Rene Vandenboom

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca(2+)/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca(2+) binding to calmodulin forming a (Ca(2+))(4)•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca(2+) results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca(2+)/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca(2+)-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.

Skeletal muscle PLIN proteins, ATGL and CGI-58, interactions at rest and following stimulated contraction

Evidence indicates that skeletal muscle lipid droplet-associated proteins (PLINs) regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN1 is thought to regulate lipolysis by directly interacting with comparative gene identification-58 (CGI-58), an activator of adipose triglyceride lipase (ATGL). Upon lipolytic stimulation, PLIN1 is phosphorylated, releasing CGI-58 to fully activate ATGL and initiate triglyceride breakdown. The absence of PLIN1 in skeletal muscle leads us to believe that other PLIN family members undertake this role. Our purpose was to examine interactions between PLIN2, PLIN3, and PLIN5, with ATGL and its coactivator CGI-58 at rest and following contraction. Isolated rat solei were incubated for 30 min at rest or during 30 min of intermittent tetanic stimulation [150-ms volleys at 60 Hz with a train rate of 20 tetani/min (25°C)] to maximally stimulate intramuscular lipid breakdown. Results show that the interaction between ATGL and CGI-58 increased 128% following contraction (P = 0.041). Further, ATGL interacts with PLIN2, PLIN3, and PLIN5 at rest and following contraction. The PLIN2-ATGL interaction decreased significantly by 21% following stimulation (P = 0.013). Both PLIN3 and PLIN5 coprecipitated with CGI-58 at rest and following contraction, while there was no detectable interaction between PLIN2 and CGI-58 in either condition. Therefore, our findings indicate that in skeletal muscle, during contraction-induced muscle lipolysis, ATGL and CGI-58 strongly associate and that the PLIN proteins work together to regulate lipolysis, in part, by preventing ATGL and CGI-58 interactions at rest.

Subcellular localization of skeletal muscle lipid droplets and PLIN family proteins OXPAT and ADRP at rest and following contraction in rat soleus muscle

Skeletal muscle lipid droplet-associated proteins (PLINs) are thought to regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN2 [adipocyte differentiation-related protein (ADRP)] is found only on lipid droplets, while PLIN5 (OXPAT, expressed only in oxidative tissues) is found both on and off the lipid droplet and may be recruited to lipid droplet membranes when needed. Our purpose was to determine whether PLIN5 is recruited to lipid droplets with contraction and to investigate the myocellular location and colocalization of lipid droplets, PLIN2, and PLIN5. Rat solei were isolated, and following a 30-min equilibration period, they were assigned to one of two groups: 1) 30 min of resting incubation and 2) 30 min of stimulation (n = 10 each). Immunofluorescence microscopy was used to determine subcellular content, distribution, and colocalization of lipid droplets, PLIN2, and PLIN5. There was a main effect for lower lipid and PLIN2 content in stimulated compared with rested muscles (P < 0.05). Lipid droplet distribution declined exponentially from the sarcolemma to the fiber center in the rested muscles (P = 0.001, r(2) = 0.99) and linearly in stimulated muscles (slope = -0.0023 ± 0.0006, P < 0.001, r(2) = 0.93). PLIN2 distribution declined exponentially from the sarcolemma to the fiber center in both rested and stimulated muscles (P < 0.0001, r(2) = 0.99 rest; P = 0.0004, r(2) = 0.98 stimulated), while PLIN5 distribution declined linearly (slope = -0.0085 ± 0.0009, P < 0.0001, r(2) = 0.94 rest; slope=-0.0078 ± 0.0010, P = 0.0003, r(2) = 0.91 stimulated). PLIN5-lipid droplets colocalized at rest with no difference poststimulation (P = 0.47; rest r(2) = 0.55 ± 0.02, stimulated r(2) = 0.58 ± 0.03). PLIN2-lipid droplets colocalized at rest with no difference poststimulation (P = 0.48; rest r(2) = 0.66 ± 0.02, stimulated r(2) = 0.65 ± 0.02). Contrary to our hypothesis, these results show that PLIN5 is not recruited to lipid droplets with contraction in isolated skeletal muscle.

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.

Skeletal muscle PLIN3 and PLIN5 are serine phosphorylated at rest and following lipolysis during adrenergic or contractile stimulation

In adipose tissue, access of adipose triglyceride and hormone‐sensitive lipases (ATGL and HSL) to the lipid droplet depends on PLIN1 phosphorylation, however, PLIN1 is not expressed in skeletal muscle and the phosphorylation of the expressed PLINs has yet to be investigated. Further, direct interactions between skeletal muscle PLINs and HSL are unknown. We investigated the isolated and combined effects of epinephrine and contraction on PLIN‐to‐lipase interactions as well as phosphorylation. Isolated rat solei were assigned to one of four 30 min in vitro conditions (25°C): (1) rest; (2) intermittent tetanic stimulation (60 Hz for 150 msec; train rate 20/min); (3) 5 nmol/L epinephrine; (4) intermittent tetanic stimulation and 5 nmol/L epinephrine. Immunoprecipitation of serine phosphorylated proteins followed by Western blotting for PLIN2, PLIN3, PLIN5, revealed that only PLIN2 is not phosphorylated under any of the experimental conditions. This is the first study to show that in whole rat skeletal muscle PLIN3 and PLIN5 are serine phosphorylated. The degree of serine phosphorylation remained unchanged following adrenergic and/or contractile stimulation. Oil red O staining of muscle sections for lipid content shows a significant decrease following each condition, confirming lipolysis occurred (P < 0.05). PLIN2, 3, and 5 all interact with HSL and ATGL, but these interactions were unchanged following treatments. Our results show that in skeletal muscle, PLIN2 is not serine phosphorylated at rest or with lipolytic stimulation and that while PLIN3, PLIN5 are serine phosphorylated at rest, the degree of phosphorylation does not change with lipolytic stimulation.

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

Juxtaposition of the changes in intracellular calcium and force during staircase potentiation at 30 and 37°C

Temperature-dependent changes in basal calcium and in the calcium transient contribute to force potentiation during repetitive stimulation.