Nitric oxide regulates skeletal muscle fatigue, fiber type, microtubule organization, and mitochondrial ATP synthesis efficiency through cGMP-dependent mechanisms

Aim: Skeletal muscle nitric oxide–cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSμ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. Results: GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSμ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1−/− muscle. Functional analyses of GC1−/− muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA–IIX fiber balance. Force deficits in GC1−/− muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. Innovation: GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. Conclusions: These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency

Dissociation of Progressive Dopaminergic Neuronal Death and Behavioral Impairments by Bax Deletion in a Mouse Model of Parkinson's Diseases

Parkinson's disease (PD) is a common, late-onset movement disorder with selective degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Although the neurotoxin 6-hydroxydopamine (6-OHDA) has been used to induce progressive degeneration of DA neurons in various animal models of PD, the precise molecular pathway and the impact of anti-apoptotic treatment on this neurodegeneration are less understood. Following a striatal injection of 6-OHDA, we observed atrophy and progressive death of DA neurons in wild-type mice. These degenerating DA neurons never exhibited signs of apoptosis (i.e., caspase-3 activation and cytoplasmic release of cytochrome C), but rather show nuclear translocation of apoptosis-inducing factor (AIF), a hallmark of regulated necrosis. However, mice with genetic deletion of the proapoptotic gene Bax (Bax-KO) exhibited a complete absence of 6-OHDA-induced DA neuron death and nuclear translocation of AIF, indicating that 6-OHDA-induced DA neuronal death is mediated by Bax-dependent AIF activation. On the other hand, DA neurons that survived in Bax-KO mice exhibited marked neuronal atrophy, without significant improvement of PD-related behavioral deficits. These findings suggest that anti-apoptotic therapy may not be sufficient for PD treatment, and the prevention of Bax-independent neuronal atrophy may be an important therapeutic target

Acoustofluidic Assembly of 3D Neurospheroids to Model Alzheimer’s Disease

Neuroinflammation plays a central role in the progression of many neurodegenerative diseases such as Alzheimer's disease, and challenges remain in modeling the complex pathological or physiological processes. Here, we report an acoustofluidic method that can rapidly construct 3D neurospheroids and inflammatory microenvironments for modeling microglia-mediated neuroinflammation in Alzheimer's disease. By incorporating a unique contactless and label-free acoustic assembly, this cell culture platform can assemble dissociated embryonic mouse brain cells into hundreds of uniform 3D neurospheroids with controlled cell numbers, composition (e.g. neurons, astrocytes, and microglia), and environmental components (e.g. amyloid-β aggregates) in hydrogel within minutes. Moreover, this platform can maintain and monitor the interaction among neurons, astrocytes, microglia, and amyloid-β aggregates in real-time for several days to weeks, after the integration of a high-throughput, time-lapse cell imaging approach. We demonstrated that our engineered 3D neurospheroids can represent the amyloid-β neurotoxicity, which is one of the main pathological features of Alzheimer's disease. Using this method, we also investigated the microglia migratory behaviors and activation in the engineered 3D inflammatory microenvironment at a high throughput manner, which is not easy to achieve in 2D neuronal cultures or animal models. Along with the simple fabrication and setup, the acoustofluidic technology is compatible with conventional Petri dishes and well-plates, supports the fine-tuning of the cellular and environmental components of 3D neurospheroids, and enables the high-throughput cellular interaction investigation. We believe our technology may be widely used to facilitate 3D in vitro brain models for modeling neurodegenerative diseases, discovering new drugs, and testing neurotoxicity

GSNOR Deficiency Enhances In Situ

Aim: Nitric oxide (NO) plays important, but incompletely defined roles in skeletal muscle. NO exerts its regulatory effects partly though S-nitrosylation, which is balanced by denitrosylation by enzymes such as S-nitrosoglutathione reductase (GSNOR), whose functions in skeletal muscle remain to be fully deciphered. Results: GSNOR null (GSNOR(−/−)) tibialis anterior (TA) muscles showed normal growth and were stronger and more fatigue resistant than controls in situ. However, GSNOR(−/−) lumbrical muscles showed normal contractility and Ca(2+) handling in vitro, suggesting important differences in GSNOR function between muscles or between in vitro and in situ environments. GSNOR(−/−) TA muscles exhibited normal mitochondrial content, and capillary densities, but reduced type IIA fiber content. GSNOR inhibition did not impact mitochondrial respiratory complex I, III, or IV activities. These findings argue that enhanced GSNOR(−/−) TA contractility is not driven by changes in mitochondrial content or activity, fiber type, or blood vessel density. However, loss of GSNOR led to RyR1 hypernitrosylation, which is believed to increase muscle force output under physiological conditions. cGMP synthesis by soluble guanylate cyclase (sGC) was decreased in resting GSNOR(−/−) muscle and was more responsive to agonist (DETANO, BAY 41, and BAY 58) stimulation, suggesting that GSNOR modulates cGMP production in skeletal muscle. Innovation: GSNOR may act as a “brake” on skeletal muscle contractile performance under physiological conditions by modulating nitrosylation/denitrosylation balance. Conclusions: GSNOR may play important roles in skeletal muscle contractility, RyR1 S-nitrosylation, fiber type specification, and sGC activity. Antioxid. Redox Signal. 26, 165–181

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Riociguat increases lung tissue cGMP levels.

<p>Riociguat significantly increased cGMP concentration in hyperoxia-exposed rats as compared with placebo treated hyperoxic rats. *<i>P</i> < 0.05 compared with hyperoxia + placebo (n = 4/group).</p

Riociguat decreases hyperoxia-induced vascular remodeling.

<p>(<b>A</b>, <b>C</b>) Double immunofluorescence staining for vWF (green signal) and α-SMA (red signal) plus DAPI nuclear stain (blue signal). (<b>B</b>) Hyperoxia exposure in the presence of placebo increased muscularization of peripheral pulmonary vessels (<50 μm in diameter) as compared with normoxia group (red arrow). Administration of riociguat decreased muscularized vessels in hyperoxia exposed lungs. (<b>D</b>) Hyperoxia increased medial wall thickness (MWT) in presence of placebo as compared with normoxia group. Riociguat administration significantly decreased MWT in hyperoxia group. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). Scale bar: 50 μm. (<b>E</b>) Double immunofluorescence staining with Ki67 (red arrow) and α-SMA (green signal) plus DAPI nuclear staining (blue signal). (<b>F</b>) Hyperoxia exposure in the presence of placebo increased vascular proliferation as compared with normoxia group. Administration of riociguat decreased vascular proliferation. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). (<b>G</b>) CTGF gene expression was up-regulated by hyperoxia and it was down-regulated by riociguat. *<i>P</i> < 0.05 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). (<b>H</b>) Representative Western blots of CTGF and β-actin. (<b>I</b>). Expression of CTGF was increased by hyperoxia, while administration of riociguat decreased CTGF expression in hyperoxia exposed lungs. ***<i>P</i> < 0.001 compared with normoxia; ⁺<i>P</i> < 0.05 compared with hyperoxia + placebo (n = 6/group). RA: room air, normaxia; O₂: hyperoxia; PL: placebo; Rio: riociguat.</p