Trial Information

Cardiac and Skeletal Muscle Energy Metabolism in Abnormal Growth Hormone States

INTRODUCTION The effect of GH and IGF-I on the heart has been demonstrated in numerous
experimental studies. GH and IGF-I receptors are expressed in cardiac myocytes, and IGF-I
causes hypertrophy of cultured rat cardiomyocytes and delays cardiomyocyte apoptosis. In
addition, GH and IGF-I have a direct effect on myocardial contractility, increasing the
intracellular calcium content and enhancing the calcium sensitivity of myofilaments in
cardiomyocytes. Clinical studies in patients with disorders of the GH/IGF-1 axis confirm the
significant relationship between GH/IGF-I and the cardiovascular system. Interestingly, both
GH excess and deficiency states are associated with abnormal cardiac function and with an
attendant increased risk for cardiovascular morbidity and mortality in both. Our contention
is that the apparent paradox of the relationship may be due to changes in the energy state
of the myocardium in GH excess (acromegaly) and deficiency (GHD in hypopituitarism) and the
inability of cellular metabolism to change appropriately between the competing demands of
oxidative stress and anabolic processes. Data from the giant GH-overexpressing transgenic
mouse demonstrate reduced creatinine phosphate-to-ATP ratio supporting an effect of GH on
cardiac energy status.

AMP-activated protein kinase (AMPK) is the energy sensor of the cell and has been shown to
be an important regulator of cell metabolism, including cardiac cells (Dyck & Lopaschuk,
2002). AMPK is activated by rising AMP/ATP ratio, and programs intracellular metabolism to
conserve energy for oxidate metabolism and to suspend anabolic processes. A substantial body
of evidence testifies to the importance of AMPK and the associated regulation of myocardial
energy status to cardiac function.

- Reduced activity of AMPK is a feature of inherited cardiomyopathies.

- Low energy status measured non-invasively is a predictor of death in dilated
cardiomyopathy.

- Activation of AMPK reduces the injury in experimental models of ischaemia

- Reduced cardiac energy reserve is a feature of type 2 diabetes mellitus (T2DM), and
cardiovascular death accounts for over 80% of mortality in T2DM.

- Drugs which are known to activate AMPK improve mortality in type 2 diabetes, such as
metformin and glitazones (Kahn et al., 2005).

- Cannabinoids and ghrelin, first identified by our group to increase cardiac AMPK
levels, have been shown to improve ischaemia-reperfusion injury (Frascarelli et al.,
2003; Underdown et al., 2005; Shibata et al., 2005).

Taking these experimental observations together, it can be inferred that in diabetic cardiac
muscle, there is impaired activation of AMPK despite low energy levels, and potentially a
failure of the normal mechanism to favour oxidative metabolism and cytoprotective functions
during ischaemia.

In patients, cardiac AMPK cannot be directly studied, although in a related animal study we
will assess the effect of GH excess and deficiency on the activation of AMPK cardiac
intracellular energy status and myocardium function. However, using 31P Magnetic Resonance
Spectroscopy (MRS), in vivo measurement of high energy phosphate molecules can be assessed
non-invasively and these have been shown in other diseases to have important clinical
consequences. Specifically, both phosphocreatine (PCr), and ATP can be determined and ADP
levels derived from the phosphocreatine:ATP ratio (Scheurmann-Freestone 2003). This
approach has been recently used to demonstrate low ambient level of myocardial ADP in
patients with T2DM. In the current study we will investigate the energy status in patients
with acromegaly and GHD before and after normalisation of their GH status. We hypothesise
that in untreated acromegaly, the unrestrained drive of anabolism will be associated with a
low energy status, and particularly in inability to respond to exercise. We propose that in
GHD, reduced anabolism will be associated with relatively high levels of energy but an
impairment of muscle mass. We further anticipate that the normalisation of hormone levels
resulting from either medical or surgical therapy will result in improvement in
energy-storing phosphate molecule ratios in the myocardium.

Acromegaly - Cardiac function Acromegalic cardiomyopathy is the specific myocardial disease
of acromegaly (Clayton, 2003; Sacca et al., 2003; Colao et al., 2004b). Active acromegaly is
associated with a 2- to 3-fold increase in mortality, mainly from cardiovascular disease,
although with effective treatment the excess mortality can be reversed (Sheppard, 2005). In
its early stages, acromegalic cardiomyopathy is characterised by increased left ventricular
mass, with eccentric remodeling and normal diastolic function with a high cardiac output
state with reduction of systemic vascular resistance (Fazio et al., 2000). In the next
stage, increased heart mass and diastolic dysfunction, attributed to direct injury of the
myocardium with GH hypersecretion, occur in the absence of associated diseases like
hypertension, diabetes mellitus, and thyroid dysfunction. These abnormalities can progress
to dilated cardiomyopathy and heart failure if acromegaly remains untreated for many years.
There are specific structural changes in the myocardium with increased myocyte size and
interstitial fibrosis of both ventricles. Left ventricular hypertrophy is common (in 64% of
newly diagnosed cases) even in young patients with short duration of disease (Colao et al.,
2003). Functionally, the main consequence of these changes is impaired left ventricular
diastolic function, particularly when exercising, such that exercise tolerance is reduced
(Colao et al., 2002). Myocardial perfusion is impaired in patients with active acromegaly as
assessed by single photon emission computed tomography (SPECT), thus representing an early
stage of cardiac involvement in acromegaly that may be directly mediated by GH excess
(Herrmann et al., 2003). Some of these structural and functional changes can be reversed by
effective treatment to lower serum GH levels to less than 1-2 ng/ml (glucose suppressed or
random, respectively) and normalize IGF-I and long-term outcome and survival is improved
(Colao et al., 2002; Jaffrain-Rea et al., 2003). Diastolic function improves with treatment,
but the effect on exercise tolerance is more variable, and more longitudinal data are
required to assess the benefits.

Acromegaly - Coronary atherosclerosis Although coronary disease has a prevalence of 3-40%
in acromegalic patients, there is considerable controversy whether this is directly related
to GH excess. According to a well-controlled study (Otsuki et al., 2001) in which intima
thickness was measured, the extent of atherosclerosis in the acromegalic patients was not
higher than that in non-acromegalic subjects, and considering their increased number of
atherosclerotic risk factors, such as lipid abnormalities, diabetes, increased homocystein,
lipoprotein a, fibrinogen and platelet activator inhibitor 1 levels which are all strong
predictors of coronary vascular disease, GH might therefore be even protective (Matta &
Caron, 2003). In a recently described study with 79 acromegalic patients with 22 age-matched
controls again no difference was observed in intima thickness (Paisley et al., 2006).
Increased concentration of IGF-I - via its vasodilatotary effect mediated by nitric oxide -
might be involved in the lack of susceptibility to atherosclerosis in some acromegalic
patients. There are no previously published studies using electron beam coronary CT in this
group of patients.

Acromegaly - Skeletal muscle It is recognised that skeletal muscle in acromegaly fatigues
rapidly but this has not been adequately explained, particularly in relation to the increase
in muscle mass. It has been suggested that this may result from associated metabolic
derangements (diabetes or thyroid abnormalities), or a direct effect of GH excess on muscle.
Increase in muscle specific creatinine kinase levels have been detected in patients with
active disease which improves with a reduction of serum GH (McNab & Khandwala, 2005) .
Microscopic examination of skeletal muscle from acromegalic patients shows type 1 fibre
hypertrophy (Nagulesparen et al., 1976).

GHD - Cardiac function Patients with GH deficiency have an increased mortality due to
cardiovascular disease. Most studies show decreases in heart function such as reduced left
ventricular mass and cardiac output, and reduced left ventricular ejection fraction
particularly during exercise; in addition there is an increased incidence of coronary artery
disease (Colao et al., 2004a; Colao et al., 2004b; Svensson et al., 2005). The mechanism of
cardiac abnormalities in GHD is debated, but most data support the hypothesis that GHD leads
to reduced cardiac mass, reduced contractility, reduced pre-load and increased after-load,
all of which could lead to reduced stroke-volume and reduced cardiac output (Colao et al.,
2004b). Treatment with GH results in improvement in cardiac function and reduction of
peripheral resistance due to direct anabolic actions of the myocardium but increased
dimensions could also be secondary to an increased cardiac output and stroke volume as a
result of increased contractility and increased pre-load. In conclusion, GHD is associated
with cardiac dysfunction related to the degree and duration of GHD and GH replacement seems
to enhance ventricular mass and cardiac function and to reverse diastolic abnormalities
(Juul, 1996).

GHD - Coronary sclerosis The evidence linking coronary sclerosis and GHD is robust although
it is unclear whether this is a direct effect of GH deficiency on cardiac endothelium, or an
indirect effect of the increase in cardiovascular risk factors documented in patients with
hypopituitarism and GH deficiency. These include increased abdominal adiposity, abnormal LDL
and triglyceride levels, high fibrinogen and plasminogen activator inhibitor activity,
increased markers of inflammation (such as interleukin-6 and C-reactive protein) and
consequently increased intima-thickness (Colao et al., 2004a; Colao et al., 2004b). In our
studies, we will use electron-beam CT (EBCT) as a non-invasive means of determining coronary
artery disease, which is a sensitive indicator of IHD. This will allow us to control for
changes in the onset and/or recovery from ischaemia that might be due to macrovascular
ischemia rather than reduced intracellular capacitance. There are no previously published
studies using electron beam coronary CT in these groups of patients.

GHD - Skeletal muscle Adult growth hormone deficiency (AGHD) is associated with fatigue,
tiredness and isometric muscle strength is reduced to 76%. After 6 months of GH therapy,
muscle mass increases 5% (Bengtsson et al., 1993; Juul, 1996). Neuromuscular dysfunction is
also observed (abnormal electromyogram and interference pattern analysis) which improves
after initiating GH therapy (Webb et al., 2003). The reduced exercise capacity observed in
GHD is multifactorial but reduced skeletal muscle mass has an important contribution to it
(Juul, 1996).

In summary, existing evidence demonstrates that patients with both GH deficiency and GH
excess have abnormal cardiac and skeletal muscle function, although the nature of the
cardiac abnormality differs. In acromegaly there is hypertrophy and impaired contractility
with dysfunction amplified during exercise. In GHD, risk factors for IHD cause premature
occlusive coronary artery disease, coupled with a poor anabolic stimulus and reduced muscle
mass. Skeletal muscle echoes these processes. . In transgenic mice overexpressing GH (an
acromegalic mouse) the phosphocreatine -to-ATP ratio measured by MRS is significantly lower
in GH overexpressing mice than controls, suggesting impaired energy level in the myocardium
(Bollano et al., 2000). We hypothesise that chronic growth hormone excess may lead to
reduced energy level within the myocardium, and that restoration of normal GH level will
ameliorate this abnormality. Our model would also predict that in GHD resting energy levels
would be normal or even high, and that the primary defect is a reduction in muscle mass due
to reduced anabolic stimulus. In patients with GH excess and GHD we will assess cardiac
energy levels with MRS, a non-invasive technique of assessing myocardial energy metabolism
before and after treatment, a technique that has been successfully used to establish
abnormal heart energy levels in patients with diabetes and heart failure
(Scheuermann-Freestone et al., 2003).

Objectives

1. Determine cardiac (at rest) and skeletal muscle (at rest, peak exercise and recovery)
energy metabolism in patients with GH excess and detect changes after normalisation of
GH and IGF-1 levels

2. Determine cardiac and skeletal muscle energy metabolism in patients with GH deficiency
and detect changes after normalisation of IGF-1 levels

3. To correlate muscle energy metabolism parameters to GH and IGF-1 status in the control
subjects and in both patient groups

4. Determine the prevalence of coronary artery calcifications in patients with GH excess
and GH deficiency and correlate with their risk factors and other surrogate markers of
CHD (serum levels of high sensitivity CRP, lipoprotein (a), homocysteine, and insulin
resistance (HOMA analysis).

5. Determine the anatomical distribution of the atheromatous plaques within the coronary
arteries and the extent of isolated plaques compared to diffuse disease.

Methods Cardiac and skeletal investigations

- MRI at 1.5T (Siemens Sonata) will be used to measure right and left ventricular
morphology and global function (Sandstede et al., 2005) (i.e. left and right
ventricular systolic and end-diastolic volumes and ejection fractions). Tissue phase
mapping will be used to measure radial and rotational tissue velocities for regional
wall motion abnormalities and abnormal contractile patterns.

- 31P MRS will be used as previously described (Crilley et al., 2000; Roest et al., 2001)
to monitor intracellular PCr and ATP in heart muscle. Acquisition will be triggered
using electrocardiographic gating. Cardiac diastolic function will be measured using
cine MRI volume-time curves and tissue phase mapping parameters.

- Heart failure severity (NYHA status) will be determined from the degree of dyspnoea (6
min walk test).

- Peak O2 uptake (MVO2) will be estimated from a metabolic gas exchange analysis
performed during maximal treadmill cardiopulmonary exercise testing.

- Skeletal muscle MR imaging and spectroscopy: The protocol for the 31P MRS
determination of calf muscle (gastrocnemius and soleus) metabolism at rest and during
dynamic exercise is well established (Scheuermann-Freestone et al., 2003). A
T1-weighted MR image will be used to determine the muscle cross-sectional area and 31P
MRS of the gastrocnemius muscle will be performed at rest, during and after exercise.
Each subject will lie in the 3T superconducting magnet with their calf overlying a 6 cm
diameter surface coil. The muscle will be exercised by plantar flexion of the right
ankle, lifting a weight of 10% lean body mass a distance of 7 cm at a rate of 30/min.
After acquisition of four spectra (each 1.25 min), the weight will be incrementally
increased by 2% of lean body mass every other minute. The subject will exercise until
stopping when fatigued and the muscle will then be studied during recovery. On
retesting after therapy, exercise will continue again until the subject is stopped by
fatigue (treatment may lengthen this time). Relative concentrations of Pi, PCr and ATP
will be measured from their signal intensities in the spectra as described previously
(Adamopoulos et al., 1999; Butterworth et al., 2000; Scheuermann-Freestone et al.,
2003).

- Electron beam coronary CT (EBCT) to assess coronary disease. As calcium is deposited in
the earliest stages of atheroma formation, EBCT can detect sub-clinical coronary
disease many years before it results in ischaemia. The extent of coronary artery
calcification reflects the overall impact of risk factors, both known and unknown, on
the end organ, the arterial wall, and as such gives a superior predictive value over
the Framingham risk assessment (Thompson & Partridge, 2004; Pletcher et al., 2004).
EBCT scanning will be performed using a GE-IMATRON 300 scanner using a conventional
protocol. Calcium deposition within coronary arteries will be assessed by total volume
and by the Agatson correction. The number of plaques and calcium score will be measured
in the left main artery, left anterior descending, left circumflex, and right coronary
artery, in addition to an overall score.

Other investigations will include: Demographic and clinical data (incl. medication) will be
obtained for all patients and control subjects; ECG, Epworth Sleepiness Scale questionnaire
and 5 point test for sleep apnoea, blood samples will be taken after a 12 h fast, after 30
min rest, for glucose, lactate, free fatty acids, ketone bodies, insulin, triglyceride,
cholesterol, HDL, LDL, ANP, BNP, noradrenalin, electrolytes, creatinine, creatinine kinase,
uric acid, TNF-alpha, leptin, Hb and glycosylated Hb determinations. Apoprotein A/B, High
sensitivity C-reactive protein, Lipoprotein (a), Homocysteine, Insulin (HOMA analysis),
biochemistry and liver function, pregnancy test. Endocrine investigations will include
Glucose tolerance tests (for acromegaly patient; for GHD if necessary), GH day-curve (a mean
of 5 estimations throughout the day), serum IGF-I, pituitary and peripheral hormones (LH,
FSH, cortisol, TFT, testo/E2, PRL), estimation of duration of the disease (the presumed
duration of acromegaly will be estimated by comparison of patients' photographs taken over a
1-3-decade span and by interviews to date the onset of acral enlargement and other clinical
symptoms (Damjanovic et al., 2002; Colao et al., 2003)) Patient selection: Patients will be
recruited at St. Bartholomew's Hospital (Dr P. Jenkins and Prof. A. Grossman), King's
Hospital (Dr S. Aylwin) and St Thomas's Hospital (Dr P. Carroll) in London, Royal Free
Hospital (Prof P. Boloux), the John Radcliffe Hospital Oxford (Prof J. Wass), Sheffield (Dr
J. Newell-Price), and Stroke-on-Trent (Prof R. Clayton) from the Endocrine Wards and
outpatient clinics. This constitutes a large recruitment base. We estimate that 45 new
acromegaly patients and 60-80 new GHD patients per year will be screened. Patients will be
selected on the basis of the clinical diagnosis of acromegaly [Acromegaly: typical signs and
symptoms and biochemical evidence (GH > 1μg/l during OGTT, IGF-I above age-related reference
range); GHD: a structural hypothalamo-pituitary defect, at least one other pituitary hormone
deficiency, AGHDA score >11 (NICE criterion) and evidence of severe biochemical GHD (usually
a peak GH response to an insulin or glucagon stress test of <3 μg/l)]. Patients will be
managed according to the clinical protocols of the referring centre.

Inclusion criteria for acromegaly

- Clinical and biochemical diagnosis of acromegaly. Thyroid and glucocorticoid
replacement if necessary stable for at least 4 weeks before the study. Gonadotrophin
status will be recorded and whenever possible patients will be studied in the same
status

- Males and females aged 18-70 years willing to give informed consent

- At least 6 months after the onset of symptoms of acromegaly and on stable medication
for heart failure treatment (if any) for at least 4 weeks prior to inclusion into the
study

- Systolic blood pressure < 180 mmHg, diastolic blood pressure < 110 mmHg.

Exclusion criteria:

- Change in medication in the preceding 4 weeks

- Patients on subcutaneous insulin therapy

- Hyperthyroidism

- Not being in sinus rhythm

- Unstable angina pectoris and decompensated heart failure (define as NYHA 3-4)

- Clinically significant valvular disease, clinically significant chronic obstructive
pulmonary disease

- History of myocardial infarction or stroke within the last 6 months, major cardiac
surgery within the last 6 months

- Significant history of drug- or alcohol abuse or unable to give informed consent

- Any other significant surgical or medical condition which would considerably affect
results in view of the identifying clinician

- Typical contraindication for MR (e.g. metal implants in delicate positions, aneurysm
clips, shrapnel injuries, pacemakers, internal defibrillators and severe
claustrophobia)

- Pregnancy

Inclusion criteria for GHD

- Clinical and biochemical diagnosis of GHD. All hormones replaced (if clinically
necessary) except GH. Thyroid and glucocorticoid replacement if necessary stable for at
least 4 weeks before the study. Gonadotrophin status will be recorded and whenever
possible patients will be studied in the same status

- Males and females aged 18-70 years willing to give informed consent

- At least 6 months after the onset of symptoms of acromegaly and on stable medication
for heart failure treatment (if any) for at least 4 weeks prior to inclusion into the
study

- Systolic blood pressure < 180 mmHg, diastolic blood pressure < 110 mmHg.

Exclusion criteria:

- Change in medication in the preceding 4 weeks

- Previous history of acromegaly

- Child-hood onset GHD

- Patients on subcutaneous insulin therapy metformin probably an exclusion

- Hyperthyroidism

- Not being in sinus rhythm

- Unstable angina pectoris and decompensated heart failure (define as NYHA 3-4)

- Clinically significant valvular disease, clinically significant chronic obstructive
pulmonary disease

- History of myocardial infarction or stroke within the last 6 months, major cardiac
surgery within the last 6 months?

- Significant history of drug- or alcohol abuse or unable to give informed consent

- Any other significant surgical or medical condition which would considerably affect
results in view of the identifying clinician

- Typical contraindication for MR (e.g. metal implants in delicate positions, aneurysm
clips, shrapnel injuries, pacemakers, internal defibrillators and severe
claustrophobia)

- Pregnancy

POST-TREATMENT ASSESSMENT Three months is estimated to be sufficient time to reach altered
(improved) cardiac metabolism as assessed by MRS based on previous studies (K. Clarke
manuscript submitted)). Therefore the first post-treatment assessment will be 3 months after
biochemical cure.

In acromegalic patients 3 months after documented biochemical remission (IGF-1 levels
reached upper limit of age-related normal range and day-curve for GH mean < 2.5 mcg/l or GH
< 1 mcg/l during OGTT) repeat MRI, MRS and biochemical investigations. If patient is not in
biochemical remission by 24 months, then repeat investigation at 24 months after the start
of therapy will be performed as the final assessment.

In GHD patients 3 months after recorded completed dose titration (IGF-I values in the upper
third of the age-related normal range) repeat MRI, MRS and biochemical investigations. If
patient has not completed GH dose titration by 24 months, then repeat investigation at 24
months after the start of GH therapy anyway.

24 months after start of therapy repeat EBCT. There are data to suggest that for example
changing lipid status can improve EBCT after 12 months of therapy (Achenbach et al., 2002)
Age and sex matched control subjects will be recruited and studied according to the
pre-treatment protocol. Males and females aged 18 to 75 years willing to give informed
consent will be selected without endocrine or coronary disease, with further exclusion
criteria as above.

Power calculation:

The primary endpoint will be a change in cardiac energetics, as assessed by 31P magnetic
resonance spectroscopy (MRS) (expressed as PCr/ATP ratio). Power calculations using data
from our cardiac 31P MRS study (Scheuermann-Freestone et al., 2003) showed a highly
significant reduction in cardiac PCr/ATP from 2.49 +/- 0.31 to 1.55 +/- 0.37, a difference
of 0.94. In order to find a biologically significant difference between groups of one
standard deviation (SD) with 90% power at P< 0.05, we would need to include 21 subjects in
each group. For a paired study, in which the effects of therapy were tested on the same
patient, we would need to include 11 patients in each group to find a significant difference
between groups of 1 SD with 90% power at P< 0.05, while for unpaired comparison with control
subjects 15 patients are necessary. To allow for patient drop-outs we will include a
minimum of 30 subjects in the studies. Secondary endpoints for all studies will include
cardiac function (MRI) and skeletal muscle function and energetics (MRI and MRS,
respectively).