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

Reviewer #1 (Public Review):

Summary:
Using a state-of-the-art image analysis pipeline the authors report that muscle cell hypertrophy in mice and humans occurs primarily through an increase in the number of myofibrils (myofibrillogenesis) and not myofibril hypertrophy.

Strengths:
A strength of the study is the development and validation of an automated image analysis pipeline to quantify myofibril size and abundance in mouse and human muscle cells. In addition to the pipeline, which requires relatively readily available microscopy equipment (an additional strength) is the development of a methodology to optimally prepare muscle samples for high-resolution imaging.

Weaknesses:
A weakness of the study was that only one time-point was assessed during hypertrophy. As mentioned by the authors, this precluded an assessment of the myofibril splitting mechanism. The second weakness was the criteria (aspect ratio of <2.5:1) used to identify a myofibril which excluded a significant number of myofibrils from analysis. How might the inclusion of these odd-shaped myofibrils impact the outcome of the study?

Reviewer #2 (Public Review):

Summary:
In this work, the authors sought to 1) establish a method for measuring muscle fiber subcellular structure (myofibrils) using common, non-specialized laboratory techniques and equipment, and 2) use this method to provide evidence on whether loading-induced muscle fiber growth was the result of myofibril growth (of existing myofibrils) or myofbrillogenesis (creation of new myofibrils) in mice and humans. The latter is a fundamental question in the muscle field. The authors succeeded in their aims and provided useful methods for the muscle field and detailed insight into muscle fiber hypertrophy; specifically, that loading-induced muscle fiber hypertrophy may be driven mostly by myofibrillogenesis.

Strengths:

1. The usage of murine and human samples to provide evidence on myofibril hypertrophy vs myofibrillogenesis.
2. A nice historical perspective on myofibrillogenesis in skeletal muscle.
3. The description of a useful and tractable IHC imaging method for the muscle biology field supported by extensive validation against electron microscopy.
4. Fundamental information on how myofiber hypertrophy ensues.

Weaknesses:

1. The usage of young growing mice (8-10 weeks) versus adult mice (>4 months) in the murine mechanical overload experiments, as well as no consideration for biological sex. The former point is partly curtailed by the adult human data that is provided (male only). Still, the usage of adult mice would be preferable for these experiments given that maturational growth may somehow affect the outcomes. For the latter point, it is not clear whether male or female mice were used.

2. Information on whether myofibrillogenesis is dependent on hypertrophy induced by loading, or just hypertrophy in general. To provide information on this, the authors could use, for instance, inducible Myostatin KO mice (a model where hypertrophy and force production are not always in lockstep) to see whether hypertrophy independent from load induces the same result as muscle loading regarding myofibrillogenesis.

3. Limited information on Type 1 fiber hypertrophy. A "dual overload" model is used for the mouse where the soleus is also overloaded, but presumably, the soleus was too damaged to analyze. Exploring hypertrophy of murine Type 1 fibers using a different model (weight pulling, weighted wheel running, or forced treadmill running) would be a welcome addition.

Reviewer #3 (Public Review):

Summary:
Radial muscle growth involves an increase in overall muscle cross-sectional area. For decades this process has been described as the splitting of myofibrils to produce more myofibrils during the growth process. However, a closer look at the original papers shows that the evidence underlying this description was incomplete. In this paper, the authors have developed a novel method using fluorescence microscopy to directly measure myofibril size and number. Using a mouse model of mechanical loading and a human model of resistance exercise they discovered that myofibrillogenesis is playing a key role in the radial growth of muscle fibers.

Strengths:
1. Well-written and clear description of hypothesis, background, and experiments.
2. Compelling series of experiments.
3. Different approaches to test the hypothesis.
4. Rigorous study design.
5. Clear interpretation of results.
6. Novel findings that will be beneficial to the muscle biology field.
7. Innovative microscopy methods that should be widely available for use in other muscle biology labs.

Weaknesses:
Supplemental Figure 1 is not very clear.