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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:28-35 (2006)
© 2006 The Gerontological Society of America

Growth Hormone Replacement Attenuates Diastolic Dysfunction and Cardiac Angiotensin II Expression in Senescent Rats

Leanne Groban, Nathan A. Pailes, Colleen D. L. Bennett, Christy S. Carter, Mark C. Chappell, Dalane W. Kitzman and William E. Sonntag

Departments of Anesthesiology, Physiology and Pharmacology, Hypertension & Vascular Disease Center, and the Sticht Center on Aging, Wake Forest University School of Medicine, Winston-Salem, North Carolina.

Address correspondence to Leanne Groban, MD, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1009. E-mail: lgroban{at}wfubmc.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
We tested the hypothesis that long-term growth hormone (GH) replacement in aged rats would preserve diastolic function and attenuate left ventricular remodeling associated with normal aging. Male Brown Norway x F344 rats were randomized to receive twice daily injections of porcine GH (200 µg/injection, subcutaneous) or saline from 24 to 30 months of age. Adult rats (6- to 9-months old) received saline injections throughout the study. Thirty-month-old, saline-treated rats exhibited low levels of insulin-like growth factor 1 (IGF-1), impaired diastolic left ventricular filling (Doppler), increased cardiac angiotensin II (Ang II), reduced plasma Ang II, and increased cardiac collagen. GH administration in old rats restored IGF-1 and diastolic indices to values comparable to those of adults. These effects were associated with reduced cardiac Ang II and attenuations in cardiac collagen. Age-related decreases in GH and IGF-1 may contribute to the decline in diastolic function of aging, in part through alterations in renin–angiotensin system-mediated ventricular remodeling.

LEFT ventricular (LV) diastolic dysfunction is a common correlate of aging. It consists of abnormal relaxation and increased chamber stiffness induced by quantitative or qualitative changes in Ca2+ regulatory proteins and extracellular matrix, respectively (1). These age-related cellular and molecular changes that contribute to diastolic impairment may represent adaptive, physiologic dysfunction or the other end of the clinical disease spectrum, diastolic heart failure. Indeed, a high proportion of patients with congestive heart failure have LV diastolic dysfunction with preserved systolic function (2,3); however, neither the pathophysiology of diastolic dysfunction nor a strategy for its treatment has been established (4,5).

One age-related physiologic change that may be partially responsible for diastolic dysfunction is a decline in the pulsatility of growth hormone (GH) and its circulating local effector, insulin-like growth factor 1 (IGF-1). The existence of a relationship between GH and IGF-1 activity and the heart has been suggested by clinical studies that demonstrate an increase in cardiac morbidity and mortality in patients with GH deficiency and GH excess (6–8). Patients with congenital GH deficiency suffer from abnormalities of LV performance, e.g., reduced diastolic filling and impaired response to peak exercise (9). These abnormalities are reversed, in part, by GH replacement therapy (9,10). Chronic overproduction in humans (as in acromegaly) (11) and overexpression of local cardiac IGF-1 in rodents (12) have also led to adverse ventricular remodeling and diastolic dysfunction, further signifying the important influence that physiologic levels of GH and IGF-1 have on cardiac structure and function. Likewise, Vasan and colleagues (13) in the Framingham Heart Study found an association between low serum IGF-I levels and increased risk of incident heart failure in elderly patients without a history of myocardial infarction. Low levels of GH and IGF-1 have been correlated with the severity of systolic impairment in some heart failure studies (14,15). In addition, acute and chronic GH replacement therapy (for 24 hours and up to 3 months) for patients with systolic heart failure has reduced symptomatology, increased exercise capacity, and attenuated cardiac remodeling (16–19). In experimental models of heart failure, GH administration has also improved contractility and LV geometry (20–22). Likewise, in senescent rats, replacement of GH has been shown to reverse the age-related decline in IGF-1 and the decline in regional coronary blood flow, myofilament contractility, and capillary density that is part of aging (23,24). Given the link between GH and IGF-1 activity and the myocardium and the fact that GH and IGF-1 decline with age (25,26), we hypothesize that long-term GH replacement in aged rats will preserve diastolic function and attenuate LV remodeling associated with normal aging.


   METHODS
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 Abstract
 Methods
Results
 Discussion
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All experimental procedures were designed in accordance with the protocols of the Institutional Animal Care and Use Committee of Wake Forest University School of Medicine and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Production of the Model
Experiments used 24-month-old (aged) and 6- to 9-month-old (adult) Brown Norway x Fisher 344 (BNxF344) rats obtained from the National Institute on Aging colony at Harlan Industries (Indianapolis, IN). Aged rats were randomly divided into two groups (n = 10 each) that received either saline (Sal) or porcine GH (200 µg/injection, subcutaneous) twice daily for 6 months. This is done to mimic the pulsatile nature of GH which appears to be essential to optimize its biological potency (25). Adult rats received saline injections throughout the study and served as additional controls (n = 11). All rats were housed 2 per cage and maintained on a 12-hour light/dark cycle at constant temperature and humidity in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Rats had ad libitum access to standard rat chow (Nestle Purina, St. Louis, MO) and tap water. Body weights were recorded monthly, and animals were assessed for signs of overt health problems such as a sudden decline in body weight, redness around the eyes and nostrils, ruffled coat, open sores, or hunched posture. Animals were palpated during these assessments to monitor for symptoms of disease and gross tumors. Recombinant porcine GH was provided by Alpharma, Inc. (Victoria, Australia). For these studies, the dose of GH was based on our data indicating that this regimen is sufficient to increase plasma IGF-1 levels in aged animals (27).

Echocardiographic Studies
Following 6 months of GH or saline treatment, transthoracic echocardiographic examinations by experienced echocardiographers (L. Groban and N. Pailes) masked to the treatment protocol were obtained on rats anesthetized with a mixture of ketamine HCl (60 mg/kg) and xylazine HCl (5 mg/kg) administered intramuscularly. Sedated, spontaneously breathing animals were placed in a shallow left lateral decubitus position with electrocardiographic adhesive electrodes applied to the paws. The left hemithorax was shaved and prepped with acoustic coupling gel to increase probe contact. Animals were secured to the surface of a warming table to maintain normothermia. Using two commercially available sector scanners equipped with a 12 MHz phased-array transducer (5500 and Envisor; Philips Medical Systems, Andover, MA), images were obtained at a 100 mm/s sweep speed and recorded on a digital storage optical disc for off-line analysis. LV M-mode images were obtained in the 2-D short-axis view close to the papillary muscles. Diastolic and systolic posterior wall thickness and LV end-diastolic and end-systolic dimensions (LVDD, LVSD) were measured according to the American Society for Echocardiography leading-edge method (28). The percentage of LV fractional shortening (%FS), an index of global systolic function, was calculated as ((LVDD – LVSD)/LVDD) x 100. Assuming spherical LV geometry, percent ejection fraction (EF%) was calculated according to the cubed method as follows: EF% = ((LVDD)3 – (LVSD)3)/LVDD3. Relative wall thickness was calculated as (2 x PWTed/LVDD), where PWTed is the posterior wall thickness at end diastole. Mitral inflow measurements of early and late filling velocities (Emax and Amax, respectively), deceleration slope (Edecslope), and time (Edectime) were obtained using pulsed Doppler, with the sample volume placed at the tips of mitral leaflets from an apical four-chamber orientation. Doppler tissue imaging to assess mitral valve septal annular velocities (e') was also obtained from the four-chamber view. All measured and calculated systolic and diastolic indices are presented as the average of at least six consecutive cardiac cycles to minimize beat-to-beat variability.

Biochemical Analysis
One week after the echocardiogram, rats were killed by rapid decapitation, blood was collected, and hearts were rapidly removed, weighed, and stored for analysis. The concentration of angiotensin II (Ang II) was determined in plasma and cardiac tissue from a subgroup of 15 animals (5 per each treatment group) by radioimmunoassay according to methods described by Allred and colleagues (29). Protein content was taken from the heart homogenate and assayed with a Bradford protein assay kit (Bio-Rad, Hercules, CA) with porcine immunoglobulin G (IgG) protein as a standard. Plasma Ang II was expressed as femtomoles (fmols) per milliliter of plasma and tissue content as fmol per milligram of protein. The remaining portion of LV was fixed in 10% buffered formalin for histopathologic examination of collagen content.

IGF-1 (Bachem, Torrance, CA) was radiolabeled using the lactoperoxidase, glucose oxidase method and purified on a Sep-Pak silica cartridge (Waters, Milford, MA). Serum was extracted in acid–ethanol (30), and IGF-1 was measured by radioimmunoassay as previously described (31).

Pathological Studies
Horizontal short-axis sections of the formalin-fixed heart were taken at three levels from base to apex through the left ventricle. Specimens were dehydrated with ethanol and embedded in paraffin. Following microtome sectioning, the 4-µm tissue specimens underwent Masson's Trichrome and Verhoeff-van Gieson (VVG) staining for assessment of collagen and elastin fibers. Sections corresponding to the short-axis echo (mid-ventricle, papillary muscle level) were examined under polarized light at a 25x objective using a Zeiss AxioCam digital camera and AxioVision software (Zeiss, München-Hallbergmoos, Germany) by an observer who had no knowledge of treatment groups and previous results. Multiple digital images of the endocardial segment corresponding to the anterior free wall of the LV and a single papillary muscle were obtained and analyzed using Adobe Photoshop. Given the intensity of the VVG staining as compared to the trichrome staining and the knowledge that collagen fibers traverse with elastin, the percent area of VVG-stained fibers for each image was calculated and used as an indicator of myocardial collagen. Perivascular collagen was excluded from this measurement.

Preparation of Sarcoplasmic Reticulum Microsomes
Sarcoplasmic reticulum (SR) membranes were prepared from frozen LV tissue obtained from a separate cohort of rats. The frozen myocardium (500 g) was minced in liquid nitrogen and then homogenized in a chilled buffer containing 20 mM histidine, 5 mM EGTA (pH 6.8), and a protease inhibitor cocktail. After centrifugation (10,000 g and 100,000 g, each for 30 minutes), the pellet was resuspended in a histidine buffer (0.15 M KCL and 0.3 M trehalose) and the protein concentration was determined according to the Bradford method (Bio-Rad).

Immunoblot Analysis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed on the isolated SR membranes using a 10% acrylamide gel. Transfer of the proteins onto a polyvinylidene difluoride membrane (Bio-Rad) was accomplished in cold transfer buffer (20% MeOH in Tris glycine) using a Bio-Rad Mini Trans-Blot Assembly. For immunoreaction, the blot was probed with sarcoplasmic endoplasmic reticulum ATPase (SERCA2) antibody (1:1000 dilution; Abcam, Cambridge, MA), followed by a secondary antibody (antirabbit IgG, 1:3000 dilution; Abcam). The blots were developed by chemiluminescence and exposed to x-ray film. To normalize the variability of protein loading, the antibody to ß-actin (Sigma Aldrich Chemical Co., St. Louis, MO) was probed onto the SERCA2-stripped membrane (Western Blot Recycling Kit; Alpha Diagnostic International, Inc., San Antonio, TX). Antimouse IgG conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) was used as secondary antibody. After incubation, the ß-actin bands were developed. The bands corresponding to SERCA2 and ß-actin were scanned and digitized (MCDI image analysis software; Imaging Research Inc., Ontario, Canada). Each SERCA2 was normalized to its own ß-actin.

Statistical Analysis
All data are expressed as mean ± standard deviation except where specified. Statistical analysis was performed using GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD). Significance of group differences was determined with one-way analysis of variance with three levels (adult saline, old saline, and old GH), followed by the Neumann-Keuls test. The least squares method was used for linear correlation between selected variables. Significance was defined as p <.05. Normal, intraobserver reproducibility was measured in eight adult rats 2 weeks apart for selected echocardiographic variables. Variability was expressed as the mean percent error (absolute difference between the two measurements divided by the mean of the two measurements).


   RESULTS
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Discussion
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GH Treatment
Prior to protocol completion, mortality averaged 30% (3/10) in the saline-treated rats; one died at 25 months and two at 28 months. These saline-treated animals were replaced by three age-matched controls. Excluding one GH-treated rat with an unexplained, transient weight loss of nearly 100 grams between 26 and 28 months of age, there were no significant differences in body weight between aged animals receiving GH and those receiving saline during the 6-month study (data not shown). Body weight and post mortem heart weights are shown in Table 1. As expected, body weight and absolute heart weights were significantly greater in old saline-treated versus adult rats, independent of GH treatment (p <.05). However, LV weight normalized to body weight was similar among groups. BNxF344 rats exhibited a nearly 26% decline in serum IGF-1 levels between 6 and 30 months of age (p <.05), whereas age-matched old rats receiving 6 months of GH replacement exhibited IGF-1 levels that were not different from those of the adults (Figure 1).


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  		Table 1. Body Weight and Post Mortem Heart Weights.

 

Figure 01
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  		Figure 1. Plasma insulin-like growth factor 1 (IGF-1) concentrations in Brown Norway x Fisher 344 (BNxF344) rats after 6 months of treatment with growth hormone (GH) or saline. Old animals received 200 µg twice daily (or saline) from 24 months of age. Adult animals (ranging from 6 to 9 months of age) received saline. Bars represent mean ± standard deviation. *p <.05 vs old saline-treated (Old Saline). Adult: n = 11; Old Saline: n = 10; old GH-treated (Old GH): n = 10

 
Echocardiography
Echocardiography was successful in every case. Within-observer reproducibility values for %FS, Emax, Amax, Edecslope, and Edectime were 13 ± 10%, 7 ± 6%, 8 ± 10%, 13 ± 7%, and 8 ± 7%, respectively. M-mode measurements of LV dimensions, wall thicknesses, and systolic function are summarized in Table 2. LV end-diastolic dimension exhibited a marked increase between adult and old rats, independent of GH treatment. No difference in end-systolic dimension was apparent with respect to advanced age and GH. It is interesting that, when normalized to body weight, end-systolic and diastolic dimensions were smaller in the old GH or saline-treated rats than in the 6- to 9-month-old adult rats. Relative wall thickness and LV posterior wall thickness at end diastole were similar among treatment groups despite greater wall thicknesses at end systole in saline-treated aged rats versus adult rats (p <.05).


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  		Table 2. M-Mode-Derived Measures of Left Ventricular Dimension, Wall Thickness, and Systolic Function.

 
Calculated indices of LV systolic function, such as fractional shortening, ejection fraction, and heart rate (adult: 263 ± 25 bpm; old saline: 233 ± 35 bpm; old GH: 248 ± 12 bpm) were not influenced by either age or long-term GH replacement. In contrast, an age-associated change in diastolic function as assessed by using Doppler flow and Doppler tissue imaging was apparent; this diastolic impairment was improved with GH treatment. The E wave-to-A wave (E/A) ratio was greater in the adult and old GH rats as compared to the old saline-treated rats. The lower E/A ratio in the old, untreated rats appeared to be a function of a 12%–22% reduction in early filling velocity of the LV (Emax) across the mitral valve and a nearly 18% increase in late filling velocity (Amax compared to the adults), suggesting a greater fraction of filling in late diastole (Table 3). A reduction in deceleration slope and an increase in deceleration time in the old saline-treated group of rats as compared to the other groups suggest a decline in LV compliance. Likewise, the peak mitral valve annular velocity measured at the septal portion of the annulus from the apical four-chamber view was 38% lower in old saline-treated as compared to the adult and old GH-treated rats (Table 3). Taken together, these findings indicate that (a) there is mild diastolic impairment in the senescent male BNxF344 rat and (b) long-term GH replacement, between 24 and 30 months of age, appears to attenuate this age-associated decline in diastolic performance.


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  		Table 3. Echocardiographic Indices of Diastolic Function.

 
Histopathology
Papillary muscle interstitial collagen was significantly increased in the old, saline-treated rats as compared to the adult rats (Figure 2A and B). Although not significant, there was a tendency for less collagen content in the GH-treated old rats as compared to the untreated old rats. As shown in Figure 3, we do find a significant inverse relationship between plasma IGF-1 levels and papillary collagen content—that is, the higher the plasma IGF-1 level, the lower the degree of collagen content in the papillary muscle. Taken together, these findings suggest that the age-related decline in circulating GH and IGF-1 may importantly contribute to an increase in interstitial fibrosis and myocardial stiffness that are particular to the senescent cardiac phenotype.


Figure 02
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  		Figure 2. Increased papillary muscle collagen in the old rats is attenuated by growth hormone (GH) treatment. A, Representative images of papillary muscle collagen were taken under fluorescence at a magnification of 25x. Following VVG (Verhoeff-van Gieson) staining, red areas represent interstitial collagen. Note the increased fluorescence in the papillary muscle section from the old saline-treated (Old saline) animal compared with the adult. B, Quantification of collagen staining revealed a marked increase in the old group that tended to decline with GH treatment. Fluorescent staining was taken as the cross-sectional area calculated as percentage of total pixels in the 25x field. **p <.01 adult vs Old Saline, old GH-treated (Old GH)

 

Figure 03
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  		Figure 3. Correlation of insulin-like growth factor 1 (IGF-1) levels to cardiac collagen expression. Chronic growth hormone treatment reduces collagen staining in the heart of old rats. Linear regression analysis shows a significant correlation between plasma IGF-1 levels and papillary muscle collagen (F = 14.6, p <.004)

 
Plasma and Cardiac Ang II
In a subset of animals (n = 15, 5 per group), plasma Ang II levels declined by over 50% in the senescent as compared to the adult rats, independent of prevailing IGF-1 levels (Figure 4A). In contrast, LV tissue Ang II levels increased by 60% in old, saline-treated rats as compared to age-matched, old, GH-treated or adult rats (p <.05) (Figure 4B).


Figure 04
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  		Figure 4. Differential expression of plasma and cardiac angiotensin II in the aged rats. A, Circulating angiotensin II levels were suppressed in both the old rats and the growth hormone (GH)-treated group. B, Cardiac tissue angiotensin II increased over 50% in the old saline-treated (Old Saline) rats, but was reduced to the Adult level following long-term GH treatment. Bars represent mean ± standard deviation. *p <.05 vs Old Saline. #p <.05 vs old GH-treated (Old GH), n = 5 for all groups

 
Cardiac SERCA2 Expression
To assess the influence of GH on SERCA2 protein content, we performed quantitative immunoblotting in hearts isolated from a separate cohort of adult (n = 6) and 30-month-old BNxF344 rats (saline: n = 3; GH-treated: n = 5). Immunoblots probed for SERCA2 and ß-actin showed single bands of molecular weight 110 kd and 70 kd, respectively (Figure 5A). The anti-SERCA2 antibody tended to be more intensely stained in the adult samples than in samples obtained from the old saline-treated rats (adult [n = 6]: 0.81 ± 0.22 vs old [n = 3]: 0.44 ± 0.27, not significant). In Figure 5B, the concentrations of SERCA2 levels normalized to ß-actin are plotted against the IGF-1 levels. The data show a positive, significant correlation between serum IGF-1 and SERCA2 expression (p <.01).


Figure 05
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  		Figure 5. Sarcoplasmic endoplasmic reticulum ATPase (SERCA2) western blots of cardiac microsomes isolated from adult (n = 6) and senescent rats treated with growth hormone (GH; n = 5) or saline (n = 3). A, Representative SERCA2 and ß-actin blots. Each lane is a sample from a different heart. Molecular weight standards are indicated on the left (KDa). B, Protein concentration of SERCA2 (normalized to ß-actin) relative to plasma insulin-like growth factor 1 (IGF-1) levels. SERCA2 protein content decreased with decreasing IGF-1 levels (p <.01)

 

   DISCUSSION
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 Methods
 Results
 Discussion
References
 
In the present study, we show that senescent rats with low levels of GH and IGF-1 exhibit impaired LV filling as evidenced by a reduced E/A ratio, reduced early-wave mitral annular velocity, increased interstitial cardiac fibrosis, and increased intracardiac Ang II despite low circulating levels of the peptide. In contrast, aged rats on long-term GH replacement initiated at 24 months of age displayed preserved diastolic function, attenuated LV remodeling, and reduced intracardiac Ang II levels.

Our Doppler findings in the 30-month-old BNxF344 rats are in agreement with both clinical (32,33) and experimental (34,35) correlates of diastolic dysfunction of aging. Specifically, a decline in myocardial relaxation is characterized by decreases in Doppler early-wave mitral annular (e') velocities, LV filling velocities (Emax), and a concomitant increase in LV deceleration time. This loss in early diastolic distensibility likely reflects the tendency for an age-related decline in SERCA2 protein content and the increased interstitial fibrosis observed in the 30-month-old saline-treated rats as compared to the young adult rats. A decreased rate of calcium sequestration by the cardiac SR, marked by reduced SERCA2 expression and protein content (36–38), and a reduction in LV compliance, marked by an imbalance between cardiac collagen synthesis and degradation (39), have both been implicated in the pathogenesis of diastolic dysfunction. Indeed, age-related increases in arterial wall stiffness (40) may have also contributed to impairments in LV relaxation.

Multiple factors are involved in age-related increases in myocardial collagen content. For example, a loss of collagen regulatory control can reflect disturbances in transcriptional regulation through physical, neurohormonal, and growth factors (41), in posttranslational regulation, such as advanced glycation end-products (AGE)-associated collagen cross-linking (42), or in enzymatic degradation specifically involving matrix metalloproteinases (43). Although an understanding of the role of these factors in the development of cardiac interstitial fibrosis is beyond the scope of the current study, our data suggest that a neurohormonally mediated mechanism may be partly involved in the morphologic changes of the senescent heart. Specifically, in old, saline-treated rats, Ang II levels were increased in the heart and reduced in the plasma. This differential expression of Ang II was not as obvious in the adult rat. Our demonstration of greater cardiac Ang II content extends the findings of Heymes and colleagues (44), who observed increased messenger RNA levels of the renin–angiotensin–aldosterone system (RAAS) components angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin type 1 (AT1) and type 2 (AT2) receptors in hearts from senescent rats. In a subsequent study, these investigators confirmed that the increase in intracardiac RAAS was independent of the circulating RAAS (45). Indeed, our present data are consistent with previous studies that demonstrate that aging is associated with reduced circulating levels of Ang II (46–48). The observation that cardiac Ang II is elevated within the heart has important implications, as the peptide is known to promote myocyte hypertrophy (49), myocardial collagen synthesis (50), cytokine production (51), and the de novo production of cardiac aldosterone which may directly contribute to interstitial fibrosis (52).

A novel finding in this study is that long-term GH treatment, started at 24 months of age, attenuates many of the structural and functional changes of cardiac aging that were apparent at 30 months in the absence of GH treatment. Specifically, the GH treatment restored plasma IGF-1 concentrations to adult levels; this was associated with preserved diastolic function, a reduction in interstitial cardiac fibrosis, and intracardiac Ang II levels that were similar to those found in adult rats. It is well accepted that one of the most robust biomarkers of mammalian aging is a down-regulation of the GH–IGF-1 axis. Reports in humans and rodents demonstrate that age-related reductions in secretion of GH and IGF-1 contribute to age-related reductions in tissue structure and function, e.g., lean body mass, skeletal muscle mass and function, bone density, immune function, vascular density, and neurocognitive function (25–27,53). There also is a strong relation between low levels of IGF-1 and an increased risk of heart failure (54,55), most notably in elderly patients without a previous history of myocardial infarction (13). Moreover, GH nearly reversed the cardiac phenotype of senescent rodents by increasing coronary blood flow and myofilament contractility and partially restoring capillary density (23,24). In the setting of experimental postinfarct heart failure, reactivation of myocardial growth by GH has been shown to be beneficial as well (20,56,57).

Presently, we do not have an explanation for the favorable effects of GH on ventricular remodeling. The lower intracardiac Ang II levels in our old GH-replete rats as compared to the old saline-treated rats, and the knowledge that Ang II promotes myocardial collagen synthesis (50), support the concept that GH and IGF-1 may interact with RAAS to alter morphology of the senescent heart. There is also good experimental evidence that GH and IGF-1 works directly via anti-apoptotic actions (22) and through inhibition of transforming growth factor beta (TGFß) (a potent stimulator of fibronectin and collagen) (58), or indirectly by opposing Ang II-mediated elevations in TGFß to attenuate myocardial collagen production (59).

Several limitations of the present study merit mention. First, many of the variables measured with echocardiography are known to be affected by loading conditions and heart rate. However, in the present study, heart rate was not significantly altered by age or GH treatment, and echocardiograms were performed under light, general anesthesia. Also, tissue Doppler-derived mitral annular motion, a measure of LV relaxation that is independent of heart rate and LV filling pressures, was reduced in old, untreated rats relative to young adult rats and old, GH-treated rats, further supporting the functional impairment exemplified by mitral inflow Doppler measurements. Although diastolic dysfunction was noted at rest in the old, untreated rats, it is reasonable to expect that it would be exaggerated under such stressful conditions as exercise (60). Second, our data do not directly identify the role that an age-related decline in GH and IGF-1 might have on myocardial calcium handling. Abnormalities in cardiac relaxation have been attributed to a decrease in content and activity of the SR Ca2+-ATPase pump in experimental models of senescence (36–38). Moreover, in rats with postinfarction heart failure, SERCA2 has been shown to increase with GH treatment (61). Although we show a strong association between SERCA2 expression and circulating IGF-1 levels from tissue obtained from young adult, old, and old GH-replete rats, our between-group analyses did not achieve statistical significance. Thus, further studies are warranted to determine whether the improvements observed in diastolic function after long-term GH replacement in old rats can be ascribed to preservations in SERCA2 content and function in addition to reductions in cardiac fibrosis.

Summary
Long-term GH administration to senescent rats preserved diastolic function, reduced cardiac Ang II expression, and attenuated LV remodeling. Whether the changes in Ang II expression in rats treated with GH were causative of or secondary to GH-mediated prevention of age-related structural or functional changes will require further study. To date, an established therapy for treatment of diastolic dysfunction in elderly persons has not yet been determined. The present findings coupled with the strong relationship that exists between low levels of IGF-1 and increased incidence of heart failure in elderly patients with preserved systolic function suggests that GH and IGF-1 augmentation may be a key therapeutic regimen for diastolic dysfunction in the elderly population.


   Acknowledgments
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 Results
 Discussion
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This research was supported in part by grant AG022598-01 from the National Institute on Aging, the Dennis W. Jahnigen Career Development and Paul Beeson Scholars Award (K08-AG026764-01), and a Merck Geriatric Cardiology Award (L.G.); grants HL-56973 from the National Heart, Lung, and Blood Institute (M.C.C.) and PO1 AG11370 from the National Institute on Aging (W.E.S.); and in part by grant P60AG10484 from the Claude D. Pepper Older Americans Independence Center (Pepper Center) (D.W.K.).

Alpharma, Inc. (Victoria, Australia) provided the porcine growth hormone.

Data from this manuscript were presented, in part, at the 26th Annual Meeting of the Society of Cardiovascular Anesthesiologists, May 2004, and at the Fourth Annual Meeting of the Section for Surgical and Related Medical Specialties, May 2005.


   Footnotes
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Decision Editor: James R. Smith, PhD

Received March 30, 2005

Accepted July 21, 2005


   References
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