Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca 2+ ) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca 2+ ] or blocking L-type Ca 2+ channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca 2+ signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.excitation-contraction coupling | dihydropyridine receptor | triad junction | muscle injury D ysferlinopathies are degenerative myopathies secondary to mutations in the gene encoding the protein dysferlin. These myopathies, most commonly limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are independent of motor neuron activation (1), indicating that they are myogenic in origin. Dysferlin is a 230-kDa protein composed of seven C2 domains with homology to synaptotagmin (2, 3) and a single transmembrane domain near its C terminus (4, 5). The complexity of dysferlin's potential role in muscle is highlighted by the number of its purported functions, including membrane repair (2, 3), vesicle fusion (4), microtubule regulation (5, 6), cell adhesion (7,8), and intercellular signaling (9). Understanding the contributions of dysferlin to the maintenance of normal skeletal muscle function is critical for the development of appropriate therapies for patients diagnosed with LGMD2B and MM.Recently, we demonstrated the localization of dysferlin at the A-I junction in mature muscle fibers (10). These results agree with earlier reports associating dysferlin with the dihydropyridine receptor (DHPR, L-type Ca 2+ channel), Ahnak, caveolin 3, and several other proteins involved in Ca 2+ -based signaling and the function of transverse (t-) tubules (11)(12)(13)(14). Consistent with this localization and the potential for a functional role in this specialized compartment, dysferlin-deficient murine muscle demonstrates altered transverse tubule (t-tubule) structure (15) as well as increased oxidative stress (16, 17), inflammation, and necrosis (18-20) after injury.Here we demonstrate that dysferlin is enriched in the t-tubule membrane, where it contributes to the maintenance of the t-tubule and Ca 2+ homeostasis. We show...

Cardiac contraction is initiated by the release of Ca2+ from intracellular stores in response to an action potential, in a process known as “excitation-contraction coupling” (ECC). Here we investigate the maturation of ECC in the rat heart during postnatal development. We provide new information on how proteins of the sarcoplasmic reticulum (SR) and the t-tubules (TTs) assemble to form the structures that support EC coupling during postnatal development. We show that the surface membrane protein, caveolin-3 (Cav3), is a good protein marker for TTs in ventricular myocytes and compared it quantitatively to junctophilin-2 (JP2), a protein found on the SR at sites of SR-TT junctions, or couplons. Although JP2 and Cav3 associate primarily with the SR and TTs, respectively, we found that, they occupy the appropriate sites at maturing structures in synchrony, as visualized with high resolution, quantitative 3-dimensional imaging. We also found the surprising result that while both ryanodine receptor type 2, (RyR2) and JP2 proteins are localized to the same membrane and sub-compartments, they assume their positions at very different rates: RyR2 moves to the SR membrane at the Z-disc very early in development while JP2 only appears in the SR membrane as the TTs mature. Our data suggest that, although RyR2 appears to be prepositioned at the sites ultimately occupied by dyad junctions, JP2 arrives at these sites in synchrony with the development of the TTs at the Z-discs. Finally, we report that EC coupling efficiency changes with development, in concert with these structural changes. Thus we provide the first well-integrated information that links the developing organization of proteins underlying EC coupling (RyR2, DHPR, Cav3 and JP2) to the developing efficacy of EC coupling.

X-ROS signaling is a novel redox signaling pathway that links mechanical stress to changes in [Ca2+]i. This pathway is activated rapidly and locally within a muscle cell under physiological conditions, but can also contribute to Ca2+-dependent arrhythmia in heart and to the dystrophic phenotype in heart and skeletal muscle. Upon physiologic cellular stretch, microtubules serve as mechanotransducers to activate NADPH oxidase 2 in the transverse tubules and sarcolemmal membranes to produce reactive oxygen species (ROS). In heart, the ROS acts locally to activate ryanodine receptor Ca2+ release channels in the junctional sarcoplasmic reticulum, increasing the Ca2+ spark rate and “tuning” excitation-contraction coupling. In skeletal muscle, where Ca2+ sparks are not normally observed, the X-ROS signaling process is muted. However in muscular dystrophies, such as Duchenne Muscular Dystrophy and dysferlinopathy, X-ROS signaling operates at a high level and contributes to myopathy. Importantly, Ca2+ permeable stretch-activated channels are activated by X-ROS and contribute to skeletal muscle pathology. Here we review X-ROS signaling and mechanotransduction in striated muscle, and highlight important questions to drive future work on stretch-dependent signaling. We conclude that X-ROS provides an exciting mechanism for the mechanical control of redox and Ca2+ signaling, but much work is needed to establish its contribution to physiologic and pathophysiologic processes in diverse cell systems.

Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum (SR) that are responsible for Ca 2+ release in rat ventricular myocytes. Localization of RyR2s is therefore crucial for our understanding of contraction and other Ca 2+ -dependent intracellular processes. Recent results (e.g. circular waves and Ca 2+ sparks in perinuclear area) raised questions about the classical views of RyR2 distribution and organization within ventricular cells. A Ca 2+ spark is a fluorescent signal reflecting the activation of a small group of RyR2s. Frequency and spatio-temporal characteristics of Ca 2+ sparks depend on the state of cytoplasmic and intraluminal macromolecular complexes regulating cardiac RyR2 function. We employed electron microscopy, confocal imaging of spontaneous Ca 2+ sparks and immunofluorescence to visualize the distribution of RyR2s in ventricular myocytes and to evaluate the local involvement of the macromolecular complexes in regulation of functional activity of the RyR2 group. An electron microscopy study revealed that the axial tubules of the transverse-axial tubular system probably do not have junctions with the network SR (nSR). The nSR was found to be wrapped around intermyofibrillar mitochondria and contained structures similar to feet of the junctional cleft. Treatment of ventricular myocytes with antibodies against RyR2 showed that in addition to the junctional SR, a small number of RyR2s can be localized at the middle of the sarcomere and in the zone of perinuclear mitochondria. Recordings of spontaneous Ca 2+ sparks showed the existence of functional groups of RyR2s in these intracellular compartments. We found that within the sarcomere about 20% of Ca 2+ sparks were not colocalized with the zone of the junctional or corbular SR (Z-line zone). The spatio-temporal characteristics of sparks found in the Z-line and A-band zones were very similar, whereas sparks from the zone of the perinuclear mitochondria were about 25% longer. Analysis of the initiation sites of Ca 2+ sparks within the same junctional SR cluster suggested that 18-25 RyR2s are in the functional group producing a spark. Because of the similarity of the spatio-temporal characteristics of sarcomeric sparks and ultrastructural characteristics of nSR, we suggest that the functional groups of RyR2s in the middle of the sarcomere are macromolecular complexes of ∼20 RyR2s with regulatory proteins. Our data allowed us to conclude that a significant number of functional RyR2s is located in the middle of the sarcomere and in the zone of perinuclear mitochondria. These RyR2s could contribute to excitation-contraction coupling, mitochondrial and nuclear signalling, and Ca 2+ -dependent gene regulation, but their existence raises many additional questions.