William Gittings

Myosin phosphorylation and force potentiation in skeletal muscle: evidence from animal models

The contractile performance of mammalian fast twitch skeletal muscle is history dependent. The effect of previous or ongoing contractile activity to potentiate force, i.e. increase isometric twitch force, is a fundamental property of fast skeletal muscle. The precise manifestation of force potentiation is dependent upon a variety of factors with two general types being identified; staircase potentiation referring to the progressive increase in isometric twitch force observed during low frequency stimulation while posttetanic potentiation refers to the step—like increase in isometric twitch force observed following a brief higher frequency (i.e. tetanic) stimulation. Classic studies established that the magnitude and duration of potentiation depends on a number of factors including muscle fiber type, species, temperature, sarcomere length and stimulation paradigm. In addition to isometric twitch force, more recent work has shown that potentiation also influences dynamic (i.e. concentric and/or isotonic) force, work and power at a range of stimulus frequencies in situ or in vitro, an effect that may translate to enhanced physiological function in vivo. Early studies performed on both intact and permeabilized models established that the primary mechanism for this modulation of performance was phosphorylation of myosin, a modification that increased the Ca2+ sensitivity of contraction. More recent work from a variety of muscle models indicates, however, the presence of a secondary mechanism for potentiation that may involve altered Ca2+ handling. The primary purpose of this review is to highlight these recent findings relative to the physiological utility of force potentiation in vivo.

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca2+ concentration ([Ca2+]i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca2+-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca2+ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca2+]i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca2+ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca2+]i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.

PDH activation during in vitro muscle contractions in PDH kinase 2 knockout mice: effect of PDH kinase 1 compensation.

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4 being the most predominant isoforms in skeletal muscle. Although PDK2 is the most abundant isoform, few studies have examined its physiological role. The role of PDK2 on PDH activation (PDHa) at rest and during muscle stimulation at 10 and 40 Hz (eliciting low- and moderate-intensity muscle contractions, respectively) in isolated extensor digitorum longus muscles was studied in PDK2 knockout (PDK2KO) and wild-type (WT) mice (n = 5 per group). PDHa activity was unexpectedly 35 and 77% lower in PDK2KO than WT muscle (P = 0.043), while total PDK activity was nearly fourfold lower in PDK2KO muscle (P = 0.006). During 40-Hz contractions, initial force was lower in PDK2KO than WT muscle (P < 0.001) but fatigued similarly to ∼75% of initial force by 3 min. There were no differences in initial force or rate of fatigue during 10-Hz contractions. PDK1 compensated for the lack of PDK2 and was 1.8-fold higher in PDK2KO than WT muscle (P = 0.019). This likely contributed to ensuring that resting PDHa activity was similar between the groups and accounts for the lower PDH activation during muscle contraction, as PDK1 is a very potent inhibitor of the PDH complex. Increased PDK1 expression appears to be regulated by hypoxia inducible factor-1α, which was 3.5-fold higher in PDK2KO muscle. It is clear that PDK2 activity is essential, even at rest, in regulation of carbohydrate oxidation and production of reducing equivalents for the electron transport chain. In addition, these results underscore the importance of the overall kinetics of the PDK isoform population, rather than total PDK activity, in determining transformation of the PDH complex and PDHa activity during muscle contraction.

The effect of work cycle frequency on the potentiation of dynamic force in mouse fast twitch skeletal muscle.

SUMMARY The purpose of this study was to test the hypothesis that the potentiation of concentric twitch force during work cycles is dependent upon both the speed and direction of length change. Concentric and eccentric forces were elicited by stimulating muscles during the shortening and lengthening phases, respectively, of work cycles. Work cycle frequency was varied in order to vary the speed of muscle shortening and/or lengthening; all forces were measured as the muscle passed though optimal length (Lo). Both concentric and eccentric force were assessed before (unpotentiated control) and after (potentiated) the application of a tetanic conditioning protocol known to potentiate twitch force output. The influence of the conditioning protocol on relative concentric force was speed dependent, with forces increased to 1.19±0.01, 1.25±0.01 and 1.30±0.01 of controls at 1.5, 3.3 and 6.9 Hz, respectively (all data N=9–10 with P<0.05). In contrast, the conditioning protocol had only a limited effect on eccentric force at these frequencies (range: 1.06±0.01 to 0.96±0.03). The effect of the conditioning protocol on concentric work (force × distance) was also speed dependent, being decreased at 1.5 Hz (0.84±0.01) and increased at 3.3 and 6.9 Hz (1.05±0.01 and 1.39±0.01, respectively). In contrast, eccentric work was not increased at any frequency (range: 0.88±0.02 to 0.99±0.01). Thus, our results reveal a hysteresis-like influence of activity-dependent potentiation such that concentric force and/or work were increased but eccentric force and/or work were not. These outcomes may have implications for skeletal muscle locomotor function in vivo.

Interaction of posttetanic potentiation and the catchlike property in mouse skeletal muscle

Introduction: Posttetanic potentiation (PTP) and the catchlike property (CLP) enhance contractile function in skeletal muscle. We investigated the CLP during dynamic performance in mouse hindlimb muscles with (wild‐type) and without (skMLCK‐/‐) the primary mechanism for PTP (myosin phosphorylation) (in vitro, 25°C). Methods: Extensor digitorum longus muscles of both genotypes were stimulated with constant frequency and catchlike trains (CFT and CLT), before and after a potentiating stimulus (PS). Results: Before the PS, the CLT increased concentric force/work relative to the CFT, but this effect was greater for skMLCK‐/‐ than wild‐type muscles. After the PS, the catchlike effect was reduced in wild‐type muscles but unchanged in skMLCK‐/‐ muscles that did not display PTP. Conclusions: These data suggest that PTP interferes with the CLP during concentric force development at moderate speeds of shortening. We conclude that the physiological utility of each mechanism and their interactions provide important modulations to fast skeletal muscle function. Muscle Nerve 54: 308–316, 2016

The force dependence of isometric and concentric potentiation in mouse muscle with and without skeletal myosin light chain kinase.

The isometric potentiation associated with myosin phosphorylation is force dependent. The purpose of this study was to assess the influence of a pre-existing period of isometric force on the concentric force potentiation displayed by mouse muscles with and without the ability to phosphorylate myosin. We tested isometric (ISO) and concentric (CON) potentiation, as well as concentric potentiation after isometric force (ISO-CON), in muscles from wild-type (WT) and skeletal myosin light chain kinase-deficient (skMLCK(-/-)) mice. A conditioning stimulus increased (i.e., potentiated) mean concentric force in the ISO-CON and CON conditions to 1.31 ± 0.02 and 1.35 ± 0.02 (WT) and to 1.19 ± 0.02 and 1.21 ± 0.01 (skMLCK(-/-)) of prestimulus levels, respectively (data n = 6-8, p < 0.05). No potentiation of mean isometric force was observed in either genotype. The potentiation of mean concentric force was inversely related to relative tetanic force level (P/Po) in both genotypes. Moreover, concentric potentiation varied greatly within each contraction type and was negatively correlated with unpotentiated force in both genotypes. Thus, although no effect of pre-existing force was observed, strong and inverse relationships between concentric force potentiation and unpotentiated concentric force may suggest an influence of attached and force-generating crossbridges on potentiation magnitude in both WT and skMLCK(-/-) muscles.

Musculoskeletal structure and function in response to the combined effect of an obesogenic diet and age in male C57BL/6J mice

SCOPE The effects of a long-term high fat and sucrose diet (HFS) superimposed with aging on bone and muscle structure and/or function. METHODS AND RESULTS Male C57BL/6J mice (20 weeks of age) were randomized to 1 of 3 groups: baseline (BSL, n = 12), or assigned to a control (AGE, n = 12) or HFS (HFS-AGE, n = 11) diet for 13 weeks. Trabecular bone structure, volumetric bone mineral density (vBMD), and body composition, were measured longitudinally at 20, 24, and 32 weeks of age. In vitro contractile measures were performed on isolated soleus and extensor digitorum longus (EDL) muscles for each group. Both AGE and HFS-AGE had similar declines in trabecular bone structure, while HFS-AGE resulted in increased soleus cross-sectional area (CSA) compared to AGE, but this did not translate to greater twitch or tetanic peak force. The ratio of outcomes of bone to muscle declined in both AGE and HFS-AGE compared to BSL as a result of greater declines in trabecular bone structure than muscle function. CONCLUSION Consumption of a 13-week HFS diet at 20 weeks of age did not exacerbate age-related declines in bone or muscle, but these tissues do not decline in a coordinate manner with greater declines in bone than muscle.

Myosin phosphorylation potentiates steady-state work output without altering contractile economy of mouse fast skeletal muscles

ABSTRACT Skeletal myosin light chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chain (RLC) increases (i.e. potentiates) mechanical work output of fast skeletal muscle. The influence of this event on contractile economy (i.e. energy cost/work performed) remains controversial, however. Our purpose was to quantify contractile economy of potentiated extensor digitorum longus (EDL) muscles from mouse skeletal muscles with (wild-type, WT) and without (skMLCK ablated, skMLCK−/−) the ability to phosphorylate the RLC. Contractile economy was calculated as the ratio of total work performed to high-energy phosphate consumption (HEPC) during a period of repeated isovelocity contractions that followed a potentiating stimulus (PS). Consistent with genotype, the PS increased RLC phosphorylation measured during, before and after isovelocity contractions in WT but not in skMLCK−/− muscles (i.e. 0.65 and 0.05 mol phosphate mol−1 RLC, respectively). In addition, although the PS enhanced work during repeated isovelocity contractions in both genotypes, the increase was significantly greater in WT than in skMLCK−/− muscles (1.51±0.03 versus 1.10±0.05, respectively; all data P<0.05, n=8). Interestingly, the HEPC determined during repeated isovelocity contractions was statistically similar between genotypes at 19.03±3.37 and 16.02±3.41 μmol P; respectively (P<0.27). As a result, despite performing significantly more work, the contractile economy calculated for WT muscles was similar to that calculated for skMLCK−/− muscles (i.e. 5.74±0.67 and 4.61±0.71 J kg−1 μmol−1 P, respectively (P<0.27). In conclusion, our results support the notion that myosin RLC phosphorylation enhances dynamic contractile function of mouse fast skeletal muscle but does so without decreasing contractile economy. Summary: Myosin regulatory light chain phosphorylation potentiates dynamic contractile function of mouse fast skeletal muscles in vitro without decreasing contractile economy.

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation

ABSTRACT Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK−/−) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK−/− muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK−/− muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK−/− muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK−/− muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK−/− muscles without RLC phosphorylation. Summary: Myosin regulatory light chain phosphorylation increases the contractile economy (mechanical output:metabolic input) of wild-type mouse fast muscle compared with muscles devoid of the enzyme responsible for regulatory light chain phosphorylation.

Epinephrine augments posttetanic potentiation in mouse skeletal muscle with and without myosin phosphorylation

Sympathetic tone may influence force potentiation, that is, the stimulation‐induced increase in skeletal muscle mechanical function associated with myosin phosphorylation, although the mechanism for this effect remains unknown. The purpose of this study was to examine the influence of epinephrine on concentric twitch force potentiation of wild‐type and skeletal myosin light‐chain kinase devoid mouse muscle (skMLCK−/−). To this end, concentric twitch force was assessed before and after a potentiating stimulus (PS) to determine the peak and the duration of potentiation in the absence (−EPI) and presence (+EPI) of 1 μmol/L epinephrine in both genotypes. Twitch force of wild‐type and skMLCK−/− muscles was increased by up to 31 and 35% and 18 and 23% in the −EPI and EPI conditions, respectively (all data n = 8, P < 0.05). In wild‐type muscles, the PS increased RLC phosphorylation from 0.14 ± 0.05 (rest) to 0.66 ± 0.08 mol phos mol RLC; by 480 sec RLC phosphorylation had returned to baseline (all data n = 4 each time point, P < 0.05). Neither resting nor peak levels of RLC phosphorylation were altered by +EPI, although the duration of RLC phosphorylation was prolonged. In skMLCK−/− muscles, RLC phosphorylation was not elevated above constituent levels by stimulation in either the −EPI or +EPI condition. Thus, given the similarity in potentiation responses between genotypes our data suggest that the influence of epinephrine on potentiation was independent of skMLCK catalyzed phosphorylation of the RLC, although the clinical significance of this pathway for skeletal muscle function remains to be identified.