Original Article
A Novel Interaction Between Sympathetic Overactivity
and Aberrant Regulation of Renin by miR-181a in BPH/2J
Genetically Hypertensive Mice
Kristy L. Jackson, Francine Z. Marques, Anna M.D. Watson, Kesia Palma-Rigo,
Thu-Phuc Nguyen-Huu, Brian J. Morris, Fadi J. Charchar, Pamela J. Davern, Geoffrey A. Head
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Abstract—Genetically hypertensive mice (BPH/2J) are hypertensive because of an exaggerated contribution of the sympathetic
nervous system to blood pressure. We hypothesize that an additional contribution to elevated blood pressure is via sympathetically
mediated activation of the intrarenal renin–angiotensin system. Our aim was to determine the contribution of the renin–
angiotensin system and sympathetic nervous system to hypertension in BPH/2J mice. BPH/2J and normotensive BPN/3J mice
were preimplanted with radiotelemetry devices to measure blood pressure. Depressor responses to ganglion blocker pentolinium
(5 mg/kg IP) in mice pretreated with the angiotensin-converting enzyme inhibitor enalaprilat (1.5 mg/kg IP) revealed a 2-fold
greater sympathetic contribution to blood pressure in BPH/2J mice during the active and inactive period. However, the depressor
response to enalaprilat was 4-fold greater in BPH/2J compared with BPN/3J mice, but only during the active period (P=0.01).
This was associated with 1.6-fold higher renal renin messenger RNA (mRNA; P=0.02) and 0.8-fold lower abundance of microRNA-181a (P=0.03), identified previously as regulating human renin mRNA. Renin mRNA levels correlated positively with
depressor responses to pentolinium (r=0.99; P=0.001), and BPH/2J mice had greater renal sympathetic innervation density as
identified by tyrosine hydroxylase staining of cortical tubules. Although there is a major sympathetic contribution to hypertension
in BPH/2J mice, the renin–angiotensin system also contributes, doing so to a greater extent during the active period and less
during the inactive period. This is the opposite of the normal renin–angiotensin system circadian pattern. We suggest that renal
hyperinnervation and enhanced sympathetically induced renin synthesis mediated by lower micro-RNA-181a contributes to
hypertension in BPH/2J mice. (Hypertension. 2013;62:00-00.) Online Data Supplement
•
Key Words: hypertension
■
■ kidney ■ microRNAs ■ renin–angiotensin system
sympathetic nervous system
B
PH/2J mice are a genetic model of hypertension developed by Schlager1 by crossing 8 normotensive strains
and selecting for elevated blood pressure (BP). Normotensive
BPN/3J control mice were bred concurrently by crossing
randomly selected mice from the same base population.
Recently, the mechanism of the hypertension has been recognized as neurogenic because ganglion blockade abolished
the hypertension in BPH/2J mice.2 Furthermore, spectral
analysis of BP revealed greater power in the autonomic frequency band, suggesting overactivity of the sympathetic nervous system (SNS), most prominently during the nocturnal
active period.2 BPH/2J mice also display exaggerated day–
night differences in BP, which are associated with greater
neuronal activity in regions of the hypothalamus and amygdala known to be important for cardiovascular regulation.2
Given the recent success of renal sympathetic nerve ablation
for the treatment of resistant hypertension,3 the importance of
renal influences on the expression of neurogenic hypertension
has been highlighted. Importantly, the peripheral renin–angiotensin system (RAS) is closely linked to renal sympathetic
nerve activity (RSNA) via its ability to stimulate renin secretion,4 and also through angiotensin II–mediated facilitation of
SNA.5 However, the interaction of the kidney and renal RAS
with SNS-mediated hypertension in BPH/2J mice has not
been investigated thoroughly. The role of the RAS has been
examined in a variety of ways in BPH/2J mice including by
measurement of messenger RNA (mRNA) in tissues and various pharmacological assessments.6–10 Iwao et al8 reported normal renin activity in plasma, kidney, and submandibular gland
of BPH/2J mice, although others found greater renin activity
Received May 15, 2013; first decision May 31, 2013; revision accepted July 9, 2013.
From the Neuropharmacology Laboratory (K.L.J., K.P.-R., T.-P.N.-H., P.J.D., G.A.H.), and Diabetes Complications Division - Diabetes and Kidney
Disease (A.M.D.W), Baker IDI Heart and Diabetes Research Institute, Melbourne, Victoria, Australia; Department of Pharmacology, Monash University
Melbourne, Victoria, Australia (K.L.J., G.A.H.); School of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia (F.Z.M., F.J.C.); and Basic
& Clinical Genomics Laboratory, School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia (B.J.M).
P.J.D. and G.A.H. contributed equally to this work as senior authors.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
113.01701/-/DC1.
Correspondence to Geoffrey A. Head, Neuropharmacology Laboratory, Baker IDI Heart and Diabetes Research Institute, P.O. Box 6492, St Kilda Rd
Central, Melbourne, Victoria 8008, Australia. E-mail [email protected]
© 2013 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.113.01701
1
2 Hypertension October 2013
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in the submandibular gland of BPH/2J mice7 and 1.3-fold
higher renal expression of angiotensin-converting enzyme
(ACE) mRNA compared with BPN/3J mice.6 Furthermore,
chronic angiotensin II type 1 (AT1) receptor blockade led to
comparable BP reductions in both BPH/2J and BPN/3J mice,
suggesting that the hypertension is independent of the RAS.9
However, chronic ACE inhibition caused an 8% greater hypotensive response in BPH/2J compared with BPN/3J mice.10
Thus, on the basis of these contrasting findings, it is unclear
whether the RAS contributes to hypertension in BPH/2J mice
or not. High-dose chronic losartan and chronic ACE inhibition
with captopril are capable of inhibiting both the peripheral
and the central RAS.11,12 Thus, a distinct contribution from the
peripheral RAS to BPH/2J hypertension is unclear.
The aim of the present study was to determine whether the
peripheral RAS contributes to the elevation in BP in hypertensive BPH/2J mice, either independently or through interactions
with the SNS. We addressed this using radiotelemetry to determine the relative BP effect of pharmacological inhibition of
the RAS and SNS or both. To delineate the contribution of the
RAS and SNS to hypertension in BPH/2J mice as opposed to
the contribution to normal BP maintenance, direct comparisons
were made with normotensive BPN/3J mice. Although there
may be some physiological differences between these 2 strains
that are independent of BP, the advantage of directly assessing the effect on BP of inhibiting each system in both strains
is that the contribution to hypertension can be examined. The
ACE inhibitor enalaprilat was used to determine the contribution of the peripheral RAS because enalaprilat does not readily
cross the blood brain barrier in the acute setting.13 Furthermore,
because renin is rate limiting in the RAS, Ren1 mRNA concentration was assessed as a measure of renal RAS activation.
Renin mRNA was used to reflect the state of renin production
and hence dynamic contribution to BP within the 12-hour periods rather than measurement of renal renin protein or its surrogate, renin enzyme activity, which more closely reflect renin
storage levels.14 We also measured the micro-RNA (miRNA)
miR-181a because its human homolog has been shown to
negatively regulate human renin mRNA and is reduced in the
kidney in human hypertension.15 Tyrosine hydroxylase (TH)
staining, a marker of sympathetic innervation,16 was used to
quantify renal innervation.
Methods
Animals
Cardiovascular experiments were performed with age-matched normotensive BPN/3J (n=7–13) and hypertensive BPH/2J (n=7–11)
male adult mice with an average age of 17 weeks. Experiments
were approved by the Alfred Medical Research Education Precinct
Animal Ethics Committee and conducted in accordance with the
Australian Code of Practice for Scientific Use of Animals, in line
with international standards.
Cardiovascular Measurements
BP telemetry transmitters (model TA11PA-C10; Data Sciences
International, St Paul, MN) were implanted as detailed in the onlineonly Data Supplement. Ten days after surgery, recordings of systolic
arterial pressure, diastolic arterial pressure, calculated mean arterial
pressure (MAP), heart rate, and locomotor activity were obtained in
freely moving mice in their home cage. During the light (inactive)
period and during the dark (active) period, cardiovascular parameters
were measured for 30 minutes before and for 30 minutes after intraperitoneal injections of pentolinium (5 mg/kg; Sigma-Aldrich), and
enalaprilat (1 mg/kg; Merck & Co). In addition, pentolinium (5 mg/kg)
was administered 30 minutes after pretreatment with enalaprilat
(1 mg/kg). The response to pharmacological treatment was represented by the difference between the control periods compared with
20 to 30 minutes after treatment.
Measurement of Renin mRNA and miR-181a Levels
in the Kidney
Ren1 mRNA and miRNA-181a abundance were measured in BPH/2J
and BPN/3J mouse kidneys collected during the dark (active) period
(n=6 per group) and light (inactive) period (n=3–4 per group; for further details see Methods in the online-only Data Supplement).
Kidney TH Staining
TH staining was performed on kidney sections from BPH/2J (n=4)
and BPN/3J (n=4) mice and the percentage of TH staining in the cortical tubules was semiquantitatively assessed (for further details see
Methods in the online-only Data Supplement)
Statistical Analysis
Data were expressed as mean or mean change±SEM and analyzed
by ANOVA (further details see Methods in the online-only Data
Supplement). A P value of <0.05 was considered significant.
Results
Baseline Cardiovascular Measurements
Average 24-hour MAP and heart rate were higher in BPH/2J
mice (n=10), compared with BPN/3J mice (n=11; Pstrain<0.001;
Figure S1 in the online-only Data Supplement). During the
dark (active) period BPH/2J mice had 27% higher MAP
(Pstrain<0.001). During the light (inactive) period, MAP in BPH/2
mice was 17% higher than in BPN/3J mice (Pstrain=0.001).
Effect of Pentolinium During the Dark Period
Treatment with pentolinium alone reduced MAP in both
BPH/2J (n=7; –39±4 mm Hg; P<0.001) and BPN/3J mice
(n=7; –28±3 mm Hg; P<0.001). The BP reduction was 39%
greater in the BPH/2J strain (Pstrain=0.04; Figure 1A).
Effect of Pentolinium During the Light Period
Pentolinium treatment induced depressor responses in BPN/3J
(–11±1 mm Hg; P<0.001; n=10) and BPH/2J mice (–11±4
mm Hg; P=0.001; n=8), which were comparable between
strains (Pstrain=0.9; Figure 1B).
Effect of Pentolinium After Enalaprilat
Pretreatment During the Dark Period
Administration of pentolinium after enalaprilat treatment produced
56% greater depressor responses in BPH/2J (–58±4 mm Hg; n=8)
than in BPN/3J mice (–37±4 mm Hg; n=9; PStrain<0.001; Figure 1A).
Enalaprilat pretreatment augmented the response to pentolinium
by 33% in BPH/2J mice and 49% in BPN/3J mice (Ptreatment<0.001),
and although there was an effect of strain (Pstrain<0.001), there was
no strain-by-treatment interaction (Pinteraction=0.3).
Effect of Pentolinium After Enalaprilat
Pretreatment During the Light Period
The depressor response induced by pentolinium after
enalaprilat was 53% greater in BPH/2J (–50±3 mm Hg;
Jackson et al Neural and Renin Contribution to Hypertension 3
n=7) compared with BPN/3J mice (–33±3 mm Hg; n=9;
PStrain<0.001; Figure 1B). Enalaprilat pretreatment augmented the depressor response to pentolinium by 3-fold in
BPN/3J and 4.7-fold in BPH/2J mice (Ptreatment<0.001), and
although there was an effect of strain (Pstrain=0.009), there
was no strain-by-treatment interaction (Pinteraction=0.1).
Effect of Enalaprilat During the Dark Period
After treatment with enalaprilat, MAP decreased in BPH/2J
(–11±2 mm Hg; P<0.001; n=9) but not in BPN/3J mice (–3±2
mm Hg; P=0.1; n=8; Pstrain=0.004; Figure 1C).
Effect of Enalaprilat During the Light Period
Enalaprilat treatment elevated MAP in BPH/2J (+10±4
mm Hg; P=0.004; n=7) but not BPN/3J mice (1±3
mm Hg; Pstrain=0.05; n=9; Figure 1D). Compared with the
response to vehicle, there were marked effects of enalaprilat treatment (Ptreatment<0.001), but no effect of strain
(Pstrain=0.2), and there was a strain-by-treatment interaction (Pinteraction=0.04).
Effect of Vehicle During the Dark Period
Administration of vehicle elevated MAP in BPH/2J (5±2
mm Hg; P=0.02; n=7) but not in BPN/3J mice (0±1 mm Hg;
P=0.9; n=9; Figure 1C). However, changes in MAP were similar between strains (Pstrain=0.1).
Effect of Vehicle During the Light Period
After vehicle treatment, MAP was elevated in BPN/3J (18±2
mm Hg; P<0.001; n=11) and BPH/2J mice (17±4 mm Hg;
P<0.001; n=8) to a similar extent in each strain (Pstrain=0.8;
Figure 1D).
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Figure 1. Mean arterial pressure (MAP) response to administration of agents in BPN/3J (gray) and BPH/2J (black) mice. A, Response induced
by pentolinium (left) and pentolinium after enalaprilat pretreatment (right) during the dark period and during the light period (B). C, Response
induced by saline (left) and enalaprilat (right) during the dark period and during the light period (D). Each point represents the mean value
averaged across a 5-minute period. The dashed vertical reference line represents the time-point of administration of treatment. Shaded area
represents the period analyzed for comparison of the effect of treatment. Bar graphs represent average changes in MAP in response to agents
in BPN/3J (N) and BPH/2J mice (H). Squares on the far right indicate effect of treatment (T), strain (S), and treatment by strain interaction (T×S).
Bar graphs values are mean±SEM. Significance refers to between-strain difference in response is shown as *P<0.05, **P<0.01, ***P<0.001.
4 Hypertension October 2013
Contribution of SNS and RAS to BP in BPH/2J
and BPN/3J Mice
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The relative contributions of the RAS and SNS to BP during
the inactive and active periods were calculated using the differences among the pentolinium, enalaprilat, and combination
treatments as follows. The basal, that is, RAS and SNS independent, level of BP was taken as the BP reached after pentolinium and enalaprilat. To take into consideration that injections
involved disturbing the conscious mice, which itself induced
mild increases in BP, the responses to drugs were compared relative with the effect of vehicle injection. The contribution of the
RAS was taken as the difference between the BP achieved after
enalaprilat relative to the BP reached after vehicle. The SNS
contribution was taken as the remaining difference between
this basal value and the level of BP observed after enalaprilat
(Figure 2). The calculated contribution of the SNS to BP was
1.7-fold greater in BPH/2J mice compared with BPN/3J mice
during both the inactive period (45 versus 26 mm Hg) and the
active period (55 versus 33 mm Hg). The contribution of the
RAS was calculated to be 2-fold greater in BPH/2J than BPN/3J
mice during the active period (19 versus 9 mm Hg) and 0.6-fold
during the inactive period (8 versus 14 mm Hg; Figure 2).
Ren1 mRNA and miR-181a Levels in the Kidney
Renal Ren1 mRNA in BPH/2J mice was 55% higher than in
BPN/3J mice during the active period (P=0.01) and was 42%
higher when compared with BPH/2J kidneys collected during
the inactive period (P=0.07). In contrast, in BPN/3J mice, renal
Ren1 mRNA levels were not significantly lower in the inactive period (P=0.6) and there was no difference between strains
during the inactive period (P=0.4; Figure 3A). Renal miR-181a
was 33% lower during the active period in BPH/2J compared
with BPN/3J mice (P=0.04). Furthermore, renal miR-181a in
BPH/2J mice during the active period was 53% lower than
during the inactive period (P=0.005), although miR-181a in
BPN/3J mice was comparable during the active and inactive
periods (P=0.2). Moreover, miR-181a levels were comparable
between strains during the inactive period (P=0.9; Figure 3B).
Correlations
There was a negative correlation between Ren1 and miR-181a
values for each animal (r=–0.52; P=0.04; Figure 4A). The
mean inactive and active depressor response to pentolinium in
each strain exhibited a strong positive correlation with Ren1
(r=0.99; P=0.001; Figure 4B) and a trend toward a negative
correlation with miR-181a (r=–0.85; P=0.07; Figure 4C). The
level of renal miR-181a showed a negative correlation with
resting MAP (r=–0.92; P=0.04; Figure 4D).
TH Staining
The percentage of TH staining in kidney tubules was greater in
BPH/2J (26±2%; n=4) compared with BPN/3J mice (19±1%;
n=4; P=0.03; Figure 5).
Discussion
Our study found that during the active period, when the hypertension is greatest in BPH/2J mice, acute ganglion blockade and
ACE inhibition each produced greater falls in BP. This indicated
greater contributions to BP in BPH/2J mice from both the SNS
and the peripheral RAS. In contrast, during the inactive period
when hypertension in BPH/2J mice is least evident, there remains
a greater contribution from the SNS, as determined by ganglionic blockade, but only a minimal contribution from the RAS.
Importantly, the greater contribution of the RAS during the active
period was associated with greater renal Ren1 mRNA expression
and lower levels of renal miR-181a, a negative regulator of renin
mRNA. We also observed a 1.4-fold greater abundance of renal
sympathetic nerve fibers in BPH/2J mice, as identified by greater
TH staining. Taken together, these findings suggest that although
the SNS is a major contributor to hypertension in BPH/2J mice,
the RAS also contributes to the BP elevation, doing so more during
the dark (predominantly awake) period and less during the light
(predominantly asleep) period. This is the opposite of the circadian
A
B
Figure 2. Renin–angiotensin system (RAS) and sympathetic
nervous system (SNS) independent contribution to blood
pressure (BP; black) and the contributions of the SNS (light
gray) and RAS (dark gray) to mean arterial pressure (MAP)
maintenance in BPN/3J and BPH/2J mice during the inactive
period (left) or active period (right). Numbers represent mm Hg
contribution to BP, whereas (×) represents fold difference in
BPH/2J mice compared with BPN/3J mice.
Figure 3. Renal Ren1 mRNA (A) and renal miR-181a (B) of
BPN/3J (gray) and BPH/2J (black) during the inactive period
(left) and active period (right). Bar graphs represent mean±SEM.
Between-strain difference shown as *P<0.05.
Jackson et al Neural and Renin Contribution to Hypertension 5
Figure 4. Correlations between renal miR-181a
and renal Ren1 mRNA (A), depressor response
to pentolinium (Δ mm Hg) and renal Ren1 mRNA
(B), depressor response to pentolinium (Δ mm Hg)
and renal miR-181a (C), baseline MAP (mm Hg)
and renal miR-181a (D). Data displayed from the
inactive period (◯) and active period (● ) in BPN/3J
(gray) and BPH/2J mice (black).
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pattern of normotensive rodents and humans, where the contribution of the RAS to BP is lower during the awake period and greater
during sleep.17,18 The reversed renin pattern is a likely additional
factor besides the overall greater SNS activity in contributing in
part to the hypertension in BPH/2J mice. The mechanism could
possibly involve renal hyperinnervation, as well as enhanced sympathetically induced renin synthesis mediated by a diminution in
miR-181a, a negative posttranscriptional regulator of Ren1 mRNA.
Contribution of SNS to Hypertension
in BPH/2J Mice
The present study confirms our previous finding that the SNS
drives the BP elevation in BPH/2J mice during the active
period.2 During the inactive period, the effect of ganglion blockade alone suggests comparable contributions of the SNS to BP
Figure 5. A, Micrograph showing tyrosine hydroxylase (TH)
staining (dark brown) in cortical tubules (×200 magnification;
scale bar, 50 µm) of BPN/3J (top) and BPH/2J mice (bottom).
B, Percentage area (mean±SEM) of the image positively stained
for TH in BPN/3J (gray) and BPH/2J (black) mice. Between-strain
difference *P=0.03.
maintenance between strains. However, ganglion blockade with
prior ACE inhibition revealed compensatory effects mediated
by the peripheral RAS which mask the full contribution of the
SNS to BP maintenance. Importantly, the unmasked contribution of the SNS to BP maintenance is consistently 1.7× greater
in BPH/2J compared with BPN/3J mice independent of the circadian period (dark/light), suggesting an elevated tonic sympathetic drive. In support, prior analysis of BP variability has
indicated that vasomotor SNA is elevated in BPH/2J mice.2 The
present study is also the first to demonstrate that renal sympathetic innervation is greater in BPH/2J compared with BPN/3J
mice, as indicated by TH staining in kidneys.
Contribution of RAS to Hypertension
in BPH/2J Mice
Our finding that the peripheral RAS contributed to 44% of the BP
elevation in BPH/2J mice during the dark (predominantly awake)
period, but made little contribution during the light (predominantly
asleep) period, is opposite to other findings which show peripheral
RAS activity peaks during the sleep period and decreases during
the awake period.17,18 This overactivity of the RAS in BPH/2J
mice during the active period suggests that there is not an inherent
tonic overactivity of the peripheral RAS, but more likely an abnormal regulation of the RAS during the active period. The greater
depressor response to enalaprilat is unlikely to merely reflect a
greater sensitivity of BPH/2J mice to RAS inhibition because
AT1 receptor inhibition has previously showed no discernable
difference in sensitivity of BPN/3J and BPH/2J mice when the
AT1 receptor inhibitor is administered at threshold and maximal
doses.9 Although ACE inhibition can influence BP through bradykinin accumulation, this is unlikely to be contributing to the
greater depressor response to enalaprilat in BPH/2J mice because
the vasodilatory response to bradykinin is reportedly reduced in
BPH/2J compared with BPN/3J mice.19 The greater depressor
response to enalaprilat was not the only indication of an elevated
6 Hypertension October 2013
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contribution of the peripheral RAS to BP in the active period. The
pattern was accompanied by a greater abundance of Ren1 mRNA
in the kidneys of BPH/2J mice during the active period, consistent
with a role for kidney-derived renin production in driving the elevated RAS activity. Plasma renin activity is normally elevated during sleep and reduced during the awake period.18 Although renal
Ren1 mRNA abundance was comparable between strains during
the inactive period, levels were elevated during the active period
in BPH/2J mice. This finding provides an explanation for the lack
of differential expression of Ren1 mRNA in a prior transcriptomewide array study6 and also for the similar renin expression demonstrated previously in the kidney of BPH/2J and BPN/3J mice.8
The greater contribution of the RAS in BPH/2J mice is consistent with the slightly greater hypotensive effect of chronic ACE
inhibition with captopril in BPH/2J mice.10 However, the present
findings contrast our previous finding of a similar contribution of
the RAS to 24-hour BP in BPH/2J and BPN/3J mice.9 The discrepancy could be explained by the exclusion of a contribution
from the central angiotensin system in the present study, although
this might suggest that the contribution of the central angiotensin
system to BP could also be aberrant in BPH/2J mice.
What Is the Association Between Overactivity of
RAS and SNS?
We noticed a remarkably strong relationship between renal Ren1
and SNS activity as determined by depressor responses to ganglion blockade. Although this correlation does not necessarily
indicate a direct or causal association, it does suggest some relationship. Furthermore in light of the elevated TH staining and
greater RAS contribution in BPH/2J mice, it is possible that this
relationship could reflect RSNA-driven overactivity of the RAS
or even potentiation of SNA by the RAS. Although the peripheral RAS is capable of facilitating SNS activity,5 this is unlikely
to be the case in BPH/2J mice because SNS overactivity, as
measured by the depressor response to ganglion blockade, was
apparent with and without ACE inhibition. Thus, SNS overactivity seems to be independent of RAS activity. The association
could also reflect greater RSNA-mediated renin release and subsequent natriuretic and vasoconstrictor effects. Although there is
a disparity between the apparently tonic overactivity of the SNS
and the state-dependent RAS overactivity in BPH/2J mice, this
may reflect circadian changes in regional SNA. Indeed RSNA is
greater during activities, such as exercise and grooming, compared with sleep,20,21 whereas vasomotor sympathetic activity as
measured by analysis of BP variability is greater during activity and lower during sleep.22 Renal denervation would be useful
to help validate the contribution of the RSNA to hypertension.
One would expect that it would cause a substantial reduction
in the RAS-mediated contribution to hypertension during the
dark (awake) period in BPH/2J mice. However, given the lack
of influence of the RAS on hypertension during the light period
(predominantly asleep), the influence of renal denervation would
be expected to be less profound at this time.
Is Reduced miR-181a Mediating the Ren1
Overexpression and Hypertension?
The importance of miR-181a as a potential novel mediator of
the BP elevation in hypertension has been largely unrecognized
until recently.15 That miR-181a is likely to be a negative regulator
of Ren1 mRNA expression in mice is supported by its strong
negative correlation with Ren1 mRNA level and by the ability of
human miR-181a to downregulate human renin mRNA in transfection experiments.15 Marques et al15 showed that hypertensive
individuals have marked underexpression of miR-181a in the
kidney, accompanied by marked overexpression of renin mRNA.
Such a pattern was seen in BPH/2J mice in our study. Expression
of miR-181a in monocytes has since been reported to be negatively correlated with systolic BP in obese subjects.23 Indeed our
present finding of a negative correlation between renal miR-181a
and BP supports a role for this micro-RNA in BP regulation in
mice. Production of miR-181a during the inactive period is comparable in BPH/2J and BPN/3J mice, but its underproduction in
the active period in BPH/2J mice indicates that a negative control
mechanism is switched on at this time. Downregulation of miR181a in BPH/2J, but not in BPN/3J mice, during the active period
suggests factor(s) unique to the BPH/2J strain during the active
period. Given the strong positive association between SNA and
Ren1, a possible negative regulator of miR-181a might be SNA,
either directly or indirectly. Although there was a negative correlation between miR-181a and response to pentolinium, this did
not quite reach statistical significance. Nonetheless, regardless of
how miR-181a is regulated in BPH/2J mice, given its ability to
regulate Ren1 mRNA, synthetic miR-181a mimics could represent novel therapeutics in the treatment of hypertension.
Limitations
The present study shows strain differences in Ren1 mRNA, TH
staining, and miR-181a and clear correlations among miR181a,
Ren1, and BP. Although these strain differences and correlations
do not necessarily indicate causal relationships with BP or hypertension, in the context of the pharmacological findings, these factors add support to the hypothesis that sympathetically mediated
activation of the intrarenal RAS is contributing to hypertension in
BPH/2J mice. Importantly though, further assessment to verify a
contribution to hypertension is necessary. As such these findings
are likely to motivate further interventional studies, such as renal
denervation and administration of miR-181a mimetics to confirm
an influence on hypertension in vivo. Another point to consider
is that the present findings represent the contribution of the SNS
and RAS to establish hypertension because tail-cuff measurements indicate hypertension is present in BPH/2J mice from as
young as 7 weeks of age.24 Similar pharmacological assessment
in very young BPH/2J mice would be useful to determine the
influence of the RAS and SNS during hypertension development,
but obtaining radiotelemetric BP measurements in such young
mice would be technically challenging.
Conclusion and Perspectives
Hypertension in BPH/2J mice involves tonic overactivity of the
SNS and elevated renal sympathetic innervation. During the sleep
and awake states, the elevated sympathetic drive is likely manifested differently by circadian-related changes in regional sympathetic activity. During the active state, the elevation in sympathetic
activity likely drives RSNA-induced renin production, which is
reflected by the elevated contribution of the peripheral RAS to
hypertension in BPH/2J mice at this time. However, underexpression of the negative regulator of Ren1 mRNA, miR-181a, may also
be responsible for elevation of Ren1 expression and hypertension.
Jackson et al Neural and Renin Contribution to Hypertension 7
There are significant implications for our present findings in a
mouse model of hypertension that has renal hyperinnervation and
enhanced sympathetically induced renin synthesis. Hypertensive
BPH/2J mice may prove to be an excellent animal model to
investigate the mechanism mediating the hypotensive effect of
renal sympathetic nerve ablation observed in treatment-resistant
patients with hypertension.3 Furthermore, given the present study
is the first to identify a mouse model of hypertension with aberrant renal miR-181a, akin to that seen in patients with essential
hypertension, BPH/2J mice could serve as a unique animal model
for the investigation of miR-181a–mediated BP regulation which
could lead to novel therapeutic targets for hypertension.
Sources of Funding
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
This work was supported by grants from the National Health and
Medical Research Council of Australia (NHMRC; project grant
526662), the Diabetes Australia Research Trust and the University of
Ballarat Self-sustaining Regions Research and Innovation Initiative,
an Australian Government Collaborative Research Network, and in
part by the Victorian Government’s OIS Program.
Disclosures
Investigators were supported by NHMRC and National Heart
Foundation (NHF) Postdoctoral Fellowships (NHMRC fellowship 1012881 and NHF fellowship PF10M5334 to P.J. Davern;
NHMRC fellowship 1052659 and NHF fellowship PF12M6785 to
F.Z. Marques), a NHMRC Principal Research Fellowship (1002186
to G.A. Head), and the Australian Diabetes Society Skip Martin
Fellowship (to A.M.D. Watson). The other authors report no conflicts.
References
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1974;76:537–549.
2. Davern PJ, Nguyen-Huu TP, La Greca L, Abdelkader A, Head GA. Role
of the sympathetic nervous system in Schlager genetically hypertensive
mice. Hypertension. 2009;54:852–859.
3. Esler MD, Krum H, Sobotka PA, Schlaich MP, Schmieder RE, Bohm M.
Renal sympathetic denervation in patients with treatment-resistant hypertension (The Symplicity HTN-2 Trial): a randomised controlled trial.
Lancet. 2010;376:1903–1909.
4. Zanchetti A, Stella A. Neural control of renin release. Clin Sci Mol Med
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5. Xiang JZ, Linz W, Becker H, Ganten D, Lang RE, Schölkens B, Unger
T. Effects of converting enzyme inhibitors: ramipril and enalapril on peptide action and sympathetic neurotransmission in the isolated heart. Eur J
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6. Puig O, Wang IM, Cheng P, Zhou P, Roy S, Cully D, Peters M, Benita Y,
Thompson J, Cai TQ. Transcriptome profiling and network analysis of
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7. Uddin M, Harris-Nelson N. Renin activity and angiotensin I concentration
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8.Iwao H, Nakamura N, Kim S, Ikemoto F, Yamamoto K, Schlager G.
Renin-angiotensin system in genetically hypertensive mice. Jpn Circ J.
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9. Palma-Rigo K, Jackson KL, Davern PJ, Nguyen-Huu TP, Elghozi JL,
Head GA. Renin-angiotensin and sympathetic nervous system contribution to high blood pressure in Schlager mice. J Hypertens. 2011;29:
2156–2166.
10. Leckie BJ. The action of salt and captopril on blood pressure in mice with
genetic hypertension. J Hypertens. 2001;19:1607–1613.
11. Wang JM, Tan J, Leenen FH. Central nervous system blockade by peripheral administration of AT1 receptor blockers. J Cardiovasc Pharmacol.
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12. Geppetti P, Spillantini MG, Frilli S, Pietrini U, Fanciullacci M, Sicuteri
F. Acute oral captopril inhibits angiotensin converting enzyme activity in
human cerebrospinal fluid. J Hypertens. 1987;5:151–154.
13. Cohen ML, Kurz KD. Angiotensin converting enzyme inhibition in tissues from spontaneously hypertensive rats after treatment with captopril
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Hypertension. 2007;50:933–938.
15. Marques FZ, Campain AE, Tomaszewski M, Zukowska-Szczechowska
E, Yang YH, Charchar FJ, Morris BJ. Gene expression profiling reveals
renin mRNA overexpression in human hypertensive kidneys and a role for
microRNAs. Hypertension. 2011;58:1093–1098.
16. Burgi K, Cavalleri MT, Alves AS, Britto LR, Antunes VR, Michelini LC.
Tyrosine hydroxylase immunoreactivity as indicator of sympathetic activity: simultaneous evaluation in different tissues of hypertensive rats. Am J
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efficacy of antihypertensive agents in spontaneously hypertensive rats.
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18. Brandenberger G, Follenius M, Goichot B, Saini J, Spiegel K, Ehrhart J,
Simon C. Twenty-four-hour profiles of plasma renin activity in relation to
the sleep-wake cycle. J Hypertens. 1994;12:277–283.
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of PAR-2 vasodilation despite endothelial dysfunction in BPH/2 hypertensive mice. Pflugers Arch. 2007;454:535–543.
20. Nagura S, Sakagami T, Kakiichi A, Yoshimoto M, Miki K. Acute shifts
in baroreflex control of renal sympathetic nerve activity induced by REM
sleep and grooming in rats. J Physiol. 2004;558(Pt 3):975–983.
21. Miki K, Kato M, Kajii S. Relationship between renal sympathetic nerve
activity and arterial pressure during REM sleep in rats. Am J Physiol
Regul Integr Comp Physiol. 2003;284:R467–R473.
22. Furlan R, Guzzetti S, Crivellaro W, Dassi S, Tinelli M, Baselli G, Cerutti
S, Lombardi F, Pagani M, Malliani A. Continuous 24-hour assessment of
the neural regulation of systemic arterial pressure and RR variabilities in
ambulant subjects. Circulation. 1990;81:537–547.
23.Hulsmans M, Sinnaeve P, Van der Schueren B, Mathieu C, Janssens
S, Holvoet P. Decreased miR-181a expression in monocytes of obese
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24. Schlager G, Sides J. Characterization of hypertensive and hypotensive
inbred strains of mice. Lab Anim Sci. 1997;47:288–292.
Novelty and Significance
What Is New?
• This is the first study to demonstrate that the BPH/2J mouse strain is a
mouse model of hypertension that has renal hyperinnervation and enhanced sympathetically induced renin synthesis, which seems to contribute to the hypertension, and that this is likely to be mediated by lower
levels of the microRNA, miR-181a.
What Is Relevant?
• Importantly, this is the first study to describe aberrant renal expression
of miR-181a in a hypertensive mouse model which is akin with that observed in patients with essential hypertension.
Summary
Our findings suggest that renal hyperinnervation and enhanced
sympathetically induced renin synthesis, mediated by lower levels
of the microRNA, miR-181a, are together responsible for the hypertension in BPH/2J mice.
A Novel Interaction Between Sympathetic Overactivity and Aberrant Regulation of Renin
by miR-181a in BPH/2J Genetically Hypertensive Mice
Kristy L. Jackson, Francine Z. Marques, Anna M.D. Watson, Kesia Palma-Rigo, Thu-Phuc
Nguyen-Huu, Brian J. Morris, Fadi J. Charchar, Pamela J. Davern and Geoffrey A. Head
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
Hypertension. published online July 29, 2013;
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Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
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Online Supplement
A Novel Interaction Between Sympathetic Overactivity and Aberrant
Regulation of Renin by miR-181a in BPH/2J Genetically Hypertensive
Mice
Kristy L. Jackson, Francine Z. Marques, Anna M. D. Watson, Kesia Palma Rigo, Thu-Phuc
Nguyen-Huu, Brian J. Morris, Fadi J. Charchar, Pamela J. Davern, Geoffrey A. Head
Short title: Neural and Renin Contribution to Hypertension
Footnote on title page:
From the Baker IDI Heart and Diabetes Research Institute, Melbourne (K.L.J., A.M.D.W.,
K.P.R., T.-P. N.-H., P.J.D., G.A.H.); Department of Pharmacology, Monash University
Melbourne (K.L.J., G.A.H.); School of Health Sciences, University of Ballarat, Ballarat,
(F.Z.M., F.J.C.), Victoria; Basic & Clinical Genomics Laboratory, School of Medical
Sciences and Bosch Institute, University of Sydney, Sydney, New South Wales (B.J.M),
Australia
P.J.D. and G.A.H. are co-senior authors.
Correspondence to Geoffrey A. Head, Neuropharmacology Laboratory, Baker IDI Heart and
Diabetes Research Institute, P.O. Box 6492, St Kilda Road Central, Melbourne, Victoria
8008, Australia. E-mail: [email protected]
Tel: +61-3-8532-1330 Fax: +61-3-8532-1100
Supplement Materials and Methods
Animals
The genetically hypertensive BPH/2J (n=24) and normotensive BPN/3J (n=27) male mice
used in the present study came from inbred colonies bred at the Alfred Medical Research and
Education Precinct Animal Centre (Generation 15-20) from breeders purchased at generation
20-36 from Jackson laboratories. The original breeding selection program, took place in the
1970’s for at least 23 generations and then brother sister mating followed to create these
inbred strains 1.
Animals in the present study were housed in individual cages in a room with a 12:12 hour
light-dark cycle (1am–1pm light/day) with ad libitum access to water and mouse chow
(Specialty Feeds, Glen Forrest, Western Australia; 19% protein, 5% fat, 5% fibre, 0.2%
sodium).
Telemetry transmitter implantation
Blood pressure (BP) telemetry transmitters (model TA11PA-C10; Data Sciences
International, St Paul, MN) were implanted under isoflurane open circuit anesthesia (5%
induction and 1.5-2% maintenance) (Forthane, Abbott, Botany, NSW, Australia) delivered
via oxygen. Carprofen (5mg/kg)(Rimadyl, Pfizer Australia Pty Ltd, West Ryde, NSW,
Australia) was administered subcutaneously just prior to surgery and 24 hours post-surgery
for analgesia. A lateral incision and blunt dissection were used to expose the left carotid
artery which was temporarily occluded using a non-absorbable silk tie (Dysilk 1-0, Dynek
Pty Ltd, SA, Australia). The catheter of the telemetry device was inserted into the carotid
artery and secured using silk ties and the body of the probe was positioned subcutaneously
along the right flank.2 A subcutaneous continuous stitch using an absorbable suture
(Polysorb, Covidien, Mansfield, MA) was used to close the incision. Mice were allowed at
least ten days recovery prior to BP measurement. Experiments were conducted in accordance
with the Australian Code of Practice for the Care and Use of Animals for Scientific purposes.
Cardiovascular measurements
Cardiovascular and locomotor recordings were sampled at 1000 Hz using an analog-to-digital
data acquisition card (National Instruments 6024E) as described previously.3 The beat-to-beat
arterial pressure and heart rate (HR) were detected on-line and analyzed later using a program
written in Labview.4
The cardiovascular effect of each drug or drug combination was assessed on separate days.
Baseline cardiovascular parameters were determined during the light (inactive) period or
during the dark (active) period at least 2 hours before or after lights off. The doses of drug
administered in the present study were based on those reported to elicit depressor effects.5-7
Drugs were dissolved in isotonic 0.9% saline (vehicle) and freshly prepared each day.
Statistical Analysis
All cardiovascular data were analysed by a multi-factor, split-plot analysis of variance
(ANOVA).8 A combined residual was used that pooled the between- and within-animal
variance as described previously.9 For cardiovascular responses to pharmacological
treatments the between-groups sums of squares was partitioned into main effects of
treatment, strain (BPH/2J and BPN/3J), and their interaction (treatment x strain). Renal TH
staining and RNA data were presented as mean ± SEM and Student’s t-test was used to
compare between-strains differences. A P value of <0.05 was considered significant.
2 Measurement of renin mRNA and miR-181a levels in the kidney
Collection of the tissues at different times was as described previously.10 Briefly, age
matched adult BPH/2J and BPN/3J mice (n=6/group) were killed with an overdose (100
mg/kg) of pentobarbitone (Lethobarb, Virbac Animal Health, NSW, Australia) during the
dark (active) period at the peak of the circadian variation in BP, 2 hours after lights out, when
average MAP difference between the strains is maximal (30 mmHg).11 Hypertensive BPH/2J
(n=3) and age-matched BPN/2J mice (n=4) were killed in the same way during the light
(inactive) period (2 hours after lights on) when the MAP levels of the BPH/2J and BPN/3J
mice differ by only 16 mmHg.11 TRIzol® reagent (Life Technologies, Mulgrave, Australia)
was used for RNA extraction according to the manufacturer’s recommendations.
Complementary DNA synthesis was performed using the High Capacity cDNA Reverse
Transcription Kit for total cDNA and the TaqMan® MicroRNA Reverse Transcription Kit for
miRNA cDNA (Life Technologies). Amplification reactions used the TaqMan® Fast
Advanced Master Mix (Life Technologies). TaqMan probes were used for gene expression to
assess Ren1 mRNA (assay Mm02342887, Life Technologies) and mature miRNA miR-181a
levels (assay 000480, Life Technologies), together with reference genes. Samples were run in
duplicates. A quantitative real-time PCR (qPCR) system (model ViiA™ 7 qPCR, Life
Technologies) and the ∆∆CT method were used to determine the levels of Ren1 mRNA and
miR-181a.
Kidney tyrosine hydroxylase (TH) staining
Hypertensive BPH/2J (n=4) and normotensive BPN/3J mice (n=4), were anesthetized deeply
with an i.p. injection of 100 mg/kg pentobarbitone (Lethobarb, Virbac Animal Health) in the
active period, 2 hours after lights out. Mice were perfused with 20 ml of 0.9% saline then 60
ml of 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) dissolved in 0.1 mol/L
phosphate buffer (PB), pH 7.2. Kidneys (n=4/group) were cryopreserved in 20% sucrose
overnight before embedding in paraffin. Four micrometer sections were processed as
described previously;12 briefly endogenous peroxidase was blocked (10 minutes, 3%
H2O2/TRIS-buffered saline (TBS)), sections were blocked in 10% normal horse serum/TBS,
followed by endogenous avidin-biotin blocking (Avidin-Biotin blocking kit, Vector
Laboratories, Burlingame, CA). Sections were incubated in rabbit anti-TH (Millipore
Australia, North Ryde, NSW, Australia) overnight, then incubated with biotinylated antirabbit (Vector) and visualized with 3,3′-diaminobenzidine tetrahydrochloride/H2O2 (DAB;
Sigma-Aldrich, St Louis, MO). The percentage of TH staining in cortical tubules was semiquantitatively assessed with 10 images per animal captured under identical light/exposure
(Olympus BX-50, Olympus Optical; Q-imaging MicroPublisher 3.3 RTV camera, Surrey,
BC, Canada). The percentage area of the image that stained positively was assessed in a
blinded manner as described previously12 (Image Pro-Plus 6.0 software; Media Cybernetics,
Silver Spring, MD) based on red, green and blue channels.
3 References
1.
Schlager G, Sides J. Characterization of hypertensive and hypotensive inbred strains
of mice. Lab Anim Sci, 1997;47:288-292.
2.
Butz GM, Davisson RL. Long-term telemetric measurement of cardiovascular
parameters in awake mice: a physiological genomics tool. Physiol Genomics,
2001;5:89-97.
3.
Jackson K, Head GA, Morris BJ, Chin-Dusting J, Jones E, La Greca L, Mayorov DN.
Reduced cardiovascular reactivity to stress but not feeding in renin enhancer knockout
mice. Am J Hypertens, 2007;20:893-899.
4.
Head GA, Lukoshkova EV, Burke SL, Malpas SC, Lambert EA, Janssen BJ.
Comparing spectral and invasive estimates of baroreflex gain. IEEE Eng Med Biol
Mag, 2001;20:43-52.
5.
Palma-Rigo K, Jackson KL, Davern PJ, Nguyen-Huu T-P, Elghozi J-L, Head GA.
Renin-angiotensin and sympathetic nervous system contribution to high blood
pressure in Schlager mice. Journal of hypertension, 2011;29:2156-2166.
6.
Tibirica E, Feldman J, Mermet C, Monassier L, Gonon F, Bousquet P. Selectivity of
rilmenidine for the nucleus reticularis lateralis, a ventrolateral medullary structure
containing imidazoline-preferring receptors. Eur J Pharmacol, 1991;209:213-221.
7.
Di Paola Eugenio D, Pietro G, Rosario M, De Sarro G. Effects of enalapril in rats with
pressure overload cardiac hypertrophy. Gen Pharmacol, 1997;28:531-533.
8.
Snedecor GW, Cochran WG. Statistical Methods. 7th ed. Ames, Iowa: Iowa State
University Press; 1980.
9.
Korner PI, Badoer E, Head GA. Cardiovascular role of the major noradrenergic cell
groups in the rabbit: analysis based on 6-hydroxydopamine-induced transmitter
release. Brain Res, 1987;435:258-272.
10.
Marques FZ, Campain AE, Davern PJ, Yang YHI, Head GA, Morris BJ. Genes
influencing circadian differences in blood pressure in hypertensive mice. PloS one,
2011;6:e19203 19201-19209.
11.
Davern PJ, Nguyen-Huu T, La Greca L, Head GA. Role of the sympathetic nervous
system in Schlager genetically hypertensive mice. Hypertension, 2009;54:852-859.
12.
Watson AM, Li J, Schumacher C, de Gasparo M, Feng B, Thomas MC, Allen TJ,
Cooper ME, Jandeleit-Dahm KA. The endothelin receptor antagonist avosentan
ameliorates nephropathy and atherosclerosis in diabetic apolipoprotein E knockout
mice. Diabetologia, 2010;53:192-203.
4 MAP (mmHg)
HR (b/min)
140
120
120
100
100
80
80
600
600
500
500
400
400
300
300
Activity (units)
140
***
**
3
3
2
2
1
1
0
0
0
4
8
12
16
20
24
Time of day (hrs)
***
*
**
N H
N H
Light
Dark
S1. Hourly averaged data showing the circadian variation of MAP (mmHg), HR (beats/min)
and activity (units) during the active (night) (outer panels) and inactive (day) (middle panel)
phases in BPN/3J (z; n=10) and BPH/2J mice (z; n=11). Bar graphs on right represent
average MAP, HR, and locomotor activity during the inactive (Day) and Active (Night)
periods in BPN/3J (N) and BPH/2J (H) mice.Values are mean±SEM. For comparisons
between strains across the entire 24 hours, *P<0.05; **P<0.01 and ***P<0.001.
5 MAP (mmHg)
∆ MAP 20-30 min
Pentolinium 5mg/kg
A 160
160
20
140
∆ MAP 20-30 min
Pentolinium 5 mg/kg
Enalaprilat 1 mg/kg
20
140
0
0
120
120
100
-20
80
-40
100
*
60
-60
80
-40
60
-60
***
∆ HR 20-30 min
HR (bpm)
∆ HR 20-30 min
100
800
800
100
600
-100
0
0
-100
600
*
-200
400
400
-300
-300
Activity (units)
∆ Act 20-30 min
2
8
0
6
BPN
BPH
4
2
BPN
BPH
0
4
-2
-4
-4
-6
-20
-10
0
Time (min)
10
20
-6
N H
30
-30
∆ MAP 20-30 min
Pentolinium 5mg/kg
MAP (mmHg)
B 160
140
160
20
-20
-10
0
Time (min)
Enalaprilat 1 mg/kg
10
20
∆ MAP 20-30 min
20
0
120
-20
100
80
-40
60
-60
-40
80
-60
60
∆ HR 20-30 min
100
HR (bpm)
800
600
800
100
600
-100
0
-100
-200
400
-400
∆ Act 20-30 min
2
8
BPN
6
0
4
T ***
S NS
TxS NS
-200
-300
BPH
***
∆ HR 20-30 min
0
400
Activity (units)
T ***
S **
TxS NS
-20
100
-300
-400
∆ Act 20-30 min
2
BPN
BPH
0
4
-2
-2
**
T ***
S ***
TxSNS
2
2
-4
-4
0
0
-6
-30
N H
30
Pentolinium 5 mg/kg
140
0
120
6
*
0
0
8
T NS
S **
TxS NS
-2
2
2
-30
**
-400
∆ Act 20-30 min
6
T ***
S ***
TxSNS
-200
-400
8
T ***
S ***
TxSNS
-20
-20
-10
Time0(min) 10
20
30
-6
N
H
-30
-20
-10
Time 0(min) 10
20
30
N
H
S2. Line graph represents 5-minute averages of MAP, HR, and locomotor activity before and
after administration of pentolinium (left) and pentolinium after enalaprilat pre-treatment
(right) between BPN/3J (gray) and BPH/2J (black) mice during A, the active period, and B,
the inactive period. Dashed vertical line represents time-point of administration. Shaded area
represents the response period analyzed. Bar graphs are mean response ± SEM of MAP, HR,
and locomotor activity to pentolinium (center) or pentolinium following enalaprilat pretreatment (right) for BPN/3J (N) and BPH/2J mice (H). Squares on the far right indicate
effect of treatment (T), strain (S) and treatment by strain interaction (T×S). Significance
refers to between-strain difference in response and is shown as *P<0.05; **P<0.01;
***P<0.001***P<0.001
6 ∆ MAP 20-30 min
Saline
MAP (mmHg)
A 160
∆ MAP 20-30 min
Enalaprilat 1.5 mg/kg
160
20
140
20
140
0
120
0
120
-20
-20
100
100
80
-40
60
-60
**
80
60
-60
800
100
600
-100
∆ HR 20-30 min
100
HR (bpm)
800
*
0
-200
400
Activity (units)
∆ HR 20-30 min
0
-100
600
8
6
400
-400
∆ Act 20-30 min
2
8
BPN
BPH
6
0
-300
-400
∆ Act 20-30 min
2
BPN
BPH
0
4
-2
-4
-4
0
0
-6
-20
-10
0
10
20
-6
N
30
H
-30
-20
-10
MAP (mmHg)
∆ MAP 20-30 min
Saline
B 160
140
0
10
20
Enalaprilat 1.5 mg/kg
160
20
140
0
120
0
80
60
-60
-40
80
200
800
60
-60
800
200
∆ HR 20-30 min
100
HR (bpm)
T ***
S NS
TxS
*
100
-40
∆ HR 20-30 min
100
0
600
0
600
-100
-200
400
-300
-300
-400
-400
∆ Act 20-30 min
∆ Act 20-30 min
2
8
0
6
BPN
BPH
4
T NS
S NS
TxS NS
-100
-200
400
Activity (units)
20
-20
100
2
BPN
BPH
0
4
-2
T NS
S NS
TxS NS
-2
2
2
-4
-4
0
0
-6
-30
H
∆ MAP 20-30 min
120
-20
6
N
30
Time (min)
Time (min)
8
T NS
S*
TxS NS
-2
2
2
-30
T ***
S NS
TxS*
-200
-300
4
T ***
S NS
TxS**
-40
-20
-10
0
10
Time (min)
20
30
-6
N H
-30
-20
-10
0
Time (min)
10
20
30
N H
S3. Line graph represents 5-minute averages of MAP, HR, and locomotor activity before and
after administration of vehicle (left) and enalaprilat (right) between BPN/3J (gray) and
BPH/2J (black) mice during A, the active period, and B, the inactive period. Dashed vertical
line represents time-point of administration. Shaded area represents the response period
analyzed. Bar graphs are mean response ± SEM of MAP, HR, and locomotor activity to
vehicle (center) or enalaprilat (right) for BPN/3J (N) and BPH/2J mice (H). Squares on the
far right indicate effect of treatment (T), strain (S) and treatment by strain interaction (T×S).
Significance refers to between-strain difference in response and is shown as *P<0.05;
**P<0.01; ***P<0.001
8