Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Depletion of skeletal muscle satellite cells attenuates pathology in muscular dystrophy.

Skeletal muscle can repair and regenerate due to resident stem cells known as satellite cells. The muscular dystrophies are progressive muscle wasting diseases underscored by chronic muscle damage that is continually repaired by satellite cell-driven regeneration. Here we generate a genetic strategy to mediate satellite cell ablation in dystrophic mouse models to investigate how satellite cells impact disease trajectory. Unexpectedly, we observe that depletion of satellite cells reduces dystrophic disease features, with improved histopathology, enhanced sarcolemmal stability and augmented muscle performance. Mechanistically, we demonstrate that satellite cells initiate expression of the myogenic transcription factor MyoD, which then induces re-expression of fetal genes in the myofibers that destabilize the sarcolemma. Indeed, MyoD re-expression in wildtype adult skeletal muscle reduces membrane stability and promotes histopathology, while MyoD inhibition in a mouse model of muscular dystrophy improved membrane stability. Taken together these observations suggest that satellite cell activation and the fetal gene program is maladaptive in chronic dystrophic skeletal muscle.

Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein.

The downregulation of sclerostin in osteocytes mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation of the Sost gene hours after stimulation. Using mouse models and rodent cell lines, we describe the rapid, minute-scale post-translational degradation of sclerostin protein by the lysosome following mechanical load and PTH. We present a model, integrating both new and established mechanically and hormonally activated effectors into the regulated degradation of sclerostin by lysosomes. Using a mouse forelimb mechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation by preventing sclerostin degradation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using human Gaucher disease iPSCs. These results reveal how bone anabolic cues post-translationally regulate sclerostin abundance in osteocytes to regulate bone formation.

MK2-Deficient Mice Are Bradycardic and Display Delayed Hypertrophic Remodeling in Response to a Chronic Increase in Afterload.

Background Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is a protein serine/threonine kinase activated by p38α/ß. Herein, we examine the cardiac phenotype of pan MK2-null (MK2-/-) mice. Methods and Results Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P=0.580). At 12 weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, aging was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3, were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+ sensitivity to mitochondrial permeability transition pore opening. Eight weeks of pressure overload increased left ventricular mass in MK2+/+ and MK2-/- mice; however, after 2 weeks the increase was significant in MK2+/+ but not MK2-/- mice. Finally, the pressure overload-induced decrease in systolic function was attenuated in MK2-/- mice 2 weeks, but not 8 weeks, after constriction of the transverse aorta. Conclusions Collectively, these results implicate MK2 in (1) autonomic regulation of heart rate, (2) cardiac mitochondrial function, and (3) the early stages of myocardial remodeling in response to chronic pressure overload.

In vitro Fluid Shear Stress Induced Sclerostin Degradation and CaMKII Activation in Osteocytes.

Bone is a dynamic tissue that adapts to changes in its mechanical environment. Mechanical stimuli pressurize interstitial fluid in the lacunar-canalicular system within the bone matrix, causing fluid shear stress (FSS) across bone embedded, mechano-sensitive osteocytes. Therefore, modeling this mechanical stimulus in vitro is vital for identifying mechano-transduction cascades that contribute to the regulation of mechano-responsive proteins, such as the Wnt/ß-catenin antagonist, sclerostin, which is reduced in response to FSS. Recently, we reported the rapid post-translational degradation of sclerostin protein in bone cells following FSS. Given the fundamental nature of sclerostin to bone physiology and the nuances of studying its rapid post-translational control, here, we detail our FSS protocol, and adaptations that can be made, to stimulate Ocy454 osteocyte-like cells to study sclerostin protein in vitro. While this protocol is optimized for detecting sclerostin degradation by western blot, this protocol can be adapted to examine transcriptional changes with RT-qPCR, cellular dynamics with live cell imaging, or secreted factors in the FSS buffer. This protocol utilizes 3D-printed FSS tips that are compatible with commercially available 96-well plates, allowing for high experimental accessibility, versatility, and throughput. However, this protocol can be adapted for any FSS chamber. It can also be combined with pharmacological inhibitors or genetic manipulations to interrogate the role of specific cellular components. In all, this experimental set-up and protocol is highly adaptable to allow for many experimental outcomes to examine many aspects of cell mechano-transduction.

TRPV4 calcium influx controls sclerostin protein loss independent of purinergic calcium oscillations.

Skeletal remodeling is driven in part by the osteocyte's ability to respond to its mechanical environment by regulating the abundance of sclerostin, a negative regulator of bone mass. We have recently shown that the osteocyte responds to fluid shear stress via the microtubule network-dependent activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species and subsequent opening of TRPV4 cation channels, leading to calcium influx, activation of CaMKII, and rapid sclerostin protein downregulation. In addition to the initial calcium influx, purinergic receptor signaling and calcium oscillations occur in response to mechanical load and prior to rapid sclerostin protein loss. However, the independent contributions of TRPV4-mediated calcium influx and purinergic calcium oscillations to the rapid sclerostin protein downregulation remain unclear. Here, we showed that NOX2 and TRPV4-dependent calcium influx is required for calcium oscillations, and that TRPV4 activation is both necessary and sufficient for sclerostin degradation. In contrast, calcium oscillations are neither necessary nor sufficient to acutely decrease sclerostin protein abundance. However, blocking oscillations with apyrase prevented fluid shear stress induced changes in osterix (Sp7), osteoprotegerin (Tnfrsf11b), and sclerostin (Sost) gene expression. In total, these data provide key mechanistic insights into the way bone cells translate mechanical cues to target a key effector of bone formation, sclerostin.

Specific inhibition of myostatin activation is beneficial in mouse models of SMA therapy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of α-motor neurons, leading to profound skeletal muscle atrophy. Patients also suffer from decreased bone mineral density and increased fracture risk. The majority of treatments for SMA, approved or in clinic trials, focus on addressing the underlying cause of disease, insufficient production of full-length SMN protein. While restoration of SMN has resulted in improvements in functional measures, significant deficits remain in both mice and SMA patients following treatment. Motor function in SMA patients may be additionally improved by targeting skeletal muscle to reduce atrophy and improve muscle strength. Inhibition of myostatin, a negative regulator of muscle mass, offers a promising approach to increase muscle function in SMA patients. Here we demonstrate that muSRK-015P, a monoclonal antibody which specifically inhibits myostatin activation, effectively increases muscle mass and function in two variants of the pharmacological mouse model of SMA in which pharmacologic restoration of SMN has taken place either 1 or 24 days after birth to reflect early or later therapeutic intervention. Additionally, muSRK-015P treatment improves the cortical and trabecular bone phenotypes in these mice. These data indicate that preventing myostatin activation has therapeutic potential in addressing muscle and bone deficiencies in SMA patients. An optimized variant of SRK-015P, SRK-015, is currently in clinical development for treatment of SMA.

Chronic dietary supplementation with soy protein improves muscle function in rats.

Athletes as well as elderly or hospitalized patients use dietary protein supplementation to maintain or grow skeletal muscle. It is recognized that high quality protein is needed for muscle accretion, and can be obtained from both animal and plant-based sources. There is interest to understand whether these sources differ in their ability to maintain or stimulate muscle growth and function. In this study, baseline muscle performance was assessed in 50 adult Sprague-Dawley rats after which they were assigned to one of five semi-purified "Western" diets (n = 10/group) differing only in protein source, namely 19 kcal% protein from either milk protein isolate (MPI), whey protein isolate (WPI), soy protein isolate (SPI), soy protein concentrate (SPC) or enzyme-treated soy protein (SPE). The diets were fed for 8 weeks at which point muscle performance testing was repeated and tissues were collected for analysis. There was no significant difference in food consumption or body weights over time between the diet groups nor were there differences in terminal organ and muscle weights or in serum lipids, creatinine or myostatin. Compared with MPI-fed rats, rats fed WPI and SPC displayed a greater maximum rate of contraction using the in vivo measure of muscle performance (p<0.05) with increases ranging from 13.3-27.5% and 22.8-29.5%, respectively at 60, 80, 100 and 150 Hz. When the maximum force was normalized to body weight, SPC-fed rats displayed increased force compared to MPI (p<0.05), whereas when normalized to gastrocnemius weight, WPI-fed rats displayed increased force compared to MPI (p<0.05). There was no difference between groups using in situ muscle performance. In conclusion, soy protein consumption, in high-fat diet, resulted in muscle function comparable to whey protein and improved compared to milk protein. The benefits seen with soy or whey protein were independent of changes in muscle mass or fiber cross-sectional area.

Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes.

The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely by regulating the abundance of sclerostin, a secreted inhibitor of bone formation. We defined a microtubule-dependent mechanotransduction pathway that linked fluid shear stress to reactive oxygen species (ROS) and calcium (Ca2+) signals that led to a reduction in sclerostin abundance in cultured osteocytes. We demonstrated that microtubules stabilized by detyrosination, a reversible posttranslational modification of polymerized α-tubulin, determined the stiffness of the cytoskeleton, which set the mechanoresponsive range of cultured osteocytes to fluid shear stress. We showed that fluid shear stress through the microtubule network activated NADPH oxidase 2 (NOX2)-generated ROS that target the Ca2+ channel TRPV4 to elicit Ca2+ influx. Furthermore, tuning the abundance of detyrosinated tubulin affected cytoskeletal stiffness to define the mechanoresponsive range of cultured osteocytes to fluid shear stress. Finally, we demonstrated that NOX2-ROS elicited Ca2+ signals that activated the kinase CaMKII to decrease the abundance of sclerostin protein. Together, these discoveries may identify potentially druggable targets for regulating osteocyte mechanotransduction to affect bone quality.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation.

ß-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 µm and SL = 2.3 µm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that ß-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner.

Calcium movement in cardiac mitochondria.

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca(2+)]i) in heart. These buffers can remove up to one-third of the Ca(2+) that enters the cytosol during the [Ca(2+)]i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca(2+) movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca(2+) signals (i.e., Ca(2+) sparks and [Ca(2+)]i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨm), the role of the mitochondria in buffering cytosolic Ca(2+) signals was investigated. We show here that rapid loss of ΔΨm leads to no significant changes in cytosolic Ca(2+) signals. Second, we make direct measurements of mitochondrial [Ca(2+)] ([Ca(2+)]m) using a mitochondrially targeted Ca(2+) probe (MityCam) and these data suggest that [Ca(2+)]m is near the [Ca(2+)]i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca(2+) signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca(2+) cycling suggests that mitochondrial Ca(2+) uptake would need to be at least ∼100-fold greater than the current estimates of Ca(2+) influx for mitochondria to influence measurably cytosolic [Ca(2+)] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca(2+) uptake does not significantly alter cytosolic Ca(2+) signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca(2+)]i under physiological conditions in heart.

Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium.

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca(2+) transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 µm) and long (2.3 µm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca(2+) sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca(2+) sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

Finding the missing link: disulfide-containing proteins via a high-throughput proteomics approach.

Top-down proteomics have recently started to gain attention as a novel method to provide insight into the structure of proteins in their native state, specifically the number and location of disulfide bridges. However, previous techniques still relied on complex and time-consuming protein purification and reduction reactions to yield useful information. In this issue of Proteomics, Zhao et al. (high-throughput screening of disulfide-containing proteins in a complex mixture, Proteomics 2013, 13, 3256-3260) devise a clever and rapid method for high-throughput determination of disulfides in proteins via reduction by tris(2-carboxyethyl)phosphine. Their work provides the foundation necessary to undertake more complex experiments in biological samples.

Mitochondrial calcium uptake.

Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

Marine n3 polyunsaturated fatty acids enhance resistance to mitochondrial permeability transition in heart failure but do not improve survival.

Mitochondrial dysfunction in heart failure includes greater susceptibility to mitochondrial permeability transition (MPT), which may worsen cardiac function and decrease survival. Treatment with a mixture of the n3 polyunsaturated fatty acids (n3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is beneficial in heart failure patients and increases resistance to MPT in animal models. We assessed whether DHA and EPA have similar effects when given individually, and whether they prolong survival in heart failure. Male δ-sarcoglycan null cardiomyopathic hamsters were untreated or given either DHA, EPA, or a 1:1 mixture of DHA + EPA at 2.1% of energy intake. Treatment did not prolong survival: mean survival was 298 ± 15 days in untreated hamsters and 335 ± 17, 328 ± 14, and 311 ± 15 days with DHA, EPA, and DHA + EPA, respectively (n = 27-32/group). A subgroup of cardiomyopathic hamsters treated for 26 wk had impaired left ventricular function and increased cardiomyocyte apoptosis compared with normal hamsters, which was unaffected by n3 PUFA treatment. Evaluation of oxidative phosphorylation in isolated subsarcolemmal and interfibrillar mitochondria with substrates for complex I or II showed no effect of n3 PUFA treatment. On the other hand, interfibrillar mitochondria from cardiomyopathic hamsters were significantly more sensitive to Ca(2+)-induced MPT, which was completely normalized by treatment with DHA and partially corrected by EPA. In conclusion, treatment with DHA or EPA normalizes Ca(2+)-induced MPT in cardiomyopathic hamsters but does not prolong survival or improve cardiac function. This suggest that greater susceptibility to MPT is not a contributor to cardiac pathology and poor survival in heart failure.

X-ROS signaling in the heart and skeletal muscle: stretch-dependent local ROS regulates [Ca²âº]i.

X-ROS signaling is a novel redox signaling pathway that links mechanical stress to changes in [Ca(2+)]i. This pathway is activated rapidly and locally within a muscle cell under physiological conditions, but can also contribute to Ca(2+)-dependent arrhythmia in the heart and to the dystrophic phenotype in the 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 the heart, the ROS acts locally to activate ryanodine receptor Ca(2+) release channels in the junctional sarcoplasmic reticulum, increasing the Ca(2+) spark rate and "tuning" excitation-contraction coupling. In the skeletal muscle, where Ca(2+) 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, Ca(2+) 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 Ca(2+) signaling, but much work is needed to establish its contribution to physiologic and pathophysiologic processes in diverse cell systems.

Microtubules underlie dysfunction in duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca(2+)) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase-dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca(2+) influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca(2+) influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca(2+) influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS-related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca(2+) and ROS signaling in DMD and could be effective therapeutic targets for intervention.

Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids.

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca(2+) load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca(2+)-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca(2+) load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.