Avian cardiomyocyte architecture and what it reveals about the evolution of the vertebrate heart.

Bird cardiomyocytes are long, thin and lack transverse (t)-tubules, which is akin to the cardiomyocyte morphology of ectothermic non-avian reptiles, who are typified by low maximum heart rates and low pressure development. However, birds can achieve greater contractile rates and developed pressures than mammals, whose wide cardiomyocytes contain a dense t-tubular network allowing for uniform excitation-contraction coupling and strong contractile force. To address this apparent paradox, this paper functionally links recent electrophysiological studies on bird cardiomyocytes with decades of ultrastructure measurements. It shows that it is the strong transsarcolemmal Ca2+ influx via the L-type Ca2+ current (ICaL) and the high gain of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR), coupled with an internal SR Ca2+ release relay system, that facilitates the strong fast contractions in the long thin bird cardiomyocytes, without the need for t-tubules. The maintenance of an elongated myocyte morphology following the post-hatch transition from ectothermy to endothermy in birds is discussed in relation to cardiac load, myocyte ploidy, and cardiac regeneration potential in adult cardiomyocytes. Overall, the paper shows how little we know about cellular Ca2+ dynamics in the bird heart and suggests how increased research efforts in this area would provide vital information in our quest to understand the role of myocyte architecture in the evolution of the vertebrate heart. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'. Please see glossary at the end of the paper for definitions of specialized terms.

CaMKII activation in early diabetic hearts induces altered sarcoplasmic reticulum-mitochondria signaling.

Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca2+ leak and arrhythmias due to increased activity of the Ca2+/calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Autophagy and Endoplasmic Reticulum Stress during Onset and Progression of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a heritable, potentially lethal disease without a causal therapy. AC is characterized by focal cardiomyocyte death followed by inflammation and progressive formation of connective tissue. The pathomechanisms leading to structural disease onset and progression, however, are not fully elucidated. Recent studies revealed that dysregulation of autophagy and endoplasmic/sarcoplasmic reticulum (ER/SR) stress plays an important role in cardiac pathophysiology. We therefore examined the temporal and spatial expression patterns of autophagy and ER/SR stress indicators in murine AC models by qRT-PCR, immunohistochemistry, in situ hybridization and electron microscopy. Cardiomyocytes overexpressing the autophagy markers LC3 and SQSTM1/p62 and containing prominent autophagic vacuoles were detected next to regions of inflammation and fibrosis during onset and chronic disease progression. mRNAs of the ER stress markers Chop and sXbp1 were elevated in both ventricles at disease onset. During chronic disease progression Chop mRNA was upregulated in right ventricles. In addition, reduced Ryr2 mRNA expression together with often drastically enlarged ER/SR cisternae further indicated SR dysfunction during this disease phase. Our observations support the hypothesis that locally altered autophagy and enhanced ER/SR stress play a role in AC pathogenesis both at the onset and during chronic progression.

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography.

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Relative lipid oxidation associates directly with mitochondrial fusion phenotype and mitochondria-sarcoplasmic reticulum interactions in human skeletal muscle.

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.

Improved Calcium Homeostasis and Force by Selenium Treatment and Training in Aged Mouse Skeletal Muscle.

During aging reduction in muscle mass (sarcopenia) and decrease in physical activity lead to partial loss of muscle force and increased fatigability. Deficiency in the essential trace element selenium might augment these symptoms as it can cause muscle pain, fatigue, and proximal weakness. Average voluntary daily running, maximal twitch and tetanic force, and calcium release from the sarcoplasmic reticulum (SR) decreased while reactive oxygen species (ROS) production associated with tetanic contractions increased in aged - 22-month-old - as compared to young - 4-month-old - mice. These changes were accompanied by a decline in the ryanodine receptor type 1 (RyR1) and Selenoprotein N content and the increased amount of a degraded RyR1. Both lifelong training and selenium supplementation, but not the presence of an increased muscle mass at young age, were able to compensate for the reduction in muscle force and SR calcium release with age. Selenium supplementation was also able to significantly enhance the Selenoprotein N levels in aged mice. Our results describe, for the first time, the beneficial effects of selenium supplementation on calcium release from the SR and muscle force in old age while point out that increased muscle mass does not improve physical performance with aging.

Dynamics of triadin, a muscle-specific triad protein, within sarcoplasmic reticulum subdomains.

In skeletal muscle, proteins of the calcium release complex responsible for the excitation-contraction (EC) coupling are exclusively localized in specific reticulum-plasma membrane (ER-PM) contact points named triads. The CRC protein triadin (T95) is localized in the sarcoplasmic reticulum (SR) subdomain of triads where it forms large multimers. However, the mechanisms leading to the steady-state accumulation of T95 in these specific areas of SR are largely unknown. To visualize T95 dynamics, fluorescent chimeras were expressed in triadin knockout myotubes, and their mobility was compared with the mobility of Sec61ß, a membrane protein of the SR unrelated to the EC coupling process. At all stages of skeletal muscle cells differentiation, we show a permanent flux of T95 diffusing in the SR membrane. Moreover, we find evidence that a longer residence time in the ER-PM contact point is due to the transmembrane domain of T95 resulting in an overall triad localization.

Lingguizhugan decoction attenuates doxorubicin-induced heart failure in rats by improving TT-SR microstructural remodeling.

BACKGROUND: Lingguizhugan decoction (LGZG), an ancient Chinese herbal formula, has been used to treat cardiovascular diseases in eastern Asia. We investigated whether LGZG has protective activity and the mechanism underlying its effect in an animal model of heart failure (HF). METHODS: A rat model of HF was established by administering eight intraperitoneal injections of doxorubicin (DOX) (cumulative dose of 16 mg/kg) over a 4-week period. Subsequently, LGZG at 5, 10, and 15 mL/kg/d was administered to the rats intragastrically once daily for 4 weeks. The body weight, heart weight index (HWI), heart weight/tibia length ratio (HW/TL), and serum BNP level were investigated to assess the effect of LGZG on HF. Echocardiography was performed to investigate cardiac function, and H&E staining to visualize myocardial morphology. Myocardial ultrastructure and T-tubule-sarcoplasmic reticulum (TT-SR) junctions were observed by transmission electron microscopy. The JP-2 protein level was determined by Western blotting. The mRNA level of CACNA1S and RyR2 and the microRNA-24 (miR-24) level were assayed by quantitative RT-PCR. RESULTS: Four weeks after DOX treatment, rats developed cardiac damage and exhibited a significantly increased BNP level compared with the control rats (169.6 ± 29.6 pg/mL versus 80.1 ± 9.8 pg/mL, P < 0.001). Conversely, LGZG, especially at the highest dose, markedly reduced the BNP level (93.8 ± 17.9 pg/mL, P < 0.001). Rats treated with DOX developed cardiac dysfunction, characterized by a strong decrease in left ventricular ejection fraction compared with the control (58.5 ± 8.7% versus 88.7 ± 4.0%; P < 0.001). Digoxin and LGZG improved cardiac dysfunction (79.6 ± 6.1%, 69.2 ± 2.5%, respectively) and preserved the left ventricular ejection fraction (77.9 ± 5.1, and 80.5 ± 4.9, respectively, P < 0.01). LGZG also improved the LVEDD, LVESD, and FS and eliminated ventricular hypertrophy, as indicated by decreased HWI and HW/TL ratio. LGZG attenuated morphological abnormalities and mitochondrial damage in the myocardium. In addition, a high dose of LGZG significantly downregulated the expression of miR-24 compared with that in DOX-treated rats (fold change 1.4 versus 3.4, P < 0.001), but upregulated the expression of JP-2 and antagonized DOX-induced T-tubule TT-SR microstructural remodeling. These activities improved periodic Ca2+ transients and cell contraction, which may underly the beneficial effect of LGZG on HF. CONCLUSIONS: LGZG exerted beneficial effects on DOX-induced HF in rats, which were mediated in part by improved TT-SR microstructural remodeling.

Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative Light Electron Microscopy.

In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.

3D ultrastructural organisation of calcium release units in the avian sarcoplasmic reticulum.

Excitation-contraction coupling in vertebrate hearts is underpinned by calcium (Ca2+) release from Ca2+ release units (CRUs). CRUs are formed by clusters of channels called ryanodine receptors on the sarcoplasmic reticulum (SR) within the cardiomyocyte. Distances between CRUs influence the diffusion of Ca2+, thus influencing the rate and strength of excitation-contraction coupling. Avian myocytes lack T-tubules, so Ca2+ from surface CRUs (peripheral couplings, PCs) must diffuse to internal CRU sites of the corbular SR (cSR) during centripetal propagation. Despite this, avian hearts achieve higher contractile rates and develop greater contractile strength than many mammalian hearts, which have T-tubules to provide simultaneous activation of the Ca2+ signal through the myocyte. We used 3D electron tomography to test the hypothesis that the intracellular distribution of CRUs in the avian heart permits faster and stronger contractions despite the absence of T-tubules. Nearest edge-edge distances between PCs and cSR, and geometric information including surface area and volume of individual cSR, were obtained for each cardiac chamber of the white leghorn chicken. Computational modelling was then used to establish a relationship between CRU distance and cell activation time in the avian heart. Our data suggest that cSR clustered close together along the Z-line is vital for rapid propagation of the Ca2+ signal from the cell periphery to the cell centre, which would aid in the strong and fast contractions of the avian heart.

In Vivo Monitoring of Ca2+ Uptake into Subcellular Compartments of Mouse Skeletal Muscle.

Ca2+ regulates many functions of skeletal muscle, including excitation-contraction coupling, energy homeostasis, and fiber-type-specific gene expression. However, microscopic observation of Ca2+ signalling in live skeletal muscle tissue has been hampered, in particular, by the combination of the high speed of Ca2+ transients and the contractile properties that are inherent to muscle. The present chapter describes methods to visualize Ca2+ signals during relaxation-contraction cycles in different subcellular compartments at high spatiotemporal resolution or at the global muscle level in combination with simultaneous measurements of muscle force. These protocols employ transfection of genetically encoded ratiometric Ca2+ sensors and two-photon microscopy as well as force transducers and associated hardware for data acquisition. Information on how to determine subcellular localization of the genetically encoded Ca2+ sensors and on how to calibrate the ratiometric data in a semiquantitative manner is given in the final paragraphs.

Drosophila Hsp67Bc hot-spot variants alter muscle structure and function.

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. Here, we report the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, we observed a different impact on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.

REEP5 (Receptor Accessory Protein 5) Acts as a Sarcoplasmic Reticulum Membrane Sculptor to Modulate Cardiac Function.

BACKGROUND: Heart failure is a complex syndrome characterized by cardiac contractile impairment with high mortality. Defective intracellular Ca2+ homeostasis is the central cause under this scenario and tightly links to ultrastructural rearrangements of sarcolemmal transverse tubules and the sarcoplasmic reticulum (SR); however, the modulators of the SR architecture remain unknown. The SR has been thought to be a specialized endoplasmic reticulum membrane system. Receptor accessory proteins (REEPs)/DP1/Yop1p are responsible for shaping high-curvature endoplasmic reticulum tubules. This study aimed to determine the role of REEPs in SR membrane shaping and thus cardiac function. METHODS AND RESULTS: We identified REEP5 (receptor accessory protein 5) as more highly expressed than other REEP members in adult rat ventricular myocardium, and it was downregulated in the failing hearts. Targeted inactivation of REEP5 in rats specially deformed the cardiac SR membrane without affecting transverse tubules, and this was visualized by focused ion beam scanning electron microscopy-based 3-dimensional reconstruction. Accordingly, simultaneous recordings of depolarization-induced Ca2+ currents and Ca2+ transients in REEP5-null cardiomyocytes revealed normal L-type Ca2+ channel currents but a depressed SR Ca2+ release. Consequently, the excitation-contraction coupling gain of cardiomyocytes and consequent cardiac contractility were compromised. REEP5 deficiency did not alter the expression of major proteins involved in Ca2+ handling in the heart. CONCLUSIONS: REEP5 modulates cardiac function by shaping the SR. REEP5 defect deforms the SR architecture to depress cardiac contractility. REEP5-dependent SR shaping might have potential as a therapeutic target for heart failure.

Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice.

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms.

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic ßcyto - or γcyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific ßcyto - and γcyto -actin KO mice. We found ßcyto - and γcyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking ßcyto - and/or γcyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific ßcyto - and γcyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old ßcyto - and γcyto -actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.

Three-dimensional imaging reveals endo(sarco)plasmic reticulum-containing invaginations within the nucleoplasm of muscle.

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+ homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12 muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+ regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+ regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.

The precise subcellular localization of Dlg in the Drosophila larva body wall using improved pre-embedding immuno-EM.

Discs-large (Dlg) plays important roles in nerve tissue and epithelial tissue in Drosophila. However, the precise positioning of Dlg in the neuromuscular junction remains to be confirmed using an optimized labeling method. In this study, we improved the method of pre-embedding immunogold electron microscopy without the osmic tetroxide procedure, and we found that Lowicryl K4 M resin and low temperature helped to preserve the authenticity of the labeling signal with relatively good contrast. Dlg was strongly expressed in the entire subsynaptic reticulum (SSR) membrane of type Ib boutons, expressed in parts of the SSR membrane of type Is boutons, weakly expressed in axon terminals and axons, and not expressed in pre- or postsynaptic membranes of type Is boutons. In muscle cells and stratum corneum cells, Dlg was expressed both in the cytoplasm and in organelles with biomembranes. The precise location of Dlg in SSR membranes, rather than in postsynaptic membranes, shows that Dlg, with its multiple domains, acts as a remote or indirect regulator in postsynaptic signal transduction.

Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle.

Store-operated Ca2+ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca2+ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca2+ sensor, and Orai1, the Ca2+-permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca2+, compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca2+ with Mg2+ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca 2+ Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca2+ ions from the extracellular space during repetitive muscle activity.

Assessing the effects of mitofusin 2 deficiency in the adult heart using 3D electron tomography.

The effects of mitofusin 2 (MFN2) deficiency, on mitochondrial morphology and the mitochondria-junctional sarcoplasmic reticulum (jSR) complex in the adult heart, have been previously investigated using 2D electron microscopy, an approach which is unable to provide a 3D spatial assessment of these imaging parameters. Here, we use 3D electron tomography to show that MFN2-deficient mitochondria are larger in volume, more elongated, and less rounded; have fewer mitochondria-jSR contacts, and an increase in the distance between mitochondria and jSR, when compared to WT mitochondria. In comparison to 2D electron microscopy, 3D electron tomography can provide further insights into mitochondrial morphology and the mitochondria-jSR complex in the adult heart.