Final Report Summary - MUSC ADAP XRCS (Mechanisms of skeletal muscle adaptation to exercise and their implications in health and disease.)
WORK CARRIED OUT TO ACHIEVE THE PROJECT’S OBJECTIVES
A – To evaluate the importance of PGC-1α coactivators for how skeletal muscle adapts to different modes of exercise training.
We have shown that the PGC-1α gene can be differentially regulated by different exercise modes, and that results in the expression of different PGC-1α variants. One of these variants, which we have named PGC-1α4, is specifically induced in skeletal muscle by resistance training (such as weight-lifting) and regulates skeletal muscle hypertrophy. Transgenic mice with increased skeletal muscle levels of PGC-1α4, display moderate increase in muscle size and strength and a remarkable resistance to disuse-induced atrophy and cancer-induced cachexia.
As a follow up of this work, we have collaborated in two studies that have defined additional effects of PGC-1α4 activation in skeletal muscle. The first, identified a novel myokine under PGC-1α4 control, that is secreted from skeletal muscle and activates the immune system to induce adipose tissue “browning” and activation of a thermogenesis program. The other study characterized GPR56 as a gene under PGC-1α4 control that participates in the induction of muscle hypertrophy.
More recently, my research group has identified a novel pathway in skeletal muscle, under the control of PGC-1α1, and that mediates resistance to stress-induced depression. In this study, we have shown that exercise-mediated elevation of PGC-1α1 in skeletal muscle, activates the expression of a class of enzymes able to convert a tryptophan metabolite that accumulates under stress and pro-inflammatory conditions (kynurenine), to its acid form (kynurenic acid) that is unable to cross the blood brain barrier. Accumulation of kynurenine in the brain induces neuro-inflammation and changes associated with depression, thus the peripheral conversion to kynurenic acid alleviates the burden to the central nervous system and protects from stress-induced depression.
B – To understand how PGC-1α coactivators regulate target gene expression (and therefore affect muscle physiology).
B.1. Target gene networks
To understand which gene networks are under the control of the different PGC-1α variants we performed global gene expression analysis in myotubes expressing each isoform. Our results indicate that some of the novel PGC-1α coactivators are involved in regulation of target gene splicing, suggesting that changes in muscle physiology mediated by different PGC-1α forms might result from the transcription of alternative mRNAs from the same gene, encoding protein variants with specific biological functions. The concept that adaptive changes in muscle might be mediated not only by quantitative changes in gene expression, but by the generation of alternative transcripts form the same gene with specific functions, creates a new platform to investigate exercise physiology (or any adaptive process).
B.2. Protein binding partners.
Since PGC-1α coactivators exert their biological functions by interacting with other proteins (including DNA-binding transcription factors) we have determined the binding partners for each PGC-1α variant by using a biochemical approach. This work was done in collaboration with Prof. Robert Roeder (The Rockefeller University, New York. USA), who is a world leader and reference in the biochemical purification of transcription complexes. One of my PhD students spent some time in Prof. Roeder’s laboratory learning some of the relevant techniques, that she has since brought to our research group.
C – To analyze the function of PGC-1a2 and a3 (variants of unknown function) in vivo.
We have succeeded in generating skeletal muscle-specific transgenic mouse lines for PGC-1alpha 2 and for PGC-1alpha3. This took longer than anticipated since our initial attempt failed. To solve that, we have used a human alpha-skeletal actin (HSA) promoter and obtained overexpressor lines. All lines have been validated and are now in my laboratory under characterization.
D – To identify novel molecular pathways that control muscle adaptation to exercise.
Changes in skeletal muscle function are accompanied by several events including vascularization, oxygen transport, fuel uptake and utilization, mitochondrial function, and protein synthesis/degradation. The fact that many of these events happen in a coordinated way, suggests common regulatory nodes. We have used a mouse hindlimb unloading/reloading protocol, which allows us to evaluate skeletal muscle atrophy and hypertrophy processes in the same system, and performed global analysis of gene expression in muscle by RNA-sequencing. This led to the identification of several gene candidates to regulators of muscle mass and function that are currently being validated in vitro and in vivo. To date, our in vivo studies have been performed acutely by using viral vectors to mediate candidate gene expression in mouse muscle, by intramuscular injection.
SOCIO-ECONOMIC IMPACT OF THE PROJECT
The results from this project have increased our understanding of how skeletal muscle responds to physical exercise. In one of the initial studies, we identified a novel molecule that controls skeletal muscle mass and strength. This opens the possibility of developing pharmacological strategies to increase PGC-1α4 levels in situations linked to loss of muscle mass. Among these are age-related sarcopenia, several muscle diseases, cancer-induced cachexia, and long-term bed rest. Another of our studies resulted in the identification of a novel pathway by which exercised muscle can contribute to improving mental health. This study suggests the interesting possibility of developing a novel generation of antidepressant agents that could target muscle, instead of the brain.
A – To evaluate the importance of PGC-1α coactivators for how skeletal muscle adapts to different modes of exercise training.
We have shown that the PGC-1α gene can be differentially regulated by different exercise modes, and that results in the expression of different PGC-1α variants. One of these variants, which we have named PGC-1α4, is specifically induced in skeletal muscle by resistance training (such as weight-lifting) and regulates skeletal muscle hypertrophy. Transgenic mice with increased skeletal muscle levels of PGC-1α4, display moderate increase in muscle size and strength and a remarkable resistance to disuse-induced atrophy and cancer-induced cachexia.
As a follow up of this work, we have collaborated in two studies that have defined additional effects of PGC-1α4 activation in skeletal muscle. The first, identified a novel myokine under PGC-1α4 control, that is secreted from skeletal muscle and activates the immune system to induce adipose tissue “browning” and activation of a thermogenesis program. The other study characterized GPR56 as a gene under PGC-1α4 control that participates in the induction of muscle hypertrophy.
More recently, my research group has identified a novel pathway in skeletal muscle, under the control of PGC-1α1, and that mediates resistance to stress-induced depression. In this study, we have shown that exercise-mediated elevation of PGC-1α1 in skeletal muscle, activates the expression of a class of enzymes able to convert a tryptophan metabolite that accumulates under stress and pro-inflammatory conditions (kynurenine), to its acid form (kynurenic acid) that is unable to cross the blood brain barrier. Accumulation of kynurenine in the brain induces neuro-inflammation and changes associated with depression, thus the peripheral conversion to kynurenic acid alleviates the burden to the central nervous system and protects from stress-induced depression.
B – To understand how PGC-1α coactivators regulate target gene expression (and therefore affect muscle physiology).
B.1. Target gene networks
To understand which gene networks are under the control of the different PGC-1α variants we performed global gene expression analysis in myotubes expressing each isoform. Our results indicate that some of the novel PGC-1α coactivators are involved in regulation of target gene splicing, suggesting that changes in muscle physiology mediated by different PGC-1α forms might result from the transcription of alternative mRNAs from the same gene, encoding protein variants with specific biological functions. The concept that adaptive changes in muscle might be mediated not only by quantitative changes in gene expression, but by the generation of alternative transcripts form the same gene with specific functions, creates a new platform to investigate exercise physiology (or any adaptive process).
B.2. Protein binding partners.
Since PGC-1α coactivators exert their biological functions by interacting with other proteins (including DNA-binding transcription factors) we have determined the binding partners for each PGC-1α variant by using a biochemical approach. This work was done in collaboration with Prof. Robert Roeder (The Rockefeller University, New York. USA), who is a world leader and reference in the biochemical purification of transcription complexes. One of my PhD students spent some time in Prof. Roeder’s laboratory learning some of the relevant techniques, that she has since brought to our research group.
C – To analyze the function of PGC-1a2 and a3 (variants of unknown function) in vivo.
We have succeeded in generating skeletal muscle-specific transgenic mouse lines for PGC-1alpha 2 and for PGC-1alpha3. This took longer than anticipated since our initial attempt failed. To solve that, we have used a human alpha-skeletal actin (HSA) promoter and obtained overexpressor lines. All lines have been validated and are now in my laboratory under characterization.
D – To identify novel molecular pathways that control muscle adaptation to exercise.
Changes in skeletal muscle function are accompanied by several events including vascularization, oxygen transport, fuel uptake and utilization, mitochondrial function, and protein synthesis/degradation. The fact that many of these events happen in a coordinated way, suggests common regulatory nodes. We have used a mouse hindlimb unloading/reloading protocol, which allows us to evaluate skeletal muscle atrophy and hypertrophy processes in the same system, and performed global analysis of gene expression in muscle by RNA-sequencing. This led to the identification of several gene candidates to regulators of muscle mass and function that are currently being validated in vitro and in vivo. To date, our in vivo studies have been performed acutely by using viral vectors to mediate candidate gene expression in mouse muscle, by intramuscular injection.
SOCIO-ECONOMIC IMPACT OF THE PROJECT
The results from this project have increased our understanding of how skeletal muscle responds to physical exercise. In one of the initial studies, we identified a novel molecule that controls skeletal muscle mass and strength. This opens the possibility of developing pharmacological strategies to increase PGC-1α4 levels in situations linked to loss of muscle mass. Among these are age-related sarcopenia, several muscle diseases, cancer-induced cachexia, and long-term bed rest. Another of our studies resulted in the identification of a novel pathway by which exercised muscle can contribute to improving mental health. This study suggests the interesting possibility of developing a novel generation of antidepressant agents that could target muscle, instead of the brain.