AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 281: H915-H922, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wannenburg, T.
Right arrow Articles by Sonntag, W. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wannenburg, T.
Right arrow Articles by Sonntag, W. E.
Vol. 281, Issue 2, H915-H922, August 2001

Growth hormone reverses age-related cardiac myofilament dysfunction in rats

Thomas Wannenburg2,*, Amir S. Khan1,*, David C. Sane2, Mark C. Willingham3, Tony Faucette2, and William E. Sonntag1

1 Department of Physiology and Pharmacology, 2 Department of Cardiology, and 3 Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1083


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypotheses that aging is associated with a reduction in overall cardiac contractility and myofilament force generation that could be reversed with growth hormone (GH) replacement. Three groups of male Brown-Norway rats were studied: young (YSAL: 8 mo old, n = 13), old (OSAL: 28 mo old, n = 13), and old GH-treated (OGH: 28 mo old, n = 12; 300 µg bovine GH, twice a day for 30 days). The left ventricular (LV) pressure-volume relation was derived in isolated hearts, after which isolated trabecular muscles from these hearts were permeabilized and maximal myofilament force generation (Fmax) was measured. LV developed pressures at a LV volume of 0.3 ml were significantly depressed with age: 84 ± 6 vs. 71 ± 6 mmHg (YSAL vs. OSAL, respectively, P = 0.001) and not restored by GH (69 ± 4 mmHg). Fmax was reduced in the aged hearts: 47.5 ± 3.12 vs. 35.9 ± 3.03 mN/mm2 (YSAL vs. OSAL, respectively, P = 0.014) but was restored with GH replacement to 46.7 ± 3.12 mN/mm2 (OSAL vs. OGH, P = 0.021). Our results suggest that cellular myofilament contractility is reduced with aging and restored with GH replacement.

somatotropin; aging; myocardium; contractility; Langendorff


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OF THE MYRIAD CHANGES in physiological parameters and function associated with aging, few have been as consistently well documented as the loss of normal phasic growth hormone secretion (37, 42). It is reasonable to expect that a significant alteration in activity of such an important anabolic growth factor with its regulatory role on structure and function may partially mediate the well-described deterioration in cardiovascular performance associated with aging.

Multiple alterations in cardiovascular structure and function have been described with aging. These include an increase in left ventricular (LV) wall thickness (15), a decrease in the number of cardiac myocytes (4, 22, 31, 32), a rarefaction of coronary arterioles (3), and an increase in fibrosis (24, 35, 43) and collagen deposition (12). On a more molecular level, there are shifts in troponin and myosin isoforms (27, 34), which suggest that alterations in cardiac function during aging may be manifested at the level of the contractile proteins.

It is highly likely that a deficiency of growth hormone and its mediator insulin-like growth factor-1 (IGF-1) contribute to these structural changes. Cardiomyocytes are replete with receptors for both growth hormone and IGF-1 (5, 19, 29, 47). Data also suggest that many age-related deficiencies are caused by decreases in growth hormone and are subsequently ameliorated by growth hormone replacement. For example, growth hormone administration to aged animals and humans can increase skeletal muscle protein synthesis (41), immune function (26), lean body mass, and skin thickness (36). Further evidence that growth hormone is important in regulating cardiac structure and function are the recent findings that growth hormone improves cardiovascular function of rodents after left coronary artery ligation (46) or LV failure (11). Studies have also shown that growth hormone replacement can improve cardiac performance in growth hormone-deficient children (6) and adults (1, 39). A beneficial effect of growth hormone in the treatment of dilated cardiomyopathy (14, 38) and heart failure (46, 50) in humans has also been suggested.

Many of these effects were demonstrated after administration of pharmacological doses of growth hormone to young rats that possessed normal growth hormone secretory dynamics. Therefore, we hypothesized that growth hormone replacement therapy designed to simulate the normal phasic secretion in aged rats would reverse age-related changes in cardiac function in senescent rats. Specifically, we hypothesized that growth hormone replacement would restore coronary blood flow, improve the contractile state in the intact heart, and increase contractility of the cardiac contractile proteins at a cellular level. To test the first two elements of our hypothesis, we measured coronary blood flow and contractile state in isolated, crystalloid-perfused rat hearts using a modified Langendorff system. The third element of our hypothesis was tested by measurement of calcium-activated force generation of cardiac myofilaments from right ventricular trabecular muscles.

We found that coronary blood flow was diminished with aging and could not be restored by maximal vasodilation with adenosine, a finding that is indicative of coronary rarefaction. The contractile state of the intact heart was reduced with aging, and this reduction was also found at a subcellular level with a reduction in force generated by the contractile proteins. Growth hormone replacement therapy restored myofilament contractile function to normal levels but did not restore coronary blood flow or contractile function of the intact heart.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Brown-Norway rats were obtained from the National Institute on Aging (NIA) Colony at Harlan Industries (Indianapolis, IN). Young animals (8 mo) and old animals (28 mo) were purchased and housed for 1 mo in an animal facility that is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Rats were maintained on a 12:12-h light-dark cycle with rat chow (Purina Mills, Richmond, IN) and water available ad libitum. Body weights were monitored, and animals that demonstrated external evidence of disease or significant weight loss during the study were eliminated from the experiment. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Wake Forest University School of Medicine. Animals were divided into three groups: young, saline- (YSAL, n = 13), old saline- (OSAL, n = 12), and old, growth hormone-treated animals (OGH, n = 12; 300 µg/kg bovine growth hormone, sc BID for 30 days). Bovine growth hormone was a generous gift from National Institute of Diabetes and Digestive and Kidney Diseases, Dr. A. Parlow, and the National Pituitary Program. Apart from growth hormone supplementation, the animals were housed under identical conditions.

Isolated intact heart preparation. After 30 days, the rats were anesthetized by halothane inhalation. The chest was opened by midline sternotomy. The heart was exposed and a bolus of 1 ml iced, heparinized (50 U/ml) Krebs-Henseleit solution was injected into the right atrium. Hearts were rapidly excised and perfused using a Langendorff preparation as described previously (48). Briefly, the hearts were perfused retrograde through the aorta with a cardioplegic, modified Krebs-Henseleit solution (in mM: 140.5 Na+, 5.0 K+, 1.2 Mg2+, 127.5 Cl-, 2.0 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 19 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 15 dextrose, and 1.0 Ca2+) bubbled with 95% O2-5% CO2 and warmed to 38°C with a continuously circulating water bath. Perfusion pressure was adjusted to 80 mmHg by adjusting the height of the perfusion reservoir and remained constant throughout the experiments. The left atrium was excised, and a water-filled latex balloon connected by a fluid-filled catheter to a pressure transducer was placed in the left ventricle via the mitral annulus to measure developed intraventricular pressure and to adjust LV volume. Pacing electrodes were placed at the right ventricular outflow tract and the LV apex, and pacing stimuli were provided by a Grass SD-9 stimulator set at 300 or 20 beats/min above the intrinsic rate and kept constant throughout the experiment. Perfusate effluent was collected in a graduated cylinder to measure coronary flow.

Langendorff protocol: measurement of coronary flow and ventricular contractility. All hearts were perfused with Krebs-Henseleit solution at a fixed perfusion pressure of 80 mmHg and LV volume of 0.3 ml for 10 min to allow the systolic and diastolic pressures and coronary flow to stabilize. Coronary flow was recorded after this stabilization period. The LV volumes were then varied from 1.5 to 5.0 ml, and developed pressure measurements were recorded at each volume followed by a measurement of coronary flow at a 0.3-ml intraventricular volume. After 10 min, coronary flow measurement was repeated. The hearts were then perfused with Krebs-Henseleit solution containing 10 mM adenosine (Sigma, St. Louis, MO), bubbled with 95% O2-5% CO2, and warmed to 38°C with a continuously circulating water bath. After a 5-min equilibration period, coronary flow measurements were repeated at a LV volume of 0.3 ml to ascertain maximally dilated coronary flow values.

Skinned fiber preparation: measurement of contractile protein function. After measurements of developed pressure and coronary flow were completed, the heart was removed from the Langendorff apparatus, placed in a bath, and perfused retrograde via the aorta with Krebs-Henseleit buffer containing 20 mM butanedione monoxime (BDM) (see Skinned fiber solutions). Under a binocular microscope, thin unbranched trabecular muscles between the atrioventricular ring and right ventricular free wall were carefully excised. Right and left ventricles were weighed, and the left ventricles were rapidly frozen in isopentane at -40°C and kept at -80°C.

Trabeculae muscle dimensions were determined via an ocular micrometer mounted in the dissection microscope (~10 µm resolution). On average, the trabeculae muscles were 1.47 ± 0.1 mm long, 0.23 ± 0.02 mm wide, and 0.16 ± 0.02 mm thick (means ± SE, measured at slack length). Muscle dimensions were not different among groups. Trabeculae muscles were incubated overnight in a relaxing solution containing 1% Triton X-100 (see Skinned fiber solutions), which served to remove cell membranes and intracellular membrane-bound structures such as mitochondria and sarcoplasmic reticula. Custom-made aluminum foil clips were gently attached to the ends of the permeabilized muscles to serve as handles for mounting the preparation to the experimental apparatus. The muscles were mounted in an aluminum bath (volume 60 µl) located on the stage of an inverted microscope (Nikon). The clip on one end was hooked onto a servomotor (model 6350, Cambridge Technology; Watertown, MA; ~250 µs 90% step response), which was used to control and adjust sarcomere length. The clip on the other end was attached to a modified semiconductor strain gauge (AE801, Sensonor, Norway; resonance frequency ~2 kHz) for muscle force measurement. The design of the bath was modified from Guth and Wojciechowski (18) and Stienen et al. (44) and has been described previously (49). A small stirring rod traversed the length of the bath, parallel to the muscle and out of view of the microscope, and was driven by a small electric motor. The bath was continuously stirred and mounted on a copper base through which water was circulated for temperature control. Temperatures of the bath and of the solutions were controlled using a heater/circulator (Fisher Scientific). A thermocouple thermometer (Digi-Sense; Cole-Palmer, NJ) was used to continuously monitor bath temperature, which was maintained at 20 ± 0.1°C (range) over all experiments.

Measurement of sarcomere length. Sarcomere length (SL) was measured by laser diffraction as previously described (49). Briefly, a beam of laser light (632 nm), perpendicular to the longitudinal axis of the muscle, was directed onto the center of the specimen. The resulting first-order diffraction band was projected onto a 512-element photo diode array (Reticon), which was scanned electronically every 0.5 ms. An analog circuit converted the intensity distribution of the diffraction band into a voltage proportional to median SL. Glass gratings of known spacing were used to calibrate the system. Errors due to muscle nonhomogeneity and Bragg angle reflection artifacts are <4% using the approach by de Tombe and ter Keurs (9).

Skinned fiber solutions. In every experiment, the trabeculae were dissected during perfusion with a low-calcium Krebs-Henseleit solution containing (in mM) 140.5 Na+, 5.0 K+, 1.2 Mg2+, 127.5 Cl-, 2.0 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 19 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 15 dextrose, and 1.0 Ca2+. In addition, BDM (20 mM), a calcium-desensitizing agent, was added to minimize damage to the ends of the trabeculae during dissection (30). The trabecular muscles were then bathed overnight in a relaxing solution to which 1% Triton X-100 was added to dissolve lipid membranes. After this skinning period, the muscles were bathed in a physiological solution that simulated intracellular conditions. Calcium in this solution was highly buffered to strictly control the calcium concentration. Three types of solutions were used: relaxing solution, preactivation solution, and activating solution. The compositions of these solutions are shown in Table 1. The solute concentrations were determined using an iterative computer program as described by Fabiato and Fabiato (13), using dissociation constants of Godt and Lindley (16). We mixed various fractions of relaxing and activating solution to obtain a variety of [Ca2+] in activating solutions.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Solutions used for myofilament study

Experimental protocol. Muscles were activated or relaxed by exchanging the superfusate. Various levels of calcium activation were obtained by mixing different proportions of activating and relaxing solutions. Muscles were allowed a minimum of 4 min in relaxing and preactivating solutions between activations. SL was set at 2.1 µm and held constant throughout the experiment. Data were collected when force development reached steady state.

Collagen deposition and heart pathology. Collagen deposition was measured in myocardial sections using picrosirius red staining (10), which has been shown to be comparable to tissue hydroxyproline measurements (23). Frozen sections of the LV were cut at the apex, middle, and base (25); mounted on charged slides; and kept frozen at -20°C before being stained. Sections were thawed to room temperature, washed in distilled H2O, incubated in 0.2% phosphomolybdic acid (Sigma) for 10 min, washed in distilled H2O, and incubated for 90 min in 0.1% Direct Red 80 (Fluka; Neu-ulm, Germany) in saturated picric acid (Fisher; Pittsburgh, PA). Slides were then washed in 0.01 N HCl for 2 min, dehydrated in an increasing gradient of alcohols and xylene, and coverslipped using Permount (Fisher) diluted in xylene. Digital images of the slides under polarized light were captured with a Spot Camera (Diagnostic Instruments; Sterling Heights, MI) and a Zeiss Axioplan 2 microscope with a ×10 objective (numerical aperature 0.3 plan). Digital images were analyzed randomly with the analyzer blind to the animal group assignments. Cardiac muscle appears green and collagen appears white under polarized light. With the use of color segmentation analysis with Image Pro Plus (Media Cybernetics; Silver Spring, MD), the number of pixels per digital image was collected for four to six sections per region (apex, middle, and base) of a randomly chosen subset of animals (5 animals from each group) and corrected for image area (1.50 mm2). The interaction between heart area and treatment was analyzed. Adjacent sections were stained with hematoxylin-eosin to examine and compare changes in cellular morphology with areas of collagen deposition in each treatment group.

Data analysis. Differences in coronary blood flow, muscle dimensions, and collagen deposition were tested using analysis of variance (ANOVA). The Fisher least significant difference test was performed as a subsequent test if ANOVA revealed P < 0.05. Contractile state of the intact heart was compared between YSAL and OSAL by plotting developed pressure (systolic minus diastolic) as a function of ventricular volume. Comparisons among YSAL, OSAL, and OGH were made using multiple linear regression analysis (40)
DP<IT>=&agr;+&bgr;·</IT>V<IT>+&ggr;·</IT>age<IT>+&dgr;·</IT>age<IT>·</IT>vol (1)
where V is ventricular volume (in ml) and "age" is animal age (in months). The predicted mean slope of the ventricular pressure volume relation in this set of experiments is given by the parameter beta . Contractile state is indexed by the slope and intercept of the pressure volume relation. The effect of aging is therefore indexed by value and statistical significance of the terms gamma  and delta .

Similarly, in the aged cohorts the effect of growth hormone supplementation on ventricular contractility was assessed using multiple linear regression analysis
DP<IT>=&agr;+&bgr;·</IT>V<IT>+&ggr;·</IT>GH<IT>+&dgr;·</IT>GH<IT>·</IT>vol (2)
where GH is growth hormone and was assigned a value of 0 or 1 for the absence or presence of growth hormone supplementation. In addition, Eqs. 1 and 2 were extended to allow for interexperiment variability. Force-[Ca2+] relations were fit to a modified Hill equation
F<IT>=</IT>F<SUB>max</SUB>[Ca<SUP>2<IT>+</IT></SUP>]<SUP><IT>n</IT><SUB>H</SUB></SUP><IT>/</IT>([Ca<SUP>2<IT>+</IT></SUP>]<SUP><IT>n</IT><SUB>H</SUB></SUP><IT>+</IT>EC<SUB>50</SUB><SUP><IT>n</IT><SUB>H</SUB></SUP>) (3)
where F is steady-state force, Fmax is the maximum saturated force, EC50 is the concentration of calcium at which F is 50% of Fmax and represents a compound affinity constant, and nH represents the slope of the force-[Ca2+] relation (Hill coefficient). The nH values and EC50 were analyzed by ANOVA to determine the effect of length and [Ca2+]. Differences in Fmax and EC50 were tested using analysis of variance. In all cases, a P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight, cardiac mass, and IGF-1 levels. Body weight increased 16% with age (YSAL vs. OSAL groups, P < 0.05) and increased 4% with growth hormone administration (OSAL vs. OGH groups); however, this effect did not reach statistical significance (refer to Table 2). Both heart weight and LV mass increased ~50% with age (P < 0.05) and increased 8% in response to growth hormone, but these latter effects did not reach statistical significance. Heart weight corrected for body weight increased 29% with age (P < 0.05), but there was no statistical difference between OSAL and OGH. Plasma IGF-1 decreased 14% between 6 and 28 mo of age [P = not significant (NS)], and growth hormone administration increased plasma IGF-1 in the old animals by 36% (P < 0.05).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   General animal information at time of euthanasia

Coronary blood flow. Basal coronary flow corrected for heart mass declined 40% from YSAL to OSAL (P < 0.01) and was not significantly different between OSAL and OGH. Maximal coronary flow (induced with 0.1 mM adenosine) corrected for heart mass also declined 24% from YSAL to OSAL (P < 0.01) but was not restored with growth hormone (Fig. 1).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1.   Basal coronary flow (solid bars) and maximally dilated coronary flow (open bars) induced with 0.1 mM adenosine decreases with age (P < 0.01). No significant effects of growth hormone administration were observed. *P < 0.05 comparing young, saline-treated rats (YSAL) and old, saline-treated rats (OSAL). OGH, old, growth hormone-treated rats.

Contractility of the intact heart. Contractility of the left ventricle was assessed in the intact heart by plotting developed pressure as a function of LV volume. As shown in Fig. 2, LV developed pressure curves were displaced downwards with age. This is consistent with a significant reduction in contractile state (P = 0.001). This reduction in contractile state was not reversed by growth hormone treatment. Another measurement of cardiac contractile state is the developed pressure at a specific ventricular volume. At a ventricular volume of 0.3 ml, developed pressures were 84 ± 6 mmHg in YSAL animals and 71 ± 6 mmHg in OSAL (P < 0.01) animals, but pressures were not restored by growth hormone replacement (69 ± 4 mmHg). Interestingly, using multiple regression analysis, we could not detect any effect of age or growth hormone replacement on the slopes of the pressure-volume relations (P = 0.15).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2.   Left ventricular (LV) developed pressure curves from intact hearts of YSAL, OSAL, and OGH. LV pressure-volume relation curves show a decline with age. At an LV volume of 0.3 ml, there is a significant decline in LV developed pressure comparing YSAL and OSAL (P < 0.01); however, no differences were observed between OSAL and OGH.

Myofilament contractile function To separate the effect of aging on contractile function of myofilaments at a cellular level from possible vascular and structural changes at an organ level, we measured force development in skinned trabeculae. As shown in Fig. 3, there is a sigmoidal relationship between isometric force generation and calcium concentration with Fmax at saturating calcium concentrations. Fmax was significantly reduced from 47.5 ± 3.12 mN/mm2 in YSAL to 35.9 ± 3.03 mN/mm2 in OSAL (P = 0.014). This reduction in contractile state was fully restored to 46.7 ± 3.12 mN/mm2 with growth hormone treatment (P = 0.021). There was no significant change in EC50 (P = NS).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3.   Myofilament force corrected for trabecular muscle cross-sectional area at increasing Ca2+ concentrations. Maximal force production at a saturating Ca2+ concentration declined between YSAL and OSAL (P = 0.014) and was restored by growth hormone administration (P = 0.021).

Structural alterations with aging. Collagen deposition increased with age from 12.9 ± 3.3 pixels/mm2 in YSAL to 100.6 ± 13.0 pixels/mm2 in OSAL (P < 0.002). However, OSAL was not significantly different from OGH (79.4 ± 24.3 pixels/mm2 in the OGH group). Accumulation of collagen was colocalized with segments of myocardial fibrosis in corresponding areas of adjacent sections. Representative images depicting collagen and corresponding fibrosis are shown in Fig. 4.


View larger version (138K):
[in this window]
[in a new window]
  		Fig. 4.   Representative images from YSAL (A and B), OSAL (C and D), and OGH (E and F) demonstrating age-related myocardial pathology. Regions stained with hematoxylin-eosin depict the increase in fibrosis between YSAL (A) and OSAL (C) and no apparent effect of growth hormone (E). In adjacent sections for each group, collagen accumulation increases between YSAL (B) and OSAL (D), with no apparent effect of growth hormone administration (F).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have documented significant age-related changes in cardiac function, some of which are reversed with growth hormone replacement. We have shown that LV mass increases with age, while coronary blood flow and cardiac contractility at the level of the intact heart decreases. We also found that cardiac contractility was reduced with aging at a cellular level with a reduction in maximal calcium-stimulated myofilament force generation or Fmax. Whereas growth hormone supplementation could not reverse the age-related decreases in coronary blood flow and contractility of the intact Langendorff heart preparation, it did effectively restore cellular myofilament contractility to the same levels as a young heart. It is intriguing why restoration of myofilament contractility did not restore contractile state in the intact heart.

We found that cellular contractility was reduced in aged rat hearts as evidenced by a reduction in maximal myofilament force generation in skinned trabeculae. The most likely mechanism for a reduction in maximal force generation is a reduction in potential actin-myosin cross bridges (20). This may be due to diminished cellular protein synthesis, which is a phenomenon well documented in aging (33). This would also be an obvious target for growth hormone that has been shown to increase cellular DNA and RNA synthesis as well as protein synthesis in skeletal muscle (41), and growth hormone can also regulate myosin isoform shifts in both skeletal muscle (8) and cardiac muscle (2). Therefore, it is not surprising that growth hormone replacement therapy restored myofilament contractile function in aged rats to levels of younger rats in our study. However, an improvement in myofilament contractility would be expected to increase overall cardiac contractility. Our studies demonstrating a differential effect of growth hormone on intact and skinned muscle indicate that age-related alterations in cardiac excitation-contraction coupling or energy transduction are not sufficiently restored by growth hormone over the time period or doses used. These results suggest that additional age-related changes were involved and these alterations could not be reversed by 4 wk of growth hormone administration.

The slope and intercept of the LV pressure-volume relation have been well documented and investigated as indexes of contractile state in the intact heart (45). Our finding of a depressed pressure volume relation is compatible with 1) a reduction in cellular contractile function per se, 2) ventricular remodeling, or 3) a mismatch in energy supply and demand such as a reduction in coronary blood flow. In this study, we found evidence of all three mechanisms discussed above. The discrepancy between the myofilament and intact heart data suggests an alteration in calcium handling in intact aged hearts. Whereas previous studies demonstrated that pharmacological doses of growth hormone can increase calcium transients in rats with postinfarction heart failure (46), the effects of physiological doses of growth hormone on calcium transients in aged rats were not investigated in this study.

We found no beneficial effect of growth hormone replacement therapy on the cardiac pressure-volume relation. This is in contrast to prior in vivo studies, which have shown potential benefits of growth hormone supplementation in heart failure in rats (7, 46), hamsters (38), and humans (14, 21). Many of these studies involved pharmacological doses of growth hormone to young rats, whereas our study investigated the role of physiological doses of growth hormone to older, growth hormone-deficient rats. It is also possible that, to some extent, the lack of benefit in our intact heart study is an artifact of the crystalloid-perfused intact heart preparation or may represent a specific growth hormone effect in cardiomyopathy, which is not operative in the aged heart.

It is also possible that differences in the source of growth hormone may be a relevant factor. Previous studies (17) have used recombinant human growth hormone that is both prolactigenic and antigenic in rodents. The present study used bovine growth hormone at a lower, physiological dose to increase IGF-1. Bovine growth hormone in rodents is not prolactigenic and does not significantly cause the production of antibodies until after 30 days of treatment (A. Parlow, personal communication). Our growth hormone replacement regimen was designed specifically to simulate normal phasic growth hormone release for 30 days. Whereas a higher dose of growth hormone may have more dramatic short-term effects on cardiac function, it has the long-term adverse consequence of acromegaly in humans.

We found evidence of ventricular structural remodeling with age. There was no direct evidence of LV hypertrophy in the growth hormone-injected rats (i.e., OGH) in this study. Rats in this treatment group demonstrated an increase in heart weight, but this failed to reach statistical significance. However, this was accompanied by an increase in collagen deposition in OSAL and OGH animals, as demonstrated by the images in Fig. 4. Similar findings have been described in other strains of rats (4), cardiomyopathic hamsters (28), and humans (24). In this study, the age-related decline in contractility was not reversed with growth hormone replacement therapy. Therefore, structural alterations in the heart with age potentially represent an important pathological barrier and could clearly contribute to the lack of improvement in cardiac pressure-volume indexes in the intact heart preparation. Our findings suggest that growth hormone replacement therapy cannot rapidly reverse ventricular remodeling once it has occurred in an aged heart.

In these aged rats, coronary blood flow was reduced at baseline and in response to maximal vessel dilation. This is most compatible with an age-related rarefaction of coronary vessels and cannot be explained solely on the basis of altered autoregulation. The reduction in coronary flow was not reversed by growth hormone replacement therapy and would very likely lead to impairments in contractile function in the intact heart. This again suggests that growth hormone replacement therapy cannot rapidly reverse the established functional effects of age-related coronary vessel rarefaction.

Perhaps the most important factor determining the impact of growth hormone supplementation in our study was the timing of the intervention. In our study, rats were already 28 mo of age before we intervened with 4 wk of growth hormone replacement therapy. It is likely that certain structural changes were already in place that could not be easily reversed within 4 wk. In this regard, growth hormone replacement could rapidly restore diminished protein synthesis, and thus restore myofilament contractile state. However, it appears to be difficult to reverse myocardial fibrosis, coronary vessel rarefaction, and LV remodeling in aged hearts. Our findings of an increase in collagen deposition and decrease in basal coronary flow with age, without an effect of growth hormone on either of these endpoints, support this conclusion.

It is possible that growth hormone replacement at an earlier age with consistent maintenance of pulsatile growth hormone release could have prevented the observed changes in coronary vasculature and ventricular remodeling. Nevertheless, growth hormone replacement for 30 days increased maximal force generation in trabeculae from aged rats. This finding encourages further examination of the role of growth hormone in age-related myocardial dysfunction, and especially its early use in the course of cardiovascular disease.


    ACKNOWLEDGEMENTS

The authors thank Colleen Lynch for assistance with rodent blood sampling, Kathie Barrett for assistance with heart pathology analysis, and Sean Bennett, Rhonda Ingram, Adrienne Cashion, and Amy Ng for excellent technical assistance.


    FOOTNOTES

* T. Wannenburg and A. S. Khan contributed equally to this work.

This study was supported by National Institutes of Health Grants AG-07752 (to W. E. Sonntag), AG-11370 (to W. E. Sonntag), and HL-03255 (to T. Wannenburg).

Address for reprint requests and other correspondence: T. Wannenburg, Dept. of Cardiology, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1083 (E-mail: twannen{at}wfubmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 November 2000; accepted in final form 3 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amato, G, Carella C, Fazio S, La Montagna G, Cittadini A, Sabatini D, Marciano-Mone C, Sacca L, and Bellastella A. Body composition, bone metabolism, and heart structure and function in growth hormone (GH)-deficient adults before and after GH replacement therapy at low doses. J Clin Endocrinol Metab 77: 1671-1676, 1993[Abstract].

2.   Ameredes, BT, Daood MJ, and Watchko JF. Refeeding reverses cardiac myosin shifts induced by undernutrition in aged rats: modulation by growth hormone. J Mol Cell Cardiol 30: 1525-1533, 1998[Web of Science][Medline].

3.   Anversa, P, Li P, Sonnenblick EH, and Olivetti G. Effects of aging on quantitative structural properties of coronary vasculature and microvasculature in rats. Am J Physiol Heart Circ Physiol 267: H1062-H1073, 1994[Abstract/Free Full Text].

4.   Anversa, P, Palackal T, Sonnenblick EH, Olivetti G, Meggs LG, and Capasso JM. Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart. Circ Res 67: 871-885, 1990[Abstract/Free Full Text].

5.   Cheng, W, Reiss K, Li P, Chun MJ, Kajstura J, Olivetti G, and Anversa P. Aging does not affect the activation of the myocyte insulin-like growth factor-1 autocrine system after infarction and ventricular failure in Fischer 344 rats. Circ Res 78: 536-546, 1996[Abstract/Free Full Text].

6.   Cittadini, A, Cuocolo A, Merola B, Fazio S, Sabatini D, Nicolai E, Colao A, Longobardi S, Lombardi G, and Sacca L. Impaired cardiac performance in GH-deficient adults and its improvement after GH replacement. Am J Physiol Endocrinol Metab 267: E219-E225, 1994[Abstract/Free Full Text].

7.   Cittadini, A, Grossman JD, Napoli R, Katz SE, Stromer H, Smith RJ, Clark R, Morgan JP, and Douglas PS. Growth hormone attenuates early left ventricular remodeling and improves cardiac function in rats with large myocardial infarction. J Am Coll Cardiol 29: 1109-1116, 1997[Abstract].

8.   Daugaard, JR, Laustsen JL, Hansen BS, and Richter EA. Growth hormone induces muscle fibre type transformation in growth hormone-deficient rats. Acta Physiol Scand 164: 119-126, 1998[Web of Science][Medline].

9.   De Tombe, PP, and ter Keurs HE. Force and velocity of sarcomere shortening in trabeculae from rat heart. Effects of temperature. Circ Res 66: 1239-1254, 1990[Abstract/Free Full Text].

10.   Dolber, PC, and Spach MS. Picrosirius red staining of cardiac muscle following phosphomolybdic acid treatment. Stain Technol 62: 23-26, 1987[Web of Science][Medline].

11.   Duerr, RL, McKirnan MD, Gim RD, Clark RG, Chien KR, and Ross J, Jr. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic left ventricular failure in the rat. Circulation 93: 2188-2196, 1996[Abstract/Free Full Text].

12.   Eghbali, M, Robinson TF, Seifter S, and Blumenfeld OO. Collagen accumulation in heart ventricles as a function of growth and aging. Cardiovasc Res 23: 723-729, 1989[Web of Science][Medline].

13.   Fabiato, A, and Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 75: 463-505, 1979[Medline].

14.   Genth-Zotz, S, Zotz R, Geil S, Voigtlander T, Meyer J, and Darius H. Recombinant growth hormone therapy in patients with ischemic cardiomyopathy: effects on hemodynamics, left ventricular function, and cardiopulmonary exercise capacity. Circulation 99: 18-21, 1999[Abstract/Free Full Text].

15.   Gerstenblith, G, Frederiksen J, Yin FC, Fortuin NJ, Lakatta EG, and Weisfeldt ML. Echocardiographic assessment of a normal adult aging population. Circulation 56: 273-278, 1977[Abstract/Free Full Text].

16.   Godt, RE, and Lindley BD. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol 80: 279-297, 1982[Abstract/Free Full Text].

17.   Groesbeck, MD, and Parlow AF. Highly improved precision of the hypophysectomized female rat body weight gain bioassay for growth hormone by increased frequency of injections, avoidance of antibody formation, and other simple modifications. Endocrinology 120: 2582-2590, 1987[Abstract/Free Full Text].

18.   Guth, K, and Wojciechowski R. Perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres. Pflügers Arch 407: 552-557, 1986[Web of Science][Medline].

19.   Haro, LS, Bustamante J, Hernandez P, Flores R, Aguilar R, Lopez-Guajardo C, and Martinez AO. Biochemistry and pharmacology of rabbit cardiac growth hormone (GH) receptors. Mol Cell Endocrinol 152: 179-187, 1999[Web of Science][Medline].

20.   Huxley, HE. The crossbridge mechanism of muscular contraction and its implications (Review). J Exp Biol 115: 17-30, 1985[Abstract/Free Full Text].

21.   Johannsson, G, Bengtsson BA, Andersson B, Isgaard J, and Caidahl K. Long-term cardiovascular effects of growth hormone treatment in GH-deficient adults. Preliminary data in a small group of patients. Clin Endocrinol (Oxf) 45: 305-314, 1996[Medline].

22.   Kajstura, J, Cheng W, Sarangarajan R, Li P, Li B, Nitahara JA, Chapnick S, Reiss K, Olivetti G, and Anversa P. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol Heart Circ Physiol 271: H1215-H1228, 1996[Abstract/Free Full Text].

23.   Kitamura, M, Shimizu M, Kita Y, Yoshio H, Ino H, Misawa K, Matsuyama T, and Mabuchi H. Quantitative evaluation of the rate of myocardial interstitial fibrosis using a personal computer. Jpn Circ J 61: 781-786, 1997[Medline].

24.   Klima, M, Burns TR, and Chopra A. Myocardial fibrosis in the elderly. Arch Pathol Lab Med 114: 938-942, 1990[Web of Science][Medline].

25.   Kuschinsky, W, Bunger R, Schrock H, Mallet RT, and Sokoloff L. Local glucose utilization and local blood flow in hearts of awake rats. Basic Res Cardiol 88: 233-249, 1993[Web of Science][Medline].

26.   LeRoith, D, Yanowski J, Kaldjian EP, Jaffe ES, LeRoith T, Purdue K, Cooper BD, Pyle R, and Adler W. The effects of growth hormone and insulin-like growth factor I on the immune system of aged female monkeys. Endocrinology 137: 1071-1079, 1996[Abstract].

27.   Long, X, Boluyt MO, O'Neill L, Zheng JS, Wu G, Nitta YK, Crow MT, and Lakatta EG. Myocardial retinoid X receptor, thyroid hormone receptor, and myosin heavy chain gene expression in the rat during adult aging (see comments). J Gerontol A Biol Sci Med Sci 54: B23-B27, 1999[Abstract].

28.   Masutomo, K, Makino N, Sugano M, Miyamoto S, Hata T, and Yanaga T. Extracellular matrix regulation in the development of Syrian cardiomyopathic Bio 14.6 and Bio 5358 hamsters. J Mol Cell Cardiol 31: 1607-1615, 1999[Web of Science][Medline].

29.   Mathews, LS, Enberg B, and Norstedt G. Regulation of rat growth hormone receptor gene expression. J Biol Chem 264: 9905-9910, 1989[Abstract/Free Full Text].

30.   Mulieri, LA, Hasenfuss G, Ittleman F, Blanchard EM, and Alpert NR. Protection of human left ventricular myocardium from cutting injury with 2,3-butanedione monoxime. Circ Res 65: 1441-1449, 1989[Abstract/Free Full Text].

31.   Nitahara, JA, Cheng W, Liu Y, Li B, Leri A, Li P, Mogul D, Gambert SR, Kajstura J, and Anversa P. Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats. J Mol Cell Cardiol 30: 519-535, 1998[Web of Science][Medline].

32.   Olivetti, G, Melissari M, Capasso JM, and Anversa P. Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy. Circ Res 68: 1560-1568, 1991[Abstract/Free Full Text].

33.   Parrado, J, Bougria M, Ayala A, Castano A, and Machado A. Effects of aging on the various steps of protein synthesis: fragmentation of elongation factor 2. Free Radic Biol Med 26: 362-370, 1999[Web of Science][Medline].

34.   Raizada, V, Pathak D, Nakouzi A, and Malhotra A. Prevention of age-related V1 myosin isozyme decrement in the adult rat heart. J Mol Cell Cardiol 26: 293-296, 1994[Web of Science][Medline].

35.   Robert, V, Besse S, Sabri A, Silvestre JS, Assayag P, Nguyen VT, Swynghedauw B, and Delcayre C. Differential regulation of matrix metalloproteinases associated with aging and hypertension in the rat heart. Lab Invest 76: 729-738, 1997[Web of Science][Medline].

36.   Rudman, D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L, Rudman IW, and Mattson DE. Effects of human growth hormone in men over 60 years old (see comments). N Engl J Med 323: 1-6, 1990[Abstract/Free Full Text].

37.   Rudman, D, Kutner MH, Rogers CM, Lubin MF, Fleming GA, and Bain RP. Impaired growth hormone secretion in the adult population: relation to age and adiposity. J Clin Invest 67: 1361-1369, 1981.

38.   Ryoke, T, Gu Y, Mao L, Hongo M, Clark RG, Peterson KL, and Ross J, Jr. Progressive cardiac dysfunction and fibrosis in the cardiomyopathic hamster and effects of growth hormone and angiotensin-converting enzyme inhibition. Circulation 100: 1734-1743, 1999[Abstract/Free Full Text].

39.   Sartorio, A, Ferrero S, Conti A, Bragato R, Malfatto G, Leonetti G, and Faglia G. Adults with childhood-onset growth hormone deficiency: effects of growth hormone treatment on cardiac structure. J Intern Med 241: 515-520, 1997[Web of Science][Medline].

40.   Slinker, BK, and Glantz SA. Multiple linear regression is a useful alternative to traditional analyses of variance (Review). Am J Physiol Regulatory Integrative Comp Physiol 255: R353-R367, 1988[Abstract/Free Full Text].

41.   Sonntag, WE, Hylka VW, and Meites J. Growth hormone restores protein synthesis in skeletal muscle of old male rats. J Gerontol 40: 689-694, 1985[Abstract/Free Full Text].

42.   Sonntag, WE, Steger RW, Forman LJ, and Meites J. Decreased pulsatile release of growth hormone in old male rats. Endocrinology 107: 1875-1879, 1980[Abstract/Free Full Text].

43.   Steer, A, Nakashima T, Kawashima T, Lee KK, Danzig MD, Robertson TL, and Dock DS. Small cardiac lesions. Fibrosis of papillary muscles and focal cardiac myocytolysis. Jpn Heart J 18: 812-822, 1977[Medline].

44.   Stienen, GJ, Papp Z, and Elzinga G. Calcium modulates the influence of length changes on the myofibrillar adenosine triphosphatase activity in rat skinned cardiac trabeculae. Pflügers Arch 425: 199-207, 1993[Web of Science][Medline].

45.   Suga, H, Yamada O, Goto Y, and Igarashi Y. Peak isovolumic pressure-volume relation of puppy left ventricle. Am J Physiol Heart Circ Physiol 250: H167-H172, 1986.

46.   Tajima, M, Weinberg EO, Bartunek J, Jin H, Yang R, Paoni NF, and Lorell BH. Treatment with growth hormone enhances contractile reserve and intracellular calcium transients in myocytes from rats with postinfarction heart failure (see comments). Circulation 99: 127-134, 1999[Abstract/Free Full Text].

47.   Tiong, TS, and Herington AC. Tissue distribution, characterization, and regulation of messenger ribonucleic acid for growth hormone receptor and serum binding protein in the rat. Endocrinology 129: 1628-1634, 1991[Abstract/Free Full Text].

48.   Wannenburg, T, de Tombe PP, and Little WC. Effect of adenosine on contractile state and oxygen consumption in isolated rat hearts. Am J Physiol Heart Circ Physiol 267: H1429-H1436, 1994[Abstract/Free Full Text].

49.   Wannenburg, T, Janssen PM, Fan D, and de Tombe PP. The Frank-Starling mechanism is not mediated by changes in rate of cross-bridge detachment. Am J Physiol Heart Circ Physiol 273: H2428-H2435, 1997.

50.   Yang, R, Bunting S, Gillett N, Clark R, and Jin H. Growth hormone improves cardiac performance in experimental heart failure. Circulation 92: 262-267, 1995[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 281(2):H915-H922
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 J Gerontol A Biol Sci Med SciHome page
L. Groban, H. Jobe, M. Lin, T. Houle, D. A. Kitzman, and W. Sonntag
Effects of Short-Term Treadmill Exercise Training or Growth Hormone Supplementation on Diastolic Function and Exercise Tolerance in Old Rats
J Gerontol A Biol Sci Med Sci, September 1, 2008; 63(9): 911 - 920.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
R. Rabkin, I. Awwad, Y. Chen, E. A. Ashley, D. Sun, S. Sood, W. Clusin, P. Heidenreich, G. Piecha, and M.-L. Gross
Low-Dose Growth Hormone is Cardioprotective in Uremia
J. Am. Soc. Nephrol., September 1, 2008; 19(9): 1774 - 1783.
[Abstract] [Full Text] [PDF]

Home page
 J Gerontol A Biol Sci Med SciHome page
L. Groban, N. A. Pailes, C. D. L. Bennett, C. S. Carter, M. C. Chappell, D. W. Kitzman, and W. E. Sonntag
Growth Hormone Replacement Attenuates Diastolic Dysfunction and Cardiac Angiotensin II Expression in Senescent Rats
J Gerontol A Biol Sci Med Sci, January 1, 2006; 61(1): 28 - 35.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. S. Khan, D. C. Sane, T. Wannenburg, and W. E. Sonntag
Growth hormone, insulin-like growth factor-1 and the aging cardiovascular system
Cardiovasc Res, April 1, 2002; 54(1): 25 - 35.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wannenburg, T.
Right arrow Articles by Sonntag, W. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wannenburg, T.
Right arrow Articles by Sonntag, W. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online