Gymnastik- och idrottshögskolan, GIH

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Skeletal muscle fiber types in man: With special reference to anabolic signaling and mitochondrial bioenergetics
Swedish School of Sport and Health Sciences, GIH, Department of Physiology, Nutrition and Biomechanics.ORCID iD: 0000-0003-2921-833x
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human skeletal muscle consists of a mixture of slow-twitch (type I) and fast-twitch (type II) fibers. The type I fibers are endurance-oriented, with a metabolic system and infrastructure that supports aerobic metabolism. This entails a well-developed capillary grid and a mitochondrial network proportioned to the number of contractile units within the fiber. These fibers generally have slower and less forceful contraction mechanics and more limited muscle growth as a resource-efficient metabolic energy system is prioritized over increasing the number of contractile units. By contrast, type II fibers prioritize contractile capabilities and force generation at the cost of resource efficiency. These fibers have a substantially lower mitochondrial volume but prioritize structures and organelles that benefit muscle contraction instead. 

It is well known that resistance exercise combined with dietary protein intake stimulates the growth of contractile proteins leading to an increased muscle mass over time. Muscle mass accumulation is primarily driven by the amplification of muscle protein synthesis, which in turn is largely governed by the mTORC1 signaling pathway within the muscle cell. Little is known about how mTORC1 signaling regulates growth in the different fiber types. Furthermore, it is unknown whether blunted anabolic signaling in type II fibers of the elderly may explain why losses of muscle mass occur primarily in these fibers with advancing age.

Endurance exercise, on the other hand, primarily stimulates a prioritization to synthesize new mitochondria to support the high demand for sustainable aerobic energy output. However, it remains to be determined if mitochondria created within type I and type II fibers are equal, or whether they have adapted to their respective milieu in any way. 

Therefore, the aim of the current thesis was to investigate how the mTORC1 pathway in type I and type II fibers responds to resistance exercise and nutritional stimuli in the form of essential amino acids (EAA), and to determine if this response is influenced by age. Fiber type-specific mitochondrial populations, including their respiratory capacity, were also investigated. To facilitate these investigations, a new and improved method for muscle fiber type identification was developed.

In paper I, the phosphorylation of mTORC1 in response to resistance exercise and EAA intake was examined in 684 individual muscle fibers. Unsurprisingly, a significant increase in mTORC1 signaling was seen following the combination of resistance exercise and EAA intake, whereas the rise following resistance exercise alone was more modest. However, no evidence of a discrete response in the different fiber types was found. 

In paper II, a new method was developed to facilitate the work surrounding fiber type-specific muscle physiology by limiting the extreme time requirements of fiber type identification of large sample sets of muscle fibers. The novel method, which was named THRIFTY, allows an experienced technician to classify over 800 fibers in under 11h.

Paper III utilizes the high throughput of the THRIFTY method described in paper II to create the most extensive study to date on individually dissected muscle fibers with 27 602 included fibers. Here, the aim was to investigate whether the fiber type-specific muscle atrophy of the type II fibers in aging could be explained by an onset of anabolic resistance in these fibers. For this investigation, ten young and ten elderly men were recruited to perform a unilateral resistance exercise session followed by ingestion of EAA. This paper showed a slightly elevated mTORC1 signaling response in type I fibers. However, there were no signs of blunted mTORC1 signaling in the elderly. 

In paper IV, the high speed of the THRIFTY method was utilized to analyze the mitochondrial respiratory function of permeabilized type I and type II muscle fibers. In addition, the intrinsic protein expression of mitochondria in the type I and type II muscle fibers was analyzed. As expected, a higher volume of mitochondria and a greater respiratory rate in the type I fibers were found. However, on a per mitochondria basis, a higher maximal respiratory rate was observed in type II fibers together with increased levels of proteins in the electron transport chain. Likewise, proteins regulating mitochondrial fission and fusion were more highly expressed in the type II fiber mitochondria, which may be a compensatory mechanism for the low volume. 

In conclusion, both fiber types show robust increases in mTORC1 signaling in response to exercise and EAA ingestion. The results indicate that the response is slightly stronger in the type I fibers, which is contrary to what was predicted. Moreover, the highly specific type II fiber atrophy seen with aging cannot be explained by a blunted anabolic response in these fibers. Surprisingly, the mitochondria of type II fibers possess a higher respiratory capacity. However, this discrepancy is concealed by the vast difference in mitochondrial volume favoring type I fibers, ultimately leading to an overall greater respiratory rate in the type I fibers.

Place, publisher, year, edition, pages
Stockholm: Gymnastik och idrottshögskolan, GIH , 2022. , p. 107
Series
Avhandlingsserie för Gymnastik- och idrottshögskolan ; 27
Keywords [en]
mTORC1, THRIFTY, fiber type identification, muscle growth, aging, sarcopenia, mitochondrial function, oxphos, metabolism
National Category
Sport and Fitness Sciences
Research subject
Medicine/Technology
Identifiers
URN: urn:nbn:se:gih:diva-7394ISBN: 978-91-986490-6-2 (print)OAI: oai:DiVA.org:gih-7394DiVA, id: diva2:1711869
Public defence
2022-12-16, Aulan, Lidingövägen 1, Stockholm, 09:00 (English)
Opponent
Supervisors
Available from: 2022-11-21 Created: 2022-11-18 Last updated: 2022-11-25Bibliographically approved
List of papers
1. mTORC1 Signaling in Individual Human Muscle Fibers Following Resistance Exercise in Combination With Intake of Essential Amino Acids
Open this publication in new window or tab >>mTORC1 Signaling in Individual Human Muscle Fibers Following Resistance Exercise in Combination With Intake of Essential Amino Acids
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2019 (English)In: Frontiers in Nutrition, E-ISSN 2296-861X, Vol. 6, article id 96Article in journal (Refereed) Published
Abstract [en]

Human muscles contain a mixture of type I and type II fibers with different contractile and metabolic properties. Little is presently known about the effect of anabolic stimuli, in particular nutrition, on the molecular responses of these different fiber types. Here, we examine the effect of resistance exercise in combination with intake of essential amino acids (EAA) on mTORC1 signaling in individual type I and type II human muscle fibers. Five strength-trained men performed two sessions of heavy leg press exercise. During exercise and recovery, the subjects ingested an aqueous solution of EAA (290 mg/kg) or flavored water (placebo). Muscle biopsies were taken from the vastus lateralis before and 90 min after exercise. The biopsies were freeze-dried and single fibers dissected out and weighed (range 0.95-8.1 mu g). The fibers were homogenized individually and identified as type I or II by incubation with antibodies against the different isoforms of myosin. They were also analyzed for both the levels of protein as well as phosphorylation of proteins in the mTORC1 pathway using Western blotting. The levels of the S6K1 and eEF2 proteins were similar to 50% higher in type II than in type I fibers (P < 0.05), but no difference was found between fiber types with respect to the level of mTOR protein. Resistance exercise led to non-significant increases (2-3-fold) in mTOR and S6K1 phosphorylation as well as a 50% decrease (P < 0.05) in eEF2 phosphorylation in both fiber types. Intake of EAA caused a 2 and 6-fold higher (P < 0.05) elevation of mTOR and S6K1 phosphorylation, respectively, in both type I and type II fibers compared to placebo, with no effect on phosphorylation of eEF2. In conclusion, protein levels of S6K1 and eEF2 were significantly higher in type II than type I fibers suggesting higher capacity of the mTOR pathway in type II fibers. Ingestion of EAA enhanced the effect of resistance exercise on phosphorylation of mTOR and S6K1 in both fiber types, but with considerable variation between single fibers of both types.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
muscle fiber type, protein expression, S6K1, single muscle fiber, EAA
National Category
Physiology
Research subject
Medicine/Technology
Identifiers
urn:nbn:se:gih:diva-5807 (URN)10.3389/fnut.2019.00096 (DOI)000472610200002 ()31294029 (PubMedID)
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2022-11-18
2. THRIFTY: a novel high-throughput method for rapid fibre type identification of isolated skeletal muscle fibres.
Open this publication in new window or tab >>THRIFTY: a novel high-throughput method for rapid fibre type identification of isolated skeletal muscle fibres.
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2022 (English)In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 600, no 20, p. 4421-4438Article in journal (Refereed) Published
Abstract [en]

Fibre type-specific analyses are required for broader understanding of muscle physiology, but such analyses are difficult to conduct due to the extreme time requirements of dissecting and fibre typing individual fibres. Investigations are often confined to a small number of fibres from few participants with low representativeness of the entire fibre population and the participant population. To increase the feasibility of conducting large-scale fibre type-specific studies, a valid and rapid method for high-throughput fibre typing of individually dissected fibres was developed and named THRIFTY (for high-THRoughput Immunofluorescence Fibre TYping). Employing THRIFTY, 400 fibre segments were fixed onto microscope slides with a pre-printed coordinated grid system, probed with antibodies against myosin heavy chain (MyHC)-I and MyHC-II and classified using a fluorescence microscope. The validity and speed of THRIFTY was compared to a previously validated protocol (dot blot) on a fibre-to-fibre basis. Fibre pool purity was evaluated using 'gold standard' SDS-PAGE and silver staining. A modified THRIFTY-protocol using fluorescence western blot equipment was also validated. THRIFTY displayed excellent agreement with the dot blot protocol, κ = 0.955 (95% CI: 0.928, 0.982), P < 0.001. Both the original and modified THRIFTY protocols generated type I and type II fibre pools of absolute purity. Using THRIFTY, 400 fibres were typed just under 11 h, which was approximately 3 times faster than dot blot. THRIFTY is a novel and valid method with high versatility for very rapid fibre typing of individual fibres. THRIFTY can therefore facilitate the generation of large fibre pools for more extensive mechanistic studies into skeletal muscle physiology. KEY POINTS: Skeletal muscle is composed of different fibre types, each with distinct physiological properties. To fully understand how skeletal muscle adapts to external cues such as exercise, nutrition and ageing, fibre type-specific investigations are required. Such investigations are very difficult to conduct due to the extreme time requirements related to classifying individually isolated muscle fibres. To bypass this issue, we have developed a rapid and reliable method named THRIFTY which is cheap as well as versatile and which can easily be implemented in most laboratories. THRIFTY increases the feasibility of conducting larger fibre type-specific studies and enables time-sensitive assays where measurements need to be carried out in close connection with tissue sampling. By using THRIFTY, new insights into fibre type-specific muscle physiology can be gained which may have broad implications in health and disease.

Place, publisher, year, edition, pages
The physiological society, 2022
Keywords
MyHC, SDS-PAGE, fibre typing, high-throughput, immunofluorescence
National Category
Physiology
Research subject
Medicine/Technology
Identifiers
urn:nbn:se:gih:diva-7147 (URN)10.1113/JP282959 (DOI)000858344500001 ()36069036 (PubMedID)
Available from: 2022-10-10 Created: 2022-10-10 Last updated: 2022-12-06
3. Fiber type-specific signaling responses to nutritional and contractile stimuli in young and aged human skeletal muscle
Open this publication in new window or tab >>Fiber type-specific signaling responses to nutritional and contractile stimuli in young and aged human skeletal muscle
(English)Manuscript (preprint) (Other academic)
Keywords
Sarcopenia, fast- and slow twitch, muscle fibers, anabolic signaling, mTORC1
National Category
Sport and Fitness Sciences Physiology Cell Biology
Research subject
Medicine/Technology
Identifiers
urn:nbn:se:gih:diva-7392 (URN)
Funder
EU, Horizon 2020, n°707336
Note

At the time of Sebastian Edman's dissertation this paper was a manuscript.

Available from: 2022-11-18 Created: 2022-11-18 Last updated: 2022-11-21Bibliographically approved
4. Need for Speed: Human fast-twitch mitochondria favour power over efficiency
Open this publication in new window or tab >>Need for Speed: Human fast-twitch mitochondria favour power over efficiency
(English)Manuscript (preprint) (Other academic)
Keywords
Respiration, oxphos, single fibre, fibre types, fast- and slow-twitch fibres, mitochondira, bioenergetics
National Category
Physiology Cell and Molecular Biology Sport and Fitness Sciences
Research subject
Medicine/Technology
Identifiers
urn:nbn:se:gih:diva-7393 (URN)
Funder
Swedish National Centre for Research in Sports
Note

At the time of Sebastian Edman's dissertation this paper was a submitted manuscript.

Available from: 2022-11-18 Created: 2022-11-18 Last updated: 2022-12-06Bibliographically approved

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