Gene Expression of Mitochondrial Enzymes from Exercise

Induced Change in Gene Expression of Mitochondrial Enzymes as a Result of Intense Exercise
Abstract
Exercise-induced changes in gene expression of mitochondrial enzymes has become a leading target for sports medicine research. Previous indirect testing of delayed-onset muscle soreness and changes in rotation of motion do not provide a sufficient explanation of the induced changes to the genome (Hubal, et al., 2010). Biomarker testing has allowed for detecting levels of proteins within a sample. Blood and serum levels, taken before and after exercise, have been analyzed and tested for changes in protein activity. Calf-raises show an increase in creatine kinase (CK) and aldolase (ALD) activities (Kanda, et al., 2014). MicroRNA arrays along with telomere extension mRNA arrays and quantitative real-time PCR on RNA taken from white blood cells have shown to down-regulate telomeric repeat binding factor 2 (Chilton, et al., 2014). Biochemical testing at the genomic level will provide a better understanding of the long-term effects of intense exercise. Knowing these high-intensity induced gene expressions in mitochondrial DNA aids in knowing what causes diseases such as Rhabdomyolysis.
Introduction
Centuries ago staying physically fit was accentual to stay alive. Those that were not fit were not able to kill prey and therefore would be less likely to survive. In today’s world, being physically fit is not a necessity, but rather something that many people strive for to live a healthy lifestyle. Living an active life lowers the chance of many diseases, such as kidney and Alzheimer’s, and cancers, such as colon and breast. It is therefore crucial to understand the biochemistry behind exercise as a helpful preventative measure for health problems.

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When exercising, the body is put through tasks that disrupt homeostasis. The body wants to eliminate wide spread deviants of homeostasis. However, after and during exercise the body needs more oxygen and energy to be able to complete the tasks one is putting on the body. These demands, the increase in affinity for oxygen and energy, require metabolic responses that disrupt homeostasis.
To test these metabolic responses, many scientists use biomarker testing on whole blood and serum samples rather than pieces of skeletal muscle. Biomarkers are used to measure the presence of a physiological state. These markers have biological properties that measure the blood and serum.
There are many different changes in the mitochondrial genome during and immediately following exercise. This paper will focus on an overview of some endurance training biomarkers, but will mainly focus on high intensity exercise and the induced gene expression in the mitochondrial genome. It is important to study the effects of exercise on gene expression to know at what levels of various genes, like lactate dehydrogenase, aldose, etc., come dangerous to human health as to prevention diseases such as Rhadomyolysis (Heled, et al., 2005).
Proliferator-activated Receptors association with Exercise-Induced Mitochondrial Biogenesis
Transcriptional cofactors peroxisome proliferator-activated receptors (PGC) regulate gene expression (Lin, et al., 2005). PGC-1a regulates gene expression in the mitochondrial genome. This coactivator interacts with other proteins to regulate contraction. Overexpression of PGC-1a results in an increase in mitochondrial gene function (Lin, et al., 2002). Calcium/Calmodulin-dependent kinase 2 (CaMKII), AMP-activated protein kinase (AMPK), and nitrogen-activated protein kinases (MAPKs) all have important signaling that help regulate PGC-1a (Hawley, et al., 2010).
Calcium is important for energy due to its role in the sarcoplasmic reticulum. Contractions of skeletal muscle are dependent on high levels of calcium. The calcium then binds to troponin, which moves the myosin fibers resulting in muscle contractions. Calcium also plays a major role in calcium-calmodulin-dependent kinases. Calcium/Calmodulin-dependent kinase 2 (CaMKII) activation increases the transport of glucose in skeletal muscle (Rose, A. and Hargreaves, M., 2003). AMPK and MAPks activate PGC-1a, Figure 1, by phosphorylating transcription factors myocyte enhancer factor 2 and ATF-2, respectively (McGee and Hargreaves, 2010).

Figure 1. Schematic of the Major Signaling Pathways Involved in the Control of Skeletal Muscle Hypertrophy and Mitochondrial Biogenesis (Hawley, et al., 2010). The right side of this figure shows the correlation of the AMPK, CAMK, SIRT1, MAPK pathways that effect PGC-1a which regulates mitochondrial biogenesis.
Another inhibitor of PGC-1a is a deactelyase silent mating type-information regulation 2 homolg 1 (SIRT1) (HIgashida, et al., 2013). Since SIRT1 is NAD⁺-dependent, Figure 1, changes in concentration of NAD⁺ change the SIRT1 activity in the cell (Gurd, 2011). Many studies have shown that the protein content of SIRT1 and the activity are independent, and that because of that it is thought that the SIRT1 activity is what subsidizes to PGC-1a activity (Gurd 2011).
Test For Correlation between SIRT1 and PGC-1a.
The following experiment was performed by Chabi and coworkers to examine SIRT1’s role in muscle during muscle use (Chabi, et al., 2009).
Rats were placed into a control group and a running group. The running group were able to run on a loaded wheel. Weight was added to the wheel during weeks one through four, but maintained a 200 g weight from weeks five through eight. The plantaris (PL) and soleus (SOL) muscles were taken as well as the tibialis anterior (TA) and extensor digitorum longus (EDL) for analysis.
Once the proteins were extracted, the SIRT1 activity and deacetylase was found by fluorescence. A cycling assay was used to determine the NAD nucleotides. Immunoblotting was done to test expression of PGC-1a, cytochrome c, SIRT1 and GAPDH. SIRT1 expression showed to be the highest in the liver and slow-twitch muscle while PGC-1a immunoreactivity was highest in the heart muscle.
This experiment did not show a correlation between SIRT1 and PGC-1a expression, like the scientists hoped, but it did bring up the question if SIRT1 activity is altered by acute exercise instead of high-intensity.
Eccentric Exercise and Muscle Damage Markers
The following was performed by Kazue Kanda and coworkers to see if eccentric exercise affects muscle damage markers (Kanda, et al., 2014).
Participates in this study each performed right calf-raises on a force plate to add 0.5 Hz to each lift. With 3 min for rest, forty repetitions for 10 sets were completed with half of their weight along with the added force. Immediately following these muscle contractions, the medial and lateral gastrocnemius and soleus were measured for tenderness using a FP meter. The meter rated based on a visual analogue scale from no pain to extremely sore. The ankle was tested for range of motion (ROM) along the dosiflexion position (-20 ) to the plantar flexion position (15). Both of tenderness of the calf and the ROM was tested at 24 h increments until 168 h after the repetitive muscle contractions. After 72 h the tenderness of the right calf increased significantly (pSubsequently, samples from the blood and urine were taken both before and at various times after the exercise. Various proteins, creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), aldolase (ALD) and alanine aminotransferase (ALT) were tested from the serum. An Enzyme-linked immunosorbent assay (ELISA) was used to test for fatty acid-binding proteins (FABP).
The activities of CK and AST increased significantly post 72 h. The activities of ALT and ALD also increased for each sample, however, it was not significant. LDH activity had a significant increase at 96 h after exercise, but not really before. The results for ALD did correlate with the tenderness of the medial gastrocnemius at the 72 h mark and could suggest that ALD might be a better muscle damage indicator because the other proteins tested did not correlate with tenderness. The ELISA testing on FABP resulted in no detectable changes. This experiment used two methods of testing muscle damage and had correlated results for ALD.
Exercise-Induced biochemical changes in Quadriceps and Gastrocnemius in Mice
The following was performed by L. Toti and coworkers to see the changes in blood lactate as well as mitochondrial enzymes as a result of two different exercises; intense activity with recovery periods and moderate activity with no recovery periods (Toti, et al., 2013). Immunoblotting and immunohistochemistry were used to assess the two muscle groups, the quadriceps and gastrocnemius, to see expression of enzymes correlated with oxidative metabolism.
Mice were divided into three different groups based on similar maximal velocities. Mice with higher maximal velocities partaked in the intense activity: running at 90% for 2 min followed by 1 min of recovery. The mice with lower mamximal velocities partaked in continuous running at 60% velocity. Data was collected for 40 sessions, where each mouse ran a distance of 1000 meters. Blood samples were taken at sessions 1, 20 and 40.
Blood lactate was tested and showed a decrease in both groups by session 40, with the higher-velocity group measuring significantly lower than the lower-velocity group. Immunochemistry testing resulted in an increase in response for the high-velocity mice for both the quadriceps and the gastrocnemius. This experiment showed that high-intensity training impacted the biochemistry more so than the low-velocity training.
MicroRNA Expression and Telomere-Associated Genes After Acute Exercise
The following was performed by W. Chilton and coworkers to see mechanisms the correlation between white blood cell (WBC) telomere length and exercise (Chiltion, et al., 2014).
Participants in this study ran on a treadmill for 30 min at 80% of maximum oxygen uptake. Blood samples were taken before and immediately following the running as well as an hour post-running. MicroRNA expression arrays that could measure a whole genome, were used on the samples. TERT mRNA expression levels were then tested by qPCR. Telomerase reverse transcriptase (TERT) mRNA and Sirtuin-6 (SIRT6) were two of the genes tested.
This experiment was able to show supporting evidence that the transcriptional regulation of key telomeric genes can be affected by exercise. TERT mRNA was upregulated as well as the SIRT6. The qPCR testing on TERT and SIRT6 showed the increase in binding miRNA. Chilton did express that the increases in both the SIRT6 and the TERT mRNAs could have been upregulations from the extra-telomeric pathways instead of just the telomeric roles since there was no definitive way to differentiate between the two in this experiment.
It is important to understand exercise’s effects on telomeres and its corresponding proteins to gain an insight on how physical health improves telomere homeostasis, keeping the telomeres from getting too short and the cell dying.
Chemokine Polymorphisms Association with Skeletal Muscle Damage
The following experiment was performed by M. Hubal and coworkers to see if chemokine ligand 2 (CCL2) and chemokine receptor 2 (CCR 2) are associated with biomarkers after exercising (Hubal, et al., 2010).
CCL2 is important because it recruits necessary items, such as memory T cells, dendritic cells, and monocytes, to inflammation sites in injured tissue. CCR2, the receptor molecule for CCL2, mediates with calcium mobilization. It is suggested that CCL2 and CCR3 play major roles in the repair of skeletal muscle damage.
Participants performed two, 25 contractions, sets of elbow flexor muscle contractions in the non-dominant arm. It was crucial that the participants had constant maximal effort and stayed hydrated the 10 days following the exercise. Blood samples were taken and sent for genotyping. Single nucleotide polymorphisms (SNPs) known to influence the level of CCL2 proteins were analyzed using PCR.
There were four SNPs from CCL2 and three from CCR2 that should high phenotype associations. The minor allele found in the SNPs was correlated with an increase in damage. This study was able to show that variations of the CCL2 and CCR2 genes are related to muscle damage markers caused by exercise.
Molecular and Metabolic Changes of High-intensity Interval Training
The following experiment was performed by J. Little and coworkers to assess the molecular and metabolic changes of high-intensity interval training (Little, et al., 2010).
Participants performed six cycling training sessions over the course of two weeks. Each session consisted of approximately 30 min of high intensity intervals. By the last sessions, the subjects were completing 12 intervals of 60 s high-intensity cycling followed by 75 s low intensity for recovery. Biopsies from the leg were taken before and after the two week training. The muscle lysates were taken for Western blotting and enzyme activity testing. Western blotting was used to test for glucose transporter type 4 (GLUT4), PGC-1a, and SIRT1while the mitochondrial enzyme activity was tested on cytochrome c oxidase (COX).
The subjects improved in both time and power, about 10%, for cycling during the two week training session. COX activity increased by 29%. PGC-1a increased by approximately 24%, however, the protein itself was did not have any genetic changes. GLUT4 content increased by 119%, while SIRT1 increased by approximately 56%. This experiment showed some changes, due to exercise, in regulators that are important in mitochondrial biogenesis.
Conclusion
There are many induced changes in the mitochondrial genome during and after exercise. These changes occur to help maintain cell homeostasis while the body is being put through stress during intense exercise. Blood and serum samples along with tissue extractions have provided a way to examine these changes and see how one is correlated with another (Figure 1).
PGC-1a helps muscle contractions by regulating gene expression in mitochondria biogenesis. However, PGC-1a has many cofactors helping it. CaMKII, AMPK, and MAPKs all help in providing energy to the skeletal muscles by impacting the activation of PGC-1a. Although SIRT1 deactelyation inhibits PGC-1a, it still impacts the biochemistry of the body during workouts due to deactelyation. LDH activity was shown to increase hours after high-intensity exercise because LDH it is released as pyruvate is converted into lactate. ALD, which converts sugar into energy, was found to be a good indicator because in the calf-raises experiment the results from the biomarker testing as well as the tenderness testing correlated with each other. CCL2 and CCR2 were found to have changes in SNPs that corresponded to the high-intensity exercise and most likely aide in the recruiting of the memory T cells and dendritic cells to the injured tissue.
High-intensity exercise, without time for recovery, would keep the body maintained at a stressful state of trying to bring it back to homeostasis. As proven in the calf-raise experiment, LDH levels decreased as the participants were able to come accustom to the exercises. The bodies were no longer in shock. If the bodies were did not become accustom, or the body was not given any time for recovery, the chances of obtaining diseases such as Rhabdomyolysis increases. Future experiments need to focus on what levels of these regulators will become dangerous. Research should be down to further understand the relationship between SIRT1 and PGC-1a.
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