Enhanced left ventricular systolic function early in type 2 diabetic mice: clinical implications.

Abstract

It is unclear whether the increase in availability of substrates for energy production in diabetes can lead to enhanced systolic function early in the disease, before the onset of structural changes to the myocardium. To examine this issue, BKS.Cg-m +/+ Lepr db (db/db) mice with type 2 diabetes and wild type controls had left ventricular pressure-volume relationships determined in situ. We demonstrated that the db/db mice, when compared to their wild type controls, generated greater left ventricular pressure and an enhancement of left ventricular systolic function based on enhanced power/EDV, positive dP/dt, preload recruitable stroke work, dP/dt--EDV relationship, and curvilinear end-systolic elastance. This enhancement in systolic function occurred despite the db/db mice having greater body weight, but similar preload (end-diastolic volume) and afterload (effective arterial elastance). We postulate that the previously described enhancement in renal glomerular filtration rate seen early in type 2 diabetes may be in part due to enhanced left ventricular systolic function early in this disease.

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Diabetes and Vascular Disease Research
http://dvr.sagepub.com/content/1/2/89
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DOI: 10.3132/dvdr.2004.013
2004 1: 89Diabetes and Vascular Disease Research
Jorge A Alvarez, Maricela Reyes, Daniel Escobedo, Gregory L Freeman, Mark E Steinhelper and Marc D Feldman
Enhanced left ventricular systolic function early in type 2 diabetic mice: clinical implications


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Abstract
I
the onset of structural changes to the myocardium. To
examine this issue, BKS.Cg-m +/+ Lepr db(db/db) mice
with type 2 diabetes and wild type controls had left ven-
tricular pressure-volume relationships determined in situ.
We demonstrated that the db/db mice, when compared to
their wild type controls, generated greater left ventricular
pressure and an enhancement of left ventricular systolic
function based on enhanced power/EDV, positive dP/dt,
preload recruitable stroke work, dP/dt – EDV relationship,
and curvilinear end-systolic elastance. This enhancement
in systolic function occurred despite the db/db mice having
greater body weight, but similar preload (end-diastolic vol-
ume) and afterload (effective arterial elastance).
We postulate that the previously described enhance-
ment in renal glomerular filtration rate seen early in type 2
diabetes may be in part due to enhanced left ventricular
systolic function early in this disease.
Diabetes Vasc Dis Res 2004;1:89–94
t is unclear whether the increase in availability of sub-
strates for energy production in diabetes can lead to
enhanced systolic function early in the disease, before
Key words: type 2 diabetes, left ventricular pressure-
volume relationships, systolic function, haemodynamics,
mouse models of diabetes.
Introduction
Type 2 diabetes is associated with myocardial disease. This is
in part due to atherosclerosis and hypertension. There is also
evidence for diabetes having a direct impact on the
myocardium, with documentation of diabetes-induced dias-
tolic dysfunction based on echocardiographic studies in
patients.1However, whether diabetes can cause myocardial
dilation and secondary cardiomyopathy is more controver-
sial. Since the variables of atherosclerosis and hypertension
cannot be removed in patients, investigators have focused
on animal models of type 2 diabetes to study the impact of
diabetes on myocardial disease more directly.
Both the BKS.Cg-m +/+ Lepr db(db/db) mouse and
obese Zucker rat (fa/fa) rat are well established animal mod-
els of insulin resistance.2-4Neither model develops coronary
artery disease. These animal models have been used to
investigate the myocardial impact of type 2 diabetes.5,6Using
isolated perfused hearts, the Zucker rat showed increased
left ventricular pressure (LVP) and its first derivative dP/dt
(contractility).7The increase in myocardial function was fur-
ther enhanced with the addition of insulin to the perfusate,
which more closely approximated the in situ insulin concen-
tration.7In contrast, isolated hearts of db/db mice showed
decreased cardiac function.8The reduced function was con-
firmed in db/db hearts in situ using echocardiography.9Thus,
it is not clear from the literature whether, early in diabetes,
there is increased or decreased LV systolic function.
The explanation for these differences in systolic function
in the isolated hearts may be related to whether the per-
fusate simulated the in situ diabetic milieu. The weaker iso-
lated db/db hearts were placed in a perfusate, which had no
insulin and low concentrations of free fatty acids. In contrast,
the stronger isolated Zucker rat hearts were placed in a per-
fusate more representative of the diabetic milieu. Further,
although the in situ db/db hearts evaluated by echocardiog-
raphy demonstrated reduced function, the investigators
used load-dependent measures of systolic function, which
could be reduced due to increased afterload. Blood pressure
and other measures of afterload were not reported in this
study.9
The diabetic heart does not demonstrate resistance to
insulin, unlike skeletal muscle.10Thus, not only is the
increased free fatty acid content of diabetic blood available
to the heart for energy generation but increased glucose is
available as well. We hypothesise that early in diabetes,
before the onset of structural myocardial damage, there is
increased energy supply to the heart in the form of increased
ORIGINAL ARTICLE
Enhanced left ventricular systolic function
early in type 2 diabetic mice: clinical
implications
JORGE A ALVAREZ, MARICELA REYES, DANIEL ESCOBEDO, GREGORY L FREEMAN,
MARK E STEINHELPER, MARC D FELDMAN
Departments of Medicine and Physiology, University of Texas Health
Science Center, San Antonio, Texas, US.
Jorge A Alvarez, Clinical Cardiology Fellow
Maricela Reyes, Master of Physiology
Daniel Escobedo, Laboratory Specialist
Gregory L Freeman, Professor of Medicine
Mark E Steinhelper, Assistant Professor of Physiology
Marc D Feldman, Associate Professor of Medicine
Correspondence to: Dr Marc D Feldman
Room 5.642 U, University of Texas Health Science Center in San Antonio,
7,703 Floyd Curl Drive, San Antonio, Texas, 78229-3900, US.
Tel: +1 210 567 4600; Fax: +1 210 567 6960
E-mail: feldmanm@uthscsa.edu
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free fatty acids and glucose. We postulate that the heart
could have enhanced systolic function, rather than reduced
function, early in diabetes due to increased energy supply.
To demonstrate this, it is important to use load-independent
measures of left ventricular function. With the recent advent
of the miniaturised mouse conductance system,11,12it is now
technically feasible to measure LV pressure-volume relation-
ships in the in situ murine heart. We chose to study the well
characterised db/db mouse model with this new instrumen-
tation.
Methods
Conductance technology
A 1.4 French miniaturised pressure-volume catheter (SPR-
719, Millar Instruments, Houston, TX) was used in these
studies. A constant excitation current (17µA root mean
square) was applied to the outermost electrodes at two fre-
quencies simultaneously, 10 and 100 kHz. The device uses
a custom signal generator/processor and bridge amplifiers
developed by us and modified by Millar Instruments
(Houston, Texas). The 10 and 100 kHz frequencies are gen-
erated with a Signametrics SM-1030 Complex DDS
Generator (Seattle, WA). The theory behind the determina-
tion of volume using the conductance catheter system in
larger animals has been described in detail.13,14The use of
dual frequency to determine instantaneous parallel conduc-
tance in mice has also been described.11The only addition-
al instrumentation used in conjunction with the dual fre-
quency conductance system was an aortic flow probe, used
to correct for electrical field inhomogeneity (α).
Husbandry
The Institutional Animal Care and Use Committee at the
University of Texas Health Science Center at San Antonio
approved all experiments. Female BKS.Cg-m +/+ Leprdb
(db/db) mice (n=7), as well as their female non-diabetic lit-
termates (db/+ or +/+) (n=9), were used in these studies.
Breeder pairs (db/+ x db/+) were purchased from Jackson
Laboratory (Bar Harbor, ME) and used to generate offspring,
who were all studied at 11 weeks of age. All mice were given
access to food and water ad libitum.
Haemodynamic protocol
Mice were anaesthetised with a combination of urethane
(1,000 mg/kg, i.p.) and etomidate (25 mg/kg, i.p.). Blood
glucose concentrations were obtained using tail blood sam-
ples and measured with a Life Scan One Touch Basic,
Johnson & Johnson (Milpites, CA). Body weight was then
recorded. Mice were placed on a temperature-controlled
operating table for small animals (Vestavia Scientific, Ill.) and
then mechanically ventilated with a rodent ventilator set at
150 breaths/min (100 % O2). In half the mice, physiological
temperatures were utilised (37°C, n=8), while the other half
were studied at room temperature (31°C, n=8). The db/db
and wild type (WT) mice were equally distributed between
these two temperatures.
The chest was entered via an anterior thoracotomy. An
apical stab was made in the heart with a 30-G needle, and
the miniaturised conductance catheter (Millar Instruments,
Houston, TX) was advanced retrograde into the left ventricle
(LV) along the long axis, with the proximal electrode just
within the myocardial wall at the apex. The inferior vena
cava (IVC) was isolated immediately below the diaphragm
for transient occlusion.
Baseline pressure-conductance relationships at both 10
and 100 kHz were acquired and stored for offline conver-
sion to pressure-volume relationships. Similar data were
acquired during transient occlusion of the IVC. To derive α
at the end of the experiment, a small animal blood flow
meter T 106 (Transonic Systems Inc., Ithica, NY) was
placed around the ascending aorta. Simultaneous LV con-
ductance at 10 and 100 kHz and aortic flow were record-
ed.
Myocardial resistivity protocol
It has been previously shown that the myocardial resistivity
of the in vivo db/db myocardium is different from the wild
type myocardial resistivity.15It has also been shown that if the
wild type myocardial resistivity is used to calculate db/db
absolute LV volumes with the dual frequency conductance
system, non-physiological (negative) volumes are obtained.15
Thus, we also sought to determine myocardial resistivity in
additional mice (db/db, n=6; wild type, n=7), also at 11
weeks of age. The myocardial resistivity was determined at
1, 10 and 100 kHz and then used with published algorithms
to extract true instantaneous LV volume.11
The protocol used to determine db/db and wild type
myocardial resistivity was similar to the haemodynamics
studies described above. It differed in that, once the anteri-
or thoracotomy was complete, a custom four-electrode epi-
cardial resistivity probe was applied to the in situ beating
murine epicardium. Frequencies of one, 10 and 100 kHz
were applied to electrodes one and four, and the instanta-
neous voltage between electrodes two and three was
recorded. The voltage to conductivity relationship had pre-
viously been determined in saline solutions spanning the
conductivity of the murine myocardium. In this manner, the
real time myocardial voltage signal was converted to con-
ductivity. Since the inverse of conductivity is resistivity, the
data are expressed in resistivity (ohms-cm).
Data analysis
The pressure-volume data were analysed with software
developed by us, and licensed to and modified by Millar
Instruments, Inc. (PVAN©). The algorithms used for dual fre-
quency were developed by us and have been published.16
Absolute volume measurements from the conductance
catheter were calibrated with a correction for gain (α). α was
defined as the ratio of flow probe stroke volume to the con-
ductance stroke volume. Each mouse was analysed with its
individual α.
Statistics
A linear mixed model to compare the association of fre-
quency and myocardial resistivity between db/db and wild
type was performed. The general linear mixed model is an
extension of analysis of variance (ANOVA). The db/db and
wild type haemodynamic parameters were compared by
ORIGINAL ARTICLE
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Student’s paired t test. A p value less than 0.05 was consid-
ered significant. Data are presented as means+SE.
Results
Myocardial resistivity
A comparison of db/db and WT myocardial resistivity is
shown in table 1. At lower frequencies (1 and 10 kHz) the
db/db resistivity was higher than the WT.
Haemodynamics and weights
Table 2 is a comparison between db/db and WT mouse
haemodynamics and weights. The diabetic mice have larger
body size compared to the WT mice at 11 weeks. Despite the
increase in body weight, the afterload (as measured by effec-
tive arterial elastance) was similar in both groups. The heart
weights and left ventricular volumes were the same. Left ven-
tricular pressure was higher in the db/db mice compared to the
wild type mice. Not shown in table 2, the db/db mice had a
mean glucose of 246+36 (n=4) and their WT controls had a
mean glucose of 82+19 (n=5), which is statistically different.
Systolic function
Table 3 is a comparison between db/db and WT mouse
measures of systolic function. Using relatively load-indepen-
dent measures of systolic function, four of five measures
show an increase in left ventricular function in the db/db
mice compared to WT controls.
Diastolic function
Table 4 is a comparison between db/dband WT mice measures
ORIGINAL ARTICLE
Table 1. Myocardial resistivity in study mice
Wild type
(n=7)
Diabetic
(n=6)
p value
1 kHz 692+27
855+41 < 0.001
10 kHz 615+21
715+36< 0.01
100 kHz 602+20
606+25ns
Values are mean+SE. Units of resistivity are ohm-cm
Table 2. Haemodynamics and weights in study mice
Wild type
(n=9)
Diabetic
(n=7)
p value
BW (g) 23+1
48+1<0.001
HW (mg)114+3
110+5ns
HR (bpm)471+16
495+13ns
Ved(µl)
Ves(µl)
Pmax(mmHg)
Pes(mmHg)
Ea(mmHg/µl)
SV (µl)
19+219+4 ns
10+2 9+3ns
82+4121+5 <0.001
75+4 115+6 <0.001
10+3 15+4ns
11+2
12+2ns
CO (µl/min)5,238+874
5,724+837 ns
SW (mmHg*µl) 758+173
1,108+181 ns
Values are mean+SE.
Key: BW = body weight; HW = heart weight; HR = heart rate;
Ved= LV end-diastolic volume; Ves= LV end-systolic volume;
Pmax= maximum LV pressure; Pes= LV end-systolic pressure;
Ea= effective arterial elastance; SV = stroke volume; CO = cardiac
output; SW = stroke work; ns = not significant
Table 3. Systolic function in study mice
Wild type
(n=9)
Diabetic
(n=7)
p value
EF (%)53+5
64+5NS
Power/EDV2
(mWatts/µl2)
91+24189+32
11,669+902
<0.05
<0.01 6,531+1,126
dP/dt max
(mmHg/sec) 69+11
107+9<0.05
PRSW (mmHg)
dP/dt-EDV
(mmHg x µl/sec)
457+132
1,020+251 ns
E’max (mmHg/µl) 20+6
137+43 <0.05
V’o (µl) 2+6
8+2ns
Values are mean+SE.
Key: EF = ejection fraction; Power/EDV2= Power-normalised log EDV2;
dP/dtmax= first derivative of left ventricular pressure rise;
PRSW = preload recruitable stroke work; dP/dt-EDV = linear index of
left ventricular contractile function; E’max = curve linear end-systolic
elastance; V’o = volume intercept of E’max; ns = not significant
Table 4. Diastolic function in study mice
Wild type
(n=9)
Diabetic
(n=7)
p value
B-diastolic
compliance (µl-1)
0.033+0. 02
0.11+0.05 ns
Tau Glantz(msec)
Tau Weiss(msec)
Ped(mmHg)
12+312+1 ns
10+28+1 ns
10+29+1 ns
Values are mean+SE.
Key: B-LV diastolic compliance = a measure of the stiffness of
myocardium; Tau Glantz and Tau Weiss are measures of isovolumetric
relaxation time; Ped= left ventricular end-diastolic pressure;
ns = not significant
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of diastolic function. Although not statistically significant,
there is a trend toward reduced diastolic compliance in the
db/db mice.
A typical example of the haemodynamic results for a
db/db and WT mouse is shown in figures 1a and 1b. As is
visibly evident, the db/db mouse had higher pressure, steep-
er E’max slope, similar diastolic compliance and effective
arterial elastance.
Discussion
This study has demonstrated that, early in type 2 diabetes,
there is an enhancement of left ventricular systolic function
as detected by multiple load-independent measures of ven-
tricular function and greater pressure generation. This
enhancement in function was despite no change in loading
conditions and the obesity of the diabetic mice i.e. effective
arterial elastance and end-diastolic volume were no different
between the diabetic and control mice. There was also no
evidence of cardiac hypertrophy early in the diabetic mice
to account for our findings since the heart weights were sim-
ilar between the diabetic and control mice. Thus, this study
provides the unexpected finding of enhanced ventricular
function early in diabetes when the variable of coronary ath-
erosclerosis is removed.
The diabetic mouse model we studied has been
described in the literature since 1979. These diabetic mice
were created by a spontaneous mutation in the leptin
receptor in the central nervous system,17so have been avail-
able to the scientific community since long before the
development of transgenic mice. However, the increase in
left ventricular systolic function is only now being
described. This is because the miniaturisation of conduc-
tance technology to determine load-independent measures
of ventricular function in vivo via pressure-volume analysis
is a recent development,11,12and has not been applied to
this mouse model in the past.
Previous echocardiographic evaluation of this intact
mouse model demonstrated the opposite finding of
decreased function in the diabetic mouse. However, frac-
tional shortening as determined by echocardiography is
known to be a load-dependent measure of ventricular func-
tion. We have demonstrated increased left ventricular pres-
sure early in diabetes in this model, which is known to
reduce fractional shortening. Blood pressure was not mea-
sured in the echocardiographic study.9Thus, previous
echocardiographic studies of this mouse model are not nec-
essarily in conflict with our findings.
The isolated perfused db/db murine heart has been
shown to generate the same left ventricular pressure, and a
reduction in cardiac output, when compared to isolated
hearts of wild-type mice.8However, the perfusate used in
this study contained a marked reduction in the concentra-
tions of glucose, insulin and free fatty acids compared to
those known to be present in the intact diabetic animal.
Therefore, the isolated heart studies published to date are
not representative of the true energy precursor milieu nor-
mally found in the db/db model. For future studies, isolated
diabetic hearts will need to be perfused with physiological
ORIGINAL ARTICLE
Figure 1. Example of pressure-volume loops during transient inferior vena cava occlusion for both diabetic (figure 1a) and wild
type (figure 1b) mice. Line identified by (+) represents E’max. Diabetic mice have a steeper E’max (curve linear end-systolic
elastance) slope consistent with greater contractility compared to the wild type mice (91 vs. 7 mmHg/µl, respectively). Left
ventricular pressure generation is greater in the diabetic mice compared to the wild type mice. Diastolic compliance in the
diabetic and wild type mice was similar (0.12 vs. 0.06 µl-1, respectively)
125
100
75
50
25
0
0
6 30 12 1824
Volume (µI)
Pressure (mmHg)
0
0 186
12
24
Volume (µl)
Pressure (mmHg)
25
50
75
100
125
30
a
b
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