Download Increased Efferent Cardiac Sympathetic Nerve Activity

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Diabetes Volume 64, August 2015
H.P. Aye Thaung,1 J. Chris Baldi,2 Heng-Yu Wang,1 Gillian Hughes,1 Rosalind F. Cook,1
Carol T. Bussey,1 Phil W. Sheard,1 Andrew Bahn,1 Peter P. Jones,1 Daryl O. Schwenke,1
and Regis R. Lamberts1
Increased Efferent Cardiac Sympathetic
Nerve Activity and Defective Intrinsic
Heart Rate Regulation in Type 2 Diabetes
PATHOPHYSIOLOGY
Diabetes 2015;64:2944–2956 | DOI: 10.2337/db14-0955
Elevated sympathetic nerve activity (SNA) coupled with
dysregulated b-adrenoceptor (b-AR) signaling is postulated as a major driving force for cardiac dysfunction in
patients with type 2 diabetes; however, cardiac SNA has
never been assessed directly in diabetes. Our aim was
to measure the sympathetic input to and the b-AR responsiveness of the heart in the type 2 diabetic heart. In
vivo recording of SNA of the left efferent cardiac sympathetic branch of the stellate ganglion in Zucker diabetic fatty rats revealed an elevated resting cardiac SNA
and doubled firing rate compared with nondiabetic rats.
Ex vivo, in isolated denervated hearts, the intrinsic
heart rate was markedly reduced. Contractile and
relaxation responses to b-AR stimulation with dobutamine were compromised in externally paced diabetic
hearts, but not in diabetic hearts allowed to regulate their
own heart rate. Protein levels of left ventricular b1-AR
and Gs (guanine nucleotide binding protein stimulatory)
were reduced, whereas left ventricular and right atrial
b2-AR and Gi (guanine nucleotide binding protein inhibitory regulatory) levels were increased. The elevated
resting cardiac SNA in type 2 diabetes, combined with
the reduced cardiac b-AR responsiveness, suggests
that the maintenance of normal cardiovascular function requires elevated cardiac sympathetic input to
compensate for changes in the intrinsic properties of
the diabetic heart.
Diabetes is a strong independent risk factor for the
development of cardiovascular complications and congestive heart failure (1–3). Cardiac autonomic dysfunction
(CAD) is an undervalued, but significant, cause of morbidity
and mortality in diabetes, most likely because CAD
1HeartOtago,
Department of Physiology, Otago School of Medical Sciences,
University of Otago, Dunedin, New Zealand
2Department of Medicine, Dunedin School of Medicine, University of Otago,
Dunedin, New Zealand
Corresponding author: Regis R. Lamberts, [email protected].
Received 20 June 2014 and accepted 9 March 2015.
pathology is poorly understood (4). Among the characteristics of CAD, diabetic patients have an elevated resting
heart rate (HR), but a paradoxically lower peak HR, which
may result from impaired cardiac sympathetic innervation
(4–6). Uptake of the sympathetic neurotransmitter norepinephrine (NE), using metaiodobenzylguanidine, is reduced in type 2 diabetic versus nondiabetic subjects who
have been screened for ischemic heart disease (7). Moreover, metaiodobenzylguanidine uptake is lower in type 2
diabetic patients with CAD than in those without CAD
(7). These data suggest that diabetes progressively reduces
sympathetic nervous input to the heart, limiting cardiac
reserve.
An alternative explanation is that sympathetic nervous
input is normal or elevated in diabetes, but that the
b-adrenoceptor (AR) responsiveness of the heart is reduced, a mechanism that has been well described in heart
failure (8,9). Isolated heart preparations from type 1 diabetic rat models describe a reduced inotropic (contraction), lusitropic (relaxation), and chronotropic (rate) b-AR
responsiveness (10–13), which is associated with the
downregulation of cardiac b1-ARs (11,14–16). Increased
cardiac NE content and spillover in type 1 diabetic rats
also suggest elevated sympathetic nervous activity (SNA)
in diabetes (17,18). Moreover, diabetes in humans is associated with augmented activation of SNA in other
organs, such as skeletal muscle (19,20). However, central
control of SNA to peripheral organs is differentially regulated (21–23). Importantly, obese humans with insulin
resistance show increased skeletal muscle SNA and renal
NE spillover, but reduced cardiac NE spillover (24).
Thus, although these studies suggest a key role for the
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
See accompanying article, p. 2708.
diabetes.diabetesjournals.org
sympathetic nervous system (SNS) in cardiac dysfunction
in diabetes, direct measurements of the sympathetic nervous input to the diabetic heart have not yet been reported.
Furthermore, it is unclear whether diabetic cardiac dysfunction results from changes in sympathetic nervous input to
the heart (cardiac SNA), a reduction in b-AR responsiveness
of the heart, or both. Identifying the underlying processes
of diabetic autonomic dysfunction is important, as the traditional treatment of patients with type 2 diabetes with
b-blockers is of benefit, although to a lesser extent than in
the treatment of nondiabetic patients (25), and it is even
more critical for the development of potential new therapeutic targets that interrupt myocardial autonomic signaling, such as b-ARKct (26), b3-AR agonists (27), or
renal denervation (28). This study uses direct in vivo
recordings of efferent cardiac SNA and b-AR responsiveness of isolated denervated hearts from Zucker type 2
diabetic fatty (ZDF) rats to establish whether type 2 diabetes 1) increases cardiac SNA and 2) reduces cardiac
b-AR responsiveness. Finally, we tested whether type 2
diabetes reduced the expression of b1- and b2-ARs and
their downstream signaling G-proteins.
RESEARCH DESIGN AND METHODS
Animals
All experiments were approved and conducted in accordance
with the guidelines of the Animal Ethics Committee of the
University of Otago, New Zealand. Experiments were conducted on 20-week-old male type 2 diabetic ZDF (fa/fa, n =
26) rats and their nondiabetic littermates (+/+, n = 26).
Recording of Cardiac SNA and b-AR Responsiveness
In Vivo
Recordings of cardiac SNA, arterial blood pressure (ABP),
and left ventricular (LV) cardiac function were performed
in 12 nondiabetic and 12 type 2 diabetic ZDF rats in vivo,
as previously described (29). In brief, animals were anesthetized with urethane (1.5 g/kg i.p.), intubated, and ventilated (tidal volume ;3.5 mL; breathing rate ;80 breaths/
min). A blood sample was taken to measure blood glucose
and insulin levels. A left thoracotomy was performed between the first and second rib, exposing the stellate ganglion. The cardiac sympathetic nerve was identified as
a branch from the stellate ganglion and was dissected free
of surrounding connective tissue. After cutting the nerve, the
proximal section was placed on a pair of platinum recording
electrodes to measure nerve activity. Consequently, the nerve
activity being measured was from the efferent sympathetic
nerves. The recorded signal was filtered (low cutoff 0.1 kHz;
high cutoff 1 kHz), amplified, and subsequently passed
through an amplitude discriminator to quantify nerve
discharge frequency (impulse frequency). The raw signal
was rectified and integrated (1-s resetting interval)
online, and the integrated nerve signal was displayed
in real time. At the end of the experiments, a postmortem nerve activity measurement was performed for
background subtraction of noise level, which was not
different between groups.
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Systemic ABP was measured through a femoral artery
cannula, and HR was derived from the arterial systolic
peaks. The right carotid artery was cannulated with a 1.5-F
Millar pressure-volume catheter (model SPR-869), which
was then advanced into the left ventricle for the
continuous measurement of LV pressure (LVP) and LV
volume, which combined provided LV end-diastolic pressure, LV end-systolic pressure, LV end-diastolic volume
(LVEDV), and LV end-systolic volume. From these
measurements, stroke volume (SV), cardiac output (CO),
maximum rate of contraction (+dP/dtmax), and maximum
rate of relaxation (2dP/dtmax) were derived. The Millar
pressure-volume catheter was calibrated using a sphygmomanometer (pressure), volumetric cuvettes (volume), and
hypertonic saline injections (for volumetric corrections),
as previously described in detail (30).
Baseline cardiac SNA was recorded, and administration
of dobutamine (b1-agonist; 0.6–10 mg/kg/min) tested the
in vivo cardiac response to b-AR stimulation.
Immunohistochemistry
After measuring the cardiac SNA, frozen sections of
isolated left sympathetic cardiac nerve of two nondiabetic and three diabetic ZDF rats that had been fixed
in 4% paraformaldehyde were cut at 12 mm and processed as described previously (31). Sections were incubated with anti-neurofilament (NF) 160 polyclonal
primary antibody (dilution 1:500 in Tris immunodiluent;
ab64300; Abcam) for axon staining, or with anti–tyrosine
hydroxylase (TH) polyclonal primary antibody (dilution
1:1,000; AB152; Millipore) for the staining of sympathetic axons. Primary antibodies were detected using
incubation with anti-species secondary antibody (dilution 1:500 in Tris immunodiluent; Alexa Fluor 488–
conjugated goat anti-rabbit IgG; Life Technologies).
The total axon number (NF) and the number of sympathetic axons (TH) per nerve were determined for each
of the nerves.
Ex Vivo Cardiac b-AR Responsiveness
Intrinsic cardiac function and responsiveness to b-AR agonist stimulation were determined using Langendorffperfused isolated hearts in 14 nondiabetic and 14 type
2 diabetic ZDF rats ex vivo as previously described (32).
In brief, animals were anesthetized with pentobarbital
(60 mg/kg), the heart was excised and mounted on a Langendorff apparatus, and the aorta was retrograde perfused with Krebs-Henseleit buffer at 37°C a constant
pressure of 80 mmHg. The Krebs-Henseleit buffer contained (in mmol/L) 118.5 NaCl, 4.7 KCl, 1.2 MgSO40.7H2O,
1.2 KH2PO4.H2O, 1.4 CaCl2, 25.0 NaHCO3, and 11.0
glucose and was continuously equilibrated with 95% O2
and 5% CO2 (pH 7.4). A custom-made balloon-tipped
catheter connected to a hydrostatic pressure transducer
was inserted into the left ventricle to measure isovolumetric LVP. Two stimulating electrodes, one at the apex
and the other on the right atrium, were used for
pacing of the heart when required. The HR, developed
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LVP (LVPdev), +dP/dtmax, 2dP/dtmax, and time constant
of relaxation (Tau) were calculated from the LVP trace.
Diabetic and nondiabetic rats were divided into the
following two groups: unpaced and paced. Isolated hearts
from the unpaced group were allowed to beat at their own
intrinsic rate, enabling the simultaneous determination of
inotropic (contraction), lusitropic (relaxation), and chronotropic (rate) properties. The hearts from the paced (to exclude
the influence of rate) group were paced at 5 Hz (physiological
resting HR ;300 bpm) to exclude chronotropic properties.
Hearts from both the unpaced and paced groups were exposed to dobutamine (b1-agonist; 1 3 1029 to 1 3 1026
mol/L, 5 min/dose) to assess responsiveness to b-AR agonist
stimulation. Maximal developed pressure was determined
with a post–extra-systolic rest potentiation protocol (33).
Determination of G-Protein and b-AR Subtype Protein
Expression
Protein lysates from LV and right atrial (RA) tissue of in
vivo and ex vivo hearts were separated on 12% SDS-PAGE
gels and were transferred onto polyvinylidene fluoride
membranes for b1-AR and nitrocellulose for b2-AR Gs (guanine nucleotide binding protein stimulatory) and Gi (guanine nucleotide binding protein inhibitory regulatory), as
described previously (34), which were subsequently probed
with specific polyclonal antibodies against b1-AR (rabbit,
1:500 dilution in 2% BSA, Novus Biologicals; or 1:1,000
dilution in 2% BSA, GeneTex), b2-AR (rabbit, 1:5,000 dilution; Badrilla), voltage-dependent anion-selective channel
protein (VDAC) 1 (1:10,000 dilution in 2% milk; Novus
Biologicals), Gs (rabbit, 1:1,000 dilution; Abcam), Gi (rabbit,
1:1,000 dilution; Santa Cruz Biotechnology), or GAPDH
(rabbit, 1:25,000 dilution; Badrilla). Proteins were visualized using an enhanced chemiluminescence detection system. The b1- and b2-AR protein expression was normalized
to VDAC1 or GAPDH and the Gs and Gi protein expression
was normalized to GAPDH to correct for protein loading.
Statistical Analysis
Unpaired t tests were used to test for differences between
groups for the in vivo and ex vivo variables and b1-AR and
b2-AR protein expression levels. A nonparametric MannWhitney test was used to test for differences in nerve
cross-sectional area (CSA) and the number of axons per
nerve. Two-way ANOVA with repeated measures was used
to test for in vivo and ex vivo b-AR responsiveness group
comparisons, followed by a Bonferroni post hoc analysis. A
P value ,0.05 was considered statistically significant.
Results are presented as the mean 6 SEM.
RESULTS
ZDF (fa/fa) rats had higher nonfasting blood glucose levels,
plasma insulin levels, and body mass compared with their
ZDF(+/+) littermates, confirming their type 2 diabetes and
obese status (Table 1). Type 2 diabetic ZDF rats showed no
structural LV hypertrophy (similar to normalized heart
mass), but they did show decreased LV filling (e.g., a significant 16 6 3% decrease in LVEDV; P , 0.05) and decreased
Diabetes Volume 64, August 2015
Table 1—Animal characteristics and in vivo hemodynamics
in 20-week-old male nondiabetic ZDF+/+ and type 2 diabetic
ZDF (fa/fa) rats
Nondiabetic
ZDF+/+
rats (n = 12)
Type 2 diabetic
ZDF (fa/fa) rats
(n = 12)
Body weight (g)
338 6 6
412 6 16**
Blood glucose
level (mmol/L)
8.0 6 1.2
23.8 6 2.9***
Plasma insulin
level (ng/mL)
3.3 6 0.5
20.5 6 4.5**
Heart weight (g)
1.83 6 0.14
1.75 6 0.05
Heart weight/tibia
length ratio
0.064 6 0.003
0.070 6 0.003
Tibia length (mm)
27.2 6 1.3
26.0 6 0.6
63 6 2
60 6 3
EF (%)
SV (mL)
109 6 7
84 6 6*
HR (bpm)
379 6 10
378 6 8
CO (mL/min)
41.0 6 1.8
35.6 6 4.2
LVEDV (mL)
121 6 7
101 6 4*
LVESV (mL)
48 6 4
44 6 4
LVESP (mmHg)
136 6 6
130 6 6
LVEDP (mmHg)
16 6 4
12 6 4
LV +dP/dtmax (mmHg/s)
9,812 6 549
11,228 6 1,009
LV 2dP/dtmax (mmHg/s)
25,513 6 217
25,263 6 357
12.3 6 1.2
13.1 6 2.1
80 6 8
85 6 7
Tau (ms)
MABP (mmHg)
Data are presented as the mean 6 SEM. EF, ejection fraction;
LVEDP, left ventricular end-diastolic pressure; LVESP, left ventricular end-systolic pressure; LVESV, left ventricular end-systolic volume. *P , 0.05; **P , 0.01; ***P , 0.001, significantly
different from nondiabetic rat, unpaired t test.
SV (21 6 4% decrease in SV; P , 0.05), suggesting diastolic
dysfunction. There was no difference in in vivo resting HR,
CO, and mean arterial blood pressure (MABP) between type
2 diabetic and nondiabetic animals (Table 1).
In Vivo Cardiac SNA
A representative direct recording of cardiac SNA in vivo in
nondiabetic and type 2 diabetic ZDF rats is shown in Fig. 1A.
The direct cardiac SNA recordings revealed a 45% increase in
resting cardiac SNA in the type 2 diabetic group, as shown
from the averaged integrated cardiac SNA values (1.25 6
0.17 and 1.87 6 0.18 mV $ s21, nondiabetes vs. type 2
diabetes, respectively; P , 0.05; Fig. 1B). Moreover, the
mean nerve firing rate was twice as high in diabetic versus
nondiabetic rats (21.1 6 4.1 and 45.0 6 8.7 impulses/s,
nondiabetes vs. type 2 diabetes, respectively; P , 0.05; Fig.
1C). This elevated SNA, recorded in vivo from the left efferent cardiac sympathetic nerve, shows that the sympathetic
input to the heart is increased in a type 2 diabetic rat model.
Cardiac Sympathetic Nerve
The CSA of the left cardiac sympathetic nerve was not
different between the nondiabetic and type 2 diabetic
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Figure 1—Direct in vivo recordings of cardiac SNA (cSNA): increased sympathetic input to the heart in type 2 diabetic ZDF rats. A:
Representative LabChart recordings showing ABP, raw efferent cardiac SNA, and the derived integrated cardiac SNA in nondiabetic
and type 2 diabetic ZDF rats. B: Increased integrated resting cardiac SNA. C: A trend toward an increased nerve firing rate in type 2
diabetic ZDF rats. *P < 0.05, significantly different from nondiabetic rats, unpaired t test, n = 11 per group. Data are presented as the
mean 6 SEM.
groups (4,399 6 397 and 4,268 6 620 mm2, nondiabetes
[n = 2] vs. type 2 diabetes [n = 3], P . 0.05; Fig. 2B).
Moreover, the immunohistochemical NF and TH staining
of the nerves revealed that neither the total number of
axons (Fig. 2C) nor the number of sympathetic axons
(Fig. 2D) per nerve was different between the nondiabetic
and diabetic groups (1,087 6 196 and 989 6 213 NF
axons per nerve and 984 6 93 and 928 6 181 TH axons
per nerve; nondiabetes [n = 2] vs. type 2 diabetes [n = 3],
P . 0.05).
In Vivo Hemodynamic Responses to b-AR Stimulation
Both the nondiabetic and type 2 diabetic ZDF animals
responded to the b-AR agonist dobutamine with increases
in HR, SV, and CO (Fig. 3A–C). No significant acceleration
of the cardiac contractility (+dP/dtmax) or relaxation
(2dP/dtmax and Tau) parameters were observed (Fig.
3D–F), and the MABP and total peripheral resistance
(TPR) both decreased (Fig. 3G and H) compared with
saline injection. During incremental dobutamine infusion,
the increase in SV was attenuated in type 2 diabetic vs.
nondiabetic rats (Δ21.7 6 3.3 vs. Δ6.7 6 3.9 mL at
10 mg/kg/min dobutamine; respectively; P , 0.05; Fig.
3B), indicating a reduced inotropic response to b-AR
stimulation in rats with type 2 diabetes in vivo. There
were no differences in the response of any other hemodynamic variables between the two groups.
Ex Vivo Animal and Cardiac Characteristics
The type 2 diabetic rats had elevated nonfasting blood
glucose levels (Table 2), whereas their heart weight and
maximum LV volume were not different, which is indicative of type 2 diabetes without cardiac structural remodeling. Interestingly, resting intrinsic HR was 32% lower in
the unpaced isolated hearts of type 2 diabetic rats. Consistent with the in vivo data, the LVPdev, the +dP/dtmax,
and the 2dP/dtmax were unchanged in type 2 diabetic
animals, whereas late Tau was prolonged in the paced
type 2 diabetic hearts. The maximal LVPdev was not different between the two groups. No differences in animal
characteristics and resting cardiac function were observed
between the unpaced and paced groups (Table 2).
Ex Vivo Cardiac Responses to b-AR Stimulation—
Paced Versus Unpaced
A greater b-AR agonist concentration was required to
achieve a target HR in unpaced type 2 diabetic versus
nondiabetic rat hearts (Fig. 4A). In addition, diabetic
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Diabetes Volume 64, August 2015
Figure 2—A: Examples of left cardiac sympathetic nerves from nondiabetic (control) and type 2 diabetic ZDF rats immunohistochemically
stained for NF and TH. Scale bars are 50 mm. B: The CSA of the cardiac sympathetic nerves. The total number of axons per nerve (stained
with NF) (C) and the total number of sympathetic axons per nerve (stained with TH) (D) were not different between nondiabetic and type 2
diabetic ZDF rats. Nondiabetic (n = 2) vs. type 2 diabetic (n = 3) rats, P > 0.05, nonparametric Mann-Whitney test. Data are presented as the
mean 6 SEM.
hearts reached a lower peak HR than nondiabetic hearts
at the highest dobutamine dose used. In contrast, the LVP
for a given b-AR agonist concentration was not different
between the groups (Fig. 4C), suggesting altered chronotropic, but preserved inotropic, b-AR responsiveness.
When HR and LVP values were normalized (as a percent
of maximum), the relative chronotropic and inotropic b-AR
responsiveness was not different between the groups (Fig.
4B and D). Moreover, the normal physiological lusitropic
(relaxation) response to b-adrenergic stimulation was observed in both groups by an increased 2dP/dtmax (Fig. 4E)
and an accelerated late Tau (Fig. 4F).
When the dobutamine protocol was performed at
a paced HR of 300 bpm (to exclude the influence of
rate), type 2 diabetic rat hearts developed less LVP at
higher concentrations of the b-AR agonist (Fig. 5A), and
the normalized LVP curve shifted to the right (Fig. 5B),
confirming reduced inotropic responsiveness to b-AR
stimulation. When paced, the changes in 2dP/dtmax
(Fig. 5C) were greater during b-AR stimulation in the
nondiabetic hearts compared with the unpaced hearts,
whereas the change in late Tau was absent in the diabetic
heart (Fig. 5D).
b-AR Subtype and G-Protein Expression Levels
b1-AR protein expression was 23% lower in the left ventricle
(Fig. 6A and B) and 17% higher in the right atrium (Fig. 6E
and F) of type 2 diabetic rat hearts compared with the nondiabetic rat hearts (LV 1.41 6 0.11 vs. 1.09 6 0.07 arbitrary
units [A.U.]; RA 0.88 6 0.02 vs. 1.02 6 0.04 A.U.; nondiabetes vs. type 2 diabetes, P , 0.05 for both). The b2-AR
protein expression level was 41% higher in the left ventricle
(Fig. 6C and D) and 20% higher in the right atrium (Fig. 6G
and H) of the type 2 diabetic hearts compared with the
nondiabetic hearts (LV expression 1.26 6 0.10 vs. 1.78 6
0.19 A.U.; RA expression 0.82 6 0.03 vs. 0.99 6 0.2 A.U.;
nondiabetes vs. type 2 diabetes, P , 0.05 for both).
The Gs expression level was 28% lower in the left ventricle (Fig. 7A and B) and 39% lower in the right atrium (Fig.
7E and F) of type 2 diabetic rat hearts compared with the
nondiabetic rat hearts (LV expression 0.89 6 0.04 vs.
0.64 6 0.07 A.U.; RA expression 0.90 6 0.07 vs. 0.54 6
0.03 A.U.; nondiabetes vs. type 2 diabetes, P , 0.05 for
both). The Gi expression level was 30% higher in the left
ventricle (Fig. 7C and D) and 27% higher in the right atrium
(Fig. 7G and H) of the type 2 diabetic rat hearts compared
with the nondiabetic rat hearts (LV expression 0.52 6 0.03
diabetes.diabetesjournals.org
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Figure 3—Preserved hemodynamic responses to b-AR stimulation in vivo in type 2 diabetic ZDF rats. In both the nondiabetic (closed
circles) and type 2 diabetic (open circles) ZDF rats, incremental doses of b-AR agonist (dobutamine) resulted in an increase in HR (A), SV
(B), and CO (C) compared with saline injection. No changes in the cardiac contractile parameter speed of contraction (+dP/dtmax) (D), the
relaxation parameters speed of relaxation (2dP/dtmax) (E), and Tau (F) were observed compared with saline injection, whereas the MABP
(G) and the calculated total peripheral resistance (TPR) (H) both decreased with b-AR agonist stimulation compared with saline injection.
The type 2 diabetic rats only had a lower SV compared with the nondiabetic animals. *Significant vs. saline (baseline), #significant vs.
nondiabetes, both P < 0.05, two-way repeated-measures ANOVA; n = 7 per group. Data are presented as the mean 6 SEM.
vs. 0.68 6 0.06 A.U.; RA expression 0.90 6 0.04 vs. 1.14 6
0.05 A.U.; nondiabetes vs. type 2 diabetes, P , 0.05
for both).
DISCUSSION
This study showed that resting and b-AR–stimulated hemodynamics of type 2 diabetic and nondiabetic rats were
similar in vivo, despite the diabetic heart receiving
a greater efferent SNA and having reduced b1-AR expression. Ex vivo experiments (e.g., denervated hearts)
revealed that the diabetic heart had a lower intrinsic HR
and required a greater b-AR stimulation to achieve a given
HR or contractile state (reduced b-AR responsiveness).
These findings suggest that the maintenance of normal
cardiovascular hemodynamics requires elevated cardiac
sympathetic input to compensate for changes in the intrinsic properties of the type 2 diabetic heart.
Previous studies (17–20) have suggested a key role for
increased sympathetic input to the diabetic heart in modulating cardiac dysfunction; however, findings were all
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Table 2—Animal characteristics and ex vivo cardiac function in unpaced and paced isolated hearts of 20-week-old male
nondiabetic and diabetic ZDF rats
Unpaced
Paced
Nondiabetic
ZDF+/+ rats (n = 7)
Type 2 diabetic
ZDF (fa/fa) rats (n = 7)
Nondiabetic
ZDF+/+ rats (n = 7)
Type 2 diabetic
ZDF (fa/fa) rats (n = 7)
357 6 7
401 6 13*
347 6 7
384 6 15*
Blood glucose level (mmol/L)
10.5 6 0.5
25.8 6 2.7**
9.4 6 0.6
28.7 6 1.8**
Heart weight (g)
1.33 6 0.14
1.5 6 0.02
1.37 6 0.06
1.43 6 0.02
LVVmax (mL)
343 6 16
357 6 30
328 6 13
350 6 11
LVPdev (mmHg)
142 6 6
135 6 9
125 6 8
111 6 7
+dP/dtmax (mmHg/s)
3,577 6 194
4,099 6 177
3,235 6 141
3,335 6 228
2dP/dtmax (mmHg/s)
22,080 6 65
Body weight (g)
22,353 6 127
22,230 6 106
22,344 6 143
Tau (ms)
74 6 12
73 6 18
67 6 13
82 6 5*
Pmax (mmHg)
338 6 16
330 6 23
382 6 11
350 6 22
HR (bpm)
245 6 10
165 6 9**
300 6 1
300 6 1
Data are presented as the mean 6 SEM. LVVmax, maximum LV volume; Pmax, maximal developed pressure. *P , 0.05, **P , 0.01,
significantly different from nondiabetic rats, unpaired t test.
based on indirect measurements of cardiac SNA. This
study, using direct recording of the left efferent cardiac
sympathetic nerve, is the first to provide direct evidence
that SNA is elevated in the diabetic heart. Ganguly and
colleagues (17,18) demonstrated a nearly twofold increase
in cardiac NE concentrations, enhanced catecholamine
turnover, and increased initial rate of NE uptake after 8
weeks of type 1 diabetes in rats. On the other hand, the
reduced inotropic response of electrically stimulated left
atria of type 1 diabetic rats has been related to an impairment of NE release from sympathetic nerve terminals (35).
Cardiac NE spillover provides an estimation of cardiac
SNA; however, the relationship between actual SNA and
the NE spillover is not linear, and high rates of nerve
discharge produce a plateau in the neurotransmitter release (36). In addition, only a variable fraction (estimated
at 20%) of the NE released from the nerve terminals
actually enters the plasma, while the majority of the NE
returned to the nerve varicosity via the NE transporter
(37). Elevated “low-frequency” HR variability (HRV) is
reported in diabetic patients and has been associated
with increased cardiac sympathetic activity and cardiovascular morbidity and mortality (38). However, others have
shown (39,40) that atropine abolishes most of the lowfrequency HRV, calling into question the association of
HRV with cardiac SNA. Our data, therefore, are the first
to directly quantify the increase in sympathetic input to
the type 2 diabetic heart, revealing a 45% increase in integrated efferent cardiac nerve activity and a doubling of
its firing rate in diabetes. Chidsey and Braunwald (41)
noted a loss of cardiac sympathetic innervation in failing
hearts, whereas Levin et al. (42) showed a marked lowering
of cardiac NE levels in obese Zucker rats, both suggesting
reduced cardiac sympathetic innervation. We were unable
to demonstrate a difference in the nerve CSA or in the
number of NF-positive or TH-positive axons within the
left cardiac sympathetic nerve between nondiabetic and
type 2 diabetic ZDF rats. This suggests that the increase
in cardiac SNA in diabetes may not be due to atrophy or
partial denervation or to hypertrophy of the cardiac
nerve. Overall, these data suggest that type 2 diabetes
does not affect the numbers of sympathetic axons (structural input) within the cardiac sympathetic nerve, but
increases its sympathetic nerve activity (throughput).
The increase in cardiac SNA may have been a consequence of cellular changes in the diabetic heart. The
intrinsic HR was 32% lower in the isolated type 2 diabetic
hearts at rest, whereas the chronotropic (rate), inotropic
(contraction), and lusitropic (relaxation) responses to
b-AR stimulation were the same as those in nondiabetic
hearts. This lower intrinsic HR at rest is observed in many
experimental rodent models of diabetes (12,43,44). Increased sympathetic input to the diabetic heart, which
accelerates spontaneous depolarization and increases conduction velocity through the heart, could explain the observed changes in function and b-AR and G-protein
expression in the diabetic heart and why, under in vivo
conditions, the HR was not different between nondiabetic
and diabetic animals.
However, changes in the intrinsic HR are primarily
determined by intracellular mechanisms within the sinoatrial node cells (vs. autonomic nervous system). Recently,
it was shown (45) in rodents that the training-induced
decrease in HR was not a consequence of changes in the
activity of the autonomic function, but was caused by
training-induced intrinsic electrophysiological changes
within the sinoatrial node. Metabolic changes occurring
during diabetes are also known to affect the modulation
of the intrinsic HR and, therefore, might provide an
alternative explanation for our finding that the intrinsic
HR of the diabetic heart was reduced. For example, carnitine supplementation corrected the reduced free carnitine levels and normalized intrinsic HR in type 1 diabetic
rats, suggesting that myocardial substrate availability
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Figure 4—Altered chronotropic but preserved inotropic and lusitropic b-AR responsiveness in unpaced isolated type 2 diabetic ZDF hearts.
A: Unpaced isolated type 2 diabetic hearts (open circles) required a higher b-AR agonist (dobutamine) concentration to achieve a given
absolute HR compared with nondiabetic hearts (closed circles). B: However, after normalization, the HR response was not different
between the groups. LVPdev increased with b-AR agonist stimulation in both groups, but showed no difference between nondiabetic
and type 2 diabetic hearts (C), even after normalization (D). Both nondiabetic and type 2 diabetic hearts showed increased speed of early
relaxation (2dP/dtmax) (E) and a faster late Tau (F) due to b-adrenergic stimulation. Thus, there was altered chronotropic but preserved
inotropic and lusitropic b-AR responsiveness in unpaced isolated hearts of 20-week-old male type 2 diabetic ZDF rats. *P < 0.05 vs.
baseline, #P < 0.05 vs. nondiabetes, two-way repeated-measures ANOVA; n = 7 per group. For normalization, data were fitted with a fourparameter sigmoidal dose-response curve with a variable slope. Data are presented as the mean 6 SEM.
plays an important role in HR regulation beyond autonomic tone (46).
When isolated hearts were paced to remove the
confounding effects of rate, we found that the diabetic
hearts had reduced inotropic and lusitropic responsiveness
to b-AR stimulation. Interestingly, in the diabetic rats under in vivo conditions (no difference in HR at rest) and in
their unpaced isolated hearts (HR reduced at rest), both
the inotropic and lusitropic responses to b-AR stimulation
were preserved. This suggests that changes in the intrinsic
functions of the diabetic heart, by means of a reduction in
absolute intrinsic HR, would ensure normal inotropic b-AR
responsiveness (potentially by improving its lusitropic window). These findings, therefore, may indicate that metabolically altered chronotropic incompetence, rather than
inotropic impediment, is an important, and undervalued,
feature of the diabetic heart (47).
The reduced inotropic and lusitropic responses to b-AR
stimulation in the paced diabetic hearts have been observed in other experimental diabetes models (10–13,15)
and have been attributed to reductions in mRNA and
protein levels of b1-ARs (11,14–16), the most predominant b-AR subtype in the healthy heart. b1-AR stimulation increases cAMP levels via Gs-coupled proteins, which
augment contraction by increasing intracellular calcium
flux during systole. Therefore, the reductions in b1-AR
(23%) and Gs (28%) in the diabetic left ventricle are consistent with a blunted stimulatory response from the increased efferent SNA and likely contributed to the observed
changes in cardiac function of our diabetic hearts. Surprisingly, the b1-AR expression in the right atrium was increased (17%), with an associated reduction of Gs
expression (39%). This could indicate specific uncoupling
of the b1-AR from its downstream signaling proteins (48).
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Diabetes Volume 64, August 2015
Figure 5—Altered inotropic and lusitropic b-AR responsiveness in paced isolated type 2 diabetic ZDF hearts. A: In isolated nondiabetic
(closed circles) and type 2 diabetic (open circles) hearts paced at a fixed HR of 300 bpm, LVPdev increased with b-AR agonist stimulation,
but less so in the type 2 diabetic group. B: Consequently, the normalized dose-response curve shifted to the right in the type 2 diabetic
hearts, which is indicative of reduced inotropic b-AR responsiveness. The arrow indicates significant rightward shift in the b-AR responsiveness. b-AR agonist stimulation increased the speed of early relaxation (2dP/dtmax) (C) and accelerated late Tau (D) in the nondiabetic
hearts. In the type 2 diabetic hearts, the change in early relaxation was impaired, and the change in late relaxation was absent. *P < 0.05 vs.
baseline, #P < 0.05 vs. nondiabetes, two-way repeated-measures ANOVA; n = 7 per group. For normalization, data were fitted with a fourparameter sigmoidal dose-response curve with a variable slope. Data are presented as the mean 6 SEM.
Alternatively, b2-ARs have a higher relative expression
level in the sinoatrial node region (49) and, as a consequence of the colocalization of b2-AR with the funny
channels in the caveolae, have been shown to contribute
more to chronotropic changes compared with b1-ARs
(50). We found that b2-AR protein expression was increased in the left ventricle (40%) and in the right atrium
(20%) of the diabetic hearts, with concomitant increases
of Gi expression (30% and 27%, respectively), which, to
the best of our knowledge, is the first report in a type 2
diabetic rodent model. It is unclear what the functional
relevance of this shift from b1-AR to b2-AR in type 2
diabetes is, although similar relative results have been
observed during heart failure (9). In the healthy heart,
it is believed that b2-ARs do not directly contribute to
changes in contraction because their colocalization with
Gi-activated phosphodiesterases inhibits the cAMP pathway and prevents the resultant increase in calcium fluxes.
Interestingly, in heart failure redistribution of b2-ARs
changes this compartmentation of cAMP and might contribute to the failing myocardial phenotype (51). Moreover, recent evidence indicates that b2-ARs modulate
intracellular oxygen availability (52) and are linked to
increases in metabolic kinases, such as AMPK (52,53).
AMPK is considered a critical sensor of cellular energy,
activated by biguanide drugs (metformin and phenformin), and an attractive metabolic target for novel therapies in the treatment of type 2 diabetes (54). Therefore,
the relation of b2-ARs, their coupling (and uncoupling) to
Gs and Gi, and their links to functional and metabolic
challenges in type 2 diabetes, especially related to chronotropic modulation of cardiac function, warrants further
research.
Limitations
Surgery and anesthetic agents modulate the SNS and
therefore caution is needed when interpreting data from
anesthetized animals during surgery. While direct assessment of cardiac SNA in conscious sheep is possible (55),
no ovine diabetic model exists. Unfortunately in rodents,
because of the limited accessibility of the cardiac nerves,
telemetric recordings are not feasible. We used urethane
because of its minimal effects on autonomic, cardiovascular, and respiratory systems (56).
SNA measurements in other cardiac diseases, such
as myocardial infarction (29,57) or more severe heart
failure (58,59), have found larger changes in cardiac
SNA. Nevertheless, our relatively small, but significant,
change in cardiac SNA in type 2 diabetes, if maintained
over long periods during the progression of diabetes,
may still have very detrimental effects on cardiac
function.
Short-term central administration of leptin activates
the SNS (60), increases lumbar and renal SNS activity
(61), and increases ABP (62), all of which are consistent
with the increased cardiac sympathetic activity. However,
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Figure 6—LV and RA b1-AR and b2-AR protein expression levels in type 2 diabetic ZDF rats. A, C, E, and G: Representative images of
b1-AR and b2-AR Western blots in the left ventricle and right atrium. B, D, F, and H: Quantitative analysis of the protein expression
(mean 6 SEM; 3 replications). Shown are reduced b1-AR protein expression levels in the left ventricle (A and B) and increased b1-AR
levels in the right atrium (E and F) in the type 2 diabetic group relative to the nondiabetic group, normalized to VDAC or GAPDH protein
expression levels. *P < 0.05 vs. nondiabetes, unpaired t test. LV: nondiabetes n = 17; type 2 diabetes n = 20; RA: nondiabetes n = 7;
type 2 diabetes n = 6 (mean 6 SEM; 3 replications). The b2-AR protein expression levels were elevated in the left ventricle (C and D) and
in the right atrium (G and H) in the type 2 diabetic group relative to the nondiabetic group, normalized to VDAC or GAPDH protein
expression levels. *P < 0.05 vs. nondiabetes, unpaired t test. LV: nondiabetes n = 17; type 2 diabetes n = 17; RA: nondiabetes n = 7;
type 2 diabetes n = 6 (mean 6 SEM; 3 replications).
this suggests that a leptin-deficient animal would, if anything, have reduced cardiac sympathetic activity. Therefore, we believe that the development of type 2 diabetes
independently explains the changes in cardiac SNA and
resultant changes in myocardial b-adrenergic responsiveness in our study.
In conclusion, this is the first study to demonstrate,
with direct measurement of the efferent cardiac sympathetic nerves, elevated resting cardiac SNA in type 2
diabetes in vivo. In contrast, the intrinsic properties of
the diabetic heart showed lower HR and rate-independent
contractility. In this context, the elevated cardiac SNA
may have been a compensatory response to a diabetesinduced lower intrinsic HR, ensuring normal hemodynamics in vivo. The proportion of b1-/b2-AR and associated
presence of Gs and Gi in the left ventricle was reduced
by diabetes, which is consistent with other models of high
cardiac SNA, such as congestive heart failure. Thereby,
in our type 2 diabetic animal model without any other
cardiovascular disease present, we demonstrated that
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Diabetes Volume 64, August 2015
Figure 7—LV and RA Gs and Gi expression levels in type 2 diabetic ZDF rats. A, C, E, and G: Representative images of Gs and Gi Western
blots in the left ventricle and right atrium. B, D, F, and H: Quantitative analysis of the protein expression. Shown are reduced Gs expression
levels in the left ventricle (A and B) and right atrium (E and F) in the type 2 diabetic group relative to the nondiabetic group, normalized to
GAPDH protein expression levels. Gi expression levels are elevated in the left ventricle (C and D) and right atrium (G and H) in the type 2
diabetic group relative to the nondiabetic group, normalized to GAPDH protein expression levels. *P < 0.05 vs. nondiabetes, unpaired t
test. Nondiabetes n = 7; type 2 diabetes n = 6 (mean 6 SEM; 3 replications, except for LV Gs duplicates).
sympathetic hyperactivity and a defective intrinsic HR
regulation are associated with cardiac dysfunction and
b-AR dysregulation in type 2 diabetes.
Funding. This work was supported by grants from Healthcare Otago Charitable Trust, New Zealand National Heart Foundation grant no. 1491–NH
Taylor Charitable Trust, and the Department of Physiology of the University
of Otago.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. H.P.A.T. performed the experimental work, the
primary analysis, and the secondary analysis and drafted the manuscript. J.C.B.
designed the study and drafted the manuscript. H.-Y.W. performed the experimental work, the primary analysis, and the secondary analysis. G.H., R.F.C.,
C.T.B., and P.W.S. performed the experimental work and the primary analysis.
A.B., P.P.J., and D.O.S. managed the experimental work and edited the manuscript.
R.R.L. designed the study, performed the secondary analysis, managed the
experimental work, and drafted the manuscript. All authors read and approved
the final manuscript. R.R.L. is the guarantor of this work and, as such, had full
access to all the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Prior Presentation. Parts of this study were presented in abstract form at
2012 Sydney: Joint Australian Physiological Society/Physiological Society of New
Zealand/Australian Society for Biophysics Meeting, Sydney, New South Wales,
Australia, 2–5 December 2012.
diabetes.diabetesjournals.org
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