Skeletal Muscle Mitochondrial Abnormalities and the Metabolic Syndrome in PAH

April 17, 2023 updated by: Steeve Provencher, Laval University

Skeletal Muscle Mitochondrial Abnormalities and the Metabolic Syndrome in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by the progressive increase in pulmonary vascular resistance ultimately leading to right ventricular (RV) failure. Its prevalence is estimated at 40-60 persons per million and predominantly affects people between 20 and 60 years of age. Newly available therapies have improved the 3-year survival to >80%. This improvement in prognosis brings new challenges for clinicians: PAH has changed from a rapidly fatal disease to a chronic disorder with persistent exercise limitation and poor quality of life.

Many observations suggest that exercise limitation in PAH is not simply due to pulmonary hemodynamic impairment, but that other determinants are involved. Interestingly, even in absence of obesity or diabetes, insulin resistance (IR) and metabolic syndrome (MS) are highly prevalent amongst PAH patients and associated with worse outcomes. Indeed, lipid accumulation in skeletal muscle (a feature of IR) is observed in both human and experimental model of PAH, but its impact on skeletal muscle function and thus exercise intolerance in PAH remains elusive.

Over the past years, several pathophysiological pathways activated by MS have been identified, including the downregulation PPARg/PGC1a and the insulin signalling pathways, especially the insulin-receptor substrate 1 (IRS1)-mediated one. The decrease in these axes is associated with lipid accumulation and impaired mitochondrial function. The investigators previously reported in PAH lungs that the downregulation of these pathways contributes to the establishment of the Warburg effect. This metabolic unbalance contributes to pulmonary artery smooth muscle (PASMC) proliferation, and resistance to apoptosis contributing to PA remodelling. The investigators recently documented that PAH skeletal muscles are less perfused and are also characterized by the presence of a Warburg effect. These features were independent of daily life physical activity. Nonetheless, the origin of these abnormalities and their impact on skeletal muscle function have never been studied. The investigators propose to determine whether or not MS seen in PAH patients impairs mitochondrial functions through an IRS1/PPARg/PGC1-dependent mechanism, which will ultimately decrease skeletal muscle function and perfusion, and thus overall exercise capacity.

Study Overview

Status

Completed

Detailed Description

AIM 1:

To determine whether MS is associated with intramuscular lipid accumulation and impaired skeletal muscle metabolism and perfusion in human PAH.

Rationale: MS and IR are highly prevalent amongst PAH patients even in the absence of obesity and diabetes. There are several lines of evidence in the literature that IR develops with the accumulation of fatty-acid metabolites within insulin-responsive tissues, especially intramyocellular lipid deposition within skeletal muscles. Although the mechanism accounting for lipid accumulation remains elusive, a reduction in lipid oxidation as a result of reduction in mitochondrial density has been proposed. The objectives of Aim 1 are 1) to confirm that PAH patients have increased intramuscular lipid accumulation; 2) to determine whether intramuscular lipid accumulation is associated with impaired skeletal muscle metabolism; 3) to demonstrate that these abnormalities correlate with MS and IR and skeletal muscle function amongst PAH patients.

Experimental approaches: The proposed experiments will be performed on PAH patients (n=10-20) vs. 10 healthy but sedentary subjects matched for age, gender, height and weight (definition based on current recommendations), excluding patients with clinically relevant conditions (e.g. diabetes). These individuals are continuously identified through our systematic plasma biobanking process at the time of right heart catheterization (CER#20735), in which roughly 40% of PAH patients with no obesity/diabetes have MS. In addition to routinely performed analyses: A) blood sample will be drawn for Apolipoprotein A1, Apolipoprotein B, glycated hemoglobin, fasting blood glucose, insulin, adiponectin and leptin. B) MR imaging will be used to assess fat infiltration within the quadriceps muscle, liver and heart (see appendix for details). C) Volitional and non volitional strength and endurance of the dominant quadriceps and VO2peak on cycle ergometer will be assessed, as previously described. D) Percutaneous biopsy specimens of the vastus lateralis muscle of the nondominant leg will be taken. Part of the specimen (≈100mg) will be used for immunohistochemistry fiber typing (ethanol modified technique), capillarisation (quantitative IF using CD31-antibody) and intramyocellular lipid accumulation (Oil red O staining, which stains only the most hydrophobic and neutral lipids, as the investigators previously described. The extracellular flux analyzer Seahorse XF24 will be used on the remaining tissues for real time measurements of oxygen consumption and extracellular acidification rates (glycolysis). To ensure that physical inactivity is not responsible for skeletal muscle lipid accumulation, subjects' daily life physical activities will be objectively quantified during one week using a physical activity monitor (SenseWear® armband).

Interpretation: This multimodality approach will provide comprehensive information to confirm: 1) PAH patients exhibit significant increases in quadriceps muscle lipid accumulation compared to controls; 2) lipid accumulation is increased within the skeletal muscle of PAH patients with MS compared to PAH without MS despite similar levels of physical activity; 3) Lipid accumulation is associated with a reduction in lipid oxidation in vivo; 4) MS/IR and quadriceps muscle function correlate with muscle lipid accumulation/glucose oxidative phosphorylation capacity.

Sample size and analysis: Comparisons between groups will be performed using one-way ANOVA followed by a Tukey-Kramer post-test, after confirmation of normality/equal variances (Levene's test). 10 subjects/group will allow detecting a 1.5±0.5 fold increase in quadriceps muscle lipid accumulation assessed by MRI (primary outcome) with type 1 and 2 errors of 5% and 15%. Based on our preliminary data (Fig.3C), these estimates are conservative.

Alternative approach: Insulin action in the liver has many similarities with insulin action in muscle. Although our proposal focuses on skeletal muscles, ectopic lipid accumulation in the liver is also increasingly recognized as contributing to MS and IR. Since MRI sequences to assess fat infiltration take few minutes only, liver and abdominal adiposity will be assessed during the same exam MR study, as previously described.

AIM 2:

To assess whether IR and MS is related to defects in insulin signalling within PAH skeletal muscles.

Rationale: Numerous studies confirmed a reduction in expression of the peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α in the muscles of patients with type 2 diabetes mellitus, reducing the mitochondrial fatty acid oxidation that promotes the accumulation of diacylglycerol within the muscle. In skeletal muscles, insulin binds to its receptor, activating the receptor tyrosine kinase activity, with subsequent phosphorylation and activation of insulin-receptor substrate 1 (IRS1), ultimately promoting the docking and fusion of glucose transporter (GLUT4)-containing vesicles to the plasma membrane. Accumulation of intracellular diacylglycerol has been shown to specifically activate protein kinases C (PKC) θ, resulting in reduction in tyrosine phosphorylation of IRS1. Consistently, activation of muscle PKCθ and increased serine (inactivation) phosphorylation of IRS1 has been noted in the muscles of individuals with type 2 diabetes mellitus and IR. More recently, the activation of the nuclear respiratory factor-2 (NRF2)-Keap1 pathway (improving mitochondrial oxygen consumption, ATP production and beta-oxidation of fatty acids) has been shown to reduce glucose uptake and IR.

Experimental approaches: The same experimental groups and experimental design as described in aim 1 will be used. A) In order to examine the mechanisms responsible for the reduction of mitochondrial activity in PAH skeletal muscles, the expression of several key transcriptional factors and coregulators that are known to regulate mitochondrial biogenesis will be examined, including PPARγ coactivator 1α (PGC-1α), NRF-2, and mitochondrial transcription factor A (WB and immunoprecipitation assay). Mitochondrial oxidative (citrate synthase, hexokinase) and glycolytic (lactate dehydrogenase, phosphofructokinase) enzymes activity (spectrophotometric techniques) will also be assessed. B) In order to assess the potential role of IRS-1 serine phosphorylation in the pathogenesis of IR, the investigators will also examine IRS-1 serine phosphorylation on several serine residues (Ser307, Ser312, Ser616, Ser636) that have been implicated to interfere with insulin signaling in vitro (WB). PKCθ expression and activity will be assessed using isoform-specific PKC antibodies (WB) and a PKC enzyme assay kit.

Interpretation: The investigators expect to demonstrate that: 1) PAH patients exhibit reduced expression/activation of PPARγ1α, and NRF-2, increased phosphorylation of IRS-1 on critical serine sites and PKCθ activation, leading to a metabolic shift toward glycolysis; 2) these abnormalities dominate amongst PAH-MS patients compared to PAH without MS.

Alternative approach: PKCθ activation has been predominantly associated with MS. However, the same experiments could be done for other members of the PKC gene family. In the event that "classical MS pathways" described above do not account for IR/MS in PAH, the role of skeletal muscle Uncoupling Protein-2 and Sirtuin-3, which have recently been implicated in both IR/MS and PAH, will be explored.

Study Type

Observational

Enrollment (Actual)

30

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Locations

    • Quebec
      • Quebec City, Quebec, Canada, G1V 4G5
        • IUCPQ-UL

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

20 years to 60 years (Adult)

Accepts Healthy Volunteers

N/A

Sampling Method

Non-Probability Sample

Study Population

The proposed experiments will be performed on PAH patients (n=10-20) vs. 10 healthy but sedentary subjects matched for age, gender, height and weight (definition based on current recommendations), excluding patients with clinically relevant conditions (e.g. diabetes). These individuals are continuously identified through our systematic plasma biobanking process at the time of right heart catheterization (CER#20735), in which roughly 40% of PAH patients with no obesity/diabetes have MS.

Description

Inclusion Criteria:

  • PAH patients: Male and female subjects, patients presenting with metabolic syndrome (MS).
  • Sedentary healthy patients: Male and female subjects. Healthy but sedentary subjects.

Exclusion Criteria:

  • Presence of obesity/diabetes

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Observational Models: Case-Control
  • Time Perspectives: Prospective

Cohorts and Interventions

Group / Cohort
PAH patients
Male and female subjects, aged between 20 and 60 years old. Absence of obesity/diabètes. PAH patients presenting with metabolic syndrome (MS).
Sedentary healthy patients
Male and female subjects, aged between 20 and 60 years old. Absence of obesity/diabètes. Healthy but sedentary subjects.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Concentration of Intramuscular lipid
Time Frame: Through study completion, an average of 1 year
MR imaging will be used to assess fat infiltration within the quadriceps muscle, liver and heart.
Through study completion, an average of 1 year
Level of physical activity
Time Frame: During 1 week
Subjects' daily life physical activities quantified using a physical activity monitor (SenseWear® armband).
During 1 week
Level of mitochondrial activity in PAH skeletal muscles
Time Frame: Through study completion, an average of 1 year
The expression of several key transcriptional factors and coregulators that are known to regulate mitochondrial biogenesis will be examined, including PPARγ coactivator 1α (PGC-1α), NRF-2, and mitochondrial transcription factor A (WB and immunoprecipitation assay). Mitochondrial oxidative (citrate synthase, hexokinase) and glycolytic (lactate dehydrogenase, phosphofructokinase) enzymes activity (spectrophotometric techniques) will also be assessed.
Through study completion, an average of 1 year
Change in serine residues (Ser307, Ser312, Ser616, Ser636) due to IRS-1 serine phosphorylation
Time Frame: Through study completion, an average of 1 year
Differences in phosphorylation of IRS-1 on critical serine residues (Ser307, Ser312, Ser616, Ser636) that have been implicated to interfere with insulin signaling in vitro will be assessed on skeletal muscle biopsies by Western Blot.
Through study completion, an average of 1 year
Level of PKCθ activation/activity
Time Frame: Through study completion, an average of 1 year
Will be assessed on skeletal muscle biopsies using isoform-specific PKC antibodies (WB) and a PKC enzyme assay kit.
Through study completion, an average of 1 year

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Investigators

  • Principal Investigator: Steeve Provencher, MD, MSc, IUCPQ - Université Laval

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

June 1, 2019

Primary Completion (Actual)

April 15, 2023

Study Completion (Actual)

April 15, 2023

Study Registration Dates

First Submitted

May 24, 2019

First Submitted That Met QC Criteria

June 5, 2019

First Posted (Actual)

June 7, 2019

Study Record Updates

Last Update Posted (Actual)

April 18, 2023

Last Update Submitted That Met QC Criteria

April 17, 2023

Last Verified

April 1, 2023

More Information

Terms related to this study

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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