Ignite Creation Date:
2024-05-06 @ 1:15 PM
Last Modification Date:
2024-10-26 @ 1:11 PM
Study NCT ID:
NCT03979482
Status:
COMPLETED
Last Update Posted:
2023-04-18
First Post:
2019-05-24
Brief Title:
Skeletal Muscle Mitochondrial Abnormalities and the Metabolic Syndrome in PAH
Sponsor:
Laval University
Organization:
Laval University
Study Overview
Official Title:
Skeletal Muscle Mitochondrial Abnormalities and the Metabolic Syndrome in Pulmonary Arterial Hypertension
Status:
COMPLETED
Status Verified Date:
2023-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
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 PPARgPGC1a 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 IRS1PPARgPGC1-dependent mechanism which will ultimately decrease skeletal muscle function and perfusion and thus overall exercise capacity
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 PPARgPGC1a 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 IRS1PPARgPGC1-dependent mechanism which will ultimately decrease skeletal muscle function and perfusion and thus overall exercise capacity
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 n10-20 vs 10 healthy but sedentary subjects matched for age gender height and weight definition based on current recommendations excluding patients with clinically relevant conditions eg diabetes These individuals are continuously identified through our systematic plasma biobanking process at the time of right heart catheterization CER20735 in which roughly 40 of PAH patients with no obesitydiabetes 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 MSIR and quadriceps muscle function correlate with muscle lipid accumulationglucose 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 normalityequal variances Levenes test 10 subjectsgroup will allow detecting a 1505 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 Fig3C 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 expressionactivation 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 IRMS in PAH the role of skeletal muscle Uncoupling Protein-2 and Sirtuin-3 which have recently been implicated in both IRMS and PAH will be explored
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 n10-20 vs 10 healthy but sedentary subjects matched for age gender height and weight definition based on current recommendations excluding patients with clinically relevant conditions eg diabetes These individuals are continuously identified through our systematic plasma biobanking process at the time of right heart catheterization CER20735 in which roughly 40 of PAH patients with no obesitydiabetes 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 MSIR and quadriceps muscle function correlate with muscle lipid accumulationglucose 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 normalityequal variances Levenes test 10 subjectsgroup will allow detecting a 1505 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 Fig3C 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 expressionactivation 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 IRMS in PAH the role of skeletal muscle Uncoupling Protein-2 and Sirtuin-3 which have recently been implicated in both IRMS and PAH will be explored
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None