fig. 4E).
No difference was detected in the gain of the baroreex
measured by sequence analysis (NP: 0.86
0.06 vs. LP:
0.72
0.07), as shown in Fig. 5.
Discussion
The LP group presented mainly an increase in the cardio-
vascular sympathetic tone and increased LF bands of the
SAP and PI, suggesting sympathetic predominance on the
cardiovascular system of these animals. It is known that
the rhythmicity of the sympathetic nervous system can

Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
Maternal low-protein diet induces changes in the cardiovascular autonomic modulation 5

Figure 3 Sympatheticecardiac vagal tone and the intrinsic heart rate (iHR). Representative recordings of the cardiovascular parameters after
methylatropine (panel A) and propranolol (panel B). The sympathetic tone (DHR after propranolol; panel C), cardiac vagal tone (DHR after atropine;
panel D) and iHR (panel E) of the normoproteic (NP, black bars; n Z 14) and low-protein (LP, grey bars; n Z 14) groups. The values are expressed as
the mean
SEM. (*) p < 0.05 compared with the NP group (unpaired Students t-test).

Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
6 M.A.V. Barros et al.

Figure 4 Assessments after hexamethonium. Representative recordings of the pulsatile arterial pressure (PAP) and mean arterial pressure (MAP)
after hexamethonium (panel A). The delta change of the MAP (panel B), the spectral analysis of the SAP (panel C) and PI (panel D), and the symbolic
analysis (panel E) after hexamethonium of the normoproteic (NP, black bars; n Z 14) and low-protein (LP, grey bars; n Z 14) groups. The values are
expressed as the mean
SEM. (*) p < 0.05 compared with the NP groups (unpaired Students t-test).

modulate the AP and the PI (time between beats, RR) at
regular frequencies [16], and these rhythmic uctuations
in the cardiovascular variables suggests a measurement of
cardiovascular autonomic balance [17].
Accordingly, the LF oscillations of the SAP are typically
enhanced during states of sympathetic activation and can
provide an indirect measurement of sympathetic nerve
activity [18]. In a model of renovascular hypertension, an
increase in the LF component of the SAP was shown,
suggesting increased sympathetic activity to the periph-
eral vessels [19]. In this study, the LP animals showed an
increase in the LF component in the SAP spectrum,
suggesting a close relationship between a protein-
restricted diet during the perinatal period and a possible
increase in vasoconstrictor sympathetic activity of the
offspring. Regarding the HF component of the SAP, the LP
animals also showed an increase in this parameter when
compared with the NP animals, indicating that the AP of
these animals may be under a bigger respiratory modu-
lation, which might contribute to the increased AP in these
animal [2].
Moreover, the PI variability showed an increase in the
LF component, in this moment, accompanied by a decrease
in the HF component in the LP animals, resulting in a

Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
Maternal low-protein diet induces changes in the cardiovascular autonomic modulation
1.0
NP Group
0.8 LP Group
Gain baroreflex
(ms/mmHg)
0.6
0.4
0.2
0.0
Figure 5 Spontaneous baroreex sensitivity. Gain baroreex in 90-
day-old rats of the normoproteic (NP, black bars; n Z 14) and low-
protein (LP, grey bars; n Z 14) groups. The values are expressed as
the mean
SEM. (*) p < 0.05 compared with the NP group (unpaired
Students t-test).
bigger LF/HF ratio, without differences in the absolute
values of these components. Knowing that the LF and HF
components represent more accurately the autonomic
modulation when presented in normalised units or by
means of the LF/HF ratio [20], our data demonstrate an
imbalance in the cardiac autonomic control of these ani-
mals and suggests an increased activity of the sympathetic
nervous system associated with a decreased activity of the
parasympathetic nervous system. Thus, this sympathetic
predominance may be contributing to the increase in AP in
LP animals.
How the dynamics of the cardiovascular variables can
also exhibit nonlinear characteristics [21], we analysed the
behaviour of HR variability also by this point of view.
Through symbolic analysis, we observed that the LP group
showed an increase in the percentage of the 0V sequences,
which corresponds to sympathetic cardiac modulation,
and a decrease in the percentage of the 2V sequences,
which corresponds to parasympathetic cardiac modula-
tion. These data corroborate with the spectral analysis of PI
and reinforce the hypothesis that this group of animals
may present cardiac autonomic dysfunction.
In addition to these methods for estimating cardiovas-
cular autonomic modulation, we used experimental pro-
tocol involving the selective autonomic blockade. In this
evaluation, after antagonism of the b-adrenergic receptors,
LP animals showed higher cardiac sympathetic tone. After
the antagonism of the muscarinic receptors, no changes
were observed in the cardiac parasympathetic tone. This
change in the cardiac autonomic balance with predomi-
nance of the sympathetic over the parasympathetic tone
was observed in animal models of hypertension [19] and in
postnatal protein-restricted animals [22]. Another ana-
lysed variable was the iHR, considered after the dual
pharmacological blockade. In this study, a signicant
decrease in the iHR of the LP animals was observed. Ac-
cording to Machado (1989), iHR can be altered by changes
in the centrally mediated sympathetic and/or cardiac
parasympathetic ow. Furthermore, iHR contributes
signicantly to the basal HR and provides an important
compensatory mechanism to maintain normal HR during
an increase in the sympathetic ow or in a decrease in the
parasympathetic activity ow [23]. It is possible that the
Please cite this article in press as: Barros MAV, et al., Maternal low-protein diet induces changes in the cardiovascular autonomic
modulation in male rat offspring, Nutrition, Metabolism & Cardiovascular Diseases (2014), http://dx.doi.org/10.1016/
j.numecd.2014.07.011
7
iHR of the LP animals acts to offset the excess sympathetic
activity observed in these animals and contributes to the
maintenance of basal HR levels.
Concerning the evaluation of the contribution of the
sympathetic nervous system for the maintenance of AP by
infusion of hexamethonium, it was possible to observe
that the delta AP variation was larger in the LP group,
suggesting that sympathetic vascular activity in these an-
imals has an increased participation in the maintenance of
AP. Further this important fact, in the spectral analysis
after the ganglionic blocker, it also was possible to observe
a larger variation delta of the LF band in the SAP in the LP
animals. Thus, we conrmed the results obtained in the
spectral analysis of baseline AP and considered the
increasing uctuations in the LF band as an index of the
sympathetic modulation of the vascular tone. The results
of this study support our hypothesis that animals on a LP
diet during the perinatal period have an imbalance in
autonomic cardiovascular control at 90 days of life, with a
predominance of sympathetic nervous system activity that
might contribute to an increase in AP observed in these
animals.
To verify whether this sympathetic overactivity
observed in the LP animals could be secondary to a
decreased baroreex function, we evaluated the cardiac
baroreex gain in these animals. In this analysis, no dif-
ferences were found by the spontaneous baroreex
sensitivity between the groups, suggesting that the cardiac
baroreex control, at least within the narrow range of the
physiological variations of AP, is not altered in adult rats
that suffered malnutrition during pregnancy and lactation.
Different from our results, at studies with post weaning
malnutrition model has been found attenuation of the
baroreex gain [24] as well as alteration in the response
pattern to central baroreex stimulation [25]. However, it
is known that respiratory neurons located in the brainstem
can modulate the cardiovascular system and play an
important role in the modulation of the sympathetic ac-
tivity contributing to arterial hypertension the central
pathways [7]. Then, it is possible that the changes pro-
moted in cardiovascular autonomic control by protein
malnutrition in this study, as well as the changes in the
respiratory system observed previously [2], reect a sce-
nario of sympathetic overactivity and contribute to the
increased AP of the LP animals. Further, studies have
indicated that in some pathological conditions, this sym-
pathetic overactivity could be due to changes in the
excitability of the neurons in the rostral ventrolateral
medulla (RVLM), which are responsible for generate
sympathetic activity and are strongly modulated by glu-
tamatergic neurotransmission and the renineangiotensin
system [26e28]. In this way, studies involving maternal
protein malnutrition have suggested that their offspring
could present changes in gene expression of different and
important pathways associated with the
renineangiotensin system [29] and glutamatergic neuro-
transmission and metabolism [24,30]. Thus, one hypothe-
sis is that sympathetic overactivity observed in the LP
animals could be due to epigenetic modications in the
8 M.A.V. Barros et al.
signalling pathways involved in the control of the sym-
pathetic nervous system at the brainstem.
In conclusion, our data showed that the long-term ef-
fects induced by protein restriction during the perinatal
period include increased LF oscillations and increased
cardiac sympathetic tone, indicating an imbalance of
autonomic control and sympathetic overactivity. These
results strongly suggest that the predominant activity of
the sympathetic nervous system in the cardiac and
vascular systems might be a key contributor to the AP
increase observed in the adult LP rats.
Acknowledgements
We thank Fundao de Amparo a Cincia e Tecnologia de
Pernambuco (FACEPE, grant n 1365-2.07/10) and the
Conselho Nacional de Desenvolvimento Cientco e Tec-
nolgico (CNPq, grant n 484452/2011-8) for nancial
support.
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