Piperine disrupts the OFF state of the resting thick filament of rat skeletal muscle, enhancing dynamic contractility in a fibre‐type‐dependent manner

Figures

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  Figure 1. Experimental setup and main protocol for whole‐muscle contractility experiments A, contralateral pairs of intact fast (extensor digitorum longus, EDL) or slow (soleus) rat skeletal muscles were dissected and mounted in the illustrated setup. B, the main experimental protocol used to test the isolated effects of piperine (vs. vehicle), and the ability to wash‐out the effects of piperine, on dynamic contractility via two distinct incubation sequences: (a) and (b). Dynamic contractility was assessed by performing force–velocity (FV) tests at baseline (FV1), after a 1 h incubation (FV2), and after a second 1 h incubation (FV3). The illustrations are made using bioRender.com and Microsoft PowerPoint.

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  Figure 2. Schematic of the resting thick filament structure assessed by fibre small‐angle X‐ray diffraction (MyoXRD) experiments A, representative MyoXRD patterns with reflections originating from longitudinally (meridional) and transversely (equatorial) repeating sarcomeric structures. The representative image is split vertically, showing a baseline (pre) image on the left and a post‐piperine image on the right, from a single soleus and a single extensor digitorum longus (EDL) bundle in the top and bottom halves, respectively. Each quadrant of the representative image is a merge of 25–50 diffraction images from the same bundle, acquired during a line scan with 100 ms exposures and a sample‐to‐detector distance of 9.8 m. B, an illustration of the sarcomeric structures from which the diffractions arise. Red lines show the primary origin of the reflection. The following outcomes were extracted: the equatorial intensity ratio (I 1,1/I 1,0), the spacings of the M3 and M6 reflections (S M3 and S M6), the intensity of the M3 reflection (I M3) and the inter‐thick‐filament lattice spacing (d 1,0). The 1,0 reflection arises from the myosin‐containing thick filament (blue) in isolation, where the…

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  Figure 3. Effects of piperine on resting thick filament structure in bundles of fast (extensor digitorum longus, EDL) rat skeletal fibres Values are shown from individual bundles from testing at baseline (black squares) and after 20 min exposure to piperine (blue circles). Repeated measurements from the same bundle are shown by connecting lines, and the model‐based mean ± observed SD are shown as red lines with error bars. The following outcomes are shown: the equatorial intensity ratio (I 1,1/I 1,0) (A), the spacing and intensity of the M3 reflection (S M3 and I M3) (B and C), the spacing of the M6 reflection (S M6) (D) and the inter‐thick‐filament lattice spacing (d 1,0) (E). Finally, we present a schematic illustrating the interpretation of the piperine‐induced changes in sarcomeric structure (F). P values show paired comparisons of baseline and piperine values from 4–13 bundles using linear mixed‐effects models. The illustration in (F) is made using bioRender.com.

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  Figure 4. Effects of piperine on resting thick filament structure in bundles of slow (soleus) rat skeletal fibres Values are shown from individual bundles from testing at baseline (black squares) and after 20 min exposure to piperine (blue circles). Repeated measurements from the same bundle are shown by connecting lines, and the model‐based mean ± observed SD are shown as red lines with error bars. The following outcomes are shown: the equatorial intensity ratio (I 1,1/I 1,0) (A), the spacing and intensity of the M3 reflection (S M3 and I M3) (B and C), the spacing of the M6 reflection (S M6) (D) and the inter‐thick‐filament lattice spacing (d 1,0) (E). Finally, we present a schematic illustrating the interpretation of the piperine‐induced changes in sarcomeric structure (F). P values show paired comparisons of baseline and piperine values from 6–11 bundles using linear mixed‐effects models. The illustration in (F) is made using bioRender.com.

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  Figure 5. Effects of piperine on isometric contractility of intact fast (extensor digitorum longus, EDL) rat skeletal muscles Exemplary force traces are shown from EDL muscles exposed to vehicle and piperine at the start (0 min) and end (60 min) of the first incubation. Force traces (n = 1 muscle per treatment) from twitch (2 Hz) (A), unfused tetanic (50 Hz) (B) and fused tetanic (150 Hz) (C) contractions are shown. For each contraction, the full force trace is shown together with inserts highlighting the phases of force development and relaxation. Force relaxation in (C) is illustrated starting from the time point of the final stimuli and shows the inflection points (group averages) distinguishing the slow (isometric) and fast (chaotic) phases of force relaxation after fused tetani.

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  Figure 6. Effects of piperine on isometric muscle function of intact slow (soleus) rat skeletal muscles Exemplary force traces are shown from soleus muscles exposed to vehicle and piperine at the start (0 min) and end (60 min) of the first incubation. Force traces (n = 1 muscle per treatment) from twitch (2 Hz) (A), unfused tetanic (20 Hz) (B) and fused tetanic (80 Hz) (C) contractions are shown. For each contraction, the full force trace is shown together with inserts highlighting the phases of force development and relaxation. Force relaxation in (C) is illustrated starting from the time point of the final stimuli and shows the inflection points (group averages) distinguishing the slow (isometric) and fast (chaotic) phases of force relaxation after fused tetani.

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  Figure 7. Assessment of low‐ and high‐frequency dynamic contractility by force–velocity testing A, exemplary low‐frequency (purple) and high‐frequency (black) contractions (n = 1 extensor digitorum longus (EDL) muscle) at relative loads of 5%, 40% and 100% of maximal low‐ and high‐frequency isometric force (F max) with corresponding displacements of the lever arm. Measured steady forces and shortening velocities were used to construct force–velocity (FV) and power curves. B and C, baseline (FV1) low‐frequency (purple squares) and high‐frequency (black circles) EDL and soleus FV (filled symbols, solid lines) and power (open symbols, dotted lines) curves, respectively. Points on the FV (left y‐axis) and power curves (right y‐axis) are shown as mean values with vertical error bars showing SD on the y‐axis. Horizontal error bars show the SD of the measured force as a percentage of the maximal isometric force (F max). Corresponding Hill curves are shown as mean lines; n = 6 EDL and 6 soleus muscles.

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  Figure 8. Effects of piperine on low‐frequency dynamic contractility of fast (extensor digitorum longus, EDL) and slow (soleus) rat skeletal muscles Top: curves show absolute low‐frequency EDL (60 Hz) (A) and soleus (20 Hz) (D) force–velocity (FV) curves (filled symbols, solid lines) and power curves (open symbols, dotted lines) after contralateral piperine (blue squares) and vehicle (orange circles) treatments. Curves show group means as absolute values. Individual points on the FV curves (left y‐axis) and power curves (right y‐axis) are shown as absolute mean values with vertical error bars showing SD on the respective y‐axis. Horizontal error bars show the SD of the measured absolute force for each contraction. The boxes show P values from statistical analyses of the main effects of treatment (piperine and vehicle), load (5%, 15%, 27.5%, 40%, 60%, 80% and 100% of initial F max) and their interaction (treatment × load) by two‐way ANOVAs (soleus) and mixed‐effects models (EDL). An asterisk (*) and hash symbol (#) with the associated P value denotes a significant post hoc test showing a difference in contractile power and isometric force (paired t test), respectively, between treatments at…

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  Figure 9. Effects of piperine on high‐frequency dynamic contractility of fast (extensor digitorum longus, EDL) and slow (soleus) rat skeletal muscles Top: curves show absolute high‐frequency EDL (150 Hz) (A) and soleus (80 Hz) (D) force–velocity (FV) curves (filled symbols, solid lines) and power curves (open symbols, dotted lines) after contralateral piperine (blue squares) and vehicle (orange circles) treatments. Curves show group means as absolute values. Individual points on the FV curves (left y‐axis) and power curves (right y‐axis) are shown as absolute mean values with vertical error bars showing SD on the respective y‐axis. Horizontal error bars show the SD of the measured absolute force for each contraction. The boxes show P values from statistical analyses of the main effects of treatment (piperine and vehicle), load (5%, 15%, 27.5%, 40%, 60%, 80% and 100% of initial F max) and their interaction (treatment × load) by two‐way ANOVAs (soleus) and mixed‐effects models (EDL). An asterisk (*) with the associated P value denotes a significant post hoc test showing a difference in contractile power between treatments at the given relative load when normalised to baseline (FV1). Mean r 2…

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  Figure A1. Example force‐ and length traces from each low‐ and high‐frequency contraction used to construct force–velocity (FV) curves Data from extensor digitorum longus (EDL) (n = 1) and soleus (n = 1) muscles. To determine the low‐ and high‐frequency force–velocity (FV) curves, each muscle performed electrically induced contractions against varying external loads (5%–100% of low‐ and high‐frequency maximal isometric force). The approach was to determine the FV curve by isotonic contractions using the afterload method. Hence, an isometric force was developed until the desired force was achieved (as a percentage of the baseline‐defined maximal isometric force indicated above each panel), after which the muscle was allowed to shorten against the relative load.