Sarcomerogenesis: Evidence, context and key stimuli—Response to the commentary by Power et al.

We thank Power et al.¹ for their interest in our review² and for contributing to this important scientific discussion. We welcome their commentary and acknowledge the merit of continuing to scrutinize and refine interpretations in this evolving field. Given that much research time and financial investment is being given to the study of the effects of eccentric training in both athletic and clinical contexts, it is incumbent on our field to demonstrate whether eccentric contractions are a key (or the key) stimulus for sarcomerogenesis (increases in serial sarcomere number [SSN]). To clarify, eccentric contractions are defined as those in which the muscle’s fascicles only, or at least predominantly, work eccentrically, without significant concentric action and regardless of whether the whole muscle-tendon unit lengthens or shortens (note that fascicles may shorten or remain isometric even when the whole muscle-tendon lengthens).

Readers might be surprised to find that no randomized controlled trials exist in either animals or humans comparing eccentric with concentric training while matching joint excursion, velocity, and torque, isolating fiber/muscle strain direction as a factor to explore whether training using only eccentric contractions is the key to sarcomerogenesis. One study in rats³ might be considered the nearest attempt, since sonomicrometry and electromyography (EMG) data showed that vastus lateralis (VL) was active predominantly during eccentric contractions in downhill running (their Fig. 2³) but active during concentric contractions in uphill running (their Fig. 1³), and also operated at similar (relatively short) lengths during the same (stance) phase of gait. As shown in their Fig. 3B,³ no difference was detected between uphill and downhill groups after 10 days of exercise, with mean SSN changes of –3% and –4%, respectively, compared to non-training controls. Morais et al.⁴ used a similar training strategy for 4 weeks in mice so their VL data could be included too, as we have done in Fig. 7C of our review,² showing +1.8% SSN for downhill running and –1.0% for uphill across the studies, with no significant difference between conditions or from control animals. Based on these data, it is not possible to conclude that the eccentric contraction mode itself is a principal stimulus for increases in SSN.

With this knowledge at hand, and given the limited available direct evidence whilst keeping in mind that “absence of evidence is not evidence of absence,” we searched for other evidence of an effect. We found that, when including all studies (to May, 2024) claiming to involve eccentric training regardless of the lack of concentric control comparison, or in the absence of definitive proof of their eccentric nature, or even in cases where evidence contradicts such a classification (discussed below), a significant increase in SSN could be detected (our Fig. 7A²). However, the mean effect of ∼4%, which is similar to the value previously obtained by 2 authors (GAP and AH) of the commentary and shown in Fig. 10.7 of their recent book chapter,⁵ is much smaller than the ∼10%–20% increases found after immobilization at long length across a range of muscles and species (also consistent with their Fig. 10.7⁵) and >30% increases observed after electically-stimulated isometric training at long muscle lengths in rats.⁶ Based on these data, we stand by our conclusions that “current evidence does not support eccentric training as an effective stimulus for sarcomerogenesis,” but instead would consider it, at best, relatively ineffective. Nonetheless, we reminded readers in our review that “eccentric exercises might still trigger SSN adaptations even if the eccentric contraction mode itself is not the primary stimulus.”

We will now reply to specific comments made in the commentary by Power and colleagues.¹

1. On selective inclusion of studies and the problem of confirmation bias

The commentary authors advocated that “one should ideally focus on interventions that were optimized to alter that outcome measure.” Consistent with this point, we limited our conclusions to the results of the analyses that included all studies in which muscles (or the fascicles themselves) were proven to work selectively, or at least predominantly eccentrically. They also questioned why both the 5-day and 10-day downhill running protocols from Butterfield et al.³ were included in the analyses, and suggested that the 5-day intervention should be considered as “acute” but the 10-day intervention as “chronic.” We disagree that the 5-day data set assesses “acute” effects, which should describe changes evoked by a single session, but agree that the study duration may be too short to produce large effects. In that case, however, we should also remove studies in which only 2 additional sessions were included (7 days total), ruling out much of the data of Lynn et al.^7,8). Importantly, even the 10-day training data showed no difference between conditions, and the 4-week data of Morais et al. ⁴ also revealed no difference. Thus, additional training time did not affect the outcome of studies in which VL (the muscle for which sonomicrometry data exist) was examined. As a reminder to the reader, the 5- and 10-day trained rats are independent groups,³ so they can be treated as distinct samples.

The authors also (i) argued that only the “optimal” eccentric protocol from Butterfield and Herzog⁹ (pre-activation before lengthening to long muscle lengths; stimulation preceeding stretch at long length [SPL_c] protocol) should be included, while removing other conditions (stimulation on stretch [SOS_c] and stimulation preceeding stretch at short length [SPS_c]) from the same study to prevent “skewing the results,” and (ii) state that the researchers “…found that performing an eccentric contraction to a long muscle length was more optimal to induce sarcomerogenesis than to a short muscle length.”⁹ However, (1) if eccentric training was a key stimulus for increasing SSN, then we should not expect reductions in sarcomere number across muscles and muscle regions, regardless of the force, strain or fiber length in training, and (2) we are unable to find the data showing, or the study author’s conclusion that, contraction to a long length was more optimal for sarcomerogenesis. In relation to Point (ii), Butterfield and Herzog¹⁰ do conclude in another study that muscles that were active at longer lengths showed more evidence of “acute damage.” But in the cited study,⁹ the group training to the longest length (SPL_c) showed a statistical increase in SSN in 2 of 4 muscle regions and a decrease in one region, while the groups training at shorter lengths (SOS_c and SPS_c) showed an increase SSN in 1 region and decrease SSN in 2 regions (one region remained unchanged in each group). Furthermore, the mean sarcomere number changes across regions were –1.63% and +0.65% for the shorter-length groups (SOS_c and SPS_c) and +1.03% for the longer-length group (SPL_c), indicating small overall changes with no discernible differences between conditions. Based on these data, Butterfield and Herzog⁹ concluded that “…fiber strains are a more powerful stimulus for adaptation than MTU stress and strain…” as “…different architecture of the individual fibers in different regions of the muscle (caused) different strains in fibers from different regions….” This argument aside, the call to include only the longer-length training group (SPL_c) in our analyses tends to suggest that the authors of the commentary agree that muscle length is a potent factor influencing SSN, and likely more important than the contraction mode itself.

Another point raised in the commentary was that the age of the animals is a confounding factor. We agree, as noted in our review: “Given that previous studies reported age-related blunting of sarcomerogenesis, an additional analysis was done without aged animals to determine whether they masked important results. No effect was detected (Δ = 1%; effect size (ES) = 0.36, 95% confidence interval (95%CI): –0.13 to 0.84, p = 0.153).”²

The authors additionally highlighted that the data of Hinks et al.,¹¹ using submaximal eccentric training in older rats, were not included. We would like to clarify that this paper was published in October 2024, after our systematic literature search concluded (May 2024), and was therefore not included. However, we can incorporate these data¹¹ into both the neuromuscular electrical stimulation (NMES)-evoked eccentric (NMES-Ecc) and “NMES-Ecc + downhill VL” analyses, and re-run the analyses without the maximal-training data from aged animals¹² to account for the “context-dependent” factors highlighted in the commentary. Even with this arguably more favorable dataset, no statistically significant effect emerged; the ES (95%CI) increased (from 0.36 [–0.13 to 0.84] to 0.57 [–0.03 to 1.16]) and the p-value almost reached significance (from 0.153 to 0.062). One could argue that this effect indicates a trend toward a meaningful effect, however this produced a weighted average change of just +2% (Fig. 1), and the heterogeneity of the results also increased (from 44% to 66%). This is an important validation, not only because of the need to consider potential confounding variables in the analyses but also because it is important to test whether exclusion or inclusion of any single data set is sufficient to alter the overall conclusion over the effect significance (i.e., “leave-one-out” sensitivity analysis); if this had been the case, then a robust conclusion could not be drawn.

Fig. 1.

Percentage differences in serial sarcomere number following various stimuli in animal models, compared to controls. The values for immobilization at neutral length (2%) and electrical stimulation added to immobilization at long length (20%) are derived from Fig. 10.7 of the book chapter by Hinks and Power.⁵ The result for immobilization at long length (15%) is consistent with the findings discussed in our review (10%–20%)² and presented in the book chapter by Hinks and Power (14%).⁵ The 1%–5% range for eccentric training reflects variability in mean reported outcomes. In our meta-analysis,² a 4% increase was observed when considering all suggested forms of eccentric training, even when eccentric contractions were not directly verified for several specific conditions (as discussed here in the review²). A 4.8% increase was reported in the authors’ commentary,¹ and a 4% increase was reported in their book chapter.⁵ In contrast, our re-analysis, following Power et al.’s recommendations¹ to remove maximal training data in aged animals and include the data from the submaximal training study,¹¹ yielded a 2% increase. The result for electrically stimulated isometric training at long length (32%) corresponds to that observed by Uçar et al.⁶ and discussed in our review.² The current evidence does not support eccentric training as an effective stimulus for sarcomere addition when compared to other chronic (including exercise) interventions, such as contractions at long muscle lengths or passive immobilization.

Finally, we note the comment that “We would thereby warrant caution when interpreting a conclusion that “current evidence does not support eccentric training as an effective stimulus for sarcomerogenesis in animal models” based primarily on downhill running studies in one muscle.”¹ We would like to point out that we have not at any stage drawn such a conclusion only from the downhill running studies alone. Instead, our conclusions were based on the analysis of all data for which we were sure the muscles (or their fascicles) operated eccentrically or predominantly eccentrically (this included downhill running in VL plus all NMES-based eccentric studies), which showed no statistical effect (our Fig. 7E²), and from the analysis of all studies suggested to be eccentric regardless of whether we can confirm that muscles operated eccentrically or predominantly eccentrically (our Fig. 7A²). The authors of the commentary also ran another analysis using only the eccentric exercise interventions they considered optimum to increase SSN (Power et al.’s Fig. 1¹), despite the issues we have raised above. Although we disagree with this approach, it is important to note that analysis led to a 5.1% SSN increase after downhill running and 4.2% after NMES-Ecc (4.8% pooled). Again, these changes are much smaller than the ∼10%–20% increases found after immobilization at long length and >30% increases observed after electrically-stimulated isometric training at long lengths (Fig. 1).² We therefore stand by our conclusions that “current evidence does not support eccentric training as an effective stimulus for sarcomerogenesis.” The effect of the eccentric contraction mode itself appears to be either small or explicable by other factors such as end-contraction fiber length and force.^2,9

2. On the mechanics of the plantarflexors: Quasi-isometric, not sustained eccentric, during locomotion

The authors questioned our omission of rat and mouse vastus intermedius data “…simply off the basis that there is no published sonomicrometry data….”¹ In fact, we can confirm that this was the basis of our decision, as outlined in detail in our review.² Given that several other data sets are available for which we can be confident that eccentric-only or predominantly eccentric contractions were performed, we believe it to be inappropriate to include muscles for which we have no certainty around the predominant contraction mode.

The authors also questioned the omission of plantarflexor muscle data, since “…while no sonomicrometry data during downhill exercise has been collected for the rat soleus, others have shown it undergoes eccentric contractions during its regular gait cycle.”^1,14,15 We thank the authors for bringing these papers to the attention of the reader, and accept that their inclusion would have proved useful in the original paper. Hodson-Tole and Wakeling¹³ (Reference 14 in Power et al.’s commentary¹) measured fascicle behaviors during both level and inclined running, concluding that “Peak-to-peak strains in both the soleus and plantaris were low (<0.1) (Fig. 6A), indicating that both of these muscles worked close to isometrically in all conditions. This agrees with previous work in other animal species (Biewener and Baudinette, 1995; Fukunaga et al., 2001; Griffiths, 1991; Hoffer et al., 1989; Lichtwark and Wilson, 2006; and Roberts et al., 1997)….”¹³ This conclusion was reached despite the fascicles working predominantly concentrically during stance in 7 of 9 conditions, as shown in their Fig. 4.¹³ Based on this evidence, we cannot conclude that the muscles worked eccentrically or predominantly eccentrically, which justifies their exclusion from the final analysis. Furthermore, medial gastrocnemius fascicles lengthened briefly just before foot contact but under low force, and then contracted concentrically during stance when force was higher. Thus, this muscle also does not satisfy the inclusion criteria. Bernabei et al.¹⁴ (Reference 15 in Power et al.’s commentary¹) identified a clear stretch–shorten cycle pattern in soleus fibers during level and incline running (80 cm/s), where the eccentric strain was smaller than the subsequent concentric strain, which also occurred predominantly during the higher-force propulsive phase. By contrast, lateral gastrocnemius fibers exhibited quasi-isometric contractile behavior. Such behaviors do not allow one to test whether eccentric contractions themselves are a stimulus for sarcomerogenesis, suggesting that this model is not appropriate to test the hypothesis set out in our review.²

Note, however, that while fascicle operating behaviors have not been determined during downhill locomotion in these studies, such studies may be done in the future. If eccentric-only or predominant eccentric patterns are observed in such studies, then data using downhill running as an exercise stimulus will need to be included in future analyses. Nonetheless, based on data across animal populations showing that the long Achilles tendon (and elastic plantar foot surface) provide an excellent mechanism for maintaining quasi-isometric and concentric plantarflexor behaviors during gait,¹⁵ we would be surprised if this were the case.

3. Clarifying the conclusion—The main point stands

It is crucial to emphasize that we have not stated that sarcomerogenesis cannot occur after periods of eccentric training. The works done by the commentary authors and others have shown such outcomes under some protocols. But the important point remains: Eccentric exercise does not seem to be a key promoter of sarcomereogenesis, thus current evidence does not support eccentric training as an effective stimulus for sarcomere addition when compared to other chronic (including exercise) interventions, such as contractions at long muscle lengths or passive immobilization. However, we do agree with the commentary authors that: “To make the biggest impact going forward, we should focus on the breadth of training interventions that can leverage this stimulus to induce sarcomerogenesis and improve performance.”¹

We look forward to seeing the results of many and varied studies in the future.

Authors’ contributions

AJB wrote the first draft; JPN performed additional analyses, wrote parts of the first draft, and made substantial contributions to the original work; WH made substantial contributions to the original work, including input of novel information. All authors have read and approved the final version of the manuscript, and agree with the order of presentation of the authors.

Declaration of competing interest

The authors declare that they have no competing interests. Given his role as Editor-in-Chief, WH had no involvement in the peer review of this article and had no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to another journal editor.