A preliminary randomized study of growth hormone administration in Becker and Duchenne muscular dystrophies

Antonio Cittadinia,*, Lucia Ines Comib, Salvatore Longobardia, Vito Rocco Petrettab, Cosma Casaburia, Luigia Passamanob, Bartolomeo Merolac, Emanuele Durante-Mangonid, Luigi Saccàa and Luisa Politanob

a Department of Internal Medicine and Cardiovascular Sciences, University Federico II,Via Sergio Pansini 5, Naples 80131, Italy
b Department of Clinical and Experimental Medicine, Second University, Naples, Italy
c Department of Endocrinology, University Federico II, Via Sergio Pansini 5, Naples 80131, Italy
d Department of Internal Medicine, Second University, Naples, Italy

* Corresponding author. Tel.: +39-081-746-4375; fax: +39- 081-746-3199
E-mail address: cittadin{at}unina.it

Received 22 July 2002; revised 8 October 2002; accepted 9 October 2002


    Abstract
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Aim Since growth hormone (GH) has proven beneficial in experimental heart failure, and the natural history of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is frequently complicated by the development of dilated cardiomyopathy, we administered GH to six patients with DMD and 10 with BMD, with the evidence of cardiac involvement.

Methods and results Patients were randomized to receive for 3 months either placebo or recombinant human GH, in a double-blind fashion. In GH-treated patients, left ventricular (LV) mass increased by 16% in BMD and by 29% in DMD (both ), with a significant increase of relative wall thickness (+19%). Systemic blood pressure remained unchanged, while LV end-systolic stress fell significantly by 13% in BMD and by 33% in DMD, with a slight increase of systolic function indexes. No changes were observed related to cardiac arrhythmias and skeletal muscle function in the patient groups during the treatment period, nor any side effects were observed. Brain natriuretic peptide, interleukin-6, and tumor necrosis factor-{alpha} circulating levels were elevated at baseline. While brain natriuretic peptide decreased by 40%, cytokine levels did not exhibit significant variations during the treatment period.

Conclusions The 3-month GH therapy in patients with DMD and BMD induces a hypertrophic response associated with a significant reduction of brain natriuretic peptide plasma levels and a slight improvement of systolic function, no changes in skeletal muscle function, and no side effects.

Key Words: Growth hormone • X-linked muscular dystrophy • Cytokines


    1. Introduction
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Duchenne and Becker muscular dystrophies (DMD and BMD) are hereditary, X-linked degenerative disorders caused by mutation of the dystrophin gene.1 Since dystrophin plays a pivotal role inmaintaining sarcolemmal integrity and functionin skeletal and cardiac muscles, cardiomyopathyrepresents a prominent clinical feature in both DMD and BMD.2,3 A clinically evident cardiomyopathy occurs in 73% of BMD patients over 40 years. Dilated cardiomyopathy represents the most frequent type of cardiac involvement and the primary cause of death.3 In DMD, even though cardiac involvementis very common and virtually present in all the patients over 18 years, motor and respiratory dysfunctions progress more rapidly representing the first cause of death.2

The role of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis in the modulation of cardiac structure and function under normal circumstances and in diseased states has recently been delineated in a series of animal and human studies.4–10 However, the use of GH as an adjunctive therapy in heart failure is still controversial. In fact, while several uncontrolled studies have documented a major improvement of clinical status, myocardial function, and energetics in patients with idiopathic dilated cardiomyopathy, recent randomized trials did not show significant changes in baseline left ventricular (LV) function despite confirming the growth-promoting effect of GH therapy.8–10

In addition to cardiac muscle, skeletal muscle is a typical target tissue for the anabolic and growth-promoting actions of GH, and its involvement is prominent in all muscular dystrophies.1 Moreover, it has been documented that the well-knownbeneficial effects of corticosteroids in DMD are associated with increased circulating IGF-1 levels, suggesting a link between augmented GH/IGF-1 axis activity and clinical improvement.11 Further support for the use of GH in muscular dystrophies comes from two recent animal studies performed in the Syrian cardiomyopathic hamster. Takeshi et al. demonstrated that 3-week GH treatment prevented the development of heart failure, significantly attenuated LV dilation, and improved cardiac function.12 More recently, Ross Jr.'s group showed decreased end-systolic wall stress and improved systolic function in young cardiomyopathic hamsters after GH treatment.13

The aim of the current study was to evaluate the effects of a 3-month GH therapy on cardiac structure and function in patients with X-linked muscular dystrophy. We also measured circulating levels of brain natriuretic peptide (BNP) and cytokines, since they have been shown to correlate with heart failure severity and prognosis.14 In view of the lack of data about the safety of GH administration in this specific patient population, we planned a pilot study, with a double-blind, placebo-controlleddesign, on 16 subjects.


    2. Methods
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
2.1. Study protocol
Ten consecutive patients with BMD and six consecutive patients with DMD, with documented cardiac involvement were selected, after written informed consent, from a large cohort attending the Cardiomyology and Myology Center of the Naples Second University. Immunohistochemical analysis of dystrophin from skeletal muscle biopsy specimens was carried out in all patients to confirm the diagnosis according to previously described methods.15 The study protocol was approved by the Ethics Committee of the Federico II University. Patients were randomized to receive either placebo or recombinant human GH at a replacement dose for patients with GH deficiency. Specifically, the GH dose was 0.23 and 0.07mg/kg/week in Duchenne and Beckerpatients, respectively, self injected s.c. at bedtime for 3 months. GH dose was chosen taking intoaccount the substitution dose routinely employed in GH-deficient subjects, the physiological decline of GH secretion with age, the impairment of GH secretion after stimulation tests occurring in severalpatients, and previous experience in patientswith chronic heart failure. Background therapyremained unchanged in all patients. In BMD, this included an angiotensin converting enzyme inhibitor (Fosinopril, 20–30mg/day), Warfarin (to keep ratio between 1.5 and 3), Magnesium pidolatum (1.5g/day), antioxidant agents (Vitamin E, 900mg/day; Vitamin C, 1g/day; Glutathione, 600mg/day for 10 days per month; Ubiquinone, 50mg/day), Furosemide (25–50mg/day), Deflazacort (15mg/day). One patient in the placebo and one in theGH group were taking Digoxin, 0.2mg/day and Amiodarone, 600mg/week. Standard therapyfor DMD included: Deflazacort, 0.5mg/kg/day;Fosinopril, 10mg/day; and antioxidant agents(Vitamin E, 600–900mg/day; Glutathione, 300–600mg/day for 10 days per month; Ubiquinone, 50mg/day). All BMD patients were in NYHA class II, except three patients who were in class III.

2.2. Laboratory evaluation
Before study entry, all subjects underwent the following pharmacological tests to evaluate GHsecretion: (1) Growth hormone-releasing hormone (GHRH); and (2) Arginine tolerance test (ATT), as previously described.16 In addition, routine analysis, creatine kinase, IGF-1, thyroid hormone, testosterone, and insulin levels were measured at baseline and after the treatment phase wereassayed using commercial kits.17 Serum GH and plasma IGF-I levels were assessed by IRMA, using DSL kits (Diagnostic System Laboratories, Inc,Webster, TX, USA). The normal IGF-I range in our laboratory for age- and sex-matched individuals was 215–290µg/l in adults and 245–365µg/l in prepubertal children. Serum IGF-I showed an intra-assay cv of 3.4, 3.0, and 1.5%, and inter-assay cv of 8.2, 1.5, and 3.7% at 10.4, 53.8, and 255.9µg/l. Testosterone and insulin were assayed by RIA method using a commercial kit (DSL, Diagnostic System Laboratories, Inc) Evaluation of plasma TSH levels was performed by an ultrasensitive immunoradiometric assay (Bouty, Milan, Italy) with a detection limit of 0.05mU/l. The intra- and inter-assay variability were 3.1 and 3.8%, respectively, at 0.25–50mU/l. Serum FT4and FT3were measured using the Lisophase Kits (Bouty, Milan, Italy). The intra-and inter-assay variations and sensitivities were 2.9%, 4.7%, and 0.8pmol/l, respectively, for FT3and 4.1%, 5.9%, and 1.0pmol/l, respectively, for FT4. Reference ranges in our laboratory were: TSH, 0.2–3.0 mU/l; FT3, 4.0–9.2pmol/l; and FT4, 7.7–20.6pmol/l. Plasma levels of BNP weremeasured with a commercially available specific immunoradiometric assay kit for human BNP. Plasma interleukin-6 (IL-6) and tumor necrosis factor-{alpha} (TNF-{alpha}) were determined by enzymeimmunoassay using commercial kits (MedgenixDiagnostics SA), with detection limits of 2 and 3ng/l, respectively. Values of BNP, IL-6, and TNF-{alpha} in age- and sex-matched normal individuals in our laboratory were 13±3pg/ml, 0.85±0.2ng/l, and 19±2ng/l, respectively. Laboratory staff were blinded as to the treatment code.

2.3. Cardiac evaluation
ECG included cardiomyopathic index (the QT:PQ ratio, normal values 2.2–4.6s) which was assessed as previously described.3 Twenty-four-hour ECGmonitoring was performed to detect rhythm disturbances. The print-outs were analyzed blindlyby two observers. The complexity of ventricular premature beats was graded according to theLown classification.18 Complete M-mode, two-dimensional (2D), and Doppler echocardiographic analysis was performed according to the standardization of the American Society of Echocardiography,19 as previously described.8,17 LV volumeswere calculated according to a biplane area–length method. The investigator reading the echoes was blinded as to whether the recordings hewas interpreting were of placebo or GH-treatedpatients.

2.4. Skeletal muscle function evaluation
The parameters assessed were: (1) timed functional testing—tests included measurement of the time needed to rise to a standing position from a lying position on the floor (Gowers time), to climb four standard-size stairs, and travel 10m as fastas possible; and (2) Dynamic index, which was assessed as previously described.3

2.5. Pulmonary-function tests
Assessment included measurements of forcedvital capacity, maximal voluntary ventilation, and maximal expiratory pressure.

2.6. Statistical analysis
All values are given as mean±SE. Statistical analysis was performed using the STATISTICA package. Between-group comparisons of echocardiographic indexes were performed using the two-way ANOVA with repeated measures in one factor (time). One-way ANOVA was used for the other comparisons. Where appropriate, comparisons to determine the significance of changes within the same group over time and between the groups at each time point were performed with Neuman–Keuls test. Linear regression analysis was used as appropriate. A value of was considered significant. Due to the small sample size of the Duchenne group (three patients in both the GH and the placebo group), we did not perform ANOVA analyses. The use of corresponding nonparametric tests yielded similar levels of significance.


    3. Results
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
3.1. Laboratory evaluation
Baseline values of anthropometric and hormonal parameters were comparable in the placebo and the GH group. There were no significant changesin the assessed hematochemical parameters(Table 1). Seven out of 16 patients displayed an impairment of the GH/IGF-1 axis, as shown by low circulating IGF-1 levels and no response to GH stimulatory tests (Tables 1 and 2). Plasma IGF-1 levels increased by 82%, compared to baseline, in the active treatment group, while decreased by 9% in the placebo group (Table 2). Thyroid hormone levels did not change significantly during the treatment period. Plasma levels of BNP, which were elevated when compared with normal values,decreased by 40% in the active treatment group, while no significant changes were detected in the placebo group (Table 2). IL-6 and TNF-{alpha} plasma levels were significantly elevated when compared with an age- and sex-matched control group, while no differences were observed during the treatment period (Table 2). BNP and cytokine plasma levels were similar in the Becker and Duchenne population during the treatment period (data not shown). Consequently, in Table 2, data from both study populations are pooled. In the entire population of X-linked dystrophic patients, linear regression analysis revealed a significant direct correlation between IL-6 and TNF-{alpha} levels (, ), while baseline TNF-{alpha} plasma levels and percent changes of IGF-1 (baseline vs. 3 months) wereinversely correlated (, ).


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Table 1 Individual clinical and endocrinological characteristics of patients with X-linked muscular dystrophies

 

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Table 2 IGF-1, BNP, and cytokine plasma levels in X-linked muscular dystrophies

 
3.2. Cardiac evaluation
No significant variations were observed in cardiomyopathic index (which was abnormal in five BMD and three DMD) in both groups of patients during the entire study period. All patients but one had less than 100 atrial premature beats in 24h; in one patient (GH group) the number of atrial premature beat decreased by 45% in 24h. In BMD patients, Lown class ranged from 0 to 5. In the GH-treated group, there was a reduction from Lown class 4A to 1A in one patient; in the other five, the class remained unchanged. In the placebo group, one patient had a decrease of Lown class from 4B to 4A while in the other three it remained unchanged.All Duchenne patients were in Lown class 0 to 1and remained in the same class during the entire treatment period. As expected, LV volumes tended to be larger and ejection fraction lower in BMDvs. DMD (see Table 3 and Fig. 1;comptd;226336n;center;stack;;;;;6;;;;;best> for EF values), reflecting the more common occurrence of dilated cardiomyopathy in Becker patients.2,3 Baseline echocardiographic parameters did not differ significantly between the two BMD groups (see Table 3). In the GH-treated BMD patients, LV mass increased by approximately 42g, while slightly decreased in the placebo group. This hypertrophic responsewas accounted for by a trend toward increased LV posterior and anterior wall thicknesses in the active treatment group. There was a concentric remodeling of the LV cavity, with a significant increase of the relative wall thickness in the GH-treated group by 12%, while no changes were observed in the placebo group. The concentric growth, without concomitant changes in systemic blood pressure, led to a significant fall in end-systolic wall stress by 13%. The 2D calculated end-diastolic volumes did not change significantly after GH therapy, while there was a tendency toward decreased end-systolic volumes in the active treatment group (138±15 vs. 113±15ml, ) with a 6% increase of LV ejection fraction. In the placebo group, 2Dderived ejection fraction did not show any change during the treatment period. There was a slight increase of stroke volume and cardiac output with GH therapy (). Heart rate did not show significant changes. Total systemic resistance fell by 18% in the GH-treated patients due to the increased cardiac output and unchanged systemic blood pressure (). The results from the Duchenne group mirrored those from the Becker group. A myocardial growth-promoting effect was evident in the GH-treated group, with a 29% increase in LV mass, a 33% decrease in end-systolic stress, and a tendency of fractional shortening to increase (Fig. 1).


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Table 3 Cardiac and skeletal muscle, and pulmonary parameters in Becker patients

 


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Fig. 1 Effect of growth hormone administration on LV mass, end-systolic stress, and ejection fraction in patients with DMD.

 
Fig. 2;comptd;;center;stack;;;;;6;;;;;width> shows a significant correlation between change in LV end-systolic stress and change in IGF-1 levels in the entire patient population (,). The pattern of LV diastolic filling, asassessed by mitral flowmetry, did not change among the four study groups during the treatment period, despite the hypertrophic response displayed by the GH-treated patients.



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Fig. 2 Relationship between changes in LV end-systolic stress and changes in serum IGF-1 concentrations.

 
3.3. Skeletal muscle function evaluation
Not all the patients were able to perform timed functional tests. No significant differences were observed in timed functional tests in Beckerand Duchenne patients during the entire period of observation (Table 3).

3.4. Pulmonary-function tests
No changes were observed among the four study groups during the treatment period.


    4. Discussion
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
The current study provides the first evidence that GH activates myocardial growth in BMD and DMD. Such hypertrophic response was associated with a significant reduction of circumferential wall stress, a tendency toward enhanced systolic function,and no changes in diastolic filling and skeletalmuscle function. Systemic vascular resistance was slightly decreased. Such structural and hemodynamic changes were associated with a significant reduction of BNP plasma levels.

These cardiovascular effects are reminiscent of those observed after GH administration in human dilated cardiomyopathy.8–10 The effects of theactivation of the GH/IGF-1 axis on the loading conditions and myocardial contractility5–7 mayindeed underlie these findings. In fact, LV concentric remodeling with consequent reduction of wall stress and GH's mediated vasodilatory actions may both have contributed to after-load reduction.Interestingly, both DMD and BMD displayed similar response to GH's growth-promoting and hemodynamic properties, despite the different muscle dystrophin content and clinical progression.

In patients treated with GH, no significantimprovement of exercise capacity could bedetected as assessed by timed functionalindexes. Since, in addition to cardiovascular status, respiratory and skeletal muscle functions are also major determinants of exercise capacity, such finding might reflect GH's inability to act on these target tissues. This may be owing to the more advanced damage of skeletal compared to heart muscle usually found at the histological level in muscular dystrophies. Alternatively, the short treatment duration might have precluded an effect of GH. This latter speculation is supported by previous studies in GH-deficient patients treated with GH replacement therapy, in whom 6-month period is usually the minimum time-range employed in most clinical trials to observe changes in cardiac and skeletal muscle function.20 In GH deficiency, cardiovascular function is generally restored sooner than skeletal muscle function, clinical indexes of which show initial modifications after at least1 year of GH treatment.17 In this regard, most of our patients display abnormalities of the GH/IGF-1 axis similar to those observed in GH deficiency.

Whether the reactivation of cardiac growth in the setting of muscular dystrophies may improve survival remains to be addressed by futurelonger term investigations. On one hand, such hypertrophic response might be viewed as anunpropitious component of LV remodeling, since LV hypertrophy is associated with increasedcardiovascular mortality. On the other hand,experimental data support the notion that, at variance with pathologic hypertrophy, GH-induced cardiac growth displays unique features, such as preserved capillary density and diastolic function, improvements of intracellular calcium dynamics, and systolic performance despite less energetic cost.5–7 The unchanged diastolic filling in GH-treated patients despite the increased LV mass appears congruent with this concept. The significant reduction of BNP plasma levels (–40%) lends support to the hypothesis that GH-induced structural and functional changes in X-linked muscular dystrophies may indeed be beneficial. BNP is a ventricular hormone whose circulating levels are directly related to LV end-diastolic pressures, to the degree of LV damage, and to LV mass, andhas recently been demonstrated to be a strong prognostic indicator of morbidity and mortality in patients with heart failure following optimized treatment.14 Therefore, the novel finding related to elevated baseline BNP circulating levels inpatients with X-linked muscular dystrophy, herein reported, supports the known impairment of LV structure and mechanics of this patient population, and, probably, its adverse prognosis despite optimal medical treatment. On the other hand, BNP reduction after GH therapy may reflect an improved hemodynamic profile and, possibly, a better outcome of the active treatment group, and appears particularly relevant insofar as itoccurs in the setting of increased LV mass induced by GH.

Patients with X-linked muscular dystrophy presented with elevated plasma levels of cytokines, that were not significantly changed after 3 months of GH therapy. Such pattern of chronic inflammation is commonly seen in chronic heart failure and is probably causally related to its pathogenesis and progression,21 as well as to muscle dysfunction in DMD.22 Moreover, since experimental modelsof inflammation also inhibit GH signaling,23 it is possible that high circulating levels of IL-6 and TNF-{alpha} may contribute to the observed impairment of the GH/IGF-1 axis (vide infra). This contentionis supported by our finding that the response toGH therapy, expressed as percent change in IGF-1 circulating levels, was inversely correlated with baseline TNF-{alpha} levels. At variance with BNP, the absence of IL-6 and TNF-{alpha} plasma levels changes after 3-month GH therapy either suggests theinability of GH therapy to modify the cytokine pattern in this clinical stetting or that a longer treatment period would be needed. This lastspeculation is congruent with a previous report showing that changes of inflammatory cytokines usually occur only after at least 6 months of therapy.24

Forty-four percent of our patient population displayed abnormalities of the GH/IGF-1 axis, similar to those observed in GH deficiency, i.e. low IGF-1 circulating levels and an altered response to GH stimulatory tests. Possible explanations for this finding include indirect mechanisms, such as physical inactivity or concomitant corticosteroid treatment, or alternatively it may reflect a primary unknown disorder of GH secretion.

Previous studies have addressed the role of GH/IGF-1 axis in patients with muscular dystrophies, in particular DMD.25–27 Conflicting results have been reported. The observation of a slower progression of DMD in a patient with dwarfism lead to the hypothesis that a low activity of the GH/IGF-1 axis might be beneficial in muscular dystrophy.25 Two lines of evidence have challenged this initialspeculation. First, the frequent finding of impaired GH secretion, particularly in patients with DMD, was not associated with a milder form of disease, in keeping with our findings.26 Second, clinical trials with a GH inhibitor, mazindolol, have been unable to show any beneficial effect of GH-reduced secretion on clinical status and natural history of DMD.27

An obvious limitation of the current preliminary trial is the limited sample size, which hampers definite conclusions related to GH efficacy in DMD and BMD. However, while larger and/or long-term trials are needed to confirm and expand on our findings, the presence of borderline statistical significance or trends toward significance, despitethe small number of patients studied, might point toward a strong biological cardiovascular effectof GH.

The 3-month therapy in DMD and BMD did not cause any clinical relevant side effect. The safety of GH therapy was also documented by the absence of cardiac arrhythmias at Holter monitoring, and by the stability of all hematological parameters.


    Acknowledgments
 
This work was supported in part by grant C61 (L.P.) by Telethon ITALY.


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 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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