Pulsatile Myofilament Activity in Myotrem Myopathy Associated with Myogenic Tremor

Figures

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  Figure 1. Stretch activation responses of permeabilized WT and E248K KI soleus fibers. (A–C). Representative sarcomere length and force traces during a stretch activation maneuver of a 1% sarcomere length stretch of permeabilized WT (A,B) and E248K KI (C) soleus fibers. Stretch activation is quantified as the delayed transient force overshoot (PTO) after sarcomere length stretch. On each trace, properties of the stretch-activated response are indicated, which include pre-stretch tension (Po), an initial force spike that coincides with stretch (P1), minimum of quick force decay (P2), rapid force decay rate (krel), delayed force re-development rate (kdf), and delayed force overshoot or stretch activation (PTO), before resolving to a new stretch-activated steady state tension (PSS). Fibers that did not exhibit delayed force redevelopment (B) had PTO and kdf values of zero. (D) Stretch activation was significantly increased in E248K KI soleus muscle fibers, as indexed by delayed force overshoot after stretch (PTO/PO). Values are calculated from n = 38 stretch maneuvers of n′ = 8 fibers from n″ = 5 WT mice and n = 53 stretch maneuvers of n′ = 11 fibers from n″ = 5 E248K KI mice. Data are…

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  Figure 2. Stretch activation properties of permeabilized WT and E248K KI soleus fibers. Stretch activation properties of soleus fibers undergoing stretch maneuvers during submaximal Ca2+ activation (pCa 6.2–6.5). (A) Fibers underwent similar step stretches ranging from 1–4% sarcomere length (p = 0.94). (B) Pre-stretch steady state tension was similar between WT and E248K KI fibers, (p = 0.17). During the stretch maneuver, the calculated maximum force peak (C; *** p = 0.0004), minimum tension of force decay (D; **** p < 0.0001), rapid force decay rate (E; ** p = 0.0046), and delayed forced development rates (F; **** p < 0.0001) were all depressed in E248K KI fibers compared to WT. After the stretch, the resolved stretched-induced steady state tension remained similar between WT and E248K KI fibers (G; p = 0.10). Values are calculated from n = 38 stretch maneuvers of n′ = 8 fibers from n″ = 5 WT mice and n = 53 stretch maneuvers of n′ = 11 fibers from n″ = 8 KI mice. Data are expressed as mean ± SEM and statistical significance was determined by Welch’s t-test (C), or Mann–Whitney test if normality failed (A,B,D,E); ns: not significant.

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  Figure 3. Pulsatile SPOC-like activity in permeabilized E248K KI soleus fibers. (A,B) A sub-population of E248K KI permeabilized soleus fibers exhibited pulsatile sarcomere length oscillations during submaximal Ca2+ activation. The pulses oscillated rhythmically at ~0.5 Hz and persisted throughout the stretch activation protocol. Pulsatile fibers were not observed in the WT group under any tested condition. (A) Representative sarcomere length and force traces of a pulsatile E248K KI fiber undergoing two stretch maneuvers at pCa 6.2. Data are shown over a 55 s period with a sampling rate of 250 Hz and 1000 Hz for sarcomere length and force, respectively. (B) Sarcomere length traces of E248K KI fibers that exhibit unsolicited pulsatile activity in submaximal Ca2+ solutions (pCa 6.2–6.5). Navy, periwinkle, and bright blue represent traces from different (n = 3) pulsatile soleus fibers from three E248K KI mice. Data shown are over a 10 s period of steady-state equilibrium with a sampling rate of 250 Hz.

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  Figure 4. Fiber typing and stretch activation properties of WT and E248K KI soleus fibers. (A) Single fiber SDS-gel electrophoresis and Western blotting were performed on WT, non-pulsatile E248K KI, and pulsatile E248K KI fibers after mechanical measurements. Lysates from whole soleus muscles from WT and E2348K KI mice were included as positive controls, and SYPRO Ruby was used to visualize total protein levels. Blots were probed for slow myosin heavy chain (MHC) and sMyBP-C. Slow-MHC was positively identified in 2/7 (29%) WT, 1/6 (17%) non-pulsatile E248K KI, and 3/3 (100%) pulsatile E248K KI fibers by Western blotting. (B,C) Post-hoc analysis of stretch activation properties were stratified according to fiber type and pulsatile activity. Fiber type was defined by mechanical properties with slow fibers exhibiting a ktr value less than 7 s−1 and fast fibers displaying a ktr values greater than or equal to 7 s−1. (B) Stretch activation (PTO/PO) was comparable among WT, non-pulsatile E248K KI, and pulsatile E248K KI type-I slow fibers. Data are expressed as mean ± SEM. Values are calculated from n = 8 stretch maneuvers of n′ = 2 WT & n′ = 2 non-pulsatile E248K KI fibers, and n = 17 stretch…

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  Figure 5. Proposed model of enhanced stretch activation in the presence of the MYBPC1 E248K Myotrem variant. (A) In submaximal Ca2+ conditions, stretch may allow for lateral displacement of thick and thin filaments, promoting crossbridge formation as part of the stretch activation response, which is (at least partially) regulated by sMyBP-C. (B) Introduction of the MYBPC1 E248K variant, enhances binding of sMyBP-C to myosin. This potentially allows sMyBP-C to provide structural support, thereby upon stretch, myosin is more likely to engage in crossbridge formation leading to enhanced stretch activation.