Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegß deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegß but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg ß (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it's low levels, HA-Spegß immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegß binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegß display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

MyoSight-semi-automated image analysis of skeletal muscle cross sections.

BACKGROUND: Manual analysis of cross-sectional area, fiber-type distribution, and total and centralized nuclei in skeletal muscle cross sections is tedious and time consuming, necessitating an accurate, automated method of analysis. While several excellent programs are available, our analyses of skeletal muscle disease models suggest the need for additional features and flexibility to adequately describe disease pathology. We introduce a new semi-automated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses. RESULTS: We describe staining and imaging methods that generate high-quality images of immunofluorescent-labelled cross sections from mouse skeletal muscle. Using these methods, we can analyze up to 5 different fluorophores in a single image, allowing simultaneous analyses of perinuclei, central nuclei, fiber size, and fiber-type distribution. MyoSight displays high reproducibility among users, and the data generated are in close agreement with data obtained from manual analyses of cross-sectional area (CSA), fiber number, fiber-type distribution, and number and localization of myonuclei. Furthermore, MyoSight clearly delineates changes in these parameters in muscle sections from a mouse model of Duchenne muscular dystrophy (mdx). CONCLUSIONS: MyoSight is a new program based on an algorithm that can be optimized by the user to obtain highly accurate fiber size, fiber-type identification, and perinuclei and central nuclei per fiber measurements. MyoSight combines features available separately in other programs, is user friendly, and provides visual outputs that allow the user to confirm the accuracy of the analyses and correct any inaccuracies. We present MyoSight as a new program to facilitate the analyses of fiber type and CSA changes arising from injury, disease, exercise, and therapeutic interventions.

The role of myostatin and activin receptor IIB in the regulation of unloading-induced myofiber type-specific skeletal muscle atrophy.

Chronic unloading induces decrements in muscle size and strength. This adaptation is governed by a number of molecular factors including myostatin, a potent negative regulator of muscle mass. Myostatin must first be secreted into the circulation and then bind to the membrane-bound activin receptor IIB (actRIIB) to exert its atrophic action. Therefore, we hypothesized that myofiber type-specific atrophy observed after hindlimb suspension (HLS) would be related to myofiber type-specific expression of myostatin and/or actRIIB. Wistar rats underwent HLS for 10 days, after which the tibialis anterior was harvested for frozen cross sectioning. Simultaneous multichannel immunofluorescent staining combined with differential interference contrast imaging was employed to analyze myofiber type-specific expression of myostatin and actRIIB and myofiber type cross-sectional area (CSA) across fiber types, myonuclei, and satellite cells. Hindlimb suspension (HLS) induced significant myofiber type-specific atrophy in myosin heavy chain (MHC) IIx (P < 0.05) and MHC IIb myofibers (P < 0.05). Myostatin staining associated with myonuclei was less in HLS rats compared with controls, while satellite cell staining for myostatin remained unchanged. In contrast, the total number myonuclei and satellite cells per myofiber was reduced in HLS compared with ambulatory control rats (P < 0.01). Sarcoplasmic actRIIB staining differed between myofiber types (I < IIa < IIx < IIb) independent of loading conditions. Myofiber types exhibiting the greatest cytoplasmic staining of actRIIB corresponded to those exhibiting the greatest degree of atrophy following HLS. Our data suggest that differential expression of actRIIB may be responsible for myostatin-induced myofiber type-selective atrophy observed during chronic unloading.