H-01036-2006
1
Cardiac Specific Overexpression of Insulin-Like Growth Factor-1 (IGF-1)
Attenuates Aging-Associated Cardiac Diastolic Contractile Dysfunction and
Protein Damage
Qun Li1, Shan Wu1, Shi-Yan Li1, Faye L. Lopez1, Min Du2, Jan Kajstura3, Piero Anversa3
and Jun Ren1
1Division of Pharmaceutical Sciences & Center for Cardiovascular Research and
Alternative Medicine; 2Department of Animal Sciences, Laramie, WY 82071; 3Department
of Medicine, New York Medical College, Valhalla, NY 10595
Running head: IGF-1 overexpression attenuates cardiac aging
Correspondence to:
Dr. Jun Ren
Division of Pharmaceutical Science & Center for Cardiovascular Research and Alternative
Medicine, University of Wyoming, Box 3375, Laramie, WY 82071-3375.
Tel: (307)-766-6131; Fax: (307)-766-2953
E-mail: [email protected]
Page 1 of 25
Copyright Information
Articles in PresS. Am J Physiol Heart Circ Physiol (November 3, 2006). doi:10.1152/ajpheart.01036.2006
Copyright © 2006 by the American Physiological Society.
H-01036-2006
2
ABSTRACT
Aging is associated with hepatic growth hormone resistance resulting in a fall in serum insulin-
like growth factor-1 (IGF-1) level. However, whether loss of IGF-1 contributes to cardiac aging
is unclear. This study was designed to examine the effect of cardiac overexpression of IGF-1 on
cardiomyocyte contractile function in young (3 mo) and old (26-28 mo) mice. Cardiomyocyte
contractile function was evaluated including peak shortening (PS), time-to-90% PS, time-to-90%
relengthening (TR90) and maximal velocity of shortening/relengthening (± dL/dt). Levels of
advanced glycation endproduct (AGE), protein carbonyl, sarco(endo)plasmic reticulum Ca2+-
ATPase (SERCA2a), phospholamban (PLB) and Na+-Ca2+ exchanger (NCX) were assessed by
Western blot. SERCA activity was measured by 45Ca2+ uptake. Aging induced a decline in
plasma IGF-1 levels. Aged cells exhibited depressed ± dL/dt, prolonged TR90 and a steeper PS
decline in response to increasing stimulus frequency compared with young myocytes. IGF-1
transgene alleviated aging-induced loss in plasma IGF-1 and aging-induced mechanical defects
with little effect in young mice. The beneficial effect of IGF-1 transgene on aging-associated
cardiomyocyte contractile dysfunction was somewhat mimicked by short-term in vitro treatment
of recombinant IGF-1 (500 nM). AGE and protein carbonyl levels were higher in aged mice
which were not affected by IGF-1. Expression of SERCA2a (but not NCX and PLB) and
SERCA activity were reduced with aging, which was ablated by the IGF-1 transgene.
Collectively, our data suggest beneficial role of IGF-1 in aging-induced cardiac contractile
dysfunction, possibly related to improved Ca2+ uptake.
Key words: IGF-1, cardiomyocytes, aging, contractile function, Ca2+ regulatory protein.
Page 2 of 25
Copyright Information
H-01036-2006
3
INTRODUCTION
Cardiac aging is a continuous and irreversible biological process contributing to the high
morbidity and mortality in the elderly (4; 10; 11). Several theories have been made for cardiac
aging including prolonged action potential duration, myosin heavy chain isozyme switch,
impaired intracellular Ca2+ homeostasis, altered membrane structure and permeability and
accumulation of reactive oxygen species (ROS), which may lead to abnormal cardiac contractile
function (8-10). In addition, aging induces reduction in growth hormone (GH) and subsequent
insulin-like growth factor-1 (IGF-1) deficiency, both of which essential for the maintenance of
normal cardiac structure and function. GH/IGF-1 deficiency has been reported to be associated
with altered body composition, cytokine and neuroendocrine activation, cardiac atrophy and
impaired cardiac function. The risk of cardiovascular disease is increased in subjects with IGF-1
deficiency. IGF-1, the mediator of many of GH-associated effects in peripheral tissues, improves
myocardial function in the setting of both healthy and failing hearts (22). The therapeutic
potential of IGF-1 has been implicated in cardiac disorders related, but not limited, to heart
failure, myocardial infarction and diabetes (22). Nonetheless, the role of IGF-1 in cardiac aging
remains largely obscure. Therefore, the present study was designed to determine if cardiac-
specific overexpression of IGF-1 alleviates aging-associated cardiomyocyte contractile function
and affects expression/function of key Ca2+ regulatory proteins.
MATERIALS AND METHODS
Experimental animals
All animal procedures used in this study were approved by the Animal Care and Use
Committees at the University of North Dakota (Grand Forks, ND) and the University of
Page 3 of 25
Copyright Information
H-01036-2006
4
Wyoming (Laramie, WY). Male FVB and IGF-1 heterozygous transgenic mice at young (3
month-old) and old (26-28 month-old) ages were used. The pigmentation of fur color was used
as a marker for heterozygous IGF-1 (light brown) or wild-type FVB (white) mouse identification
as described (20). All animals were kept in our institutional animal facility at the University of
Wyoming with free access to standard lab chow and tap water. At the time of sacrifice, blood
glucose and plasma IGF-1 levels were measured using a glucose monitor (Accu-ChekII, model
792, Boehringer Mannheim Diagnostics, Indianapolis, IN, USA) and an ELISA commercial kit
from Diagnostic System Laboratory (Webster, TX, USA), respectively.
Isolation of mouse ventricular myocytes
Hearts were rapidly removed from anesthetized mice and mounted onto a temperature-
controlled (37°C) Langendorff system. After perfusing with a modified Tyrode solution (Ca2+ free)
for 2 min, the heart was digested for about 10 min with 0.9 mg/ml collagenase D (Boehringer
Mannheim Biochemicals) in the modified Tyrode solution. The modified Tyrode solution (pH 7.4)
contained the following (in mM): NaCl 135, KCl 4.0, MgCl2 1.0, HEPES 10, NaH2PO4 0.33,
glucose 10, butanedione monoxime 10, and the solution was gassed with 5% CO2-95% O2. The
digested heart was then removed from the cannula and left ventricle was cut into small pieces in the
modified Tyrode solution. Tissue pieces were gently agitated and pellet of cells was resuspended.
Extracellular Ca2+ was added incrementally back to 1.20 mM over a period of 30 min. Isolated
myocytes were used for experiments within 8 hours of isolation. Only rod-shaped myocytes with
clear edges were selected for mechanical and intracellular Ca2+ studies (3).
Cell shortening/relengthening
Page 4 of 25
Copyright Information
H-01036-2006
5
Mechanical properties of ventricular myocytes were assessed using a SoftEdge MyoCam®
system (IonOptix Corporation, Milton, MA, USA) (3). In brief, cells were placed in a Warner
chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused (∼1
ml/min at 25oC) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose,
10 HEPES, at pH 7.4. The cells were field stimulated with suprathreshold voltage at a frequency of
0.5 Hz (unless otherwise stated), 3 msec duration, using a pair of platinum wires placed on opposite
sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The myocyte being
studied was displayed on the computer monitor using an IonOptix MyoCam camera. An IonOptix
SoftEdge software was used to capture changes in cell length during shortening and relengthening.
In the case of altering stimulus frequency from 0.1 Hz to 5.0 Hz, the steady state contraction of
myocyte was achieved (usually after the first 5-6 beats) before PS was recorded. Cell shortening and
relengthening were assessed using the following indices: peak shortening (PS), time-to-90% PS
(TPS90), time-to-90% relengthening (TR90), half width duration (HWD) – indicating late cell
shortening and early relengthening, as well as maximal velocity of shortening (+ dL/dt) and
relengthening (-dL/dt). To examine the acute effect of IGF-1 on cardiomyocyte contractile function,
cohorts of cardiomyocytes from young and aged FVB mice were treated with recombinant IGF-1
(500 nM) for 2 hrs at 37oC with 100% humidity and 5% CO2 as described previously (14).
Western blot analysis
Protein levels of SRECA2a, PLB, NCX, AGE and protein carbonyl were examined by
standard Western blot. Left ventricular tissues were homogenized and centrifuged at 70,000 x g
for 20 min at 4°C. The supernatants were used for immunoblotting of SERCA2a, PLB, NCX,
AGE and carbonyl. The extracted proteins were separated on 10 - 15% SDS-polyacrylamide gels
Page 5 of 25
Copyright Information
H-01036-2006
6
and transferred to polyvinylidene difluoride membranes. After blocking the membrane was
incubated with mouse anti-AGE monoclonal (1:1000, Trans Genic Inc., Kumamoto, Japan),
rabbit anti-NCX polyclonal (1:1000, Swant, Bellinzona, Switzerland) and mouse anti-PLB
monoclonal antibody (1:2000, Abcam Inc, Cambridge, MA), rabbit anti-SERCA2a (1:1000,
Affinity BioReagents Inc., Golden, CO) and rabbit anti-DNP (dinitrophenyl, 1:150, Chemicon
International, Temecula, CA) overnight at 4°C followed by incubation with second antibodies.
The antigens were detected by the luminescence method. Quantification of band density was
determined using Quantity One software (Bio-Rad, version 4.4.0, build 36) and reported in
optical density per square millimeter (16).
SERCA activity measured by 45Ca2+ uptake
Cardiomyocytes were sonicated and solubilized in a Tris-sucrose homogenization buffer
consisting of 30 mM Tris-HCl, 8% sucrose, 1 mM PMSF and 2 mM dithioithreitol, pH 7.1. To
determine SERCA-dependent Ca2+ uptake, samples were treated with and without the SERCA
inhibitor thapsigargin (10 µM) for 15 min. The difference between the two readings was deemed
the thapsigargin-sensitive uptake through SERCA. Uptake was initiated by the addition of an
aliquot of supernatant to a solution consisting of (in mM) 100 KCl, 5 NaN3, 6 MgCl2, 0.15
EGTA, 0.12 CaCl2, 30 Tris-HCl pH 7.0, 10 oxalate, 2 ATP and 1 µCi 45
CaCl2 at 37°C. Aliquots
of samples were injected onto glass filters on a suction manifold and washed 3 times. Filters
were then removed from the manifold, placed in scintillation fluid and counted. SERCA activity
was expressed as cpm/mg protein (15).
Statistical analysis
Page 6 of 25
Copyright Information
H-01036-2006
7
Data were presented as Mean ± SEM. Statistical significance (p < 0.05) for each variable
was determined by analysis of variance (ANOVA) or t-test, where appropriate.
RESULTS
General feature of young and old experimental animals
General features of young and aged FVB and IGF-1 transgene mice are shown in Table 1.
At young age, transgenic IGF-1 overexpression did not elicit any notable effect on body, liver and
kidney weights compared with age-matched FVB mice. However, IGF-1 significantly increased
heart weight and heart size (heart-to-body weight ratio) in young mice. The enlarged heart weight
and size seen in young IGF-1 mice prevailed through the older age. Aged mice had heavier body
and organ weights (although not organ size) compared with young counterparts with the exception
of a large kidney size. Aging significantly reduced plasma IGF-1 levels. IGF-1 overexpression
significantly elevated plasma IGF-1 levels in young mice and nullified aging-induced decline in
plasma IGF-1 levels. Fasting blood glucose levels were not affected by either IGF-1 transgene or
age.
Mechanical properties of cardiomyocytes from young or aged FVB and IGF-1 mice
Mechanical properties obtained at a pace frequency of 0.5 Hz revealed that resting cell
length and PS amplitude were similar in ventricular myocytes among young and aged FVB and
IGF-1 mice with the exception of a reduced resting cell length in aged IGF-1 mice. However,
myocytes from aged FVB mice displayed significantly reduced maximal velocity of
shortening/relengthening (± dL/dt) compared with myocytes from young FVB mice. The reduced ±
dL/dt was associated with normal shortening duration (TPS90), prolonged relengthening duration
Page 7 of 25
Copyright Information
H-01036-2006
8
(TR90) and half-width duration (HWD) in aged FVB myocytes. Interestingly, these aging-induced
mechanical dysfunctions were not observed in aged IGF-1 transgenic mice, suggesting a beneficial
role of IGF-1 against aging-associated cardiac dysfunction. IGF-1 itself did not affect mechanical
contractile properties in young mice. To explore if IGF-1 transgene-elicited beneficial effect against
cardiac aging was due to IGF-1-induced cardiac hypertrophy or positive inotropicity as described
previously (20-22), cohorts of cardiomyocytes from young and aged FVB mice were treated in vitro
with recombinant IGF-1 (500 nM) for 2 hrs before mechanical property was recorded. Our data
revealed that IGF-1 treatment alleviated aging-induced decrease in ± dL/dt, prolongation in TR90
and HWD, in a manner similar to endogenous IGF-1 overexpression. In vitro IGF-1 treatment did
not significantly affect cardiomyocyte mechanical function in young mice with the exception of
significantly shortened HWD (Fig. 1).
Effect of increasing stimulation frequency on myocyte shortening
To evaluate the impact of aging on cardiac contractile function under various frequencies,
stimulus frequency was raised from 0.1 Hz to 5.0 Hz and the steady-state peak shortening was
recorded. Cells were initially stimulated to contract at 0.5 Hz for 5 min to ensure steady-state
before commencing the frequency response study. All recordings were normalized to PS at 0.1
Hz of the same myocyte. Fig. 2 shows a steeper negative staircase in PS in aged FVB myocytes
with increased stimulus frequency (at 0.5 Hz or higher) compared with young FVB myocytes,
suggesting reduced stress intolerance under advanced aging. Interestingly, IGF-1 transgene
ablated aging-induced stepper reduction in PS amplitude at high stimulating frequencies.
Protein expression of intracellular Ca2+ regulatory proteins, AGE and carbonyl formation
Page 8 of 25
Copyright Information
H-01036-2006
9
As depicted in Fig 3, Western blot analysis demonstrated a significantly reduced expression
of SERCA2a in aged FVB group which was nullified by IGF-1. IGF-1 itself did not affect the
expression of SERCA2a. Neither aging nor IGF-1 affected expression of Na+-Ca2+ exchanger and
phospholamban (monomer). IGF-1 but not aging significantly reduced the expression of pentamer
phospholamban. Our data further revealed that significantly elevated formation of AGE and protein
carbonyl in aged FVB mouse hearts. IGF-1 attenuated aging-elicited elevation of protein carbonyl
but not AGE. Interestingly, IGF-1 itself significantly reduced the levels of AGE and protein
carbonyl in young mouse hearts (Fig. 4).
Effect of IGF-1 on SERCA activity in young and aged mice
Consistent with reduced SERCA2a protein expression in aged FVB mouse hearts shown
in Fig. 3, direct measurement of SERCA activity using 45Ca uptake technique revealed that aging
significantly dampened SERCA activity which may be rescued by IGF-1. Similar to its effect on
SERCA2a protein expression, IGF-1 transgene itself did not affect SERCA activity in young
FVB mouse hearts (Fig. 5).
DISCUSSION
Our study revealed that cardiac overexpression of IGF-1 rescued aging-induced cardiac
contractile abnormalities. The IGF-1-induced cardiac protection against aging-induced cardiac
defects may be underscored by alteration in SERCA protein expression and function as well as
protein carbonyl formation. Furthermore, cardiac-specific overexpression of IGF-1 helped to
alleviate aging-induced decline in plasma IGF-1 levels. Since IGF-1 itself did not significantly
affect cardiac contractile function in young mice, its protective role against aging-induced
Page 9 of 25
Copyright Information
H-01036-2006
10
cardiac dysfunction may implicate certain clinical potential of this growth factor in delaying
cardiac aging process and minimize senescence-associated high cardiovascular mortality.
An ample of evidence has confirmed dysregulated cardiac function in senescence (4; 9;
13). Our results revealed reduced maximal velocity of contraction and relaxation, prolongation of
relaxation and half-width duration in aged FVB cardiomyocytes. We found that IGF-1 attenuated
aging-induced cardiac contractile dysfunction, SERCA expression/function and protein carbonyl
formation, indicating possible contribution of facilitated intracellular Ca2+ re-sequestration and
reduced protein damage from IGF-1 transgene. The fact that IGF-1 itself did not affect myocyte
function, SERCA expression and function in young mouse hearts indicates the excessive amount
of this growth factor is not innately harmful to cardiac function. It was demonstrated previously
that IGF-1 overexpressing transgenic mice exhibit protection against diabetes-induced cardiac
pathology, oxidative damage, activation of oncogene p53 and cardiac contractile dysfunction (7;
18). IGF-1 is known to trigger cardiac hypertrophy (20; 22), as evidenced by significantly
increased cardiac mass and heart-to-body weight ratio (Table 1). It is possible that the IGF-1
overexpression-associated changes in cardiomyocyte function and protein expression under
aging could be consequences of cardiac hypertrophy. Nonetheless, data from our short-term in
vitro treatment of recombinant IGF-1 indicated that IGF-1-induced cardiac hypertrophy is less
likely responsible for the IGF-1-elicited protective effect against cardiac aging. The observation
that short-term IGF-1 incubation did not significantly affect young cardiomyocyte contractile
function (with the exception of shortened HWD) is consistent with our previous report (14),
indicating a possibly sufficient IGF-1 levels in young cardiomyocytes. The fact that recombinant
IGF-1 short-term treatment shortened HWD without affecting TPS90 and TR90 in young
cardiomyocytes indicates that IGF-1 may predominantly facilitate late phase contraction and
Page 10 of 25
Copyright Information
H-01036-2006
11
early phase relaxation (which comprises HWD). However, such response possibly elicited by
acute IGF-1 treatment may be adapted with sustained high IGF-1 levels in IGF-1 overexpression
condition.
SERCA and NCX are considered as the main machineries to remove Ca2+ from cytosolic
space for cardiac relaxation to occur. However the relative contribution for intracellular Ca2+
extrusion by SERCA and NCX are still debatable, and may vary among different species (1; 5;
12). Bers indicated that SERCA is responsible for 92% Ca2+ removal while NCX only
contributes to <10% in mouse hearts (1). Our data revealed down-regulation of SERCA but not
NCX or phospholamban, an endogenous inhibitor of SERCA, in aged FVB hearts. The reduced
SERCA2a protein expression is supported by reduced SERCA activity in aged FVB hearts,
which may account for, at least in part, diastolic dysfunction (prolonged relaxation and half-
width duration) in aged myocytes. One rather surprising finding from our study was that IGF-1
transgene reduced phospholamban pentamer expression without affecting that of phospholamban
monomer. Although no precise mechanism regarding IGF-1-elicited response on phospholamban
subunit can be offered at this time, two scenarios may be considered. First, the phosphorylation
status of phospholamban may affect the affinity of phospholamban antibody (6). Although our
earlier study did not reveal phosphorylation of phospholamban with short-term treatment of IGF-
1 (14), the potential that sustained IGF-1 overexpression alters phospholamban phosphorylation
and thus antibody affinity for unphosphorylated phospholamban cannot be ruled out. Secondly, it
was depicted that phospholamban monomer rather than pentamer is deemed the “active” SERCA
inhibitor (23). Therefore, the IGF-1-elicited downregulation on phospholamban pentamer may be
non-specific and trivial to the overall effect of IGF-1 on SERCA function, given that
phospholamban monomer is unaffected by IGF-1.
Page 11 of 25
Copyright Information
H-01036-2006
12
IGF-1 is a peptide growth factor structurally and functionally similar to insulin. It is
synthesized by various cell types including cardiomyocytes and acts as an autocrine/paracrine
factor (20; 22). IGF-1 regulates myocardial growth and function under both physiological and
pathophysiological conditions. It improves myocardial function in post-infarction rat heart, in
patients with chronic heart failure and in healthy humans and experimental animals (2; 17; 22).
Advanced age is associated with insufficient production of GH levels and hepatic GH resistance.
This reduction in GH secretion is sufficient to cause a fall in the serum IGF-1 level, abnormal body
composition and metabolism (19). These changes are distinct from those associated with the
hyposomatotropism of the elderly, but are less severe than those seen in younger adults with organic
GH deficiency. Evidence has indicated that GH and/or IGF-I are involved in the regulation of
cardiovascular function. Patients with GH/IGF-1 deficiency displayed comparable cardiac
dysfunctions to aging, manifested primarily as reduced left ventricular mass, ejection fraction, and
diastolic filling. IGF-1 is believed to mediate, to a large extent, the action of GH in cardiac
growth and contraction. It improves cardiac contractility, tissue remodeling, glucose metabolism,
insulin sensitivity and lipid profile (22). Data from our present study suggest that cardiac-specific
overexpression of IGF-1, which may be secreted from cardiomyocytes into circulation (7; 20),
may compensate for the reduced circulating IGF-1 levels and subsequently impaired SERCA
expression/function under aging.
In conclusion, our study revealed that cardiac specific overexpression of IGF-1 rescues
aging-induced cardiomyocyte mechanical dysfunction possibly through improvement in SERCA
expression and function, in addition to its anti-protein damage capacity. It should be stated that
other known beneficial effect of IGF-1 including anti-oxidant property should not be ruled out at
Page 12 of 25
Copyright Information
H-01036-2006
13
this time. These data have convincingly demonstrated the clinical potential of IGF-1 in the
prevention and treatment of aging-associated cardiac dysfunction.
ACKNOWLEDGMENTS
The authors would like to thank Bonnie H. Zhao and Nicholas S. Aberle II for their
assistance in data acquisition and analysis. This work was supported in part by National Institute
of Health grants to JR (NIA: 1 R03 AG21324-01 and NIAAA: 1R15 AA13575-01). This work
was presented in the 15th World Congress of Pharmacology in Beijing, P.R. China in July 2006.
No disclosure is declared from any of the authors.
Page 13 of 25
Copyright Information
H-01036-2006
14
FIGURE LEGENDS
Fig. 1: Contractile properties of cardiomyocytes from young or old FVB and IGF-1 transgenic
mice. A cohort of cardiomyocytes from young and old FVB mice was treated in vitro with IGF-1
(500 nM) for 2 hrs prior to assessment of mechanical function. A: Resting cell length; B: Peak
shortening (PS, normalized to resting cell length); C: Half-width duration (HWD); D: Maximal
velocity of shortening/relengthening (± dL/dt); E: Time-to-90% PS (TPS90); and F: Time-to-90%
relengthening (TR90). Mean ± SEM, n = 62-64 cells/group, * p < 0.05 vs. FVB young group, # p
< 0.05 vs. FVB-old group.
Fig. 2: Peak shortening (PS) amplitude of cardiomyocytes from young or old FVB and IGF-1
mice at different stimulus frequencies (0.1 – 5.0 Hz). PS was shown as % change from PS at 0.1
Hz of the same cell. Mean ± SEM, n = 38 – 40 cells per group, * p < 0.05 vs. FVB young group,
# p < 0.05 vs. FVB-old group.
Fig. 3: Western blot analysis exhibiting expression of SERCA2a, Na+-Ca2+ exchanger (NCX)
and phospholamban (PLB) in ventricles from young and old FVB and IGF-1 mice. A: SERCA2a
expression; B: Na+-Ca2+ exchanger expression, C: phospholamban monomer expression; and D:
phospholamban pentamer expression. Inset: Represent gel blots depicting expression of above
mentioned proteins using specific antibodies. Mean ± SEM, n = 6 -8 samples per group, * p <
0.05 vs. FVB young group, # p < 0.05 vs. FVB-old group.
Fig. 4: Western blot analysis exhibiting expression of AGE (panel A) and protein carbonyl
(Panel B) in ventricles from young and old FVB and IGF-1 mice. Inset: Represent gel blots
Page 14 of 25
Copyright Information
H-01036-2006
15
depicting expression of above mentioned proteins using specific antibodies. Mean ± SEM, n = 5
-8 samples per group, * p < 0.05 vs. FVB young group, # p < 0.05 vs. FVB-old group.
Fig. 5: SERCA2 activity measured by SERCA-dependent 45Ca2+ uptake in ventricles from young
and old FVB and IGF-1 mice. Mean ± SEM, n = 6, * p < 0.05 vs. FVB young group, # p < 0.05
vs. FVB-old group.
Page 15 of 25
Copyright Information
H-01036-2006
16
Table I: General features of young- and old-FVB and IGF-1 mice.
FVB-young FVB-old IGF-1-young IGF-1-old
Body Weight (g) 19.1 ± 0.5 28.7 ± 0.8* 20.7 ± 0.9 30.2 ± 0.6*
Heart Weight (mg) 98 ± 4 156 ± 8* 126 ± 6** 185 ± 10*,**
HW/BW (mg/g) 5.12 ± 0.18 5.31 ± 0.22 6.12 ± 0.17** 6.19 ± 0.19**
Liver Weight (g) 0.89 ± 0.03 1.51 ± 0.07* 0.98 ± 0.05 1.40 ± 0.06*
LW/BW (mg/g) 47.6 ± 1.4 51.3 ± 1.2 47.6 ± 1.3 46.1 ± 1.4
Kidney Weight (g) 0.23 ± 0.01 0.42 ± 0.02* 0.25 ± 0.02 0.45 ± 0.02*
KW/BW (mg/g) 12.2 ± 0.4 14.6 ± 0.6* 12.1 ± 0.4 15.0 ± 0.5*
Blood glucose (mg/dl) 97.0 ± 5.6 99.1 ± 6.4 95.2 ± 6.4 100.8 ± 7.0
Plasma IGF-1 (ng/ml) 101.8 ± 6.0 48.6 ± 4.2* 154.4 ± 5.3** 87.3 ± 5.7**
HW = heart weight; LW = liver weight; KW = kidney weight; Mean ± SEM, * p < 0.05 vs.
corresponding young group, ** p < 0.05 vs. corresponding FVB group n = 17 – 23 mice/group.
Page 16 of 25
Copyright Information
H-01036-2006
17
Li et al., Fig. 1
A. B.
0
25
50
75
100
125
Re
sti
ng
Ce
llL
en
gth
(µm
)
FVB-Young
FVB-Old
IGF-O
ld
IGF-Y
oung
* #
FVB-Young
FVB-Old
+ IGF-1 (500 nM)
0
2
4
6
8
10
12
Pea
kS
ho
rten
ing
(%C
ellL
eng
th)
FVB-You
ng
FVB-Old
IGF-O
ld
IGF-Y
oung
FVB-Y
oung
FVB-Old
+IGF-1 (500 nM)
0
100
200
300
400
Hal
fW
idth
Du
rati
on
(mse
c )
FVB-Youn
g
FVB-Old
IGF-O
ld
IGF-Y
oung
*
* *
#
FVB-Young
FVB-O
ld
+IGF-1 (500 nM)
-150
-100
-50
0
50
100
150
+/-
dL
/dt
(µm
/sec
)
FVB-Youn
g
FVB-O
ld
*
IGF-
Old
IGF-Y
oung
*
#
##
FVB-Y
oung
FVB-Old
+IGF-1 (500 nM)
0
50
100
150
200
TP
S90
(mse
c)
FVB-You
ng
FVB-Old
IGF-O
ld
IGF-Y
oung
FVB-Y
oung
FVB-Old
+IGF-1 (500 nM)
C. D.
E. F.
0
50
100
150
200
250
300
TR
90
(mse
c)
FVB-Y
oung
FVB-O
ld
#
IGF-O
ld
IGF-Y
oung
*
#
FVB-Y
oung
FVB-Old
+IGF-1 (500 nM)
Page 17 of 25
Copyright Information
H-01036-2006
18
0.5 1.0 3.0 5.0-90
-60
-30
0
%C
hang
efr
omC
ontr
ol(0
.1H
z)
Stimulus Frequency (Hz)
FVB-Young FVB-Old
*
*
*
*
IGF-Young IGF-Old
#
# #
Li et al., Fig. 2
Page 18 of 25
Copyright Information
H-01036-2006
19
Li et al., Fig. 3
0
0.5
1.0
SE
RC
A2a
(Arb
itra
ryD
en
sity
)
FVB-Young
FVB-Old
*
IGF-Y
oung
IGF-O
ld0
0.2
0.4
0.6
Na+
-Ca2+
exc
han
ge
r(A
rbit
rary
De
nsi
ty)
FVB-Young
FVB-Old
*
IGF-Y
oung
IGF-O
ld
0
0.2
0.4
0.6
0.8
PL
B-6
Exp
ress
ion
(Arb
itra
ryD
en
sity
)
FVB-Young
FVB-Old
IGF-O
ld
IGF-Y
oung
*
A. B.
C. D.
0
0.5
1.0
1.5
FVB-Young
FVB-Old
IGF-O
ld
IGF-Y
oung
*
**
PL
B-3
0E
xpre
ssio
n(A
rbit
rary
De
nsi
ty)
SERCA2a 110KD
6KDPLB 30KDPLB
120KDNCX
Page 19 of 25
Copyright Information
H-01036-2006
20
Li et al., Fig. 4
A.
0
0.2
0.4
0.6
0.8
To
talA
GE
s(A
rbit
rary
De
nsi
ty)
FVB-Young
FVB-Old
*
IGF-O
ld
IGF-Y
oung
#
*
0
0.5
1.0
1.5
2.0
FVB-Young
FVB-Old
IGF-O
ld
IGF-Y
oung
*
#
Pro
tein
Car
bo
nyl
(Arb
itra
ryD
en
sity
)
B.
AGE100 KD50 KD37 KD
Protein Carbonyl
98 KD
64 KD
50 KD
36 KD
Page 20 of 25
Copyright Information
H-01036-2006
21
FVB-Young
1SE
RC
A a
ctiv
ity-
-[45
]Ca2+
up
take
(CP
M/ m
g p
rote
in/ 3
0 m
in)
0
2000
4000
6000
8000
10000
12000
FVB-Old
IGF-Y
oung
IGF-O
ld
*
Li et al., Fig. 5
Page 21 of 25
Copyright Information
H-01036-2006
22
Reference List
1. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198-205, 2002.
2. Delafontaine P and Brink M. The growth hormone and insulin-like growth factor 1 axis
in heart failure. Ann Endocrinol (Paris) 61: 22-26, 2000.
3. Duan J, McFadden GE, Borgerding AJ, Norby FL, Ren BH, Ye G, Epstein PN and
Ren J. Overexpression of alcohol dehydrogenase exacerbates ethanol-induced contractile
defect in cardiac myocytes. Am J Physiol Heart Circ Physiol 282: H1216-H1222, 2002.
4. Fleg JL, Shapiro EP, O'Connor F, Taube J, Goldberg AP and Lakatta EG. Left
ventricular diastolic filling performance in older male athletes. JAMA 273: 1371-1375,
1995.
5. Hove-Madsen L and Bers DM. Sarcoplasmic reticulum Ca2+ uptake and thapsigargin
sensitivity in permeabilized rabbit and rat ventricular myocytes. Circ Res 73: 820-828,
1993.
6. Huke S and Periasamy M. Phosphorylation-status of phospholamban and calsequestrin
modifies their affinity towards commonly used antibodies. J Mol Cell Cardiol 37: 795-
799, 2004.
7. Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F,
Nadal-Ginard B, Leri A and Anversa P. IGF-1 overexpression inhibits the
development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress.
Diabetes 50: 1414-1424, 2001.
Page 22 of 25
Copyright Information
H-01036-2006
23
8. Kass DA, Shapiro EP, Kawaguchi M, Capriotti AR, Scuteri A, deGroof RC and
Lakatta EG. Improved arterial compliance by a novel advanced glycation end-product
crosslink breaker. Circulation 104: 1464-1470, 2001.
9. Lakatta EG. Cardiovascular aging research: the next horizons. J Am Geriatr Soc 47:
613-625, 1999.
10. Lakatta EG. Cardiovascular aging in health. Clin Geriatr Med 16: 419-444, 2000.
11. Lakatta EG, Mitchell JH, Pomerance A and Rowe GG. Human aging: changes in
structure and function. J Am Coll Cardiol 10: 42A-47A, 1987.
12. Li L, Chu G, Kranias EG and Bers DM. Cardiac myocyte calcium transport in
phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am J
Physiol 274: H1335-H1347, 1998.
13. Li SY, Du M, Dolence EK, Fang CX, Mayer GE, Ceylan-Isik AF, LaCour KH, Yang
X, Wilbert CJ, Sreejayan N and Ren J. Aging induces cardiac diastolic dysfunction,
oxidative stress, accumulation of advanced glycation endproducts and protein
modification. Aging Cell 4: 57-64, 2005.
14. Li SY, Fang CX, Aberle NS, Ren BH, Ceylan-Isik AF and Ren J. Inhibition of PI-3
kinase/Akt/mTOR, but not calcineurin signaling, reverses insulin-like growth factor I-
induced protection against glucose toxicity in cardiomyocyte contractile function. J
Endocrinol 186: 491-503, 2005.
Page 23 of 25
Copyright Information
H-01036-2006
24
15. Li SY, Yang X, Ceylan-Isik AF, Du M, Sreejayan N and Ren J. Cardiac contractile
dysfunction in Lep/Lep obesity is accompanied by NADPH oxidase activation, oxidative
modification of sarco(endo)plasmic reticulum Ca(2+)-ATPase and myosin heavy chain
isozyme switch. Diabetologia 49: 1434-1446, 2006.
16. Li SY, Yang X, Ceylan-Isik AF, Du M, Sreejayan N and Ren J. Cardiac contractile
dysfunction in Lep/Lep obesity is accompanied by NADPH oxidase activation, oxidative
modification of sarco(endo)plasmic reticulum Ca(2+)-ATPase and myosin heavy chain
isozyme switch. Diabetologia 49: 1434-1446, 2006.
17. Lombardi G, Di Somma C, Marzullo P, Cerbone G and Colao A. Growth hormone
and cardiac function. Ann Endocrinol (Paris) 61: 16-21, 2000.
18. Norby FL, Aberle NS, Kajstura J, Anversa P and Ren J. Transgenic overexpression
of insulin-like growth factor I prevents streptozotocin-induced cardiac contractile
dysfunction and beta-adrenergic response in ventricular myocytes. J Endocrinol 180:
175-182, 2004.
19. Paolisso G, Ammendola S, Del Buono A, Gambardella A, Riondino M, Tagliamonte
MR, Rizzo MR, Carella C and Varricchio M. Serum levels of insulin-like growth
factor-I (IGF-I) and IGF-binding protein-3 in healthy centenarians: relationship with
plasma leptin and lipid concentrations, insulin action, and cognitive function. J Clin
Endocrinol Metab 82: 2204-2209, 1997.
20. Reiss K, Cheng W, Ferber A, Kajstura J, Li P, Li B, Olivetti G, Homcy CJ, Baserga
R and Anversa P. Overexpression of insulin-like growth factor-1 in the heart is coupled
Page 24 of 25
Copyright Information
H-01036-2006
25
with myocyte proliferation in transgenic mice. Proc Natl Acad Sci U S A 93: 8630-8635,
1996.
21. Ren J. Attenuated cardiac contractile responsiveness to insulin-like growth factor I in
ventricular myocytes from biobreeding spontaneous diabetic rats. Cardiovasc Res 46:
162-171, 2000.
22. Ren J, Samson WK and Sowers JR. Insulin-like growth factor I as a cardiac hormone:
physiological and pathophysiological implications in heart disease. J Mol Cell Cardiol
31: 2049-2061, 1999.
23. Zhao W, Yuan Q, Qian J, Waggoner JR, Pathak A, Chu G, Mitton B, Sun X, Jin J,
Braz JC, Hahn HS, Marreez Y, Syed F, Pollesello P, Annila A, Wang HS, Schultz
JJ, Molkentin JD, Liggett SB, Dorn GW and Kranias EG. The presence of Lys27
instead of Asn27 in human phospholamban promotes sarcoplasmic reticulum Ca2+-
ATPase superinhibition and cardiac remodeling. Circulation 113: 995-1004, 2006.
Page 25 of 25
Copyright Information
Top Related