Making Mice Mighty: recent advances in translational models of load-induced muscle hypertrophy

Abstract

The ability to genetically manipulate mice allows for gain- and loss-of-function in vivo, making them an ideal model for elucidating mechanisms of skeletal muscle mass regulation. Combining genetic models with mechanical muscle loading enables identification of specific factors involved in the hypertrophic response as well as the ability to test the requirement of those factors for adaptation, thereby informing performance and therapeutic interventions. Until recently, approaches for inducing mechanically mediated muscle hypertrophy (i.e., resistance-training analogs) have been limited and considered “nontranslatable” to humans. This mini-review outlines recent translational advances in loading-mediated strategies for inducing muscle hypertrophy in mice, and highlights the advantages and disadvantages of each method. The skeletal muscle field is poised for new breakthroughs in understanding mechanisms regulating load-induced muscle growth given the numerous murine tools that have very recently been described.

INTRODUCTION

Key regulators of muscle mass discovered using genetic or pharmacological gain- and loss-of function approaches in mice are often subsequently shown to be altered by loading in humans (i.e., acute resistance exercise and/or training), likely contributing to muscle hypertrophy (5, 12, 38, 39, 48, 49). The development of murine muscle-specific Cre/Lox and Tet-On technologies over the last decade now allows for unprecedented insight into the modifiable factors affecting muscle growth in vivo (24, 37, 40). Moving beyond association and correlation, the utility of mouse models for mechanistically understanding skeletal muscle mass regulation is inarguable and invaluable (55).

To identify targets associated with load-mediated muscle hypertrophy, as well as test the requirement of those targets for growth in vivo, a surgical model of overload in mice has historically been the standard (18, 54). This method, termed synergist ablation, involves the excision of one large muscle or a group of muscles to elicit a compensatory growth response from a smaller muscle through ambulation. Most often, the tibialis anterior (dorsiflexor) or gastrocnemius/soleus complex (plantarflexors) are partially or completely removed to overload the extensor digitorum longus or plantaris, respectively. Employed for almost 50 years in mice (26, 50), synergist ablation induces rapid hypertrophy (<2 wk) and has been leveraged by our laboratory and others to understand cellular and molecular aspects of acute and chronic mechanically-induced muscle hypertrophy (8, 29, 35, 36, 44, 56, 59). While widely adopted, synergist ablation has distinct drawbacks not limited to its invasiveness, tendency to cause damage/degeneration-regeneration and/or muscle fiber splitting, extreme rate and magnitude of growth, isolated functional and metabolic profiles of the muscles being overloaded, continual and irreversible loading stimulus, and ambiguity with respect to the signal that triggers growth (e.g., stretch versus tension) (1, 27, 42, 54). Although the surgical approach has recently been modified to attenuate muscle damage (16, 44, 60), translational models of skeletal muscle hypertrophy that more closely reflect human resistance training are still necessary to obtain an accurate understanding of load-induced adaptations; this has proved challenging since mice are resistant to operant conditioning for exercise.

Very recently, an explosion of alternatives to synergist ablation for enhancing muscle mass through loading have been described for mice. All approaches are comparatively more translatable to human exercise than surgical methods and may have specific clinical implications. Each stimulus is also unique and involves different aspects of load-induced adaptation that could be used in combination to provide a broader view of muscle hypertrophic adaptation. The purpose of this mini-review is to highlight these recent advancements and address the advantages and disadvantages of each method. Our hope is that this information will assist muscle researchers in selecting the optimal loading approach to study muscle hypertrophy, and ultimately facilitate the discovery of genes and therapies that preserve or enhance muscle mass.

PROGRESSIVE WEIGHTED WHEEL RUNNING

Mice are highly active creatures with an affinity for wheel-running. With respect to muscle hypertrophy, unweighted wheel running in mice may or may not elicit muscle and/or fiber type-specific hypertrophy (3, 6, 33). In our hands, at least in the plantaris muscle, eight weeks of voluntary wheel running (8–10 km/night) was insufficient to elicit muscle growth in adult (>4 mo old) female mice (25), although we recently reported that 13 mo of unweighted running may ultimately generate hypertrophy (14). Early work from the Edgerton laboratory illustrated how gradually adding weight to a running wheel over 8 wk can elicit muscle hypertrophy in rats (23). It follows that by adding resistance to a murine running wheel, one can leverage the mouse’s innate drive to run and induce hypertrophy in multiple muscles. Initial attempts yielded some success in generating hypertrophy across various muscles, but the constant loading of the wheel ultimately results in reduced training volume at higher loads, thereby diminishing the efficacy of this approach (7, 22, 31, 47, 52, 57). Our laboratory recently developed a progressive weighted wheel running (PoWeR) strategy that circumvents this issue (13). By loading standard running wheels progressively with 1 g magnets situated asymmetrically, which encourages repeated bouts of activity as opposed to continuous running, adult female mice run 10–12 km/night for 8 wk with up to 6 g on the wheel (~20% of body weight) (13); male mice will run similar volumes but slightly less, while older mice (22–24 mo) will run about half as much as younger mice (unpublished data). Both the plantaris (glycolytic) and soleus (oxidative) muscles grow 15–30% according to whole muscle and muscle fiber cross-sectional area measures (13, 42), concomitant with a substantial glycolytic-to-oxidative fiber type shift and myonuclear accretion; the gastrocnemius muscle also experiences fiber type-specific growth (unpublished data). Furthermore, weighted wheel running can be adapted to simulate an acute resistance exercise-like stimulus (11).

As with all nonsurgical methods for inducing hypertrophy, the adaptations can be reversible with detraining, thus allowing for study of the loss of adaptations in addition to the gain of adaptations (13). Other advantages of PoWeR are that it does not involve specialized equipment (only standard running wheels), requires minimal oversight, is high-throughput (i.e., entire cohorts of mice can train at once), and induces robust cardiac adaptations (13). PoWeR involves high volumes of moderate-velocity dynamic contractions, is akin to resistive cycle training or concurrent training in humans (15, 41, 43), and simulates an “athlete-like” phenotype in mice (i.e., strength/power production along with fatigue resistance). On balance, PoWeR is not strictly hypertrophic and may not suit the needs of researchers interested in muscle growth alone; specifically, the “hybrid” stimulus that features potentially large alterations in metabolism may complicate the interpretation of results related to growth. The lack of control over training volume may also present an issue; in our hands, female mice will consistently run 10–12 km per night, but this may vary significantly from laboratory to laboratory and present challenges for comparison across sex and age. Alternatively, varied running volume within large cohorts can be leveraged to study the effects of training dosage on a desired outcome (14). Collectively, PoWeR is a simple and effective means for inducing hypertrophy across various muscles in mice, but should be used with the understanding that training volume is high and that the stimulus is a combination of resistance and endurance.

WEIGHTED-VEST MOTORIZED WHEEL RUNNING

Graber, Fendray, and Thompson in 2019 developed a method of training specifically to enhance muscle power output (19), since the loss of whole muscle power-producing capacity is a major contributor to age-related muscle dysfunction (46). This approach involves outfitting mice with a bespoke weighted harness (vest) and locking them into a custom motorized wheel for progressive training over the course of 12 wk (19). The stimulus involves a controlled dose of high-velocity dynamic contractions, administered in ~60 s bouts to failure, that enhances muscle contractile function in younger adult and aged mice (much like resistance training in humans). The strategy, which can be considered involuntary since the mice are placed into a wheel that is then fastened shut, also induces hypertrophy of the soleus and quadriceps muscles of younger mice. Advantages of this approach are that resistance (mass of the harness), intensity (velocity of wheel), and frequency (days per week) can be tightly controlled and individualized, making it similar to human resistance training. Aged mice can also perform it (albeit with less hypertrophy than younger mice), and the quadriceps muscle grows, which yields ~200–400 mg of tissue for diverse analyses from a single mouse. The disadvantages are that it requires specialized equipment, is low-throughput, and is only modestly hypertrophic in young plantaris muscles (historically a highly-studied muscle in mice). While a very specialized mode of training, this is an effective approach for inducing muscle hypertrophy (and power output) in the mouse hindlimb via bouts of loaded dynamic contraction.

HIGH-INTENSITY INCLINE TREADMILL RUNNING

Simultaneous with the publication of PoWeR and weighted vest running to promote hypertrophy in 2019, two laboratories developed involuntary high-intensity interval inclined treadmill running protocols that involve high-velocity dynamic contractions to induce muscle hypertrophy in mice (17, 51). These protocols generally entail increasing speed and grade during 3 separate bouts per week over 8–16 wk, which results in increased muscle mass in lower extremity dorsi- and plantarflexors in both young and aged mice. This approach is advantageous because exercise volume, frequency, and intensity can be closely monitored and controlled, and standard rodent treadmills can be employed. Conversely, this method can cause undue stress on the mice (an electric shock grid motivates the mice to run and excessive shocking occurs at higher workloads), is labor-intensive and not high throughput, and is also associated with endurance-type adaptations in addition to hypertrophic adaptations. High-intensity treadmill running can induce hypertrophy across multiple muscle groups using progressive bouts followed by rest, which is similar to human exercise, but may also be considered a hybrid approach that develops an athlete-like phenotype, similar to PoWeR.

ELECTRICAL STIMULATION

A popular technique in rats (4, 58), involuntary electrical stimulation under anesthesia to maximally activate lower-extremity muscles has been employed on a limited basis to induce hypertrophy in mice (2, 21). This technique involves stimulation of the sciatic nerve, causing the plantarflexor and dorsiflexor complexes to contract. The plantarflexors are stronger, so they contract concentrically while the dorsiflexors experience eccentric muscle action. Electrical stimulation generally results in hypertrophy of the dorsiflexors and not the plantarflexors after 7 bouts over 2 wk (21), but has been reported to induce modest whole-muscle hypertrophy in the gastrocnemius in healthy and cachectic mice after 14 sessions (53). The advantages of this approach are that there is complete control over the training parameters and exercise dosage (making load and work highly quantifiable), it can be modified to study acute responses to eccentric loading (20, 45), it elicits modest but significant hypertrophy in a matter of weeks (i.e., more rapidly than other models discussed here), and can be deployed in unloaded, aged, and diseased populations where voluntary training may not be possible. The disadvantages are that this approach requires specialized equipment and expertise, is relatively labor intensive and low-throughput, involves maximal motor unit recruitment for every contraction (which is unlike traditional resistance training), necessitates frequent exposure of the mice to deep anesthesia, and while a strong hypertrophic stimulus, the eccentric nature has the potential to damage the dorsiflexors. Electrical stimulation is unique from the other training modalities outlined in this mini-review since it is eccentrically driven and targets the dorsiflexors (as opposed to plantarflexors), so it could be used in combination with other training modes to explore hypertrophy mediated by a novel loading stimulus in muscles of different function.

SQUAT-LIKE EXERCISE

Another popular technique for inducing hypertrophy in rats that is difficult to execute in mice is squat-like exercise (9, 30, 34, 54). This approach involves operant conditioning of the animal to push voluntarily against an object, often for a food reward. Recently, the Yan laboratory cleverly and effectively adapted this model for usage in mice (10). Utilizing a specialized cage that is substituted for a normal cage during the dark cycle, mice push off on a ramp to displace a resistance-adjustable lid equipped with a magnetic sensor to record activity. Pushing against the lid involves high volumes of concentric and eccentric muscle actions that provide access to food. The motion required to move the lid and access the food is similar to a squat motion in humans, and over the course of eight weeks while performing ~400 repetitions per night with progressively increased weight, hypertrophy of the soleus, plantaris, and gastrocnemius muscles was reported (10). Importantly, mice did not eat less food or lose weight during the training, which is sometimes reported in other species using similar training models (9). This method involves specialized equipment and daily replacement of the customized lid, making it modestly labor intensive, and it is unknown whether mice under different conditions (e.g., aged or diseased) can or would perform the squat activity. Otherwise, this model can be adapted for an acute exercise bout (10), can be high-throughput, is primarily hypertrophic (not hybrid), and is perhaps the closest available analog to “traditional” concentric/eccentric resistance training in humans that involves voluntary repetitions followed by rest (albeit at high frequency and volume).

ALTERNATIVE APPROACHES

There are a few alternative approaches for resistance-training murine skeletal muscle. Weighted ladder climbing, where a weight is attached to a mouse’s tail while it climbs a vertical ladder, may result in modest growth of the plantaris muscle at the whole muscle level, but not according to fiber cross-sectional area (28). A similar approach, unloaded tower climbing, does not result in muscle hypertrophy (47). Being difficult to execute and minimally effective, vertical climbing has not been widely adopted. Another interesting strategy of resistance training in the mouse forelimb is voluntary burrowing through resistant material within a burrowing tube, but this only increased grip strength and muscle hypertrophy was not reported (47). These alternative approaches may be appropriate for specific applications, but are not the most robust training modality for inducing hypertrophy. One investigation reported on the efficacy of unweighted vertical hanging from a wire grid in mice as a method of isometric strength training (32). In this model, mice hang from a metal wire grid for 3-min sets repeated multiple times, 5 days per week. Fast-twitch muscle fiber diameter increased in the rectus femoris and gastrocnemius after 10 wk of training with this strategy, without a shift in muscle fiber type composition (32). While inducing modest fiber type-specific hypertrophy in two muscles, the training does not involve dynamic muscle action, which is a component of traditional strength training in humans, and is very labor-intensive. A curious finding from this investigation was that forced treadmill running 5 days per week was reported to be equally, if not more hypertrophic at the muscle fiber level than the isometric hanging. Introduced in 2013, wire hanging has not yet been widely adopted.

CONCLUSIONS

Selecting the best training modality for inducing hypertrophy in mice is context-specific and highly contingent on the research question. There are clear tradeoffs between the effective translatable strategies for inducing muscle hypertrophy: PoWeR, involuntary weighted-vest wheel running, involuntary incline treadmill running, involuntary electrical stimulation, and voluntary squat-like exercise (summarized in Table 1). Worth mentioning is that most models of murine hypertrophy have focused on muscle adaptation resulting from ankle joint action, but adaptation in every muscle of the lower limb is usually not characterized, nor are upper limb adaptations considered; in other words, muscle and limb-specific adaptations for each modality deserve further investigation. Enhanced translatability to humans afforded by newly-developed murine exercise models, along with the ability to detrain, provide the opportunity to study muscle growth and deloading while minimizing confounding variables associated with a surgical approach, and can be combined with genetically modified mice to provide detailed mechanistic insight into muscle mass regulation.

Table 1.

Effective translatable strategies for inducing muscle hypertrophy: PoWeR, involuntary weighted-vest wheel running, involuntary incline treadmill running, involuntary electrical stimulation, and voluntary squat-like exercise

Model	Voluntary	Non-Standard Equipment	Volume Control	Throughput	Hypertrophied Muscle(s)	Fiber Size	Fiber-Type Shift	Refs.
Progressive Weighted Wheel Running (PoWeR, 8 wk)	Yes	No	No	High	Plantaris (15%)	↑ (17%)	Yes (oxidative)	Dungan et al. 2019 (13)
					Soleus (NR)	↑ (22%)	NR	Murach et al. 2019 (42)
Weighted-Vest Motorized Wheel Running (12 wk)	No	Yes	Yes	Low	Soleus (15%); Quadriceps (22%)	↑ (7–10%, Soleus)	NR	Graber et al. 2019 (19)
High-Intensity Incline Treadmill Running (8–16 wk)	No	Yes	Yes	Low Moderate	Aged mice: Soleus (29%), EDL (13%)	↑ (18%, glycolytic TA)	Yes (oxidative)	Seldeen et al. 2018 (51)
					Gastrocnemius, Quadriceps, TA, EDL (10–20%)#	↑	NR	Goh et al. 2019 (17)
Electrical Stimulation (≤4 wk)	No	Yes	Yes	Low	TA (7%)	↑	NR	Hardee et al. 2016 (21)
					Gastrocnemius (5%)	NR	NR	Tatebayashi et al. 2018 (53)
Squat-Like Exercise (8 wk)	Yes	Yes	No	High	Soleus, Plantaris, Gastrocnemius (10–20%)#	NR	NR	Cui et al. 2020 (10)

Data from young adult mice are shown in the table (except where noted), and values reported are statistically significant. NR, not reported; EDL, extensor digitorum longus; TA, tibialis anterior.

^#Estimated from figures).

GRANTS

This study was supported by National Institutes of Health Grant K99 AG063994 to K. A. Murach.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.A.M. and C.M.D. conceived and designed research; K.A.M. drafted manuscript; K.A.M., J.J.M., C.A.P., and C.M.D. edited and revised manuscript; K.A.M., J.J.M., C.A.P., and C.M.D. approved final version of manuscript.