Fig. 4.

Effects of the hcTnT RCM mutation on IFM myofibrillar properties. (A) Fluorescence (10×) and confocal micrographs (63×, 100×) show no gross structural differences in IFMs (Top) or myofibrils (Middle and Bottom) between control and up^E87K Drosophila. Top (10×) depicts hemithoraces that were flash-frozen and sagittally bisected to reveal IFM fiber morphology. Preparations were stained with Alexa-568 phalloidin to label actin. Z discs were distinguished by the expression of GFP-tagged Zasp52, a protein that binds to α-actinin, thereby restricting GFP fluorescence to Z discs. Dotted rectangular boxes highlight the regions imaged below. Sarcomeres were visualized along myofibrils of the hemithoraces, in situ, by confocal microscopy (Middle, 63×). Myofibrils were also isolated for ex vivo imaging (Bottom, 100×). (Scale bars, 5 μm.) (B) up^E87K sarcomeres, measured from hemithoraces (in situ) or along isolated myofibrils (ex vivo), were significantly shorter than control (n = 220 to 240). Hence, myofibril removal and isolation from IFMs did not affect mutant sarcomere length. (C) Representative image of an IFM myofibril held between a glass microtool and cantilevered force probe for mechanical assessment. (D) up^E87K IFM myofibrils exhibited significantly higher resting tension relative to control, which was restored to WT values postincubation with BDM. Control myofibrils showed no difference in stiffness pre- and post-BDM incubation (n = 7 to 16). Data are presented as mean ± SEM (ns > 0.05; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001).