Gymnastik- och idrottshögskolan, GIH

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.

Maintaining muscle mass is crucial for health and physical activity. Around age 40, muscle mass begins to decline, potentially leading to sarcopenia, a condition associated with frailty and increased fall risk. Age-related muscle loss is complex and multifactorial. The prevailing view is that this loss is driven by anabolic resistance, which is a reduced capacity to increase muscle protein synthesis (MPS) after anabolic cues, i.e., essential amino acids (EAA) or resistance exercise (REx). Mechanistically, this is thought to be underpinned by dysregulation of the mTORC1 signaling pathway. However, it is unclear whether anabolic resistance contributes to muscle loss in healthy, physically active older adults or if studies supporting this have been confounded by other factors, e.g., inactivity and adiposity. Aging also induces changes at the myocellular level, such as satellite cell loss and morphological alterations, but whether these changes are due to aging itself or lifestyle factors is still being debated.           

This thesis examined how anabolic cues impact MPS, mTORC1 signaling, and markers of protein degradation in young and older men. Emphasis was on performing analyses on whole muscle samples and in type I and type II fibers separately. Further aims were to investigate features of muscle fibers in young and older men, focusing on morphology, satellite cells, capillarization, and denervation-reinnervation cycles. The final aim was to develop a valid and fast method for fiber type identification of isolated fibers.           

In paper I, the MPS and mTORC1 signaling response was examined in young and older men after EAA intake alone and combined with REx. The results showed comparable rates of MPS across age groups in response to EAA intake, both alone and with REx. Additionally, mTORC1 signaling was similar to or more pronounced in older men compared to younger men. Notably, older men displayed higher levels of amino acid transporters, nutrient sensors, and mTORC1 activators. In paper II, older men had a lesser proportion of type II fibers, smaller and misshaped type II fibers, and fewer satellite cells and capillaries surrounding their type II fibers. Additionally, older men had more denervated and “grouped” muscle fibers compared to young. In paper III, a new method (THRIFTY) for fiber typing individual fibers was developed, proving valid and more time-efficient than reference methods. In paper IV, the THRIFTY method was implemented, and the cell signaling response to intake of EAA alone and combined with REx was examined in pooled type I and type II fibers. The anabolic signaling response was similar or even more pronounced in old compared to young, with a more robust response observed in type I than in type II fibers. No deficits or alterations in autophagic signaling or E3 ligase expression were observed in older adults after EAA intake alone and combined with REx.           

In conclusion, healthy, lean, physically active, older men did not display deficits in MPS and mTORC1 signaling after anabolic cues, assessed in whole muscle and pooled type I and type II fibers. This indicates that anabolic resistance is not inherently linked to aging per se. However, older men showed increased expression of amino acid transporters, nutrient sensors, and mTORC1 activators, which may help maintain anabolic sensitivity. Despite exhibiting decrements specifically in type II fibers, such as atrophy and altered shape, there was no impairment in mTORC1 signaling or signaling related to autophagy and proteasomal degradation in these fibers after anabolic stimulation. Other factors, such as denervation and satellite cell deficits, may contribute to muscle loss in this population, but their relative impact remains unclear.

Det övergripande syftet med avhandlingen var att öka förståelsen för de cellulära och molekylära mekanismer som ligger bakom muskelförlust vid åldrande, med särskilt fokus på anabol resistens. Detta tillstånd kännetecknas av en nedsatt förmåga att stimulera proteinsyntesen vid anabola stimuli, såsom intag av proteinrik mat eller fysisk träning. En viktig bakomliggande faktor för anabol resistens är minskad aktivering av mTOR, en signalväg essentiell för cellens tillväxt. Vidare undersöktes hur åldrande påverkar egenskaperna hos snabba (typ II) och långsamma (typ I) muskelfibrer. I avhandlingen utvecklades också en ny metod för att förenkla framtida studier på enskilda muskelfibrer.           

Studierna omfattade friska, fysiskt aktiva, män i åldrarna 18–35 år och 65–74 år som genomförde ett styrketräningspass samt intog essentiella aminosyror (EAA). Muskelprover analyserades för proteinsyntes, mTOR-signalering och markörer för proteinnedbrytning. Ytterligare analyser i typ I och typ II muskelfibrer utfördes för att studera morfologi, stamceller, kapillärer och tecken på denervering för att öka förståelsen av åldrandets påverkan på dessa olika fibertyper.           

Resultatet från studie I visade att proteinsyntesen ökade efter intag av EAA, en effekt som förstärktes efter styrketräning, men inga skillnader fanns mellan unga och äldre män. Den äldre gruppen hade däremot högre mTOR-signalering och ökade nivåer av proteiner relaterade till aminosyraupptag och aktivering av mTOR-signalvägen. Studie II visade att äldre män hade en högre andel typ I fibrer, mindre och missformade typ II fibrer, samt färre stamceller och kapillärer kring typ II fibrerna. Den äldre gruppen hade också fler denerverade fibrer och en högre andel grupperade typ I fibrer. I studie III utvecklades en ny metod, THRIFTY, för snabb och effektiv fibertypning av enskilda muskelfibrer. Metoden möjliggjorde, bland annat, tillförlitlig identifiering av hybridfibrer. Studie IV visade att muskelfibrer från äldre män inte hade nedsatt mTOR-signalering eller förändringar i signalvägar relaterade till proteinnedbrytning i respons till anabola stimuli.           

Sammanfattningsvis, proteinsyntes och mTOR-signalering efter EAA-intag, med eller utan styrketräning, inte är nedsatt hos friska, fysiskt aktiva äldre män. Detta tyder på att åldrande i sig inte är en huvudorsak till anabol resistens. Friska äldre har ett högre innehåll av proteiner som aktiverar mTOR-signalvägen, vilket kan hjälpa till att bevara anabol känslighet. Muskelvävnaden från friska äldre uppvisar dock förändringar som fiberatrofi och förlust av stamceller, främst i typ II fibrer, samt tecken på denervering. Muskelatrofin som specifikt drabbar typ II fibrer vid åldrande verkar inte bero på akuta förändringar i signalvägar för proteinsyntes eller proteinnedbrytning vid anabolt stimuli, men kan potentiellt förklaras av ökad denervering eller förlust av stamceller.