Effect of Physical Therapy on NLRP3 Inflammasome Activation and Muscle Atrophy in Critical Illness Myopathy (PT-NLRP3-CIM).
Phase: NA
Conditions: Critical Illness Myopathy
Sponsor: University of Chile

 
 
   
 
 
"Effect of Physical Therapy on NLRP3 Inflammasome Activation 
and Muscle Atrophy in Critical Illness Myopathy" 
Óscar Leonardo Arellano Pérez 
 
Doctoral Thesis Project Submitted in Candidacy for the Degree of Doctor in 
Biomedical Sciences 
 
 
 
 
 
 
 
Aug 11, 2025 
 

 
 
   
Table of Contents 
 
Abstract ....................................................................................................................................... 4 
1. 
Background .......................................................................................................................... 5 
1.1. 
Critical illness myopathy ............................................................................................... 5 
1.2. 
Potential Mechanisms and Pathogenesis of CIM............................................................ 6 
1.3. 
Muscle Atrophy, Inflammation, and the NLRP3 Inflammasome ..................................... 6 
1.4. 
NLRP3 Inflammasome Activation in CIM: Priming and Oligomerization .......................... 7 
1.5. 
Physical Therapy, Inflammation, and Potential Modulation of the NLRP3 Inflammasome 
in CIM 8 
2. 
Hypothesis ........................................................................................................................... 9 
3. 
General Objective ................................................................................................................. 9 
4. 
Specific Objectives ................................................................................................................ 9 
5. 
Experimental Design........................................................................................................... 10 
6. 
Patients and Methods ........................................................................................................ 12 
6.1. 
Study Design .............................................................................................................. 12 
6.2. 
Patients ..................................................................................................................... 12 
6.2.1. 
CIM Patients (Group A, n = 16) .................................................................................................. 12 
6.2.2. 
Control Patients (Group B, n = 8) ............................................................................................... 12 
6.2.3. 
Patient Recruitment Sites .......................................................................................................... 12 
6.3. 
Sample Size Validation ............................................................................................... 13 
6.4. 
Sample Collection from Patients with CIM and Controls .............................................. 13 
6.4.1. 
Vastus Lateralis Muscle Biopsy .................................................................................................. 13 
6.4.2. 
Blood Samples............................................................................................................................ 13 
6.5. 
Evaluators .................................................................................................................. 13 
6.6. 
Histological and Biochemical Methods ........................................................................ 13 
6.6.1. 
Histology .................................................................................................................................... 13 
6.6.2. 
Immunofluorescence ................................................................................................................. 14 
6.6.3. 
Western Blot .............................................................................................................................. 14 
6.6.4. 
RT-qPCR...................................................................................................................................... 15 
6.6.5. 
ELISA........................................................................................................................................... 16 
6.6.6. 
Microarray. ................................................................................................................................ 16 
6.7. 
Demographic Variables, Severity of Critical Illness, Clinical Assessment of Muscle 
Strength, and Functionality..................................................................................................... 16 
6.8. 
Statistical Analysis ...................................................................................................... 17 
7. 
Ethical Considerations ........................................................................................................ 18 
7.1. 
Social Utility ............................................................................................................... 18 
7.2. 
Protection of Vulnerable Groups................................................................................. 18 
7.3. 
Randomization ........................................................................................................... 18 
7.4. 
Adverse Events ........................................................................................................... 19 
7.5. 
Benefit Assessment .................................................................................................... 19 
7.6. 
Protection of Privacy .................................................................................................. 20 
7.7. 
Ethical Monitoring of the Project ................................................................................ 20 

 
 
   
7.8. 
Feasibility .................................................................................................................. 20 
8. 
Discussion .......................................................................................................................... 21 
10. 
Gantt Chart .................................................................................................................... 32 
Appendix 2. Ultrasound Evaluation Procedure ............................................................................. 33 
 
 
 

 
 
   
Abstract  
Critical Illness myopathy (CIM) is a common cause of acquired weakness in the intensive care 
unit. It is defined as a proximal predominant tetra-paresis, usually symmetrical and with 
respiratory muscle involvement. This condition is a usual complication in intensive care unit 
(ICU) and has been associated with increased mortality and healthcare costs. Myopathy in the 
critically ill patient can progress to post-intensive care syndrome, which results in persistent 
cognitive, mental and physical dysfunction. In Chile, 40% of patients with critical illness due to 
COVID-19 developed this syndrome. This pathology presents muscle atrophy and alters of 
contractility, and several cellular mechanisms have been associated with its development, but 
its etiopathogenesis is still unclear. The role of the ubiquitin proteosome system and atrogenes 
expression has been linked to its genesis. In murine models of sepsis and denervation, which 
replicate the characteristics of the critically ill patient, NLRP3 inflammasome complex produces 
muscle atrophy by overexpression of atrogenes. The NLRP3 inflammasome is a multiprotein 
complex that plays a pivotal role in inflammatory signalling, and secretion of IL-1β and IL-18. 
Overexpression of this complex has been reported in CIM, but is NLRP3 inflammasome 
activation associated with muscle atrophy due to atrogen upregulation in critically ill 
patients? One of the most important risk factors for this myopathy is mechanical silencing, 
which, unlike other factors, is modifiable by early mobilization. Physical therapy has 
demonstrated different effects at the cellular level in the skeletal muscle of these patients. In 
different pathological conditions, physical therapy is a regulator of inflammation, IL-1β 
and NLRP3 inflammasome levels, but does physical therapy decrease NLRP3 
inflammasome activation in the skeletal muscle of patients with CPM, ¿thereby 
contributing to a reduction in muscle atrophy? Based on this background, the following 
hypothesis is proposed: Physical therapy decreases NLRP3 inflammasome activity and 
reduces muscle atrophy in critical illness myopathy. To test this assertion, in the early 
stage of ICU stay, patients at risk of this condition (n=16) will be randomized to be treated with 
conventional physical therapy or to additional physical therapy using servo-assisted motorized 
movement therapy device, 2 times a day for 60 minutes. In addition, patients without this 
condition will be considered as controls (n=8).  From biopsy samples of the vastus lateralis 
muscle, histology studies will be performed to determine muscle atrophy and the structure of 
cellular organelles, expression of atrogenes mRNA by RT-qPCR, protein levels of key 
mediators involved with NLRP3 inflammasome activation by Western blot, and transcriptomics 
by microarray. The above to address the following specific aims: a) To evaluate the effect of 
physical therapy on NLRP3 inflammasome activation in vastus lateralis biopsies from patients 
with CIM, and to compare this activity with control patients, b) To evaluate the effect of physical 
therapy on muscle atrophy in vastus lateralis biopsies from patients with CIM, and to relate 
these results with NLRP3 inflammasome activation, c) To evaluate the effect of physical 
therapy on the gene expression profile of signalling pathways involved in skeletal muscle 
atrophy in patients with CIM, and d) To analyze the association between gene expression and 
skeletal muscle biochemical parameters with the clinical diagnosis of CIM. Extra physical 
therapy is expected to decrease NLRP3 inflammasome activation, muscle atrophy, and 
differential expression of genes related to the development of CIM. Additionally, it is expected 
that these variables are associated with the clinical and ultrasound diagnosis of CIM. Due to 
the functional deficit suffered by patients who survive critical illness, it is importance 
to study the mechanisms underlying to this disorder. This study proposes a translational 
approach to achieve this purpose, furthermore, to explore the contribution of the non-
pharmacological intervention for the prevention and management of the critical illness 
myopathy: the physical therapy. 
 

 
 
   
1. Background 
 
1.1. Critical illness myopathy 
Critical illness myopathy is a common cause of intensive care unit-acquired weakness 
(ICUAW)(1). ICUAW is a neuropathy and/or myopathy characterized by symmetric, 
predominantly proximal muscle weakness(2,3), without involvement of facial or ocular 
muscles(3,4). Respiratory muscles may also be affected(4,5), leading to diaphragmatic 
dysfunction associated with mechanical ventilation (ventilator-induced diaphragmatic 
dysfunction, VIDD), which complicates weaning from ventilatory support(6). ICU-AW may be 
triggered by: a) a neurogenic disorder known as critical illness polyneuropathy (CIP), b) a 
myogenic disorder referred to as critical illness myopathy (CIM), and/or c) the coexistence of 
both conditions(4,7–10).  CIP is an acute, acquired sensorimotor axonal neuropathy caused 
by inflammation of the peripheral nerves (6,11). CIM is characterized by impaired muscle mass 
and contractility6,12, and presents with pathognomonic signs such as preserved sensory 
function, elevated creatine kinase levels(11), and most notably, a preferential loss of myosin 
compared to actin in muscle biopsy samples(6,12,13). Since muscle atrophy and impaired 
contractility are key features associated with increased mortality(14–18) and physical 
disability(19,20), much of the research has focused on CIM and the molecular pathways 
involved in skeletal muscle degradation(21). 
The development of critical illness myopathy (CIM) is associated with the severity of the critical 
illness(22–24). Both modifiable and non-modifiable risk factors have been identified(3,4,25). 
Modifiable factors include hyperglycaemia(8,25–28), parenteral nutrition(4,8,25,27,29), use of 
vasopressor 
agents(4,25,27), 
neuromuscular 
blockade(4,8,10,11,25,27,28,30,31), 
corticosteroid therapy(2,4,8,11,25,27,31), prolonged sedation and analgesia(4,25,31), use of 
aminoglycoside antibiotics(4,25,26,28), prolonged mechanical ventilation(2,8,25–27,29,31), 
extended ICU stay(8,29), and mechanical silencing due to prolonged immobilization(4,25,27–
29,31). Non-modifiable risk factors include specific diagnoses and life-support measures 
associated with critical illness, such as sepsis(25–27,29,32), systemic inflammatory response 
syndrome (SIRS)(4,8,25,32), multiple organ failure (MOF)(2,4,25–27,32), hyperlactatemia(4), 
and renal replacement therapy(26). Patient-related characteristics such as age(4,25,29), 
comorbidities(4), and female sex (4,25,27) have also been identified as non-modifiable 
contributors. 
Regarding the diagnosis of CIM, different strategies are employed depending on whether the 
patient is cooperative or non-cooperative. Another key aspect is the differential diagnosis 
between CIM and CIP. In awake and cooperative patients, muscle strength is clinically 
assessed using the Medical Research Council Sum Score (MRC-SS)(3,9,33). A score of 48 or 
below indicates muscle weakness, with scores below 36 denoting severe weakness(4,33,34). 
Handgrip dynamometry is another diagnostic tool in conscious patients; values below 11 kg 
for men and 7 kg for women are indicative of CIM. However, the validity of handgrip strength 
as a representative measure of global muscle strength has been questioned(35). 
Electrophysiological evaluation is also used to diagnose CIM and can be applied in 
unconscious or non-cooperative patients, as well as for differential diagnosis between CIP and 
CIM(34,36). Nerve and muscle biopsies may provide valuable mechanistic insight and enable 
differential diagnosis; however, due to their invasive nature, biopsies are recommended only 
within a research context(33,34). In non-cooperative patients, various imaging techniques 
have been employed to assess muscle mass as a surrogate for muscle strength. Computed 
tomography (CT) and magnetic resonance imaging (MRI) allow for the evaluation of muscle 
quality, detection of fatty infiltration, and quantification of lean muscle mass(16); Nevertheless, 
these methods are costly and require specialized personnel and software(16,34). Among 
imaging tools, quadriceps muscle ultrasound appears the most promising, as it permits rapid, 
bedside, and repeatable assessments of both quantitative parameters (muscle thickness, 
anatomical and physiological cross-sectional area) and qualitative features (echogenicity and 
muscle architecture)(18,34,37). Diagnosis of CIM by muscle ultrasound is based on changes 
in sonographic variables observed within 7 to 10 days of critical illness progression, including 
a ≥20% reduction in muscle thickness, ≥10% decrease in cross-sectional area, ≥5% decrease 
in pennation angle, and an 8–10% increase in echogenicity(17,18,37). Early diagnosis is of 
critical importance, as protein degradation, muscle mass loss (38,39), and electrophysiological 

 
 
   
alterations(40) occur within the first 10 days of ICU admission. iven the relevance of early 
detection, several critical illness severity scores—such as the Sequential Organ Failure 
Assessment (SOFA) and the Acute Physiology and Chronic Health Evaluation II (APACHE 
II)—have been proposed. When combined with muscle ultrasound assessment, these tools 
may be sufficient to raise suspicion and support the clinical consideration of this condition 
(22,23,41,42).  
CIM is a common complication among critically ill patients(17,31,43) and is associated with 
increased mortality(9,11,25,28,29,31), prolonged hospital stays(9,10,25,28), longer durations 
of mechanical ventilation(10,29), higher healthcare costs, and reduced long-term physical 
function and quality of life(9,10,29,31,44). Moreover, this condition may progress to post-
intensive care syndrome (PICS), which can result in persistent cognitive, mental, and physical 
dysfunction(4,28,45). A multicentre Chilean study evaluating the functional and cognitive 
sequelae of PICS in patients who experienced critical illness due to COVID-19 found that 40% 
of survivors exhibited some degree of disability after hospital discharge(46). Therefore, 
understanding the cellular and molecular etiopathogenesis of CIM is essential for developing 
both pharmacological and non-pharmacological therapeutic strategies. 
 
1.2. Potential Mechanisms and Pathogenesis of CIM 
CIM is characterized by reduced muscle mass and impaired contractility(44,47–49). In vivo 
muscle biopsies from critically ill patients, postmortem tissue samples, and animal models of 
CIM have revealed skeletal muscle fiber atrophy and degeneration(21,50–52). Biopsies from 
patients with CIM suggest that these alterations result from an imbalance between protein 
synthesis and degradation, the latter being exacerbated through: a) activation of the ubiquitin-
proteasome system (UPS), b) cytosolic proteases such as calpains and caspases, and c) 
dysregulation of autophagy pathways. Additional mechanisms described include decreased 
muscle excitability, impaired neuromuscular transmission, channelopathies, altered calcium 
homeostasis, mitochondrial dysfunction, and oxidative stress, among others(4,33,38,44,53). 
Although multiple etiological mechanisms have been proposed in the pathophysiology of CIM, 
its precise aetiology and causative pathways remain unclear. Therefore, further biomedical 
research is needed to elucidate the pathogenesis of CIM and to develop novel preventive and 
therapeutic strategies. 
 
1.3. Muscle Atrophy, Inflammation, and the NLRP3 Inflammasome 
The primary risk factors for the loss of muscle mass and strength, hallmarks of CIM, include 
sepsis, SIRS, and elevated levels of inflammatory markers(54). Among the proinflammatory 
cytokines involved in the development of muscle atrophy are tumor necrosis factor-alpha (TNF-
α), interleukin-6 (IL-6), and interleukin-1 (IL-1)(55). In addition to systemic inflammation, 
skeletal muscle is now recognized as a secretory organ capable of releasing various myokines 
(peptides, metabolites, cytokines, among others), including IL-6 and IL-1β.(55). The processes 
of gene expression, cleavage, and secretion of IL-1β are tightly regulated(56). Since IL-1β is 
translated as an inactive precursor (pro-IL-1β), its cleavage is mediated by the NLRP3 
inflammasome (55,56), a multiprotein complex essential to inflammatory signalling (57). This 
complex is composed of the NLRP3 receptor protein, the ASC adaptor protein, and pro-
caspase-1 as the effector component(57). In addition to its role as a key regulator of muscle 
metabolism(58,59), the NLRP3 inflammasome has been implicated in various myopathies(60–
62), the development of sarcopenia(63,64) and sepsis-induced muscle atrophy in C2C12 
myotubes(65). In patients with CIM, two transcriptomic analyses have reported differential 
expression of the NLRP3 gene(66) and functional enrichment of the Gene Ontology category 
“NLRP3 inflammasome complex”(67) in muscle biopsies from the tibialis anterior and vastus 
lateralis, respectively. In murine models of denervation and sepsis, NLRP3-associated muscle 
atrophy appears to be mediated by the upregulation of the E3 ubiquitin ligases MuRF1 and 
Atrogin-1, with NLRP3 gene deletion conferring a protective effect(68,69). The upregulation of 
these ligases is well documented in human CIM(12,40,42,66,70–73), and in various 
experimental models of this myopathy(74–82). Additionally, increased expression of other 
skeletal muscle E3 ligases, such as MUSA1(66,73), TRIM62(73), and TRIM32(66), has been 
described. This upregulation is associated with enhanced proteasome activity in muscle 
samples from critically ill patients with muscle atrophy.(42,71,83). Taken together, evidence 

 
 
   
from murine models of denervation and sepsis indicates that the NLRP3 inflammasome 
contributes to muscle atrophy via upregulation of the UPS(68,69). Since these models replicate 
key features of CIM and increased NLRP3 gene expression has been reported in patients with 
CIM, it is plausible to propose that the NLRP3 inflammasome contributes to muscle atrophy in 
human CIM. 
 
1.4. NLRP3 Inflammasome Activation in CIM: Priming and Oligomerization 
Canonical activation of the NLRP3 inflammasome occurs in two steps. The first signal, known 
as "priming," is triggered by the detection of pathogen-associated molecular patterns (PAMPs) 
or damage-associated molecular patterns (DAMPs) via Toll-like receptors (TLRs), or through 
the activation of tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) receptors(57). 
This in turn activates the nuclear factor kappa B (NF-κB), which upregulates the transcription 
of NLRP3, pro-IL-1β, and pro-IL-18(57,84,85).  
Given that the NLRP3 inflammasome can be activated by various inflammatory and pathogen-
associated stimuli(85,86), and that sepsis and SIRS are common risk factors for CIM(4,25–
27,29,32,87), the critical illness environment provides favorable conditions for priming. 
Supporting this notion, increased expression of TNF-α, TNFR, and IL-1β has been reported in 
the muscle tissue of patients with CIM(40,66,71,73,88), as well as in murine models of sepsis 
(89). 
Moreover, overexpression of genes related to the NF-κB signaling pathway has been 
documented in skeletal muscle from patients with CIM(90) and in a murine ICU model(77), 
suggesting a potential pivotal role of this transcription factor in CIM pathogenesis. Therefore, 
it is reasonable to hypothesize that priming of the NLRP3 inflammasome may occur during the 
development of CIM. 
Regarding the second activation signal, or the assembly of the NLRP3 inflammasome 
complex, this also occurs in response to PAMPs and DAMPs(57,84,85). Oligomerization of the 
NLRP3 protein leads to the cleavage of pro-caspase-1, generating active caspase-1(57,85). 
This enzyme subsequently processes pro-IL-1β and pro-IL-18 into their active forms. Caspase-
1 also cleaves gasdermin D (GSDMD), and the N-terminal fragment (GSDMD-NT) 
oligomerizes to form pores in the plasma membrane, facilitating the secretion of IL-1β and IL-
18 and initiating inflammatory responses, including pyroptotic cell death(57,84,85,91).  
 
A broad array of intracellular conditions and mediators contribute to this second activation 
step(57,84,85). Among them, mitochondrial dysfunction and the production of reactive oxygen 
species (ROS) are considered major stimuli for NLRP3 inflammasome activation(92–94).  
Mitochondrial dysfunction is well documented in critical illness and during the early stages of 
CIM(38). This has been evidenced by: (a) disruption of mitochondrial ultrastructure(89,95–99); 
(b) dysregulation of mitochondrial biogenesis factors—such as TFAM(95,98–100), TFBM1, 
TFBM2(98–100), PGC1α(42,88,95,96,98,100–103), PGC1β(100), PPARγ2(82), PPRC1(102), 
NRF-1(95,98,100), and NRF-2(98,100,101); (c) increased mitophagy as indicated by elevated 
Bnip3 expression(40,70,72,76,98,104); 
(d) 
reduced 
ATP 
synthesis 
and 
oxidative 
phosphorylation(88,89,105,106); (e) alterations in levels, expression, and activity of 
mitochondrial respiratory complexes(79,80,95–97,99–101,103,106–108); (f) redox imbalance 
reflected by changes in superoxide dismutase levels and reductions in various antioxidant 
enzymes(51,95,99,100,103,104); and (g) increased oxidative stress, as shown by lipid 
peroxidation, protein carbonylation, and nitration(70,97,100,103,109). Taken together, these 
findings support the hypothesis that activation and assembly of the NLRP3 inflammasome may 
occur in human CIM. Additionally, the release of lysosomal contents such as cathepsin B also 
contributes to this second signal and NLRP3 inflammasome assembly(85,110). In CIM, 
increased expression and activity of cathepsin L(40,70,72) and cathepsin B(66,72), among 
others(66), have been reported, along with an increased presence of lysosomal vacuoles and 
vesicles(13,70). Therefore, mitochondrial dysfunction, oxidative stress, and elevated cathepsin 
activity—features described in CIM—may be mechanistically linked to the second activation 
signal of the NLRP3 inflammasome. However, this association has not yet been specifically 
studied in CIM. 
 

 
 
   
1.5. Physical Therapy, Inflammation, and Potential Modulation of the NLRP3 Inflammasome 
in CIM 
Mechanical silencing due to prolonged bed rest is a key risk factor in the development of CIM. 
Importantly, this factor is modifiable through early mobilization(4,25,27–29,31). However, 
further evidence is needed to support the notion that physical therapy(3,111), active 
mobilization, and rehabilitation in critically ill patients can improve muscle strength and 
functional status. These interventions may reduce the incidence of CIM and shorten 
hospital stays(3,4,25,112–116).  
Physical therapy has demonstrated biological effects on the muscles of critically ill patients, as 
well as in experimental models of CIM. In a murine ICU model, physical therapy was shown to 
exert a protective effect against myosin loss, while increasing the expression of sarcomeric 
proteins and growth factors involved in muscle development(80). Kalamgi et al. reported that 
physical therapy increased levels of Akt, p-Akt, p-S6, as well as the expression of myogenin 
and MyoD(76), suggesting that mobilization promotes protein synthesis and muscle repair 
pathways in murine models of critical illness. In a randomized controlled trial, Hickmann et al. 
described a reduction in REDD1 expression(40), which may enhance mTOR signaling and 
protein synthesis(117). The same study reported decreased expression of Bnip3, Gabarapl1, 
and Lc3b, and increased p-ULK1Ser757, a condition that inhibits autophagy. Additionally, the 
authors observed reduced colocalization of p62 with Lamp-2A(40), suggesting a decrease in 
chaperone-mediated autophagy. In a murine ICU model, mechanical loading was found to 
induce PGC-1α4 (an exercise-induced isoform)(76), thereby promoting mitochondrial 
biogenesis. Based on current evidence, physical therapy and early mobilization in critically ill 
patients may exert effects on mitochondrial function and autophagy, potentially influencing 
NLRP3 inflammasome activation. However, this regulatory role has not yet been 
demonstrated in the skeletal muscle of critically ill patients with CIM. Regarding physical 
therapy and its relationship with systemic inflammation, several studies have described its 
modulatory effect on the inflammatory state across different health conditions(118–132). arious 
forms of exercise and physical therapy have been shown to reduce levels of IL-
1(124,131,132) andTNF-(124,128,130) in multiple pathologies. In elderly populations, 
exercise reduces levels of NLRP3 and caspase-1(133). Furthermore, physical therapy and 
exercise have been associated with reduced NLRP3 inflammasome activity in murine models 
of Parkinson’s disease(134); post-stroke depressio(135); and chronic kidney disease–
associated cachexia(136) Similarly, in a murine model of sciatic nerve injury, decreased levels 
of IL-1β and TNF-α were observed(137). However, the potential modulatory effect of physical 
therapy on the NLRP3 inflammasome and its role in the development of CIM has not yet been 
described. The pathogenesis of inflammation-associated muscle atrophy remains unclear (84), 
particularly in CIM. Therefore, understanding the molecular mechanisms underlying this 
condition is of great importance.  
 
In summary: (1) Skeletal muscle from patients with CIM shows overexpression of the NLRP3 
inflammasome gene and associated signaling components; (2) the NLRP3 inflammasome 
promotes muscle atrophy through upregulation of atrogenes in murine models of sepsis, 
denervation, and in C2C12 myotubes; (3) critically ill patients exhibit systemic inflammation, 
sepsis, and elevated levels of TNF-α and IL-1β—conditions that promote NLRP3 
inflammasome priming; (4) CIM is characterized by mitochondrial dysfunction, oxidative stress, 
and increased cathepsin B, all of which favor the second activation signal of the NLRP3 
inflammasome; (5) in various pathological contexts, physical therapy has been shown to 
reduce inflammation, IL-1β, TNF-α, and NLRP3 inflammasome levels, suggesting it may act 
as a protective factor against NLRP3-mediated muscle atrophy in CIM. 
Based on these observations, the following research questions arise: In human CIM: (a) Is 
NLRP3 inflammasome activation associated with muscle atrophy via upregulation of 
atrogenes?; (b) Does physical therapy reduce NLRP3 inflammasome activation in the skeletal 
muscle of patients with CIM, thereby contributing to the attenuation of muscle atrophy? 
Figure 1 provides a graphical summary of the proposed contribution of the NLRP3 
inflammasome to muscle atrophy in CIM, and the potential protective effect of physical therapy. 
 

 
 
   
 
Figure 1. Proposed contribution of the NLRP3 inflammasome to muscle atrophy in critical 
illness myopathy and the protective effect of physical therapy. 
 
Based on the aforementioned background, the hypothesis of this research project is: 
2. Hypothesis 
Physical therapy reduces NLRP3 inflammasome activity and mitigates muscle atrophy in 
critical illness myopathy. 
3. General Objective 
To investigate the association between the effects of physical therapy on NLRP3 
inflammasome activation and muscle atrophy in critical illness myopathy. 
4. Specific Objectives 
4.1. 
SO1: To evaluate the effect of physical therapy on NLRP3 inflammasome activation in 
vastus lateralis biopsies from patients with CIM, and to compare this activity with that of 
control patients. 
4.2. 
SO2: To assess the effect of physical therapy on muscle atrophy in vastus lateralis 
biopsies from CIM patients, and to relate these findings to NLRP3 inflammasome 
activation. 
4.3. 
SO3: To determine the effect of physical therapy on the gene expression profile of 
signaling pathways involved in skeletal muscle atrophy in CIM patients. 
4.4. 
SO4: To analyze the association between gene expression and skeletal muscle 
biochemical parameters and the clinical diagnosis of CIM. 
 

 
 
   
5. Experimental Design 
 
SO1: To evaluate the effect of physical therapy on NLRP3 inflammasome activation in 
vastus lateralis biopsies from CIM patients, and to compare this activity with that of 
control patients. 
Rationale: The NLRP3 inflammasome causes muscle atrophy in murine models of CIM 
(68,69). Prolonged bed rest, a major risk factor for CIM, is modifiable through physical therapy 
(4,25,27–29,31). This intervention has been shown to modulate inflammation(118–132) and 
the NLRP3 inflammasome(133), reduce levels of IL-1β and TNF-α(124,128), and influence 
mitochondrial function and autophagy(40), potentially leading to decreased inflammasome 
activation. However, this relationship has not been explored in human CIM. 
Experimental approach: Patients with CIM will be randomized to receive conventional or 
additional physical therapy. Non-CIM patients will serve as controls. From serum and vastus 
lateralis biopsies, the following will be assessed: 
1. NLRP3 inflammasome priming: Protein levels of phospho-p65S536 and total p65 will 
be measured via Western blot; mRNA levels of NLRP3, IL-1β, and IL-18 via RT-qPCR. 
2. NLRP3 inflammasome activity: Cleaved IL-1β, cleaved caspase-1, and GSDMD-NT 
will be quantified by Western blot relative to their precursor forms. IL-1β and IL-18 levels 
in plasma will be measured by ELISA. 
3. Second-signal activation factors: Oxidative stress markers (protein carbonylation 
and nitration) and cathepsin B levels will be evaluated by Western blot and RT-qPCR. 
In addition, mitochondrial ultrastructure and the presence and volume of lysosomal 
vacuoles will be analysed by transmission electron microscopy as a strategy to assess 
mitochondrial dysfunction and dysregulated autophagy. 
Expected outcomes: Physical therapy is expected to reduce NLRP3 inflammasome 
activation in a frequency- and duration-dependent manner. 
Pitfalls: If difficulties arise in measuring the proposed signaling pathway from muscle biopsy 
lysates, primary skeletal muscle cultures will be used to study inflammasome activity. 
Caspase-1 activity will also be assessed using a fluorometric kit (FAM-FLICA™, Bio-Rad). 
 
SO2: To assess the effect of physical therapy on muscle atrophy in vastus lateralis 
biopsies from CIM patients, and to relate these findings to NLRP3 inflammasome 
activation. 
Rationale: Muscle mass loss in CIM is partly attributed to atrogene overexpression(7). In 
murine models, NLRP3 contributes to atrophy through this mechanism (68,69). However, this 
association—and its modulation by physical therapy—has not yet been explored in humans. 
Experimental approach: The characteristics of CIM and muscle atrophy will be correlated 
with findings from SO1: 
1. Muscle atrophy: Muscle cryosections (10 μm) will be analyzed by indirect 
immunofluorescence using anti-laminin to delineate cell boundaries and quantify fiber 
diameter. mRNA levels of atrogenes—MuRF1, Atrogin-1, MUSA1, TRIM62, and 
TRIM32—will be measured by RT-qPCR. 
2. Molecular and histological features of CIM: Myosin/actin ratio will be quantified by 
Western blot using biopsy homogenates. Sarcomeric organization will be evaluated in 
10 μm cryosections using transmission electron microscopy. 
Expected outcomes: Physical therapy is expected to reduce muscle atrophy in relation to 
intervention frequency and duration. These effects are expected to be associated with changes 
in NLRP3 inflammasome activation. 
Pitfalls: If the proposed evaluations are not feasible, miRNA expression related to muscle 
atrophy will be assessed via nanopore sequencing. If immunofluorescence is not possible, 
myofiber cross-sectional area will be analyzed using H&E histology. 
 
SO3: To determine the effect of physical therapy on the gene expression profile of 
signaling pathways involved in skeletal muscle atrophy in CIM patients. 
Rationale: Transcriptomics has advanced rapidly in critical care, enabling early diagnosis, 
precision medicine(138,139), and identification of key signaling pathways associated with gene 
expression changes (138,140). Such analyses have been applied in CIM patients(66,67,141),  

 
 
   
and murine models(76,80,142). However, only one study has investigated the effect of physical 
therapy on transcriptomics in CIM, and it did not show significant effects, likely due to its 
sample size and unilateral intervention(66). More studies are needed to uncover the underlying 
mechanisms of CIM and the impact of early mobilization. 
Experimental approach: Differential gene expression related to NLRP3 inflammasome and 
skeletal 
muscle 
atrophy 
pathways 
will 
be 
assessed 
via 
microarray. 
Expected outcomes: Greater differential expression of genes associated with muscle atrophy 
is expected in CIM patients. Physical therapy is expected to modulate this expression. 
Pitfalls: If technical or interpretative issues arise with the microarray, mRNA sequencing will 
be performed using nanopore technology. 
 
SO4: To analyze the association between gene expression and skeletal muscle 
biochemical parameters with the clinical diagnosis of CIM. 
Rationale: Early diagnosis of CIM is crucial. Although tools like the SOFA score and muscle 
ultrasound have shown promise(39,40), more evidence is needed to validate their use(41). 
Experimental approach: Molecular biology findings and gene expression profiles will be 
associated with the clinical and ultrasound-based diagnosis of CIM, in conjunction with critical 
illness severity, to support early detection strategies. 
Expected outcomes: A strong association is expected between early CIM diagnosis, clinical 
markers, and biochemical/histological findings. 
 

 
 
   
6. Patients and Methods 
6.1. Study Design 
This pilot study adopts a quantitative approach, with an analytical, experimental, prospective, 
and longitudinal design. 
6.2. Patients 
6.2.1. CIM Patients (Group A, n = 16) 
Inclusión Criteria: Critically ill patients with CIM will be selected based on the following 
inclusion criteria: 
1. Medical diagnosis of sepsis upon ICU admission, 
2. Receiving invasive mechanical ventilation with a projected requirement ≥7 days, 
3. SOFA score ≥8 for three consecutive days within the first five days of ICU admission 
(22,41,42,81,141,144). 
To screen for CIM in these patients, serial quadriceps ultrasound assessments will be 
conducted at three timepoints: within the first 24 hours of ICU admission, again within the first 
72 hours, and finally 7 days after the second assessment. The ultrasound protocol is detailed 
in Annex 2. Changes in muscle thickness, cross-sectional area, pennation angle, and 
echogenicity will be evaluated. Patients meeting the following criteria within 7–10 days of ICU 
admission will be considered suspected CIM cases: ≥20% reduction in muscle thickness, 
≥10% reduction in cross-sectional area, ≥5% decrease in pennation angle, ≥8% increase in 
echogenicity(37). 
For patients who regain consciousness and are cooperative, muscle strength will be assessed 
using the Medical Research Council Sum Score (MRC-SS); a score ≤48 confirms muscle 
weakness(9,33,145). 
Exclusion criteria: Patients will be excluded if they present with: neurocritical illness, prior 
malnutrition or cachexia, pre-existing neuromuscular disease, coagulopathy (severe liver 
disease or continuous dialysis), thrombocytopenia <20,000 platelets/μL, inability to walk 
independently prior to hospitalization or low pre-admission functional status (Clinical Frailty 
Scale ≥4), lower limb amputation, lower limb fractures, ongoing chemotherapy, pregnancy, 
BMI >35, uncontrolled epilepsy, allergy to ultrasound gel, prolonged corticosteroid therapy, 
expected ICU stay <7 days, imminent death, or legal guardian refusal to provide informed 
consent. 
Group A Subgroups: 
• 
A.1: CIM patients receiving conventional physical therapy 
• 
A.2: CIM patients receiving additional physical therapy (servo-assisted lower limb 
mobilization using Motomed Letto® at 30 rpm, 2 sessions/day, 60 minutes/session for 
7 days). 
Although no specific treatment exists for CIM(66), prior studies have evaluated the effect of 
prolonged physical therapy compared to standard care (40,66), allowing all patients access to 
this intervention regardless of standardization(40,147,148). Therapy dosage may be titrated 
by adjusting rpm intensity, under safety criteria defined by Hodgson’s expert consensus(149) 
and continuous monitoring of hemodynamic and respiratory parameters. 
6.2.2. Control Patients (Group B, n = 8) 
Control patients will be recruited from those admitted for conditions unrelated to CIM and who 
are conscious and capable of providing informed consent. 
Exclusion criteria: Same as for Group A, with the addition of: laboratory evidence of systemic 
inflammation (elevated C-reactive protein and/or leukocytosis) and refusal to consent. 
6.2.3. Patient Recruitment Sites 
All Group A and B patients will be recruited from the Adult Critical Care Unit at the Hospital de 
Urgencia y Asistencia Pública (Ex Posta Central) and Clínica INDISA. Each participant will 
have a clinical file where all morbidity data will be recorded. Either the patient or their legal 
guardian will sign an informed consent form outlining all study procedures. All information will 
be treated with strict confidentiality.
 

 
 
   
6.3. Sample Size Validation 
The sample size was calculated based on the increased gene expression of IL-1β observed in 
vastus lateralis muscle biopsies from critically ill patients with sepsis, reported as 15.36 times 
higher than that of healthy controls (with a standard deviation of 4.96), according to Hickmann 
et al.(40).  This expression level is proposed as the effect size, or the variable to be reduced 
by physical therapy, under the assumption that IL-1β gene expression reflects NLRP3 
inflammasome activation in skeletal muscle. 
Assuming a statistical power of 99%, a type I error (alpha) of 5%, and an anticipated 40% 
mortality rate in critically ill patients (to account for potential loss)(150), the estimated sample 
size was 8 patients with CIM per physical therapy group. Based on the principle of equivalence, 
the control group will also include 8 patients. Sample size was calculated using R Studio with 
the following command: n.ttest(power = 0.99, alpha = 0.05, mean.diff = 15.36, sd1 = 4.96, 
design = "unpaired", variance = "unequal"). 
 
6.4. Sample Collection from Patients with CIM and Controls 
6.4.1. Vastus Lateralis Muscle Biopsy 
Two samples of the vastus lateralis muscle will be obtained from both CIM patients and 
controls: the first within the initial 3 days of ICU admission, and the second 7 days later. The 
muscle biopsy procedure is well-established in the literature (12,13,39,40,42,49,67,71–73,81–
83,88,95,99,101,104–106,141,144,151). A percutaneous needle biopsy (TPNB) will be used, 
as it is a minimally invasive technique(152) that has demonstrated good yield in critically ill 
patients for gene expression, protein, and ultrastructural analyses(40). Under local anesthesia 
with 2% lidocaine, three to five samples of the vastus lateralis (VL) muscle will be collected 
using a disposable 12-gauge coaxial biopsy needle (BARD, MAGNUM®) to obtain 
approximately 90 mg of tissue. The first sample will be taken at a 90° angle and stored at -
80°C for histological studies. The remaining samples will be cleared of blood with sterile gauze, 
snap-frozen in liquid nitrogen, and stored at -80°C for gene and protein expression analysis. 
6.4.2. Blood Samples 
Each CIM patient and control subject will donate 10 ml of peripheral blood on two occasions, 
collected in EDTA vacutainers (Becton Dickinson Ind Cir Ltda.) as anticoagulant. The sampling 
will be synchronized with the timing of muscle biopsy collection. 
 
6.5. Evaluators 
The methodology requires ultrasound assessment of the quadriceps muscle and the use of 
the MRC Sum Score and FSS-ICU scales by physical therapists from the ICU. Although these 
professionals have clinical experience with these evaluations and adequate inter-rater 
reliability has been reported in the literature(145,153–158), it is necessary to verify inter-
evaluator concordance. A prior pilot study titled “Validation of Operators and Inter-Evaluator 
Variability in Diagnostic Strategies for ICU-Acquired Weakness” will be conducted in 
cooperative patients capable of providing consent. This study is under development and has 
received ethical approval from the Scientific Ethics Committee of Clínica INDISA (Protocol No. 
176-25-24). 
 
6.6. Histological and Biochemical Methods 
6.6.1. Histology 
Transmission electron microscopy (TEM) will be performed on ultrathin 10 µm cryosections 
from muscle biopsy fragments. Sections will be fixed in 2.5% glutaraldehyde for 2 hours at 
room temperature, washed four times in 0.1 M sodium cacodylate buffer, dehydrated in graded 
acetone series, and embedded in Epon resin. Ultrathin sections will be contrasted sequentially 
with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer and 1% uranyl acetate in the 
same buffer, then examined via TEM. 

 
 
   
6.6.2. Immunofluorescence 
10 µm cryosections will be washed with TBS-T (Tris-buffered saline with 0.1% Tween20®), 
fixed with 100% cold acetone for 30 minutes at -20°C, then blocked with 3% BSA in TBS-T for 
2 hours. Permeabilization will be performed using 0.1% Triton X-100 in 3% BSA for 30 minutes. 
Primary antibody against laminin (1:30, L9393, Sigma-Aldrich) will be incubated overnight at 
4°C, followed by secondary antibody Alexa Fluor 488 Goat anti-Rabbit (1:500, Life 
Technologies) for 2 hours at room temperature in the dark. Slides will be mounted with ProLong 
containing DAPI (Life Technologies), stored at 4°C, and imaged by confocal microscopy. 
Muscle fiber diameter will be measured using ImageJ software and compared against control 
values to determine muscle atrophy. 
 
6.6.3. Western Blot 
30 mg of muscle biopsy samples will be lysed using an automated homogenizer (D1000, 
Biolab, Madrid, Spain) in a lysis buffer containing: 1X T-PER (#78510, ThermoFisher, 
Massachusetts, USA), 1 mM EGTA (Winkler, Santiago, Chile), 1 mM EDTA (Winkler, Santiago, 
Chile), 2 mM BAPTA (#A4926, Sigma Aldrich, Missouri, USA), 10 mM NaVO₄ (#S6508, Sigma 
Aldrich, Missouri, USA), 10 mM Na₂P₂O₇ (Winkler, Santiago, Chile), 1X protease inhibitor 
(#04693124001, Roche, Mannheim, Germany) and 1X phosphatase inhibitor (#04906845001, 
Roche, Mannheim, Germany). Samples will then be centrifuged at 4°C for 14 minutes at 12,000 
rpm. The supernatant will be analyzed by SDS-PAGE (8% or 10%, depending on the target 
protein). Wet electrotransfer will be performed onto polyvinylidene fluoride (PVDF) 
membranes, which will be blocked with 5% semi-skimmed milk (SVELTY®) in TRIS-buffered 
saline with 0.1% Tween 20 (TBS-T), followed by TBS-T washes. Membranes will be incubated 
with the primary antibody for 16 hours at 4°C. Subsequently, incubation with the appropriate 
HRP-conjugated secondary antibody will be carried out—either anti-rabbit (1:10,000, 7074, 
Cell Signaling) or anti-mouse (1:10,000, 516102, Santa Cruz Biotechnology). Membranes will 
be developed using enhanced chemiluminescence (ECL) reagent (sc-2048, Santa Cruz 
Biotechnology), and images will be acquired with the LI-COR C-Digit imaging system and 
Image Studio software (Lincoln, Nebraska, USA). Individual protein levels and protein-to-
protein ratios will be compared with those obtained from control patients without 
neuromusculoskeletal disease. 
6.6.3.1. 
Myosin/Actin Ratio 
Membranes will be incubated with two antibodies: anti-myosin antibody diluted 1:1000 (F59: 
sc-32732, Santa Cruz Biotechnology), and anti-actin antibody diluted 1:1000 (HUC1-1: sc-
53141, Santa Cruz Biotechnology). The myosin/actin ratio will then be calculated. A ratio below 
1.5 is considered conclusive for myopathy (6,12,13,90). 
 
6.6.3.2. 
Priming of the NLRP3 Inflammasome 
To assess the phosphorylation levels of p65 (a component of the NF-κB complex), membranes 
will be incubated with two antibodies: anti-p65S536 antibody diluted 1:1000 (sc-101752, Santa 
Cruz Biotechnology) and total anti-p65 antibody diluted 1:1000 (sc-8008, Santa Cruz 
Biotechnology). 
 
6.6.3.3. 
Activation of the NLRP3 Inflammasome 
To evaluate the activation of the NLRP3 inflammasome, the ratios of cleaved caspase-1/pro-
caspase-1, cleaved IL-1β/pro-IL-1β, and GSDMD-N/GSDMD will be determined. 
 
6.6.3.3.1. Cleaved Caspase-1/Pro-Caspase-1 Ratio 
Membranes will be incubated with two antibodies: anti-cleaved caspase-1 antibody diluted 
1:1000 (4199, Cell Signaling) and anti-caspase-1 antibody diluted 1:1000 (3866, Cell 
Signaling). 
 
 
 

 
 
   
6.6.3.3.2. Cleaved IL-1β/Pro-IL-1β Ratio 
Membranes will be incubated with two antibodies: anti-cleaved IL-1β antibody diluted 1:1000 
(83186, Cell Signaling) and total anti-IL-1β antibody diluted 1:1000 (12703, Cell Signaling). 
 
6.6.3.3.3. GSDMD-N/GSDMD Ratio 
Membranes will be incubated with two antibodies: anti-GSDMD-N antibody diluted 1:1000 
(ab215203, Abcam) and anti-GSDMD antibody diluted 1:400 ((H-11): sc-393581, Santa Cruz 
Biotechnology). 
 
6.6.3.4. 
Secondary Activation of the NLRP3 Inflammasome 
To assess conditions that favor the secondary activation of the NLRP3 inflammasome, 
oxidative stress and increased cathepsin B due to dysregulated autophagy will be evaluated. 
Oxidative stress will be assessed by measuring protein carbonylation and nitration. Protein 
carbonylation will be detected using the OxyBlot™ Protein Oxidation Detection Kit (EMD 
Millipore), according to the manufacturer’s instructions. Protein nitration will be assessed using 
anti-nitrotyrosine antibody diluted 1:2000 ((39B6): sc-32757, Santa Cruz Biotechnology). 
Cathepsin B levels will be quantified using anti-cathepsin B antibody diluted 1:1000 (H-5: sc-
365558, Santa Cruz Biotechnology). GAPDH will be used as a loading control, detected with 
anti-GAPDH antibody diluted 1:2000 ((0411): sc-47724, Santa Cruz Biotechnology). 
6.6.4. RT-qPCR  
A total of 20 mg of muscle biopsy tissue will be lysed using an automatic homogenizer (D1000, 
Biolab, Madrid, Spain) in 1 mL of TRIzol reagent (Invitrogen). The lysate will be incubated at 
room temperature for 5 minutes to ensure complete dissociation of nucleoprotein complexes. 
Then, 200 μL of chloroform per mL of TRIzol will be added to the homogenate tubes and 
vigorously shaken for 30 seconds. After 2–3 minutes of incubation, the samples will be 
centrifuged at 12,000 g for 15 minutes at 4°C. The aqueous phase will be transferred to a new 
microcentrifuge tube. To precipitate RNA, 200 μL of isopropanol per 1 mL of TRIzol used will 
be added and incubated for 2 hours at -20°C. The samples will then be centrifuged at 12,000 
g for 10 minutes at 4°C, and the supernatant will be discarded. For RNA washing, the pellet 
will be suspended in 1 mL of 75% ethanol at -20°C (same volume of TRIzol used for lysis), 
briefly vortexed, and centrifuged at 7500 g for 5 minutes at 4°C. After discarding the 
supernatant, the RNA pellet will be air-dried for 5–10 minutes. To solubilize the RNA, the pellet 
will be resuspended in 20–30 μL of RNase-free water and incubated on a heat block at 55–
60°C for 10–15 minutes. RNA concentration will be measured using the MaestroNano 
spectrophotometer. 
Reverse transcription will be performed using the iScript cDNA Synthesis Kit (Bio-Rad). 
Reactions will be carried out in a final volume of 10 μL containing 1 μg of RNA, 0.1 μL of each 
primer (100 nM final), and 5 μL of cDNA. Thermal cycling will follow this protocol: 3 minutes at 
95°C, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 
72°C. SYBR® Green (Applied Biosystems) will be used as the fluorescent probe. All samples 
will be run in duplicate. β2-microglobulin (β2M) will be used as the reference gene, as it has 
shown stable expression levels regardless of the exercise modality (159). 
 
6.6.4.1. 
Priming of the NLRP3 Inflammasome 
To assess the priming of the NLRP3 inflammasome, RT-qPCR will be performed to quantify 
mRNA levels of NLRP3, IL-1β, and IL-18. 
6.6.4.2. 
Secondary Activation of the NLRP3 Inflammasome 
To evaluate dysregulated autophagy favoring the secondary activation of the NLRP3 
inflammasome, RT-qPCR will be used to assess Cathepsin B mRNA expression. 
6.6.4.3. 
Atrogenes 
To evaluate the ubiquitin-proteasome system, RT-qPCR will be performed to quantify mRNA 
levels of the ubiquitin ligases: MurF1, Atrogin-1, MUSA1, TRIM62, and TRIM32. 
 
 
 

 
 
   
Primer List: 
 
6.6.5. ELISA 
EDTA vacutainer tubes will be centrifuged for 10 minutes at 4°C and 4000 rpm. Plasma and 
serum will be aliquoted into microcentrifuge tubes with 500 μL per sample. Measurements will 
be performed according to the manufacturer’s instructions using the following kits: IL-1β: 
Human IL-1β ELISA Kit (Invitrogen), IL-18: Human IL-18 ELISA Kit (Invitrogen). 
 
6.6.6. Microarray.  
Total mRNA will be extracted from samples of control and CIM (Critical Illness Myopathy) 
patients. Using reverse transcriptase (RT), complementary DNA (cDNA) libraries will be 
synthesized from the extracted RNA. These cDNA libraries will be labeled with fluorescent 
probes incorporated during synthesis. The labeled cDNA samples will then be hybridized to a 
commercial microarray chip containing oligonucleotides with complementary sequences. This 
hybridization process results in a stable DNA double helix. After hybridization, the chip will be 
washed several times to remove any unbound cDNA. The chip will then be scanned using a 
laser to excite the fluorescent tags. The fluorescence signal from each spot will be captured 
and analyzed with specialized software that generates a colorimetric image of the microarray. 
Expression profiles will be obtained using Illumina HT-12 v4 microarrays (one microarray per 
sample). 
6.6.6.1. 
Microarray Data Analysis 
Raw expression data will be processed using the R software environment and the 
"Bioconductor" package. The data will undergo background correction, quantile normalization, 
and log2 transformation using the neqc function from the limma package. Probes classified as 
"No match" or "Bad" will be removed using the illuminaHumanv4 annotation package. 
6.6.6.2. 
Differential Gene Expression 
Robust differential expression analysis of all probes will be performed using the limma package 
in R, adjusted for each patient’s sociodemographic and clinical variables. Sample correlation 
will be assessed using the duplicateCorrelation function. The decideTests function with the 
"global" adjustment will be used to estimate the false discovery rate (FDR), using a significance 
threshold of FDR < 0.05 and a minimum expression change of 1.5-fold compared to controls. 
6.6.6.3. 
Gene Ontology (GO) 
Functional enrichment analysis in Gene Ontology will be performed using the "gProfiler" tool 
in R. The statistical threshold for GO enrichment will be an FDR-adjusted p-value < 0.05, with 
a minimum of 10 detected genes. The enrichment list will include all genes represented in the 
Illumina Human HT-12 v4 array with a p-value < 0.05 in at least three samples. 
 
6.7. Demographic Variables, Severity of Critical Illness, Clinical Assessment of Muscle 
Strength, and Functionality 
The sociodemographic and clinical variables that will be recorded throughout the study include: 
biological sex, age, body mass index (BMI), degree of hypoxemia, comorbidities, route of 
nutrition, nitrogen balance, serum albumin, lactatemia, duration of mechanical ventilation, 
length of ICU stay, and total hospitalization. The severity of critical illness will be assessed 
using the SOFA (Sequential Organ Failure Assessment) score. The use and dosage of the 
following medications will also be recorded: norepinephrine, milrinone, dobutamine, propofol, 
Gene
Forward Primer
Reverse Primer
NLRP3
5'-GAGGAAAAGGAAGGCCGACA-3'
5'-CCCGGCAAAAACTGGAAGTG-3'
IL-1β 
5’AGCAACAAGTGGTGTTCTCCATGT-3’ 
5’-AGCTGTAGAGTGGGCTTATCATCTT-3’ 
IL-18
5'-AGCCTAGAGGTATGGCTGTAAC-3'
5'-AAAGGTCTCTCTCTTTTTCACAAGC-3'
Catepsina B
5'-GCGCTGGGTGGATCTAGGA-3'
5'-GTTGACCAGCTCATCCGACA-3'
MurF1
5’-AAACAGGAGTGCTCCAGTCGG-3’ 
5’-CGCCACCAGCATGGAGATACA-3’ 
Atrogina-1
5’-CGACCTCAGCAGTTACTGCAA-3’ 
5’-TTTGCTATCAGCTCCAACAG-3’ 
MUSA1
5'-GTGATCATGCCAATCCACAG-3'
5'-CATGAATGTCACCATGCACA-3'
TRIM62
5’-AAGTCCCTGTTCCAGGACATC-3’
5’-GCAGTCGTCCGACAGGAT-3’
TRIM32
5'-CCCCATAGGGGATGAGAAAT-3'
5'-GTCCTATTTGGGCAGTGCAT-3'
Fbxo21/SMART
5'-CACGTTCCTTGTAATGGCTTCA-3'
5'-ACGACTCATAGTCATCTGGCTG)-3'
β2M (Housekeeping)
5’-ATGAGTATGCCTGCCGTGTGA-3’
5’GGCATCTTCAAACCTCCATG-3’

 
 
   
ketamine, midazolam, dexmedetomidine, neuromuscular blocking agents, corticosteroids, and 
aminoglycosides. 
Regarding physical therapy and functionality, the Clinical Frailty Scale(160) will be used to 
determine the functional status prior to ICU admission. In cooperative patients (those who 
respond to at least 3 out of 5 commands in the S5Q scale—five standardized ICU questions), 
analytical muscle strength will be assessed using the Medical Research Council Sum Score 
(MRC-SS)(9,33,145). A score of ≤48 points confirm the presence of muscular weakness, which 
must be corroborated by ultrasound and SOFA scale findings. This scale also allows for the 
classification of severe muscle weakness when the score is below 36 points(33,34,41). 
Functional status will be assessed using the Functional Status Score for the ICU (FSS-
ICU)(46,158,161). Data collection will include the time elapsed until the first physical therapy 
session, the number of sessions, the modality of intervention, and its temporal distribution. 
 
6.8. Statistical Analysis 
Primary quantification results will be expressed as gene or protein expression levels relative 
to control patients. These will be reported using central tendency and dispersion measures, 
and normality will be assessed using the Shapiro–Wilk test. 
For comparisons between independent groups, Student’s t-test or the Wilcoxon rank-sum test 
will be applied, depending on the distribution of the data. To assess differences before and 
after the early physical therapy intervention, paired Student’s t-tests or Wilcoxon signed-rank 
tests will be used. 
Correlation between the outcome variables of Objective 1 (SO1) and Objective 2 (SO2) will be 
analyzed using Pearson or Spearman correlation coefficients, depending on variable 
distribution. 
For differential gene expression analysis, quantification will be relative to control patients, and 
significance will be assessed with a false discovery rate (FDR) threshold of <0.05. 
Associations between variables will be analyzed based on their nature. Multiple linear 
regression will be applied to quantitative variables. For associations between cellular and 
molecular biology findings and dichotomous variables (e.g., presence of muscle weakness), 
logistic regression will be used. For associations with ordinal variables, such as functional 
scales (e.g., FSS-ICU), Poisson regression will be applied. All statistical analyses and graph 
generation will be conducted using R Studio and Stata software. 
 

 
 
   
7. Ethical Considerations 
7.1. Social Utility 
Critical illness myopathy (CIM) is a common cause of ICU-acquired weakness (ICUAW)(1). 
This condition is characterized by muscle atrophy and impaired contractility, both of which 
contribute to increased mortality(14–18), prolonged hospital stays (9,10,25,28), (extended 
durations of mechanical ventilation(19,20), elevated healthcare costs(9,10,29,31,44), and 
long-term physical disability(19,20). CIM may progress to post-intensive care syndrome 
(PICS), potentially resulting in persistent disability(4,28). A Chilean study reported that 40% of 
critically ill patients experience some degree of disability after hospital discharge(46).  
Therefore, understanding the etiology of CIM is essential for designing both pharmacological 
and non-pharmacological therapeutic strategies. 
Several risk factors have been identified in the development of CIM(4,25), with prolonged 
immobility highlighted as a modifiable factor through physical therapy (4,25,27–29,31). This 
underscores the importance of evaluating the effects of physical therapy and supports the 
biomedical justification for dedicated clinical staff to manage and implement rehabilitation in 
critically ill patients. Since muscle protein degradation and electrophysiological abnormalities 
emerge within the first 10 days of ICU admission(39,40), early and timely rehabilitation is 
crucial. Early detection strategies for CIM are necessary. Internationally, research groups have 
implemented methods combining the Sequential Organ Failure Assessment (SOFA) score with 
ultrasound assessment to detect CIM in non-cooperative patients(22–24,41,42). While this 
method has proven effective in specialized research groups, it remains to be broadly adopted 
in Chile. Implementing this evaluation locally could facilitate early CIM detection and allow for 
better allocation of physical therapy resources, minimizing mechanical silencing — a 
modifiable risk factor. Currently, critical care units in Chile do not have full coverage for 
rehabilitation needs(162). Thus, biomedical research on CIM could benefit the general 
population by informing public policies that expand access to physical therapy and 
rehabilitation in intensive care units. 
 
7.2. Protection of Vulnerable Groups 
This study focuses on critically ill patients during the first days of admission to the intensive 
care unit (ICU), a population considered vulnerable due to the lack of consciousness resulting 
from sedoanalgesia. However, since the study specifically targets the development of critical 
illness myopathy (CIM), it can only be conducted in this population. The initial hospitalization 
period is when the risk factors associated with CIM development manifest (22,26,87,163). In 
accordance with Article 28 of Chilean Law No. 20.584, this study will include adult patients 
who "are physically or mentally incapable of expressing consent." It is important to note that 
“this physical or mental condition that prevents providing informed consent or expressing 
preference is a necessary characteristic of the target research population.” 
As such, the study protocol will be submitted for review by a scientific ethics committee. If 
approved, it will subsequently be submitted for evaluation to the Regional Health Authority 
(SEREMI). Once approved by these institutions, legal guardians will be approached to provide 
informed consent for the inclusion of patients in the research protocol. Furthermore, as 
stipulated by Article 28 of Law No. 20.584, once the patients have recovered from sedation, 
are cooperative, and demonstrate no delirium or physical incapacity, “consent or expression 
of preference will be sought from the individuals who have regained the physical or mental 
capacity to provide such consent or to express their preferences.” 
This research does not introduce any form of bias related to sex, race, or age among 
participants. 
 
7.3. Randomization 
Patients at risk of developing CIM (Group A) who meet the inclusion criteria and do not fulfill 
any exclusion criteria will be randomly assigned to receive either conventional physical therapy 
or an enhanced physical therapy protocol using a servo-assisted lower limb cycling device 
(Motomed Letto®). This strategy has been employed in previous studies to assess the effects 
of 
physical 
therapy 
by 
comparing 
extended 
interventions 
with 
conventional 
protocols(40,66,146–148,164). This design ensures that all patients have access to early 

 
 
   
physical therapy and rehabilitation, thereby avoiding comparison with a non-intervention 
group(40,147). 
Randomization will be independent of ethnicity. However, in order to ensure homogeneity 
across intervention groups, stratification will be performed based on two factors: age and sex. 
Stratified randomization will be implemented, and allocation concealment will be applied only 
at the assignment stage. Due to the nature of the intervention—physical therapy—it will not be 
possible to blind either participants or investigators to the treatment received; thus, the study 
is classified as open-label. 
Randomization will be conducted using R Studio software and the “randomizr” package. 
Stratification by age and sex will be performed using the “split” function. 
 
7.4. Adverse Events 
This study involves performing a muscle puncture to obtain tissue samples from the vastus 
lateralis. This procedure is well established in the study of CIM (12,13,39,40,42,49,67,71–
73,81–83,88,95,99,101,104–106,141,144,151). Although the most commonly used techniques 
for human muscle biopsy are the percutaneous needle biopsy (Bergström method)(49,67,70–
72,82,83,99,101,106,151), conchotome(13,39,66), and Henckel-Tilley forceps(95,105), a 
more recently described method—tiny percutaneous needle biopsy (TPNB)—has shown 
advantages. This technique is less traumatic for patients(152) and has already been applied 
in CIM studies, providing adequate yield for gene expression, protein analysis, and 
ultrastructural studies without reports of adverse events(40). 
The biopsy will be performed under local cutaneous anesthesia (2% lidocaine hydrochloride) 
with prior disinfection using topical chlorhexidine, employing a sterile technique and a coaxial 
disposable biopsy needle (BARD, MAGNUM®, C.R. Bard Inc., Covington, GA). To minimize 
the occurrence of adverse events, biopsies will be performed by intensivist physicians using 
ultrasound guidance, allowing image-assisted needle placement(165). In parallel, the clinical 
nurse of the intensive care unit, will supervise and provide nursing care for the biopsy sites. 
As previously mentioned, patients at risk of CIM will be randomized to receive either 
conventional physical therapy or more frequent and prolonged sessions using a servo-assisted 
lower-limb cycling device (Motomed Letto®). Recent studies have demonstrated the safety of 
this type of intervention during physiotherapy in intensive care settings(147). 
Ultrasound evaluation is routine in intensive care units(166).  However, some cases of contact 
dermatitis due to ultrasound gel have been reported (167–172). Therefore, the skin of the area 
assessed by ultrasound will be monitored after each session. Additionally, the exclusion criteria 
in this protocol are designed to minimize the likelihood of adverse events. 
 
7.5. Benefit Assessment 
Percutaneous muscle biopsy is recognized as an essential technique for advancing knowledge 
in muscle physiology and for the diagnosis of musculoskeletal disorders(165,173,174). 
Although general concerns exist regarding potential adverse events associated with this 
procedure, the incidence of complications is low and typically of minimal clinical relevance. 
The most commonly reported complications include pain, erythema, ecchymosis, edema, and 
bleeding(173). This study entails the inherent risks associated with muscle puncture; however, 
these are generally mild and self-limiting(173). The likelihood of significant complications is 
further reduced through the application of strict exclusion criteria. 
In the event of a complication arising from the muscle biopsy procedure, medical professional 
liability insurance will be available to cover any associated harm. It is important to note that 
participants will not receive any direct clinical benefit from undergoing the biopsy procedure. 
This will be explicitly explained during the informed consent process to the legal guardians of 
the patients, and subsequently to the patients themselves, once they regain the cognitive 
capacity necessary to either continue with or withdraw from the study. 
Physical therapy within the ICU is a standard intervention in critically ill patients and will be 
conducted in accordance with established safety criteria described in the literature(149). 
Additionally, an enhanced physical therapy regimen will be applied using servo-assisted 
cycling devices(147,148).  Importantly, all enrolled patients will receive some form of physical 
therapy—either conventional or enhanced—thus ensuring that no participant is deprived of 
therapeutic benefit. 

 
 
   
7.6. Protection of Privacy 
All collected data will be securely stored under the custody of the principal investigator. 
Participant information will be recorded using the REDCap (Research Electronic Data Capture) 
platform. The presentation of study results will always preserve participant anonymity; no 
personal identifiers or sensitive data will be disclosed to third parties. Muscle biopsy samples 
will be processed at the Muscle Metabolism Laboratory, where they will remain under the 
custodianship of Dr. Paola Llanos Vidal. 
7.7. Ethical Monitoring of the Project 
Any issues that arise during the implementation of the study or any amendments to the protocol 
will be reported to the ethics committee. In accordance with Article 10 of Law 20.120, “Any 
serious adverse event occurring during the course of the research must be reported by the 
investigator to the Committee and to the Director of the institution where the research is being 
conducted.” 
7.8. Feasibility 
This study is supported by the clinical teams of the Adult Intensive Care Units at the Hospital 
de Urgencia y Asistencia Pública (Ex Posta Central) and Clínica INDISA, which will serve as 
the recruitment centers from 2025 through the end of 2026. 
The research project is led by Dr. Lilian Jara Sosa, Dr. Paola Llanos Vidal, and Dr. Denisse 
Valladares Ide. Dr. Lilian Jara Sosa is a geneticist with extensive experience in the field, 
including research on the genetic profile of myopathies in the Chilean population. Under her 
guidance, transcriptomic analyses and differential gene expression studies will be conducted 
using advanced sequencing and bioinformatics technologies. 
Dr. Paola Llanos is a specialist in the activation pathway of the NLRP3 inflammasome in 
skeletal muscle in preclinical models of insulin resistance induced by obesity. She leads 
investigations focused on low-grade inflammation in skeletal muscle, employing biochemical, 
cellular, fluorescence and confocal microscopy techniques, as well as muscle fiber cultures. 
Dr. Denisse Valladares Ide has extensive expertise in studying the molecular mechanisms 
underlying sarcopenia using human muscle biopsy samples. She is an active researcher on 
the modulatory role of physical activity on sarcopenia in elderly populations. 
Experiments related to the quantification of markers of NLRP3 inflammasome “priming” and 
assembly, as well as transcriptomic analyses, will be carried out with funding from Fondecyt 
Regular Grant No. 1231103 awarded to Dr. Paola Llanos Vidal and Fondecyt Regular Grant 
No. 1241959 awarded to Dr. Denisse Valladares. The acquisition of the biopsy device and 
disposable 12-Gauge coaxial biopsy needles (BARD, MAGNUM®) will be financed through 
the operating expenses of the National Doctoral Scholarship ANID No. 21231808. 
Additionally, the project has the formal approval of medical leadership and rehabilitation 
coordinators of the Adult Intensive Care Units at the Hospital de Urgencia y Asistencia Pública 
and Clínica INDISA. 
The project also includes the participation of Dr. Margot Navarrete, physician, general surgeon 
and intensive care medicine resident, and Dr. Valentín López, physician and internal medicine 
specialist, who will be responsible for performing the muscle biopsies. 
Clinical nurse Valentina Burgos will assist with the monitoring of the muscle biopsy puncture 
site and associated nursing care. 
The complementary expertise of the thesis advisors, along with a multidisciplinary team, will 
allow the project to be developed with a translational (bench-to-bedside) approach. 

 
 
   
8. Discussion 
Why is it important to study Critical Illness Myopathy? CIM is a common condition among 
critically ill patients(17,31,43), associated with increased mortality (9,11,25,28,29,31), 
prolonged hospital stay(9,10,25,28), elevated healthcare costs, and reduced physical function 
and long-term quality of life(9,10,29,31,44). It may also lead to Post-Intensive Care Syndrome 
(PICS), characterized by persistent cognitive, mental, and physical dysfunction(4,28).  
In this study, an increase in NLRP3 inflammasome activation is expected in critically ill patients, 
potentially contributing to muscle atrophy. NLRP3 inflammasome activation has been linked to 
the upregulation of muscle atrophy-related genes in established murine models of the 
disease(68,69). However, this relationship has not yet been described in human CIM. We 
hypothesize an association between changes in inflammasome activation and muscle atrophy 
in patients with CIM. This connection would be highly relevant, as muscle atrophy and impaired 
contractility are key features driving poor outcomes in these patients(14–20). While 
associations do not necessarily imply causation(3), the findings of this study, derived from ex 
vivo samples, may support a link between the proposed mechanism and CIM pathogenesis. 
This could lead to the identification of novel pharmacological and non-pharmacological 
therapeutic targets, including physical therapy. 
Mechanical silencing due to prolonged bed rest is a major modifiable risk factor for the 
development of CIM(4,25,27–29,31).  Physical therapy is a non-pharmacological intervention 
that could regulate the proposed mechanism of atrophy. This intervention has shown beneficial 
effects in both CIM patients and experimental models, particularly in improving mitochondrial 
function(40,76) and autophagy processes(40).  Additionally, various modalities of exercise and 
physical therapy have been shown to reduce IL-1β and TNF-α levels in different pathological 
conditions(124,126,128,130–132,137). Therefore, physical therapy in critically ill patients 
could modulate NLRP3 inflammasome activation(57,84,85,91), and the associated muscle 
atrophy. However, to date, research groups studying CIM pathophysiology have not yet 
addressed the potential protective effects of physical therapy on this pathway. 
As an additional methodological approach to evaluate the mechanisms involved in CIM 
development and the effects of physical therapy, a genetic screening strategy is proposed. 
Transcriptomic analyses have previously been performed in both CIM patients(66,67,82) and 
in murine models of this condition(76,80,142). Nevertheless, only one study has analyzed the 
impact of physical therapy in patients with CIM, reporting no significant changes in the skeletal 
muscle gene expression profile(66). It should be noted that this study included only 7 patients 
and compared one limb with the contralateral immobilized limb(66). In contrast, the present 
study proposes to assess the effects of conventional physical therapy versus additional 
physical therapy, as previously described in the literature(40,147). Given the scarcity of such 
studies and the rationale outlined in this project, there is a clear need to expand knowledge 
regarding the effects of early mobilization and the underlying mechanisms of CIM.

 
 
   
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10. Gantt Chart 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ACTIVIDAD 
3°
4°
1°
2°
3°
4°
1°
2°
3°
4°
OE1: Evaluar el efecto de la terapia física en la activación del inflamosoma NLRP3 en biopsias del vasto lateral de 
pacientes con MPC, y comparar esta actividad con la de pacientes control. 
Priming del inflamosoma NLRP3 - NLRP3, IL-1β e IL-18 mRNA - RT-qPCR 
Priming del inflamosoma NLRP3 - p65S536/p65t - Western blot 
Actividad del inflamosoma NLRP3 - Caspasa-1/pro-caspasa-1 IL-1β/pro-IL-1β GSDMD/NGSDMD - Western blot
Actividad del inflamosoma NLRP3. IL-1β - IL-18 - ELISA 
Fact. ~ con ensamblaje I. NLRP3 - Estrés oxidativo - Dinitrofenol, Nitrotirosina - Western Blot 
Fact. ~ con ensamblaje I. NLRP3 - Catepsina B - RT-qPCR/Western Blot 
OE2: Evaluar el efecto de la terapia física sobre la atrofia muscular en biopsias de vasto lateral de pacientes con 
MPC, y relacionar estos resultados con la activación del inflamosoma NLRP3. 
Diagnóstico molecular de la MPC - Razón Miosina/Actina - Western blot
Diagnóstico molecular de la MPC - Desorganización sarcomética - TEM 
Atrofia muscular - IFI laminina 
Atrogenes- MurF1, atrogina-1, MUSA1, TRIM62 y TRIM32 mRNA - RT-qPCR 
OE3: Evaluar el efecto de la terapia física en el perfil de expresión génica de las vías de señalización involucradas 
en la atrofia del músculo esquelético en pacientes con MPC.  
Ensayo de transcriptómica por Microarray
Análisis estadístico y bioinformático: DEG, GO
OE4: Analizar la asociación entre la expresión génica y los parámetros bioquímicos del músculo esquelético con 
el diagnóstico clínico de la MPC.
Análisis estadístico de la asociación entre los hallazgos bioquímicas del músculo y el diagnóstico clínico de la MPC. 
Otros
Estandarización técnica: WB - RT-qPCR - Histología e IFI
Proyecto piloto Variabilidad inter-evaluador: MRC SS, FSS ICU, ecografía muscular y ecografía de nervio periférico
Examen de calificación
Presentación proyecto a Comité de Ética 
Presentación proyecto a SEREMI de Salud
Registro proyecto en ClinicalTrials 
Escritura artículo científico - Protocolo 
Diseño e incorporación plataforma REDCap
Avance 1
Avance 2
Escritura artículo científico 
Escritura de tesis 
Tesis final 
2024
2025
2026

 
 
   
Appendix 1. Graphical Summary of the Experimental Design and Methodology
 
 
Appendix 2. Ultrasound Evaluation Procedure 
1. Muscle Ultrasound 
  
 

 
 
   
Ultrasound assessments of the quadriceps muscle will be performed within the first 72 hours 
of ICU admission (Day 1) and again at Day 7. To identify the site for the transverse 
measurement, the patient will be placed in a supine position with the knee joint in a neutral 
resting position. A line will be drawn between the anterior superior iliac spine (ASIS) and the 
superior pole of the patella, and measurements will be taken at the junction between the middle 
and lower third of this line. At this measurement point, the thickness of the anterior quadriceps 
complex—including the rectus femoris (RF) and the vastus intermedius (VI)—as well as its 
cross-sectional area (CSA), will be assessed. Images will be acquired for echogenicity analysis 
using ImageJ software. 
For longitudinal measurements, a point 5 cm lateral to the transverse measurement site will 
be marked, where the pennation angle will be evaluated. A linear transducer with a frequency 
range of 4 to 12 MHz (Sonosite™