Investigating the Anabolic Response to Resistance Exercise After Critical Illness: The ARTIST-2 Non-Randomized Controlled Trial
Phase: NA
Conditions: Critical Illness, Muscle Loss
Sponsor: Karolinska University Hospital

Study protocol 2021-04-26
Investigating the anabolic response to resistance exercise during and
after critical illness.
Study acronym
ARTIST 1 & 2
Primary investigator
Martin Sundström Rehal, MD PhD
Version history
2022-02-15: Amendment to ethical review authority regarding screening and recruitment of
patients in Study 2.
2022-03-10: Minor amendment regarding dose of oral protein and labeled phenylalanine in
Study 2.
2022-07-10: Amendment to ethical review authority regarding exclusion criteria for therapeutic
anticoagulation in Study 2.
2023-01-17: Minor amendment regarding exclusion criteria # 13 and 14 in Study 2.
2023-05-08: Amendment to ethical review authority regarding additional data collection (SF-36
and hand grip strength).
2023-10-23: Minor amendment regarding sample size target in Study 2.
Aim
The overall aim of this project is to determine the anabolic response to resistance exercise
during and after critical illness.
Hypothesis
1) Resistance exercise, in addition to amino acid supplementation and routine physiotherapy,
results in an improved muscle protein balance in ICU patients compared to amino acid
supplementation and routine physiotherapy alone.

2) Patients recovering from critical illness have an impaired anabolic response to resistance
exercise 3-6 months after ICU stay as compared to healthy controls.
Overview of the field
The debilitating impact of critical illness has been recognized for several decades. Disability
related to intensive care is now described as a syndrome called ICU-acquired weakness
(ICUAW). ICUAW affects up to 70% of ICU patients and is most common with higher illness
severity. The pathophysiology is heterogenous, and involves muscle protein breakdown, altered
bioenergetics, axonal injury and impaired excitation-contraction coupling. Patients that develop
ICUAW require longer hospitalization and have a higher risk of death [1].
Weakness also has significant long-term consequences. In a landmark study, Herridge et al
observed that predominantly young and previously healthy survivors of acute respiratory failure
had reduced exercise capacity up to five years after ICU stay [2]. Physical function was
unrelated to residual pulmonary dysfunction. Long-term disability was associated with significant
health care costs, delayed return to work, and overall poor quality of life.
Preventing muscle atrophy is a potential way to counteract weakness. Critical illness is
associated with a rapid loss of skeletal muscle, which may exceed 10% of lean body mass in
the first week of intensive care [3,4]. Dos Santos et al found that a majority of ICU survivors had
not recovered their muscle volume up to six months after hospital discharge [5].
During critical illness there is a dramatic increase in muscle protein breakdown, while synthesis
is unaltered [6]. The result is a net loss of total body protein. Aging and comorbid conditions also
contribute toward a resistance to anabolic signals, which may be exacerbated by immobilization
and systemic inflammation [7]. Our research group has previously found that nutritional
interventions such as amino acid supplementation can improve whole-body protein balance in
ICU patients [8,9], but the effect on muscle metabolism is still unknown. Studies in exercise
physiology have demonstrated that resistance training and amino acid ingestion have
synergistic effects on muscle protein synthesis in healthy subjects [10]. It is therefore an
appealing therapy to counteract muscle wasting in ICU, and to regain muscle mass during
convalescence.
Despite several clinical trials, there remains equipoise regarding the efficacy of exercise in
improving physical function in-ICU or after discharge [11,12]. These mixed signals are
unsurprising given the heterogeneous causes of ICUAW. Only a few studies in this field assess

muscle architecture or cellular signaling in response to training [13,14]. However, the gold
standard in determining the anabolic response to exercise is to directly measure the effect on
protein synthesis and breakdown. To our knowledge there is still no published research using
this methodology in critically ill patients.
Project description
The project consists of two studies. Study 1 will be conducted in the ICU. Study 2 will recruit
study subjects who have been discharged from hospital after ICU stay.
Study populations:
Study 1 will recruit adult ICU patients suitable for active mobilization, which is already standard
care in the unit at Karolinska University Hospital Huddinge.
Inclusion criteria:
1.
Adult (≥18 years) ICU patient.
2.
Suitable for active mobilization as determined by the attending physician and
physiotherapist.
3.
Not expected to be discharged or transferred from the unit within 24 h.
4.
Functioning arterial line in situ.
Exclusion criteria:
1.
Not able to provide informed consent.
2.
Systemic anticoagulation with LMWH/UFH/DOAC in therapeutic dose range for
deep vein thrombosis or pulmonary embolism, or dual antiplatelet therapy. If
LMWH is administered twice daily, the patient is eligible for participation provided
that vascular access is performed at nadir prior to the first daily dose.
3.
Clinically significant inherited or acquired disorder of hemostasis.
4.
Morbid obesity that interferes with femoral cannulation or doppler measurements.
5.
Hemodynamic instability requiring ongoing volume resuscitation with crystalloid
solutions or blood products.
6.
Lower-limb amputee.
7.
Lower-limb artheriosclerotic disease with critical ischemia.
8.
Metastatic cancer or active hematological malignancy.
9.
Inherited disorder of amino acid metabolism.
10. Chronic muscle, neuromuscular och neurologic disease with prior documentation
of clinically significant lower-limb involvement.

11. Pregnancy.
12. Single organ failure not requiring invasive mechanical ventilation prior to
enrollment.
Study 2 will include patients 3-6 months after hospital discharge. All patients treated for ≥3 days
in an ICU at Karolinska University Hospital will be screened for eligibility.
Inclusion criteria:
1.
Adult (≥18 years) subject previously admitted to an ICU at Karolinska University
Hospital for ≥3 days and discharged from hospital (intervention group).
OR
2.
Adult (≥18 years) subject without a history of ICU admission (control group).
Exclusion criteria (applies to both intervention and control group):
1.
Not able to provide informed consent.
2.
>6 months since ICU discharge.
3.
Systemic anticoagulation with warfarin or dual antiplatelet therapy.
4.
Clinically significant inherited or acquired disorder of hemostasis.
5.
Lower-limb amputee.
6.
Lower-limb artheriosclerotic disease with critical ischemia.
7.
Recent fracture in lower limbs or significant osteoarthritis limiting movement in
knee or hip joint.
8.
Metastatic cancer or active hematological malignancy.
9.
Inherited disorder of amino acid metabolism.
10. Chronic muscle, neuromuscular och neurologic disease with prior documentation
of clinically significant lower-limb involvement.
11. Pregnancy.
12. Single organ failure not requiring invasive mechanical ventilation during ICU stay.
13. Intubated only for airway protection with no other organ failure(s) during ICU stay.
14. Planned postoperative care in ICU after elective cardiothoracic surgery.
Intervention and controls:
●
Study 1: All patients will perform a physiotherapist-led session of standardized active
mobilization. An intravenous infusion of mixed amino acids is administered concurrently.

Patients randomized to the intervention group will perform weighted resistance exercise
with the leg catheterized for blood sampling as a part of their physiotherapy session.
●
Study 2: All subjects will perform standardized lower extremity resistance exercise in
conjunction with enteral protein supplementation. An age- and sex-matched group of
healthy controls will be used for comparison.
Outcomes:
●
Study 1: The primary outcome is the difference in change between the intervention and
control groups in lower limb protein balance, from baseline to post-intervention. Other
parameters of muscle protein kinetics (protein breakdown and synthesis) will be reported
as secondary outcome measures.
●
Study 2: The primary outcome is the difference between patients and healthy controls in
fractional rate of muscle protein synthesis, from baseline to post-intervention. The impact
of exercise on anabolic/catabolic signaling pathways in muscle fibres will be reported as
secondary outcome measures. Planned post-hoc analyses include characterization of
metabolomics and hormonal profiles in responders and non-responders.
Study protocols
Study 1
At the beginning of the protocol, a primed intravenous infusion of labeled ring-2H5-phenylalanine
(bolus 2.94 µmol/kg, infusion rate 2.94 µmol/kg/h) and 2H3-methylhistidine (bolus 0.06 µmol/kg,
infusion rate 0.06 µmol/kg/h) is started to achieve a steady state of tracer dilution in arterial
blood and continued until the end of the protocol. A femoral venous catheter is then sited under
local anaesthesia for lower limb blood sampling, with the tip placed just above the junction of the
greater saphenous vein and common femoral vein. The physician placing the catheter is free to
determine the side of placement depending on the vascular anatomy on ultrasound
investigation. A standardized measurement of upper and lower limb muscle thickness is also
performed to quantify the baseline muscle mass of study subjects [15].
After 165 minutes of tracer infusion, four 2 ml samples from arterial and lower limb venous blood
and femoral artery blood flow measurements on the side of the catheterized leg are performed
at five minute intervals. Doppler blood flow measurements will be performed using a
high-frequency linear probe with a fixed probe angulation at a level immediately cranial to the

branching of the deep femoral artery. The end-diastolic inner diameter of the vessel is
determined at the same level. Immediately after the baseline measurements, an intravenous
infusion of mixed amino acids (Glavamin, Fresenius Kabi) is administered at a maximum rate of
0.1 g/kg/h.
The study subjects will then participate in a physiotherapy session of protocolized active
mobilization. While sitting on the side of the bed, patients in the intervention group will be
encouraged to perform weighted leg extensions with the leg catheterized for blood sampling,
targeting 8-12 repetitions to failure in three sets. The physiotherapist in charge will document
the number of repetitions and other items of the mobilization protocol performed.
After returning to a supine or semi-recumbent position, new blood samples and blood flow
measurements as described above are performed to determine the change in lower limb protein
balance every 30 minutes up to 90 minutes post-intervention. At the completion of the protocol
the infusions of labeled and unlabeled amino acids are stopped and the femoral vein catheter is
removed. The protocol is illustrated in Figure 1.
Figure 1. A schematic illustration of the protocol in Study 1 (not to scale).
All other aspects of treatment will be determined by the attending physician and care team, with
a request that the rate of enteral and parenteral nutrition remains unchanged from 06:00 in the
morning until the end of the protocol.

Study 2
Study subjects will arrive at the study site in the morning in a fasted state. After a baseline
physical examination, a peripheral venous catheter and an arterial line are placed in an upper
extremity under local anaesthesia. A primed intravenous infusion of ring-2H5-phenylalanine
(bolus 2.94 µmol/kg, infusion rate 2.94 µmol/kg/h) is started to achieve steady state distribution
of tracer between plasma and the intracellular precursor pool, and continued until the end of the
protocol. During the infusion period a standardized ultrasound measurement of lower limb
muscle thickness and grip strength is performed to quantify the baseline muscle mass and
function of study subjects. Charlson Comorbidity Index, Clinical Frailty Scale and RAND 36-item
Short Form Health Survey are reported through self-assessment. After 150 minutes of tracer
infusion, a baseline muscle biopsy of ~150 mg is extracted from the non-dominant vastus
lateralis muscle under local anaesthesia. Blood samples are drawn at baseline (plasma amino
acids and lipid profile, inflammatory markers, white blood cell count, hormonal profile and
metabolomics) and at 15 minute intervals during the infusion period to determine tracer
enrichment.
Subjects will then perform a short program of standardized quadriceps resistance exercise with
the dominant leg, consisting of leg extensions in a flywheel inertia training machine targeting ten
repetitions in four sets. Afterwards the subject will ingest a commercially available protein
supplement containing 24 g of hydrolyzed whey protein over 5 minutes. To avoid diluting the
ratio of labeled/unlabeled phenylalanine, 235 µmol of ring-2H5-phenylalanine is added to the
beverage. A second biopsy is extracted from the contralateral vastus lateralis muscle used in
the training session 150 minutes from the start of protein intake. The protocol is illustrated in
Figure 2.

Figure 2. A schematic illustration of the protocol in Study 2 (not to scale).
Calculations
Study 1
Lower limb protein balance will be determined using a two-pool model [6]. Net balance (NB) of
phenylalanine is derived using the Fick principle:
NB = (CA - CV)*PF
where CA = arterial content (nmol/ml); CV = venous content (nmol/ml); and PF = plasma flow
(ml/min).
The rate of appearance (Ra) approximates protein breakdown and is calculated as:
Ra = CV*[1-(EV/EA)]*PF
Where EA and EV are the arterial and venous enrichments of tracer expressed as molar
percentage excess and calculated as an average of the four blood samples in each
measurement series.
The rate of disappearance (Rd) approximates protein synthesis and is calculated as:
Rd = NB + Ra
Study 2

Fractional synthesis rate (FSR) of muscle protein is calculated as
𝐹𝑆𝑅 =  (𝐸𝐵(𝑡2) −𝐸𝐵(𝑡1))/
𝑡1
𝑡2
∫𝐸𝑃(𝑡) 𝑑 𝑡 
Where EB is the enrichment of bound amino acid, EP enrichment of the precursor pool, t1
and t2 the duration of tracer infusion at the time of the first and second biopsy respectively.
Statistical considerations
Randomization
In Study 1, randomization will be determined using a computer-generated sequence in
permuted blocks of four. Treatment allocation is concealed in opaque envelopes prepared by a
research associate not involved in the study. The envelope is opened by the physiotherapist
supervising the exercise session just before the intervention. Research staff in charge of
outcome assessment are blinded to allocation until all data analysis is complete.
Sample size
As variation in muscle protein balance between ICU patients is very large [16], only paired
comparisons will allow for studies of small-moderate size to find an effect. Unfortunately, there is
no published data that describes the variation in repeated measures of muscle protein balance
in ICU patients to support a formal power calculation. For Study 1, the best available estimate of
effect size comes from pooled results on changes in whole-body protein balance during
nutritional interventions. Using this data, we determine that 10 patients in each treatment arm
are required to observe a mean change from negative to neutral lower limb muscle protein
balance. To account for potential dropouts we will enroll a total of 24 patients.
For Study 2, there is no data from similar studies in this population on which to accurately
estimate a treatment effect for sample size calculation. A target of 20 post-ICU patients and 20
matched controls has been chosen to allow for potential characterization of responders and
non-responders in the post-intensive care group.

Statistics
Between-group differences of continuous outcomes will be analyzed using two-way repeated
measures ANOVA. Within-group differences will be analyzed using one-way repeated measures
ANOVA. A normal distribution of within-subject change in protein kinetics is assumed from
previous observations. The predetermined level of significance is p ≤0.05. Correction for
multiple comparisons will not be applied and p-values for secondary outcomes should be
considered as exploratory.
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