(Circulation. 2000;101:790.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Restoration of Diastolic Function in Senescent Rat Hearts Through Adenoviral Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase
From the Cardiovascular Research Center and the Cardiology Division (F.d.M., M.I.M., T.M., A.R., R.J.H.) and Anesthesia Department (U.S.), Harvard Medical School and Massachusetts General Hospital; and Boston University School of Medicine (J.K.G.), Boston, Mass.
Correspondence to Roger J. Hajjar, MD, Cardiovascular Research Center, Massachusetts General Hospital, 149 13th St, CNY-4, Boston, MA 02129. E-mail hajjar{at}cvrc.mgh.harvard.edu
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Abstract |
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Background—Senescent hearts are characterized by diastolic dysfunction and a decrease in sarcoplasmic reticulum (SR) Ca2+-ATPase protein (SERCA2a).
Methods and Results—To test the hypothesis that an increase in
SERCA2a could improve cardiac function in senescent rats (age 26
months), we used a catheter-based technique of adenoviral gene transfer
to achieve global myocardial transduction of SERCA2a in vivo. Adult rat
hearts aged 6 months and senescent rat hearts infected with an
adenovirus containing the reporter gene ß-galactosidase were
used as controls. Two days after infection, parameters of
systolic and diastolic function were measured in
open-chest rats. Cardiac SERCA2a protein and ATPase activity were
significantly decreased in senescent hearts compared with adult rats
(
-30±4% and -49±5%) and were restored to adult levels after
infection with Ad.SERCA2a. At baseline, left ventricular
systolic pressure and +dP/dt were unaltered in senescent
hearts; however, diastolic parameters were
adversely affected with an increase in the left ventricular
time constant of isovolumic relaxation and diastolic
pressure ( +29±9% and +38±12%) and a decrease in -dP/dt (
-26±11%). Overexpression of SERCA2a did not significantly affect
left ventricular systolic pressure but did increase
+dP/dt ( +28±10%) in the senescent heart. Overexpression of
SERCA2a restored the left ventricular time constant of
isovolumic relaxation and -dP/dt to adult levels. Infection of
senescent hearts with Ad.SERCA2a markedly improved rate-dependent
contractility and diastolic function in
senescent hearts.
Conclusions—These results support the hypothesis that decreased Ca2+-ATPase activity contributes to the functional abnormalities observed in senescent hearts and demonstrates that Ca2+ cycling proteins can be targeted in the senescent heart to improve cardiac function.
Key Words: aging • gene therapy • heart failure • sarcoplasmic reticulum • diastole
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Introduction |
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Congestive heart failure is becoming an increasingly prevalent clinical problem in the United States and now is the most frequent cause of hospitalization in people older than 65 years.1 Indeed, mortality rates from congestive heart failure continue to increase in elderly populations.1 Interestingly, up to 40% of patients older than 60 years who have symptoms of congestive heart failure have normal systolic function and abnormal diastolic function. In mammalian hearts, aging is associated with impaired cardiac relaxation.2 3 Senescent myocytes are characterized by prolonged relaxation, diminished contraction velocity, a decrease in ß-adrenergic response, and increased myocardial stiffness.4 This impairment in diastolic function contributes to the increased incidence in congestive heart failure in the elderly.5 A number of cellular and molecular mechanisms may contribute to the age-related defects. The abnormalities in cardiac relaxation have been attributed to a defect in sarcoplasmic reticulum (SR) Ca2+-ATPase pump activity, which is mainly responsible for controlling the rate at which Ca2+ is taken up into the SR during relaxation.6 7 8 The decrease in SR Ca2+ uptake during relaxation, which results in prolonged contraction, has been associated with a decrease in the content and activity of the SR Ca2+-ATPase pump in experimental models of senescence.9 More recently, SERCA2a protein levels were found to be significantly decreased in senescent human myocardium.10 This decrease in SERCA2a levels was associated with impaired myocardial function at baseline and was further accentuated by hypoxic conditions.10
Because a number of key steps of excitation-contraction coupling are altered during normal aging, it is not clear whether a decrease in the number of SR Ca2+ pumps constitutes an adaptive or a pathological process. We have recently shown that the overexpression of SERCA2a in vitro through adenoviral gene transfer results in increased contractility and a faster relaxation rate of the Ca2+ transient.11 12 These results were further confirmed in transgenic animals with SERCA2a overexpression.13 More recently, we developed a catheter-based technique to deliver adenoviruses to the heart in a homogeneous fashion.14 In addition, we used this approach to demonstrate profound physiological effects on cardiac function specifically mediated by the transduced gene.
The goal of the present study was to test the hypothesis that the restoration of the SR Ca2+-ATPase through adenoviral gene transfer in the aging heart will normalize diastolic function in vivo.
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Methods |
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Construction of Recombinant Adenoviruses
Two first-generation type 5 recombinant adenoviruses were used in these studies: Ad.ß-galactosidase (Ad.ß-Gal) and Ad.SERCA2a. Ad.ß-Gal was generously provided by Dr David Dicheck (Gladstone Institute for Cardiovascular Disease).15 The construction of Ad.SERCA2a has been described previously, and the recombinant viruses were prepared as high titer stocks through the propagation in 293 cells as described previously.11 16 The titer of stocks used for the present study measured with the use of plaque assays was 9x1010 pfu/mL for Ad.ß-Gal and 10x1011 pfu/mL for Ad.SERCA2a with a particle-to-pfu ratio of 25:1 and 40:1, respectively (viral particles/mL were determined with the relation of 1 absorbance unit at 260 nm being equal to 1012 viral particles/mL).
Experimental Protocol
Both 26-month old (senescent) and 6-month old (adult) male
Fisher 344 rats obtained from the National Aging Institute were
studied. Animals were divided into 4 groups: (1) 8 uninfected,
sham-operated senescent rats, (2) 8 senescent rats infected with
1010 pfu Ad.ß-Gal, (3) 8 senescent rats
infected with 1010 pfu Ad.SERCA2a, and (4) 8
uninfected sham-operated adult rats.
Adenoviral Delivery Protocol
The delivery of adenovirus was described previously by our group
in detail.14 Briefly, rats were anesthetized with
intraperitoneal pentobarbital (60 mg/kg) and placed
on a ventilator. The chest was entered from the left side through the
third intercostal space. A 22-gauge catheter containing 200 mL
adenovirus was advanced from the apex of the left ventricle (LV) to the
aortic root. The aorta and pulmonary arteries were clamped
distal to the site of the catheter, and the solution was injected. The
clamp was maintained for 10 seconds while the heart pumped against a
closed system (isovolumically). After 10 seconds, the clamp on the
aorta and pulmonary artery was released, the chest was closed,
and the animals were extubated and transferred back to their cages.
Pressure Measurements
Rats in the different treatment groups were anesthetized
with 60 mg/kg pentobarbital and mechanically ventilated. The chest was
then opened, and a 1.8F high-fidelity pressure transducer (Millar
Instruments) was introduced into the LV. LV systolic pressure
(LVSP), LV end-diastolic pressure, the maximal rates of
pressure rise (+dP/dt) and of pressure fall (-dP/dt), and the time
constant of isovolumic relaxation (
) were measured or derived in the
different groups. The time course of isovolumic relaxation was measured
with the following equation:
P=Poe-t/t+PB,
where P is LV isovolumic pressure, Po is pressure
at the time of peak -dP/dt, and PB is residual
pressure. A pair of 0.7-mm piezoelectric crystals (Sonometrics Co) were
attached on the LV epicardium across the short axis of the ventricle at
the level of the mitral valve to obtain intercrystal distances. LV
pressure-dimension loops were generated under different loading
conditions by transient occlusions of the inferior vena
cava. The end-systolic pressure-dimension relation was obtained
through the production of a series of pressure-dimension loops
during transient inferior vena cava occlusion and through
linear regression analysis of the end-systolic points
of the individual pressure-dimension loops to generate the
end-systolic elastance.
Isolation of Single Cardiac Myocytes
Ca2+-tolerant adult rat
ventricular myocytes were isolated as described
previously.17 The isolated myocytes were then fixed with
0.5% glutaraldehyde for 15 minutes and stained with a
solution containing 5-bromo-4-chloro-3-indolyl-ß-D
galactopyranoside (X-Gal) for 2 hours. To quantify the percentage of
cells with blue nuclei, we counted 100 cells from each heart
preparation.
Preparation of SR Membranes
We isolated SR membranes from the LVs of hearts as described
previously.14 Briefly, LV myocardium was
suspended in a buffer containing 300 mmol/L sucrose, 1 mmol/L
PMSF, and 20 mmol/L PIPES, at pH 7.4, that was
homogenized and centrifuged at 25 000g
for 60 minutes. The pellet was resuspended in a buffer containing
600 mmol/L KCl, 30 mmol/L sucrose, and 20 mmol/L PIPES;
frozen in liquid nitrogen; and stored at -70°C. Protein
concentration was determined in these preparations according to a
modification of the method of Bradford (Bio-Rad).
Western Blot Analysis
SDS-PAGE was performed on the isolated membranes from cell
cultures under reducing conditions on a 7.5% separation gel with a 4%
stacking gel in a Miniprotean II cell (Bio-Rad). For immunoreaction,
the blot was incubated with 1:2500 diluted monoclonal antibodies to
SERCA2a (MA3-919), Na+/Ca2+
exchanger (MA3-926), or ryanodine receptors (MA3-925; Affinity
BioReagents) or with 1:2500 diluted anti-cardiac phospholamban
monoclonal IgG (Upstate Biotechnology) for 90 minutes at room
temperature. The densities of the immunoreactive bands were evaluated
with the use of NIH Image. Normalization was performed by dividing
densitometric units of each membrane preparation by the protein amounts
in each of the preparations.
SR Ca2+-ATPase Activity
SR Ca2+-ATPase activity assays were
carried out based on a pyruvate/NADH coupled reactions as described
previously.11 12 With a photometer (DU 640; Beckman)
adjusted at a wavelength of 340 nm, oxidation of NADH (which is coupled
to the SR Ca2+-ATPase) was assessed at 37°C in
the membrane preparations according to the difference between the total
absorbance and basal absorbance. The activity of the
Ca2+-ATPase was calculated as
Absorbance/6.22xproteinxtime (in nmol ATP · mg
protein-1 · min-1).
Statistical Analysis
All values are presented as mean±SD. ANOVA was used to
calculate statistical differences among the different groups, and ANOVA
for repeated measures was used where appropriate. Statistical
significance was accepted at the level of P<0.05.
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Results |
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Cardiac Gene Transfer
We have previously reported that the catheter-based adenoviral technique induces global gene transfer in rat hearts with an expression pattern that is grossly homogeneous throughout the ventricles. Because these results were obtained in adult rats, we quantified the efficiency of gene transfer in senescent rat hearts. We isolated single cardiomyocytes from adult and senescent rat hearts after in vivo transduction with Ad.ß-Gal. As shown in Figure 1
, the majority of
cardiomyocytes demonstrate specific nuclear ß-Gal
activity, reflecting transgene expression in both adult and senescent
rat hearts, 2 days after in vivo transduction with Ad.ß-Gal (72±3%,
n=3 young adult; 61±2%, n=4 senescent; P<0.05).
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Quantification of Ca2+ Regulatory Proteins
To assess SERCA2a protein content in the different groups, we
performed quantitative immunoblotting. The intensity of
the labeled SERCA2a band was proportional to the amount of cardiac
membrane protein electrophoresed in the range of 5 to 50 µg (Figures 2A and 2B). The protein content of
SERCA2a was significantly decreased in 26-month-old rats compared with
6-month-old rats as shown in Figures 2C and 2D. On the other
hand, other Ca2+ regulatory proteins, such as
phospholamban, Na+/Ca2+
exchanger, and ryanodine receptors, were unchanged in the senescent
ventricles. Adenoviral gene transfer of Ad.SERCA2a induced a
significant increase in SERCA2a content in the senescent hearts
restoring its expression level to adult levels.
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, n=3) versus adult (•, n=3) hearts.
C, Immunoblots of SERCA2a, phospholamban (PLB),
Na+/Ca2+ exchanger, and ryanodine receptor from
crude membranes of LVs from adult, senescent uninfected rats, and
senescent rats 2 days after infection with either Ad.ß-Gal or
Ad.SERCA2a. D, Protein levels of SERCA2a in preparations from adult
uninfected rat hearts (n=6), preparations from senescent uninfected rat
hearts (n=8), preparations of senescent hearts infected with Ad.ß-Gal
at day 2 (n=6), and preparations of senescent hearts infected with
Ad.SERCA2a at day 2 (n=6). *P<0.05 compared with adult.
P<0.05 compared with senescent group plus
Ad.ß-Gal.
SR Ca2+-ATPase Activity
Senescent hearts had a significant decrease in
Ca2+-ATPase activity compared with adult hearts
(Figure 3). Adenoviral gene transfer with
Ad.ß-Gal did not alter Ca2+-ATPase activity in
the senescent heart. However, adenoviral gene transfer of Ad.SERCA2a
induced a significant increase in Ca2+-ATPase
activity in the senescent hearts, increasing it by nearly 2-fold to
levels comparable to those seen in adult animals.
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Hemodynamic Effects of SERCA2a Overexpression in
Senescent Hearts
As shown in Figures 4 and 5, the systolic
parameters were not altered in the senescent rat hearts
compared with adult hearts. However, overexpression of SERCA2a
increased the rate of rise of pressure but did not alter
systolic pressure. Diastolic parameters
were significantly altered in the senescent rat hearts compared with
adult hearts as demonstrated by a decrease in the maximal rate of
decline of LVSP, an increase in diastolic pressure, and a
significantly prolonged time course of pressure decline () (Figures 4 and 5). The overexpression of SERCA2a normalized both
the maximal rate of decline of LVSP and the time course of pressure
decline (); however, it did not restore diastolic
pressure back to adult levels.
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Pressure-Dimension Relationship
To further assess systolic function, pressure-dimension
analysis was performed in a subset of animals. Figure 6A shows pressure-dimension relations in
an adult heart compared with a senescent heart. Even though the slope
of the end-systolic pressure dimension relation was similar in
adult and senescent rat hearts (137±21 versus 118±31 mm Hg/mm,
n=4, P>0.1), the pressure-dimension relations were shifted
toward higher ventricular dimensions. However, as shown in
Figure 6B, the slope of the end-systolic pressure
dimension relation was higher in senescent rat hearts overexpressing
SERCA2a compared with senescent rat hearts infected with Ad.ß-Gal
(154±24 versus 112±28 mm Hg/mm, n=4, P<0.03).
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Effect on Contractile Reserve
To test whether SERCA2a overexpression augmented inotropic
reserve, we studied the effect of incremental atrial pacing on
hemodynamic parameters in adult and
senescent hearts. As shown in Figure 7, +dP/dt normalized to end-diastolic dimension (+dP/dt/LVEDD)
increased with a rise in heart rate, but this rate-dependent response
was significantly blunted in the senescent hearts. Overexpression of
SERCA2a in the senescent hearts induced an increase in +dP/dt/LVEDD at
baseline, and with increasing frequency, there was a further increase
in the maximal rate of pressure rise that paralleled the adult
situation. Atrial pacing resulted in a significantly higher increase in
diastolic pressure in senescent hearts compared with adult
hearts. Overexpression of SERCA2a attenuated the rate-dependent
increase in diastolic pressure but did not restore it to
adult levels.
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, n=6). |
Discussion |
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Functionally, aging myocardium is characterized by prolonged relaxation and action potential duration, diminished contraction velocity and ß-adrenergic response, and increased myocardial stiffness.4 18 19 20 21 22 Important cellular and molecular alterations underlie the functional abnormalities of aging myocardium; they include (1) a decrease in SR Ca2+ uptake during relaxation that is associated with a decrease in SERCA2a content, (2) diminished ß1-receptor density and uncoupling of the adrenergic receptor from adenylyl cyclase, resulting in a decrease in cAMP levels, (3) a reduction in the inactivation rate of the Ca2+ current and a decrease in the magnitude of the outwardly directed K+ current, resulting in a prolonged action potential, and (4) an age-associated (and species- dependent [occurring only in rodents]) change from the V1 isoform of the myosin heavy chain (
-MHC) to
the V3 isoform (ß-MHC), resulting in a decrease
in myosin ATPase activity.4 21 22 These age-related
changes of the various excitation-contraction coupling steps have been
widely implicated in the mechanical alterations of cardiac function in
senescence and can individually or in combination contribute to the
abnormal relaxation and diastolic function observed in
senescent myocardium. Studies have consistently
shown that the Ca2+ transient and the
simultaneously measured isometric contraction or shortening
are significantly prolonged in senescent
cardiomyocytes.5 Because SR
Ca2+-ATPase controls the rate at which
Ca2+ is taken up and is responsible for reuptake
of most of the Ca2+ into the SR, these
abnormalities in Ca2+ handling in the aged
myocardium have led to a close examination of the
regulation and function of the SR Ca2+-ATPase in
aged myocardium. We found that there was a 
40% decrease
in SERCA2a protein content in senescent myocardium compared
with adult myocardium in this rat model. This decrease in
expression was also associated with a decrease in the ATPase activity
in the senescent hearts. These results are consistent with
prior studies showing that a stable level of SERCA2a mRNA is maintained
during adulthood until 20 to 24 months, when a 40% to 50% decrease
occurs.23 24 More recently, SERCA2a protein levels were
found to be significantly decreased in senescent human
myocardium.10 However, our results have also
shown that there is no change during the aging process in the content
of other key Ca2+ cycling proteins:
phospholamban, the ryanodine receptors, and calsequestrin. Recently, we developed a catheter-based technique to deliver adenoviruses to the heart. This results in a homogeneous expression throughout the ventricle. We have used this approach to demonstrate profound physiological effects on cardiac function specifically mediated by the transduced gene.11 With this method, we were able to achieve significant expression as demonstrated by the high percentage of isolated cardiomyocytes being transduced by the reporter adenovirus Ad.ß-Gal. Interestingly, we found that the efficiency of adenoviruses at transduction of myocardial cells is lower in senescent rats hearts than in adult rat hearts. Whether this is due to a differential expression of the common receptor for coxsackievirus B and adenoviruses 2 and 525 or to altered viral DNA processing in senescent cardiac cells is unknown. However, even though adenoviral gene transfer is less efficient in senescent hearts, it does achieve a high level of myocardial infectivity, and most importantly, it is sufficient to induce protein expression, resulting in functional changes. The overexpression of SERCA2a in this model of senescence resulted in the restoration of SERCA2a protein to levels comparable to those of adult rats. Furthermore, this increase in SERCA2a content resulted in an enhancement in the SR Ca2+-ATPase activity in the senescent rat hearts. In previous studies, we and others have shown that increasing levels of SERCA2a in isolated cardiomyocytes shortened the relaxation phase of the Ca2+ transient, increased Ca2+ release, and decreased resting Ca2+.12 26 27 However, it was not clear whether these effects could be translated in the in vivo setting. In the present study, overexpression of SERCA2a resulted in an increase in +dP/dt, a decrease in the time constant for isovolumic relaxation, and an increase in both the maximal rate of rise and the maximal rate of fall of LVSP. Isovolumic relaxation, which is an index of active relaxation and reflects the removal of Ca2+ from the myofilaments into the SR, was significantly prolonged in the senescent hearts and was restored to adult levels through the overexpression of SERCA2a. The overexpression of SERCA2a did not affect LVSP in the senescent hearts. However, the load-independent parameter of LV contractility, such as the slope of the end-systolic pressure dimension relation, end-systolic elastance, was significantly increased through the overexpression of SERCA2a. Interestingly, diastolic pressure was not decreased in the senescent hearts through the overexpression of SERCA2a.
An age-associated decrease in the cardiovascular response to pacing has been well documented in the elderly and in experimental models of senescence.5 The response to increased atrial pacing was blunted in the senescent hearts compared with the adult hearts. Overexpression of SERCA2a restored to a great degree the frequency response in these senescent hearts. These results again point to a deficiency in SR Ca2+ handling in senescent hearts that is not apparent at rest but becomes relevant at higher frequencies of stimulation.
A transgenic approach to overexpression of SERCA2a has been undertaken in both mice and rats13 27 28 ; however, adenoviral transduction offers several advantages. In transgenic animals overexpressing SERCA2a, developmental adaptation to higher levels of SERCA2a occurs with upregulation of other important excitation-contraction proteins, such as phospholamban and the Na+-Ca2+ exchanger, thereby masking or diluting the effects of transgene overexpression. These compensatory alterations make it difficult to assess the specific effect of increased SERCA2a on cardiac function. In the present study, overexpression of SERCA2a did not significantly alter protein expression of the ryanodine Ca2+-releasing channels, SERCA2a, Na+/Ca2+ exchanger, or calsequestrin, all of which are involved in intracellular Ca2+ handling. However, adenoviruses have significant disadvantages, including the transient nature of overexpression of the desired gene and the immune/inflammatory response they produce, which were also present in our infected hearts. However, in older rats, there is an overall reduction in the immune response that may prolong gene expression after adenoviral infection, thereby validating the use of gene therapy in the elderly.
There are a number of limitations of the study that must be addressed. The open-chest model that we used is subject to the effects of anesthesia on cardiovascular function, both directly and via reflex responses to stress.29 Our hemodynamic measurements, however, were well within the range of those observed in previous studies of open-chest rats. For the measurement of cardiac function, LV short-axis epicardial dimensions were used. Although endocardial LV cavity dimensions would have been preferable, given the size of the piezoelectric crystal (0.7 mm diameter) relative to the normal rat LV wall thickness (1 to 2 mm), such measurements are not currently feasible. Our data add to the accumulating body of rodent physiological data and support the feasibility and applicability of LV pressure-dimension analysis in the phenotypic characterization of genetically altered models.30 31
Our results suggest that the decrease in SERCA2a content and associated decrease in SR Ca2+ uptake play a major role in the diastolic dysfunction observed in the elderly. Exercise training has also been shown to improve SR Ca2+ uptake and to improve systolic and diastolic parameters in senescent rats. However, an improvement in cardiac function after long-term exercise cannot be entirely ascribed to the observed SERCA2a changes. In the present study, the beneficial cardiac effects resulted from the somatic gene transfer of SERCA2a.
Our physiological data demonstrate the feasibility of achieving important functional cardiac effects through in vivo somatic gene transfer in a rodent model of senescence. This study demonstrates that the overexpression of SERCA2a through adenoviral gene transfer in senescent rat hearts improves diastolic function and restores contractile reserve. Therefore, targeting the SR Ca2+-ATPase pump may be an important strategy to improve diastolic function in the aging myocardium.
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Acknowledgments |
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This work was supported in part by NIH grants HL-50361 and HL-57623 (to Dr Hajjar), HL-54202 and HL-59521 (to Dr Rosenzweig), and HL-49574 and HL-60323 (to Dr Gwathmey); a Doris Duke Charitable Foundation Award (to Dr Hajjar); and an American Federation of Aging Research grant (to Dr Hajjar). Dr Rosenzweig is an Established Investigator of the American Heart Association.
Received April 20, 1999; revision received August 1, 1999; accepted August 26, 1999.
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