Dutch Limb Suspension 2006

Physical inactivity is an important and independent risk factor for
atherosclerosis and cardiovascular and a numerous additional amount of other
chronic health conditions. According to extensive scientific results, it is
evident that inactivity has a great influence on the structural and functional
vascular parameters in humans, which are (in)directly linked to cardiovascular
morbidity and mortality. To gain further insight in the factors that initiate
these vascular adaptations, we have to search for the specific underlying genes
that are responsible for the NO pathway regulation resulting in these vascular
changes. Deficient physical activity in the current sedentary culture has been
shown to lead to abnormal gene expression. The present project will combine
human in vivo and in vitro research to identify the key genes in the NO pathway
responsible for vascular adaptations to inactivity and training in humans.
The current project will be the first to identify genes in the NO pathway that
are turned on or off by inactivity in humans and that are related to vascular
adaptations. Our results will initiate follow up studies in which these
key-genes will be knocked out in animal models or by specific drugs in humans
to further delineate the relevance and redundancy of those genes. Understanding
the genetic mechanisms leading to inactivity-related cardiovascular disease may
contribute to more adequate prevention strategies of these diseases.

Primary objective
To identify specific genes in the NO pathway that are turned on or off by
inactivity. The results will disclose key-genes in the NO pathway relevant for
vascular adaptations.
Secondary objective
To correlate functional vascular adaptations to inactivity with transcriptome
analyses (achieved in the primary objective). The results will reveal specific
genes linked with vascular adaptations to either inactivity.

To achieve these objectives, healthy subjects will immobilize one leg for three
weeks. Muscle biopsy of the vastus lateralis and the gastrocnemius will be
taken before and after immmobilisation to provide information on gene
expression. The vascular parameters will be measured by echo Doppler ultrasound
and plethysmography by experienced vascular nurses. The NO pathway will be
manipulated by infusion of Ach (stimulates NO synthase) and L-NMMA (blocks NO
synthase). Functional vascular adaptations will be measured in parallel to gene
expression profiling in order to correlate the vascular functional data and the
transcriptome characterization. Additionally, musculo-skeletal function will be
evaluated.

The intervention consists of 3 weeks of immobilisation of one leg. This will be
done by elevation of the active foot by a sole and fixing of the foot by belts
to the upper body, in a way the foot does not touch the ground. During
immobilisation, the subject will walk on crutches and the limb will not be
loaded.

Using the immobilisation method described in this protocol, only a part of the
leg is immobilized, while the joints remain flexible. Except for small ankle
oedema for the first days, a relatively colder skin of the suspended leg and a
small, but controlable risk of thrombosis, no side-effects have been reported.
After the restart of the normal loading, good recovery was present at the
suspended leg and the effect of immobilisation will disappear in some weeks
according to literature. The canulation of the femoral artery can lead to a
light hematoma or some bleeding afterwards. The subjects are controled for this
very precisely. The medication is infused only locally in doses at which no
systemic effects are expected, based on comparable studies. Considering the
removal of the muscle biopt samples, the upper leg might be a bit stiff and
sensitive afterwards. In very rare cases, a light hematoma can occur, that will
disappear after some days. All tests are performed extensively on the
participating departments, without any complications.