Stretch-shortening cycles protect against the age-related loss of power generation in rat single muscle fibres

Aging is associated with impaired strength and power during isometric and shortening contractions, however, during lengthening (i.e., eccentric) contractions, strength is maintained. During daily movements, muscles undergo stretch-shortening cycles (SSCs). It is unclear whether the age-related maintenance of eccentric strength offsets age-related impairments in power generation during SSCs owing to the utilization of elastic energy or other cross-bridge based mechanisms. Here we investigated how aging influences SSC performance at the single muscle fibre level and whether performing active lengthening prior to shortening protects against age-related impairments in power generation. Single muscle fibres from the psoas major of young (~8 months; n = 31 fibres) and old (~32 months; n = 41 fibres) male F344BN rats were dissected and chemically permeabilized. Fibres were mounted between a force transducer and length controller and maximally activated (pCa 4.5). For SSCs, fibres were lengthened from average sarcomere lengths of 2.5 to 3.0 μ m and immediately shortened back to 2.5 μ m at both fast and slow (0.15 and 0.60 Lo/s) lengthening and shortening speeds. The magnitude of the SSC effect was calculated by comparing work and power during shortening to an active shortening contraction not preceded by active lengthening. Absolute isometric force was ~37 % lower in old compared to young rat single muscle fibres, however, when normalized to cross-sectional area (CSA), there was no longer a significant difference in isometric force between age groups, meanwhile there was an ~50 % reduction in absolute power in old as compared with young. We demonstrated that SSCs significantly increased power production (75 – 110 %) in both young and old fibres when shortening occurred at a fast speed and provided protection against power-loss with aging. Therefore, in older adults during everyday movements, power is likely ‘protected ’ in part due to the stretch-shortening cycle as compared with isolated shortening contractions.


Introduction
Aging is associated with impaired strength and power during isometric and shortening (i.e., concentric) contractions (Macaluso and De Vito, 2004;Narici and Maganaris, 2006;Power et al., 2013a;Wu et al., 2021), however, during lengthening (i.e., eccentric) contractions, strength is well-maintained into old age (Vandervoort et al., 1990;Roig et al., 2010;Power et al., 2012;Power et al., 2013b).During daily movements, muscles undergo stretch-shortening cycles (SSCs), in which active muscle shortening is directly preceded by active muscle lengthening (see Fukutani et al., 2021 for review).Enhanced muscle force, work, and power production are observed during the shortening phase of a SSC in comparison to a pure shortening contraction; this phenomenon is termed the SSC effect (Cavagna et al., 1968;Bosco et al., 1982;Bosco and Rusko, 1983;Komi, 2000;Seiberl et al., 2021).Recently, during multi-joint exercise, a SSC improved power production in older adults compared with only shortening contractions (Mc Dermott et al., 2023).It is unclear in older adults, however, whether this SSC effect is a cellular mechanism intrinsic to the muscle fibre or owing to some aspect of age-related muscle architecture remodeling/composition (Narici and Maffulli, 2010;Power et al., 2013a).In the present study we investigated whether the age-related maintenance of eccentric strength contributes to the SSC effect and offsets impairments in power generation in old age at the single muscle fibre (cellular) level.
In addition to neural and muscle-tendon unit level mechanisms (Fukutani et al., 2017;Fukutani and Isaka, 2019), the SSC effect also exists at the cellular level in healthy young muscle.It is believed that active lengthening-induced residual force enhancement (rFE) contributes to the SSC effect (Seiberl et al., 2015;Fukutani et al., 2017;Fortuna et al., 2018).rFE is characterized by an increase in isometric steady-state force following an eccentric contraction compared to a fixed-end isometric contraction at the same muscle length and level of activation.The increased isometric steady-state force following lengthening is likely due to the shortening of titin's free spring length, and stiffening upon activation, leading to a greater contribution of passive force to total force (Herzog, 2018;Hahn et al., 2023).rFE is greatest following large stretch amplitudes, to long muscle lengths (i.e., descending limb of the force-length relationship) (Julian and Morgan, 1979;Morgan et al., 2000;Peterson et al., 2004;Fukutani and Herzog, 2019).As SSCs also include active muscle shortening, shortening-induced residual force depression (rFD) must also be considered (Chen et al., 2019).Opposite to rFE, rFD is characterized as a decrease in isometric force following a shortening contraction owing to a reduction in the number of attached cross-bridges likely due to angular deformation of actin during shortening (Herzog et al., 2000;Chen et al., 2019;Hahn et al., 2023).Notably, rFD is reduced with increasing speeds of shortening, thereby increasing steady-state isometric force following a SSC and amplifying the presence of rFE in the SSC effect (Fortuna et al., 2017).In addition to contractile history, SSC performance also depends on shortening/ lengthening velocity due to the shape of the force-velocity relationship of muscle (Gasser and Hill, 1924;Hill, 1938), and the amortization phase (time between end of lengthening to start of shortening), with longer times associated with less performance enhancement (Bosco et al., 1981;Fortuna et al., 2017).Furthermore, power output during SSCs is amplified with increasing stretch-shortening velocity, likely due to the velocity-dependence of eccentric force development (Tomalka et al., 2021).
Natural adult aging is associated with reductions in muscle strength and power (Power et al., 2013a).Strength loss is most apparent during shortening and isometric contractions, while eccentric strength is wellmaintained (Roig et al., 2010).The age-related maintenance of eccentric strength is suggested to be due to increases in passive stiffness, owing to the accumulation of non-contractile material in the muscletendon unit, and a greater proportion of bound cross-bridges during eccentric contractions (Vandervoort et al., 1990;Roig et al., 2010;Power et al., 2012;Power et al., 2013b).With a slowing of cross-bridge kinetics in old age (Miller and Toth, 2013), there is a greater probability more cross-bridges are attached at any given point during the eccentric contraction thereby contributing to force during active lengthening.Additionally, at the joint level in humans, both rFD and rFE appear to be amplified in old compared to young individuals (Power et al., 2014;Power et al., 2012).Therefore, it seems the aged neuromuscular system would benefit from a prior lengthening contraction for power generation during a subsequent shortening contraction.Mc Dermott et al. (2023) recently showed SSCs can improve power production in older adults during multi-joint exercise.However, the underlying mechanisms are unclear.
Thus, the purpose of this study was to investigate how aging influences SSC performance in rat single muscle fibres.Specifically, we sought to determine whether performing an active lengthening contraction prior to shortening provides protection against typical agerelated impairments in force and power production.Due to the agerelated maintenance of eccentric strength and amplification of rFE, we expect to observe a greater magnitude of the SSC effect in single muscle fibres from old compared to young rats.

Animals
Single muscle fibres from six young (sacrificial age ~ 8 months; mass = 381.61± 15.71 g; n = 31 fibres) and ten old (sacrificial age ~ 32 months; mass = 480.79g ± 37.91 g; n = 41 fibres) male F344BN rats were tested (Charles River Laboratories, Senneville, QC, Canada) with approval from the Animal Care Committee (AUP: #4905) at the University of Guelph and all protocols followed CCAC guidelines.Rats were housed in groups of two or three with a 12 h light/dark cycle at 23 • C and were given unrestricted access to room-temperature water and a Teklad global 18 % protein rodent diet (Envigo, Huntington, Cambridgeshire, UK).Rats were sacrificed via isoflurane followed by CO asphyxiation then cervical dislocation prior to harvesting muscle tissue.

Tissue preparation
The right and left psoas major muscles were harvested from the rats following sacrifice and immediately transferred to a silicone elastomerplated petri dish containing chilled dissecting solution.The psoas major muscle was selected owing to its long fibres and primarily fast-type fibre composition (Edström et al., 1982;Hämäläinen and Pette, 1993;Schilling et al., 2005;Vlahovic et al., 2017).Fibre bundles were dissected from the proximal half of the psoas major, which is almost exclusively fast-type fibres (< 1 % type I) (Hämäläinen and Pette, 1993).The muscles were then sectioned into fibre bundles and transferred to a tube containing 2.5 ml of chilled skinning solution where they remained on ice for 30 min for permeabilization (Mazara et al., 2021;Hubbard et al., 2022).The bundles were then washed with fresh chilled dissecting solution and gently agitated to ensure any remaining skinning solution was removed.The bundles were then transferred to a tube containing storage solution and incubated for 24 h at 4 • C. Tubes were prepared with fresh storage solution and the bundles were placed in individual tubes and stored in a freezer at − 80 • C until mechanical testing began (Mashouri et al., 2021;Hubbard et al., 2022).

Mechanical testing and force measurements
On testing days, single muscle fibres were isolated from a fibre bundle and moved to a temperature-controlled chamber filled with relaxing solution where they were then tied to pins attached to a force transducer (403 A, Aurora Scientific, Toronto, ON, Canada) and a length controller (322C, Aurora Scientific) with nylon sutures.All experiments were performed at 12 • C to allow for a balance between force production capacity, and the number of contractions performed before experiencing force loss (Ranatunga, 1982;Bottinelli et al., 1996;Tomalka et al., 2017;Tomalka et al., 2021).Muscle contraction was initiated by first transferring the fibre from relaxing solution to a pre-activating solution with reduced Ca 2+ buffering capacity for 10 s before being transferred to an activating solution.All activations were performed in a maximal Ca 2+ concentration solution (pCa 4.5).For all tests, the average sarcomere length (SL) was measured using a high-speed camera (Aurora Scientific Inc., HVSL 901A).Total force was recorded, and active force was M.A. Patterson et al. determined by subtracting passive from total force.Before starting the testing protocol, the fibre was set to a SL of 2.5 μm (i.e., the optimal SL for rat muscle fibres (Burkholder and Lieber, 2001;Ledvina and Segal, 1995;Smith et al., 2021)) and a 'fitness' contraction was performed to ensure the ties were not loose and the condition of the fibre was sufficient for testing.Following the fitness contraction and prior to experimental testing, SL was re-measured and, if necessary was re-adjusted to 2.5 μm.Then, the cross-sectional area (CSA) of each fibre was determined.This was done by measuring the diameter of the fibre in three places (at each end and in the centre) using an eyepiece reticle.These three measurements were then averaged to calculate the CSA of the individual fibre.Given these samples are muscle fibre segments and chemically permeabilized, they are treated as cylindrical, therefore we assumed circularity in the calculation of CSA.
Instantaneous stiffness (k) tests were performed to determine the proportion of attached cross-bridges during the force plateau after 40 s of activation.This was done by rapidly lengthening the fibre (500 Lo /s) by 0.3 % of Lo and dividing the change in force during the stretch by the change in length.

Reference isometric contractions
Two reference isometric contractions were performed first, one at optimal length (average SL of 2.5 μm) and one at a long length (average SL of 3.0 μm).Fibres were set to their respective length, then activated for 45 s.The order of these contractions was randomized.

Active shortening contractions
Active shortening contractions at a slow (0.15 Lo/s) and a fast (0.60 Lo/s) speed were performed following the isometric reference contractions.Fibres were initially set to an average SL of 2.5 μm and were passively lengthened to 3.0 μm and activated 15 s later.Following 20 s of contraction, fibres were actively shortened to an average SL of 2.5 μm at 0.15 Lo/s or 0.60 Lo/s for the slow and fast speeds, respectively, and were held for 25 s before deactivation (Fig. 1).The order of these contractions was randomized.

Stretch-shortening cycle trials
Two lengthening and shortening speeds were implemented during the SSC trials, a slow speed (0.15 Lo/s) and a fast speed (0.60 Lo/s).As such, each fibre underwent four SSC trials to account for the possible speed combinations (Fig. 2).Fibres were activated at an average SL of 2.5 μm, lengthened to 3.0 μm, immediately shortened back to an average SL of 2.5 μm, and held isometrically for another 25 s (Fig. 2A).The order of the trials was randomized.

Transient aspects of force
Mechanical work of shortening was calculated as the product of the area under the curve of the force-time trace and the change in fibre length (Fig. 1).Absolute power during shortening was calculated by dividing the work of shortening by the shortening contraction duration (i.e., work divided by time).Peak eccentric force was the highest force value observed during lengthening in the SSC trials.

SSC effect
The magnitude of the SSC effect was calculated by comparing 1) work and absolute power during the SSC to an active shortening contraction not preceded by active lengthening.Shortening-induced residual force depression was determined by comparing isometric steady-state force following a purely shortening contraction and the SSC to an isometric reference contraction not preceded by a SSC.

Number of fibres
Given that the testing protocol involved nine contractions, some fibres were damaged before the completion of all nine contractions.Thus, the number of fibres varies between measures and the sample size is noted in each figure.

Statistics
Age-related differences in fibre CSA and isometric force were assessed by one-way analysis of variance (ANOVA).Age-related differences in work and power during shortening contractions with respect to different shortening speeds were assessed by two-way ANOVA with speed (fast, slow) as a within-subjects factor and age (young, old) as a between-subjects factor.A Holm-Sidak correction was used for pairwise comparisons.Where significant effects or interactions were observed, two-tailed t-tests were performed with a Holm-Sidak correction for multiple comparisons.Age-related differences in the SSC effect, work, power, peak eccentric force, and stiffness during SSCs with respect to different lengthening and shortening speeds were assessed by two-way ANOVA with protocol (fast-fast, fast-slow, slow-fast, slow-slow) as a within-subjects factor and age (young, old) as a between-subjects factor.Where significant effects or interactions were observed, two-tailed ttests were performed with a Holm-Sidak correction for multiple comparisons.Data are reported in figures as the mean ± standard error.Where significance was observed, effect sizes are reported as the partial eta squared (η p 2 ) for main effects and interactions, and Cohen's d for ttest.

Absolute force
For absolute force, there was a main effect for both age (F(1,143) = 22.640, P < 0.001) and length (F(1,143) = 95.091,P < 0.001) with an interaction between the two (F(1,143) = 4.437, P = 0.039).Pairwise comparisons showed that at both optimal and long sarcomere lengths, old produced 37 % less force than young.For both young and old rats, there were differences in absolute force production between optimal and long lengths, with 15 % less force produced at the long length compared to the optimal length for both young and old fibres (Fig. 3A).

Cross-sectional area
For cross-sectional area (CSA), there was an effect of age (F(1,71) = 16.279,P < 0.001) such that fibres from the old rats (0.00874 ± 0.000517 mm 2 ) had a 34 % smaller cross-sectional area compared to that of the young rat fibres (0.0132 ± 0.00106 mm 2 ).

Specific force
For specific force, there was an effect of length (F(1,143) = 111.995,P < 0.001) such that specific force produced at long lengths was 14 % less than the specific force at optimal length (Fig. 3B).There was no effect of age for specific force (F(1,143) = 0.409, P = 0.525), nor interaction (F(1,143) = 0.141, P = 0.708), indicating no difference in isometric force between old and young rats when accounting for differences in CSA.

Stiffness
For stiffness during the isometric contractions, there was a main effect of both age (F(1,143) = 39.405,P < 0.001) and length (F(1,143) = 135.220,P < 0.001) with an interaction between the two (F(1,143) = 6.025,P = 0.017).Pairwise comparisons showed that old had 47 % and 50 % less stiffness than young at optimal and long lengths, respectively (P < 0.001), indicating fewer attached cross-bridges in old as compared to young.For both young and old rats, there were differences in stiffness between optimal and long lengths, with 23 % and 28 % less stiffness at the long length compared to the optimal length for young and old, respectively (P < 0.001) (Fig. 3C).

Peak force during active lengthening (i.e., eccentric force)
For peak force during active lengthening in the SSC contractions, there was a main effect of age (F(1,227) = 219.457,P < 0.001) such that single fibres from the old rats produced a 42-54 % lower peak force during active lengthening than those from the young rats across all SSC conditions.There was no effect of lengthening speed on peak force during active lengthening (F(3,227) = 1.207,P = 0.308), nor an interaction (F(3,227) = 2.113, P = 0.099).

Power
For power, there was a main effect of both age (F(1,371) = 34.952,P < 0.001) and condition (F(5,371) = 20.673,P < 0.001) with an interaction between the two (F(5,371) = 20.673,P < 0.001).Pairwise comparisons showed that for all conditions, old produced lower power than young (P < 0.001-0.018).The greatest differences were for the fast-slow and fast-fast SSC speed combinations (55 %) while the smallest difference was for the slow-slow SSC speed combination (42 %) (Fig. 5).The SSC-effect was demonstrated in both young and old rats, as power output was significantly increased (75-110 %) during the fast/fast and Power output was increased by 4-27 % during the slow/slow and fast/ slow SSC trials compared to the slow active shortening contractions, however, these differences did not reach statistical significance.
Most notably, comparing the fast-fast and slow-fast conditions to the fast condition showed that adding an active lengthening contraction prior to shortening protected against age-related impairments power generation, eliminating the 48-52 % age-related loss in power by increasing power during active shortening of the old rats up to approximately the same level of power produced in the fast condition for young rats.

Work
For work, there was a main effect of both age (F(1,371) = 39.621,P < 0.001) and condition (F(5,371) = 85.853,P < 0.001) with an interaction between the two (F(5,371) = 14.232,P < 0.001).Pairwise comparisons showed that for all conditions, old performed less work than young (P < 0.001-0.022).The greatest differences were for the fast-slow and fast-fast SSC speed combinations (58 %) while the smallest difference was for the slow-slow SSC speed combination (43 %) (Fig. 6).

Residual force depression
For residual force depression, there was a main effect of both age (F (1,371) = 12.219, P < 0.001) and condition (F(5,371) = 46.169,P < 0.001) with an interaction between the two (F(5,371) = 5.950, P < 0.001).Pairwise comparisons showed that for the fast/slow and fast/fast SSC speed combinations, old fibres had more force depression than young (P < 0.001), with old having 48 % and 82 % more force depression than young during fast/slow and fast/fast, respectively.Conversely, there were no significant differences in force depression found between old and young for the fast, slow, slow/slow, and slow/ fast speeds (P = 0.096-0.300)(Fig. 7).

Instantaneous stiffness during SCCs
For instantaneous stiffness during the SSCs, there was a main effect of both age (F(1,371) = 40.030,P < 0.001) and condition (F(5,371) = 97.423,P < 0.001) with an interaction between the two (F(5,371) = 14.552,P < 0.001).Pairwise comparisons showed that for all conditions, stiffness was less in old compared to young (P < 0.001-0.003).The greatest differences were for the fast-fast SSC speed combination (63 %) while the smallest difference was for the slow-slow SSC speed combination (40 %) (Fig. 8), indicating fewer attached force producing cross-bridges in old as compared with young.

Discussion
In the present study we investigated whether SSCs could provide protection against age-related power-loss in old rats at the cellular level.In line with our hypothesis, performing an active lengthening contraction prior to a shortening contraction protected against age-related power loss, particularly when the shortening contraction was performed at a fast speed.Despite not observing an age-related maintenance of eccentric force, SSCs provided protection on power-loss at fast shortening speeds likely due to history-dependent effects, preactivation, and active lengthening-induced advantages in cross-bridge kinetics (discussed below).

Age-related impairments in force and power
Single muscle fibres from old rats produced ~37 % less isometric force than young at both optimal and long sarcomere lengths (Fig. 3A), which is consistent with previous findings (Power et al., 2013a;Grosicki et al., 2022).Additionally, muscle fibres from both young and old generated significantly less force at 3.0 μm compared to 2.5 μm, indicating that we were indeed testing on the descending limb of the forcelength relationship.When combined with instantaneous stiffness data, we can conclude there were fewer attached force-producing crossbridges at long lengths, driving the differences in force for both young and old.Furthermore, instantaneous stiffness was lower in old compared to young rats (Fig. 3C), indicating fewer attached cross-bridges are contributing to the age-related difference in force production, likely owing to a lower myofibrillar protein content as indicated by a smaller CSA in old compared to young fibres.When force was normalized to CSA to account for myofibrillar protein content, no differences were observed in specific force between old and young (Fig. 3B).Age-related changes to specific force is equivocal, some studies have shown an increase, while others show no change or a decrease as compared with young (Bruce et al., 1989;Larsson and Ansved, 1995;Larsson et al., 2019).However, several studies using rodent muscles with a predominately Type II fibre distribution, like the psoas major, have shown that specific force decreases with age, although not to the extent of absolute force (Larsson and Edström, 1986;Brooks and Faulkner, 1988;Degens et al., 1995;McBride et al., 1995;Wineinger et al., 1995;Lowe et al., 2001).In the present study we did not observe an age-related difference in specific force, therefore, the results of our study indicated that the lower absolute force production in old was attributed to a loss of myofibrillar protein content with age, not an impairment to the contractile machinery.
Despite no difference in specific force, our data support previous reports of an age-related loss of power (Macaluso and De Vito, 2004;Power et al., 2013a;Wu et al., 2021), as the single fibres from old rats were 48-52 % less powerful as compared with young across both fast and slow shortening speeds (Fig. 5).For the two shortening speeds used in this study, power output was greater when shortening occurred at the fast speed compared to slow in both young and old.

Force during active lengthening (eccentric strength) was not maintained in old
Although numerous studies have reported a preservation of eccentric strength with aging at the joint level (Vandervoort et al., 1990;Poulin et al., 1992;Hortobagyi et al., 1995;Porter et al., 1995;Porter et al., 1997;Horstmann et al., 1999;Pousson et al., 2001;Klass et al., 2005;Roig et al., 2010;Power et al., 2012;Power et al., 2013b), our results do not support the age-related maintenance of eccentric strength at the single muscle fibre level (Fig. 4).Several mechanisms have been proposed as explanations for the age-related maintenance of eccentric strength including neural and muscular contributors (see Roig et al., 2010 for review), most notably an increased contribution of passive force to total force during eccentric contractions due to age-related connective tissue accumulation (Haus et al., 2007;Kanazawa et al., 2023).
Neural contributions and intramuscular connective tissue are not present in skinned single muscle fibres, which may explain why we observed no age-related maintenance of eccentric strength.However, Ochala et al. (2006) observed preserved eccentric strength in old human vastus lateralis single fibres.The authors suggested that an increase in weakly bound cross-bridges as well as increased instantaneous stiffness in old muscle fibres contribute to increased force production during lengthening contractions (Ochala et al., 2006;Roig et al., 2010).Our results may differ from Ochala et al. (2006) because the human vastus lateralis has a larger proportion of slow Type I fibres compared to the fast rat psoas muscle (Staron et al., 2000).Additionally, their experiments examined the age-related preservation of eccentric strength during faster eccentric contractions as compared to ours.Altogether, the age-related maintenance of eccentric strength at the fibre level may only be discernible during very fast eccentric contractions and/or slower type fibres.

Active lengthening-induced power amplification and protection against age-related power loss
For our SSC conditions, single fibres from old rats were less powerful than young, regardless of lengthening or shortening speed (Fig. 5).This finding is in line with the literature, as power output is diminished with aging (Macaluso and De Vito, 2004;Power et al., 2013a;Wu et al., 2021).Additionally, our results show that regardless of age, power output is amplified during SSCs when the shortening speed is fast, as compared to pure shortening contractions, demonstrating the SSC-effect (Fig. 5).Power output for young rats increased by 110 % and 75 % during the fast-fast and slow-fast contractions, respectively.These magnitudes of the SSC effect were slightly different in old rats, with power output increasing by 84 % and 96 % during the fast-fast and slowfast contractions, respectively.A recent study that evaluated the effect of a prior eccentric lowering phase on concentric power performance in older human males during multi-joint resistance exercise had similar findings, as their results found that a prior lengthening phase increased mean power output during the shortening phase of contraction (Mc Dermott et al., 2023).However, the greatest increase in mean power output that they observed was 55 % compared to their pure shortening contractions, whereas our findings ranged from increases of 75-110 % (Mc Dermott et al., 2023).This may imply that the SSC effect is greater at the single fibre compared to the joint level.
There were no significant increases in power output for young or old rats during SSCs when the shortening speed was slow (Fig. 5).Possible explanations for why SSCs only significantly improved power output when the shortening speed was fast include two previously proposed mechanisms responsible for the SSC-effect: pre-activation and active lengthening-induced advantages in cross-bridge kinetics (Fukutani  The dashed red line depicts the main finding that implementing an active lengthening contraction prior to shortening at fast shortening speeds provided protection against age-related power loss when compared to the power output of young rats during the fast active shortening contractions.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)et al., 2021).During SSCs, shortening is directly preceded by active lengthening, hence the muscle is already activated at the onset of shortening, which results in more active force during the shortening phase compared to a purely shortening contraction (Fukutani et al., 2021).The effect of pre-activation has been demonstrated in several studies in humans (Svantesson et al., 1994;Bobbert et al., 1996;Bobbert and Casius, 2005;Fukutani et al., 2015aFukutani et al., , 2016Fukutani et al., , 2021)).Pre-activation may also explain the velocity-dependence of the SSC-effect, as the effect of pre-activation is greatest when the concentric phase of contraction is short, whether the shortening distance is small or the speed of shortening is fast (Fukutani et al., 2021).This velocity-dependence was confirmed by Svantesson et al. (1994) and Fukutani et al. (2015b) who, in line with our results, both found that in humans the SSC-effect was optimized at faster shortening speeds, likely owing to the pre-activation effect.Mc Dermott et al. ( 2023) also suggested the pre-activation effect as an explanation for their findings of increased mean power production during SSC contractions in humans.Furthermore, active lengtheninginduced force production advantages in cross-bridge kinetics during pre-activation may explain our finding of increased power production during SSCs with fast shortening speeds.When shortening occurs fast, there is less opportunity for cross-bridge attachment.When active lengthening is performed prior to shortening, however, more crossbridges are able to bind and are forcibly elongated (Flitney and Hirst, 1978;Lombardi and Piazzesi, 1990;Fukutani et al., 2021).This results in an increase in force per cross-bridge and storage of elastic energy, increasing power output during the subsequent shortening phase (Flitney and Hirst, 1978;Bosco and Komi, 1979;Lombardi and Piazzesi, 1990;Fukutani et al., 2021).When shortening occurs at a slower speed, fibres have less of a force production disadvantage as there is more opportunity for cross-bridge attachment.Therefore, when shortening occurs at slow speeds there are fewer benefits of prior lengthening compared to when shortening occurs fast, as the cross-bridges that were elongated during the lengthening phase have time to detach from actin filaments, resulting in a loss of stored energy (Huxley and Simmons, 1971;Bosco et al., 1982;Wilson et al., 1991;Fukutani et al., 2017).Thus, our results agree with previous suggestions that the mechanisms primarily responsible for the SSC-effect likely include pre-activation and cross-bridge kinetics (Fukutani et al., 2021).
Of note, the SSC-effect was not always greater in the single fibres from old rats as compared with young, likely because we observed no age-related maintenance of eccentric strength.However, our results showed that performing active muscle lengthening prior to shortening at fast shortening speeds provided protection against the ~50 % agerelated impairment in power generation, as the power output from old during the fast-fast and slow-fast SSC contractions was the same as the power output from young fibres during the fast pure shortening contractions (Fig. 5).Therefore, power production was protected in aged rats by implementing an active lengthening contraction prior to fast shortening.

History dependent effects
The magnitude of rFD only differed between young and old fibres during the SSC contractions with fast lengthening speeds, with old fibres experiencing 48 % and 82 % greater rFD than young for the fast-slow and fast-fast conditions, respectively (Fig. 7).This partially agrees with the previous findings of Power et al. (2014), who found that jointlevel rFD was greater in the dorsiflexors and plantar flexors of older men compared to younger men.Additionally, rFD usually decreases with increasing shortening velocity (Maréchal and Plaghki, 1979;Meijer et al., 1998;Power et al., 2014;Fortuna et al., 2017), however, our results showed no significant differences in rFD between shortening speeds (Fig. 7).Previous studies in humans have also found that rFD is reduced during SSCs compared to pure shortening contractions (Seiberl et al., 2015;Hahn and Riedel, 2018).Conversely, our results showed that rFD was greater following SSC contractions compared to pure shortening contractions (Fig. 7).
Our observations regarding instantaneous stiffness and work help to explain our rFD findings.Instantaneous stiffness was decreased following SSCs compared to pure shortening contractions (Fig. 8), which is consistent with previous literature stating that stiffness decreases in proportion to the magnitude of rFD, indicating a decreased proportion of attached cross-bridges (Sugi and Tsuchiya, 1988;Lee and Herzog, 2003;Power et al., 2014).rFD is also generally regarded as proportional to the mechanical work (force multiplied by displacement) performed during shortening, because greater work exacerbates actin angular deformation-the primary mechanism for rFD (Josephson and Stokes, 1999;Herzog et al., 2000).This association between work and rFD was not always observed in our study, as during our pure shortening contractions more work was performed at slow speeds, however, rFD did not differ between speeds (Figs. 6 and 7).During SSC contractions, it has been found that rFD is not dependent on the work performed and may depend on the amount of rFE produced during the lengthening phase, which offsets rFD (Fortuna et al., 2016;Fortuna et al., 2017;Fortuna et al., 2018).Since we did not assess rFE in our study, we can only speculate on this.We observed a closer association between work and rFD when comparing the SSCs to the purely shortening contractions: both work and rFD were greater for the slow-fast, fast-fast, and slowslow SSCs compared to the pure shortening contractions (Fig. 6), which is consistent with previous reports in humans (Fortuna et al., 2017;Fortuna et al., 2018).Finally, it is important to note that our SSC contractions were always performed after the initial isometric contractions and purely shortening contractions.This methodological consideration could explain why we observed more rFD during our SSC contractions compared to pure shortening contractions, as fibres may have experienced natural force loss as the number of contractions increased.

Implications for humans
Given the results from our study at the cellular level align with the findings of Mc Dermott et al. ( 2023) in humans, we agree with their conclusions that performing SSCs, as opposed to purely shortening contractions, may provide a more effective power development stimulus for older individuals.This indicates that exercise programs involving SSCs, without a pause between eccentric and concentric contractions, may be a good approach for increasing power development due to mechanisms at the single-fibre (i.e., cellular) level.

Methodological considerations
It is important to note that this study was conducted on fibres from male rats.Therefore, we acknowledge that our results may have differed if we tested female rat single fibres as there may be an aspect of female physiology and aging, such as the complex changes in hormones during menopause (Hubbard et al., 2023;Hinks et al., 2024;Mashouri et al., 2024), that could affect outcomes.As such, we suggest that future studies examine sex-differences in this area.Similarly, we tested single fibres from F334BN rats.Although our findings are in line with results of Mc Dermott et al. ( 2023) at the joint level in humans, we acknowledge that we are not able to fully extrapolate our results to humans.Additionally, the muscle chosen for this study was the proximal portion of the psoas major, which is composed almost exclusively of type II fibres.As such, our results may differ in muscles with a more slow-type composition, as the magnitude of the SSC effect has been shown to be greater in slower type muscles such as the soleus (Fukutani and Herzog, 2020).Lastly, to ensure fibre viability throughout testing it is important to note that our testing was conducted at 12 • C, therefore our results may not entirely mimic what occurs under more physiological conditions (i.e., at 37 • C).

Conclusion
The present study investigated whether SSCs could provide protection against age-related power-loss at the cellular level in aged rats.In line with our hypothesis, we found that implementing an active lengthening contraction prior to shortening protected power production in aged rats, as during SSCs with fast shortening contractions old fibres were able to reach the same level of power output as young fibres did during the fast pure shortening contractions.Despite not observing an age-related maintenance of eccentric strength at the single fibre level, SSCs with fast shortening speeds amplified power production, likely owing to pre-activation and active lengthening-induced advantages in cross-bridge kinetics.Therefore, power generation in older adults is likely 'protected' during everyday movements, in part due to the stretchshortening cycle as compared with isolated shortening contractions.).#Significant difference between indicated means (condition) among both young and old age groups (P < 0.05), demonstrating that among both young and old age groups, stiffness measurements were lower following SSC contractions compared to active shortening contractions, however, there were no significant differences within these groups.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 1 .
Fig. 1.Representative force-time (upper graphs) and fibre length-time (lower graphs) traces of a single skinned rat psoas muscle fibre for the fast (0.60 Lo/s) (A) and slow (0.15 Lo/s) active shortening conditions (B).The blue lines indicate that the fibre is being actively shortened, and the blue shaded area under the curve depicts the work performed during shortening.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .
Fig. 2. Representative force-time (upper graphs) and fibre length-time (lower graphs) traces of a single skinned rat psoas muscle fibre for the fast-fast (A), fast-slow (B), slow-fast (C), and slow-slow (D) stretch-shortening cycle conditions.The orange lines indicate that the fibre is being actively lengthened and the blue lines indicate that the fibre is being actively shortened.The orange shaded area under the curve depicts the work performed during lengthening and the blue shaded area under the curve depicts the work performed during shortening.Conditions: Fast =0.60 Lo/s and slow = 0.15 Lo/s.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .
Fig. 3. (A) Absolute force during isometric contractions at fibre lengths corresponding to optimal (2.5 μm) and long (3.0 μm) sarcomere lengths, for young (orange bars, n = 31) and old (blue bars, n = 41) rat single muscle fibres.(B) Specific force (force ÷ CSA) during isometric contractions at fibre lengths corresponding to optimal (2.5 μm) and long (3.0 μm) sarcomere lengths, for young (orange bars, n = 31) and old (blue bars, n = 41) rat single muscle fibres.(C) Stiffness during isometric contractions at fibre lengths corresponding to optimal (2.5 μm) and long (3.0 μm) sarcomere lengths, for young (orange bars, n = 31) and old (blue bars, n = 41) rat single muscle fibres.Data are presented as mean ± SEM. *Significant difference between indicated means (age group) (P < 0.05).#Significant difference between indicated means (fibre length) among both young and old age groups (P < 0.05).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .
Fig. 4. Peak force during the lengthening phase of SSC contractions at all four speed combinations: fast-fast (young: n = 22, old n = 31), fast-slow (young: n = 23, old n = 32), slow-fast (young: n = 21, old n = 36), and slow-slow (young: n = 24, old n = 39), for young (orange bars) and old (blue bars) rat single muscle fibres.Data are presented as mean ± SEM. * Main effect of age, indicating that peak force during active lengthening was lower in old as compared with young among all speed combinations (P < 0.05).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. Power output during the shortening phase of the active shortening and SSC contractions at all speeds: fast (young: n = 31, old n = 41), slow (young: n = 31, old n = 41), fast-slow (young: n = 23, old n = 32), slow-slow (young: n = 24, old n = 39), fast-fast (young: n = 22, old n = 31), and slow-fast (young: n = 21, old n = 36), for young (orange bars) and old (blue bars) rat single muscle fibres.Data are presented as mean ± SEM. *Significant difference between indicated means (age group) (P < 0.05).#Significant difference between indicated means (condition) among both young and old age groups (P < 0.05).The dashed red line depicts the main finding that implementing an active lengthening contraction prior to shortening at fast shortening speeds provided protection against age-related power loss when compared to the power output of young rats during the fast active shortening contractions.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .
Fig. 6.Work performed during the shortening contraction and shortening phase of the SSC contractions at all speeds: fast (young: n = 31, old n = 41), slow (young: n = 31, old n = 41), fast-slow (young: n = 23, old n = 32), slowslow (young: n = 24, old n = 39), fast-fast (young: n = 22, old n = 31), and slow-fast (young: n = 21, old n = 36), for young (orange bars) and old (blue bars) rat single muscle fibres.Data are presented as mean ± SEM. *Significant difference between indicated means (age group) (P < 0.05).#Significant difference between indicated means (condition) among both young and old age groups (P < 0.05).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7 .Fig. 8 .
Fig. 7. Shortening-induced residual force depression for the active shortening and SSC contractions at all speeds, fast (young: n = 31, old n = 41), slow (young: n = 31, old n = 41), fast-slow (young: n = 23, old n = 32), slow-slow (young: n = 24, old n = 39), fast-fast (young: n = 22, old n = 31), and slow-fast (young: n = 21, old n = 36), for young (orange bars) and old (blue bars) rat single muscle fibres.Data are presented as mean ± SEM. *Significant difference between indicated means (age group) (P < 0.05).#Significant difference between indicated means (condition) among both young and old age groups (P < 0.05), demonstrating that among both young and old age groups, the amount of residual force depression was greater following SSC contractions compared to active shortening contractions, however, there were no significant differences within these groups.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)