A novel imaging method (FIM-ID) reveals that myofibrillogenesis plays a major role in the mechanically induced growth of skeletal muscle.

An increase in mechanical loading, such as that which occurs during resistance exercise, induces radial growth of muscle fibers (i.e. an increase in cross-sectional area). Muscle fibers are largely composed of myofibrils, but whether radial growth is mediated by an increase in the size of the myofibrils (i.e. myofibril hypertrophy) and/or the number of myofibrils (i.e. myofibrillogenesis) is not known. Electron microscopy (EM) can provide images with the level of resolution that is needed to address this question, but the acquisition and subsequent analysis of EM images is a time- and cost-intensive process. To overcome this, we developed a novel method for visualizing myofibrils with a standard fluorescence microscope (fluorescence imaging of myofibrils with image deconvolution [FIM-ID]). Images from FIM-ID have a high degree of resolution and contrast, and these properties enabled us to develop pipelines for automated measurements of myofibril size and number. After extensively validating the automated measurements, we used both mouse and human models of increased mechanical loading to discover that the radial growth of muscle fibers is largely mediated by myofibrillogenesis. Collectively, the outcomes of this study offer insight into a fundamentally important topic in the field of muscle growth and provide future investigators with a time- and cost-effective means to study it.

Approximately 45% of human body mass is made of skeletal muscle. These muscles contract and relax to provide the mechanical forces needed for breathing, moving, keeping warm and performing many other essential processes. Both sedentary and active adults lose approximately 30-40% of this muscle mass by the age of 80, increasing their risk of disease, disability and death. As a result, there is much interest in developing therapies that can restore, maintain and increase muscle mass in older individuals. Muscles are made of multiple fibers that are in turn largely composed of smaller units known as myofibrils. Previous studies have shown that performing resistance training or other exercise that increases the mechanical loads placed on muscles stimulates muscle growth. This growth is largely due to increased girth of the existing muscle fibers. However, it remained unclear whether this was due to myofibrils growing in size, increasing in number, or a combination of both. To address this question, Jorgenson et al. developed a fluorescence imaging method called FIM-ID to count the number and measure the size of myofibrils within cross-sections of skeletal muscle. Using FIM-ID to study samples of mouse and human muscle fibers then revealed that increasing mechanical loads on muscles increased the number of myofibrils and this was largely responsible for muscle fiber growth. FIM-ID mostly relies on common laboratory instruments and free open-source software is used to count and measure the myofibrils. Jorgenson et al. hope that this will allow as many other researchers as possible to use FIM-ID to study myofibrils in the future. A better understanding of how the body controls the number of myofibrils may lead to the development of therapies that can mimic the effects of exercise on muscles to maintain or even increase muscle mass in human patients.

The Structural Adaptations That Mediate Disuse-Induced Atrophy of Skeletal Muscle.

The maintenance of skeletal muscle mass plays a fundamental role in health and issues associated with quality of life. Mechanical signals are one of the most potent regulators of muscle mass, with a decrease in mechanical loading leading to a decrease in muscle mass. This concept has been supported by a plethora of human- and animal-based studies over the past 100 years and has resulted in the commonly used term of 'disuse atrophy'. These same studies have also provided a great deal of insight into the structural adaptations that mediate disuse-induced atrophy. For instance, disuse results in radial atrophy of fascicles, and this is driven, at least in part, by radial atrophy of the muscle fibers. However, the ultrastructural adaptations that mediate these changes remain far from defined. Indeed, even the most basic questions, such as whether the radial atrophy of muscle fibers is driven by the radial atrophy of myofibrils and/or myofibril hypoplasia, have yet to be answered. In this review, we thoroughly summarize what is known about the macroscopic, microscopic, and ultrastructural adaptations that mediated disuse-induced atrophy and highlight some of the major gaps in knowledge that need to be filled.

A Novel Imaging Method (FIM-ID) Reveals that Myofibrillogenesis Plays a Major Role in the Mechanically Induced Growth of Skeletal Muscle.

An increase in mechanical loading, such as that which occurs during resistance exercise, induces radial growth of muscle fibers (i.e., an increase in cross-sectional area). Muscle fibers are largely composed of myofibrils, but whether radial growth is mediated by an increase in the size of the myofibrils (i.e., myofibril hypertrophy) and/or the number of myofibrils (i.e., myofibrillogenesis) is not known. Electron microscopy (EM) can provide images with the level of resolution that is needed to address this question, but the acquisition and subsequent analysis of EM images is a time- and cost-intensive process. To overcome this, we developed a novel method for visualizing myofibrils with a standard fluorescence microscope (FIM-ID). Images from FIM-ID have a high degree of resolution and contrast, and these properties enabled us to develop pipelines for automated measurements of myofibril size and number. After extensively validating the automated measurements, we used both mouse and human models of increased mechanical loading to discover that the radial growth of muscle fibers is largely mediated by myofibrillogenesis. Collectively, the outcomes of this study offer insight into a fundamentally important topic in the field of muscle growth and provide future investigators with a time- and cost-effective means to study it.

Protocol for quantifying the in vivo rate of protein degradation in mice using a pulse-chase technique.

The ability to measure the in vivo rate of protein degradation is a major limitation in numerous fields of biology. Here, we present a protocol for quantifying this rate in mice using a pulse-chase technique that utilizes an azide-bearing non-canonical amino acid called azidohomoalanine (AHA). We describe steps for using chow containing AHA to pulse-label the animal's proteome. We then detail the quantification of AHA-labeled proteins in whole-tissue lysates or histological sections using a copper-catalyzed azide-alkyne cycloaddition 'click' reaction. For complete details on the use and execution of this protocol, please refer to Steinert et al. (2023).1.

A novel method for visualizing in-vivo rates of protein degradation provides insight into how TRIM28 regulates muscle size.

Skeletal muscle size is controlled by the balance between protein synthesis and protein degradation. Given the essential role of skeletal muscle in maintaining a high quality of life, understanding the mechanisms that modulate this balance are of critical importance. Previously, we demonstrated that muscle-specific knockout of TRIM28 reduces muscle size and function and in the current study, we discovered that this effect is associated with an increase in protein degradation and a dramatic reduction in the expression of Mettl21c. Importantly, we also determined that overexpression of Mettl21c is sufficient to induce hypertrophy in both control and TRIM28 knockout muscles. Moreover, we developed a simple pulse-chase biorthogonal non-canonical amino acid tagging technique that enabled us to visualize the in vivo rate of protein degradation, and with this technique were able to conclude that the hypertrophic effect of Mettl21c is due, at least in part, to an inhibition of protein degradation.

Weight Pulling: A Novel Mouse Model of Human Progressive Resistance Exercise.

This study describes a mouse model of progressive resistance exercise that utilizes a full-body/multi-joint exercise (weight pulling) along with a training protocol that mimics a traditional human paradigm (three training sessions per week, ~8-12 repetitions per set, 2 min of rest between sets, approximately two maximal-intensity sets per session, last set taken to failure, and a progressive increase in loading that is based on the individual's performance). We demonstrate that weight pulling can induce an increase in the mass of numerous muscles throughout the body. The relative increase in muscle mass is similar to what has been observed in human studies, and is associated with the same type of long-term adaptations that occur in humans (e.g., fiber hypertrophy, myonuclear accretion, and, in some instances, a fast-to-slow transition in Type II fiber composition). Moreover, we demonstrate that weight pulling can induce the same type of acute responses that are thought to drive these long-term adaptations (e.g., the activation of signaling through mTORC1 and the induction of protein synthesis at 1 h post-exercise). Collectively, the results of this study indicate that weight pulling can serve as a highly translatable mouse model of progressive resistance exercise.

Identifying the Structural Adaptations that Drive the Mechanical Load-Induced Growth of Skeletal Muscle: A Scoping Review.

The maintenance of skeletal muscle mass plays a critical role in health and quality of life. One of the most potent regulators of skeletal muscle mass is mechanical loading, and numerous studies have led to a reasonably clear understanding of the macroscopic and microscopic changes that occur when the mechanical environment is altered. For instance, an increase in mechanical loading induces a growth response that is mediated, at least in part, by an increase in the cross-sectional area of the myofibers (i.e., myofiber hypertrophy). However, very little is known about the ultrastructural adaptations that drive this response. Even the most basic questions, such as whether mechanical load-induced myofiber hypertrophy is mediated by an increase in the size of the pre-existing myofibrils and/or an increase in the number myofibrils, have not been resolved. In this review, we thoroughly summarize what is currently known about the macroscopic, microscopic and ultrastructural changes that drive mechanical load-induced growth and highlight the critical gaps in knowledge that need to be filled.