Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers

Figures

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  FIGURE 1. Exchange of native slow-twitch skeletal troponin complex (ssTn) complex for IANBD-labeled fsTn (fsTn-IANBD) complex. (A) Spatial and temporal equilibration of fsTn-IANBD within the cross-section of a single slow-twitch human fiber imaged by confocal microscopy. Clear insets at the left of each picture represent mean intensity profiles in transverse direction of the fiber that were integrated over a longitudinal length of 20–25 sarcomeres. Sarcomere length is 2.4 μm. White bar: 50 μm. (B) Western blot analysis showing TnI from single skeletal muscle fibers. Lanes 1 and 5 show the TnI content in single rabbit fast-twitch psoas muscle fibers [R.P. fast-twitch skeletal troponin I subunit (fsTnI)], and lanes 2 and 6 the TnI content from single human slow-twitch soleus muscle fibers [H.S. slow skeletal troponin I subunit (ssTnI)]. Lanes 3, 4, 7, and 8 show the TnI content from single human slow-twitch soleus muscle fibers after 3.5 h incubation in 0.5 mg/mL fsTn-IANBD and performing the mechanical experiments. Note that after the troponin exchange procedure ssTnI is reduced and fsTnI is present in the human slow-twitch fibers, confirming the exchange of ssTn by fsTn-IANBD.

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  FIGURE 2. Effect of fsTn-IANBD exchange and of the experimental protocol on force generation in slow-twitch human soleus (n = 8) and fast-twitch rabbit psoas (n = 5) muscle fibers. (A) Normalized maximal force (pCa 4.5) after mounting the fiber, after troponin exchange, after measuring the pCa-force relationship (before AmBleb experiments), and after incubation in 50 μM AmBleb. 100% corresponds to maximal force before fsTn-IANBD exchange. Note that maximal force after troponin exchange is not significantly different between both preparations. (B) Same as in A for force at pCa 7.5. Note that increases in force are not statistically significant.

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  FIGURE 3. Effect of fsTn-IANBD exchange and of the experimental protocol on the rate constant of force redevelopment, ktr. (A) ktr max (pCa 4.5) after mounting the fiber, after troponin exchange, and after measuring the pCa-force relationship (before AmBleb experiments) in human soleus slow-twitch muscle fibers (n = 8). (B) Comparison of ktr max (pCa 4.5) after fsTn-IANBD exchange in slow-twitch (human soleus) and fast-twitch (rabbit psoas, n = 5) fibers. 100% corresponds to maximal ktr before fsTn-IANBD exchange. (C) Force–ktr relationship after fsTn-IANBD exchange in slow-twitch fibers. Force is normalized to force at pCa 4.5. ktr max before troponin exchange is shown for direct comparison.

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  FIGURE 4. Experimental protocol for measuring steady state activation and dynamic inactivation of the thin filament. The dotted lines show the time points of the switch from isometric to isotonic (slightly loaded) and back to isometric conditions. (A) Total length of the muscle fiber (expressed in length transducer signal voltage). (B) Force signal. Note that the fiber does not slack during the isotonic phase, since a constant, small amount of tension is kept. (C) N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) emission intensity during isometric and isotonic phases. Note the difference in intensity immediately before and immediately after the isotonic phase. (D) Sarcomere length during isometric and isotonic phases was used as a correction factor for IANBD emission intensity. (E) IANBD emission intensity multiplied by sarcomere length (SL). This corrected IANBD emission does not show any step at the transitions between isotonic and isometric contraction. Thus, this signal shows only emission changes induced by inactivation (during isotonic phase) and reactivation (during the subsequent isometric phase) of the troponin complex while excludes alterations…

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  FIGURE 5. Steady state force and thin filament activation with and without the activating effect of myosin at different calcium concentrations in human slow-twitch fibers. (A) Inhibition of active force by incubation in increasing AmBleb concentration. n = 4. (B) pCa-force relationship in exchanged muscle fibers before (empty circles) and after (filled circles) incubation in 50 μM AmBleb. (C) pCa-IANBD emission intensity relationship before (empty circles) and after (filled circles) incubation in 50 μM AmBleb. Note the clearly diminished activation of the thin filament throughout the whole pCa scale when it is induced only by calcium (after incubation in AmBleb). The vertical bars show the relative magnitude of the thin filament activation induced by calcium and strong binding cross-bridges (CB). The combination of both factors (100%) accounts for the total fluorescence signal observed between pCa 7.5 and 4.5. (D) Subtracting the fluorescence signals in C reveals that AmBleb treatment increased the fluorescence emission intensity (less activation of the thin filament) and that the myosin contribution to thin filament activation increases with the calcium concentration. n = 8 in B, C, and…

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  FIGURE 6. Unloading-induced deactivation of the thin filament. (A) Example of isotonic shortening phases of a single muscle fiber. Increasing the concentration of calcium induced a 29.6% decrease of fluorescence intensity (from 2.94 V at pCa 7.5 to 2.07 V at pCa 4.5). The dotted line shows the transition from isometric to isotonic contraction. (B) Same as in A after 10 min incubation in 50 μM AmBleb in relaxing solution. Increasing the calcium concentration induced only an 18.9% decrease of fluorescence intensity (from 2.48 V at pCa 7.5 to 2.01 V at pCa 4.5). (C) The rate constant of the thin filament deactivation induced by switching from isometric contraction to isotonic shortening phase [observed rate constant (kobs)] increased from ≈10 to ≈ 150 s– 1 between pCa 7.5 and 4.5. Note that the rate constant of force redevelopment after re-stretching the fiber back to isometric conditions (ktr) is ≈10–50-fold lower than kobs at same calcium concentration. (D) The amplitude of increase in IANBD emission induced by unloading is normalized to the delta of emission intensity between pCa 7.5 and 4.5. All traces in A and B represent averages over of six cycles. In C and D, n = 8.