Muscle Cell Death Research Articles

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle <em>Ex vivo</em> and Tibialis Anterior Muscle <em>In situ</em> in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied. The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle <em>Ex vivo</em> and Tibialis Anterior Muscle <em>In situ</em> in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Role of protein kinase C δ in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells

During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.

Inhibiting Connexin Channels Protects Against Cryopreservation-induced Cell Death in Human Blood Vessels

Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

Protective effect of melatonin on TNF‐α‐induced muscle atrophy in L6 myotubes

Muscle atrophy, characterized by decreased cell number and size, is a serious concern for patients afflicted with inflammatory diseases. Mounting evidence indicates that tumor necrosis factor alpha (TNF-α) plays a critical role in muscle atrophy in a number of clinical settings. We hypothesize that reactive oxygen species (ROS) mediate TNF-α-induced muscle cell death and hypotrophy. Recently, melatonin has attracted attention because of its free-radical scavenging and antioxidant properties. The aim of the current study was to evaluate the possible protective role of melatonin in TNF-α-induced muscle cell death and hypotrophy in rat L6 myotubes. To examine this possible role, L6 myotubes were exposed to various concentrations of recombinant TNF-α for 24 hr. We found that TNF-α at a concentration of 100 ng/mL induced ROS generation and decreased cell viability. Further analysis revealed that apoptosis, but not autophagy, may be important for TNF-α-induced cell death. Melatonin significantly attenuated TNF-α-induced ROS generation and apoptosis. In addition, decreased muscle fiber diameter and increased muscle cell proteolysis by TNF-α was highly attenuated by treatment with melatonin. The effects of melatonin were mediated neither through its plasmalemmal receptors nor by modulating the nuclear factor kappa B pathway activated by TNF-α. Taken together, these results suggest that TNF-α may mediate ROS-induced muscle cell death and hypotrophy and that melatonin may be a useful tool for protecting against muscle atrophy stemming from inflammatory diseases.

Influence of LRP-1 and Apolipoprotein E on Amyloid-β Uptake and Toxicity to Cerebrovascular Smooth Muscle Cells

Vascular deposition of amyloid-β (Aβ) leads to the death of cerebrovascular smooth muscle cells (CVSMCs) in cerebral amyloid angiopathy (CAA). Apolipoprotein E (APOE) genotype influences the severity of CAA and development of vasculopathic complications. We have studied the relationship between uptake of Aβ into human CVSMCs and cell death, and the influence of ApoE isoforms on this process. We found that both Aβ42 and Aβ40 were taken up by human CVSMCs, and that this uptake-particularly that of Aβ42-caused disruption to smooth muscle actin and cell death. Uptake of Aβ42 was partially blocked by the addition of receptor-associated protein (RAP), implicating low-density lipoprotein receptor-related protein-1 (LRP-1) as the cell surface receptor. RAP significantly reduced the death of CVSMCs exposed to Aβ42. In further experiments, CVSMCs were exposed to Aβ42 in the presence of the different isoforms of exogenous ApoE and high-density lipoprotein (HDL). All three isoforms of ApoE in the presence of HDL (HDL-ApoE) reduced the uptake of fluorescein-tagged Aβ42 but only HDL-ApoE3 significantly decreased cell death. We conclude that HDL-ApoE3, acting as an Aβ chaperone molecule, significantly reduces the death of CVSMCs that result from LRP-1-mediated uptake of Aβ. This may contribute to the differential effects of APOE genotype on severity of CAA and the risk of rupture of Aβ-laden blood vessels.

Atorvastatin Sensitises Vascular Smooth Muscle Cells, but not Endothelial Cells, to TNF-α-induced Cell Death

Stimuli activating vascular smooth muscle cell death can constrain the neointimal response to arterial damage and prevent vascular thickening. Conversely, endothelial cell death increases endothelial dysfunction and thrombosis risk. We investigated the combined effect of atorvastatin and TNF-α on vascular cell death. Cell death was investigated in cultures of human aortic smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs). Atorvastatin downregulated NF-κB and enhanced JNK activity and cell death in VSMC cultured with TNF- α. In the absence of TNF-α, percentages (mean and StDev) of annexin V positive cells were 17.4 ± 6.6%, 19.3 ± 5.9%, 22.9 ± 9.4% and 35.0 ± 20.0 % with 0, 1, 3 and 10 µM atorvastatin, respectively. The cytotoxic effect of statin was significant at the highest dose of 10 μM (p=0.001). In the presence of TNF-α, percentages of annexin V positive cells were 27.1 ± 10.6%, 34.2 ± 8.5%, 37.4 ± 14.6, and 54.1 ± 20.0% with 0, 1, 3 and 10 µM atorvastatin, respectively. The cytotoxic effect of statin was significant at each dose used (p≤0.02), in the presence of TNF-α. The cell death sensitising effect of atorvastatin was apparently mediated by down modulation of PKCβ activity, because it was reproduced by the specific PKCβ inhibitor LY317615 and prevented by the PKC activator phorbol-12-myristate-13-acetate (PMA). This effect was cell context dependent because it was not observed in HUVECs. PKCβ was found to be constitutively active in VSMCs but not in HUVECs, thereby explaining the differential effect among the two cell types. Measurement of phosphoPKCβ protein levels in arterial specimens confirmed increased activation of this kinase in the smooth muscle layer, in comparison with endothelium. We show that PKCβ provides survival signals to vascular smooth muscle cells and not the endothelium. Our study suggests that atorvastatin enhances TNF-α-induced cell death in vascular smooth muscle- but not endothelial - cells; by a cell-context-dependent mechanism, involving PKCβ inhibition.

Pre-emptive hypoxia-regulated HO-1 gene therapy improves post-ischaemic limb perfusion and tissue regeneration in mice

AimsHaem oxygenase-1 (HO-1) is a haem-degrading enzyme that generates carbon monoxide, bilirubin, and iron ions. Through these compounds, HO-1 mitigates cellular injury by exerting antioxidant, anti-apoptotic, and anti-inflammatory effects. Here, we examined the influence of HO-1 deficiency and transient hypoxia/ischaemia-induced HO-1 overexpression on post-injury hindlimb recovery.Methods and resultsMice lacking functional HO-1 (HO-1−/−) showed reduced reparative neovascularization in ischaemic skeletal muscles, impaired blood flow (BF) recovery, and increased muscle cell death compared with their wild-type littermates. Human microvascular endothelial cells (HMEC-1) transfected with plasmid vector (pHRE-HO-1) carrying human HO-1 driven by three hypoxia response elements (HREs) and cultured in 0.5% oxygen demonstrated markedly increased expression of HO-1. Such upregulated HO-1 levels were effective in conferring protection against H2O2-induced cell death and in promoting the proangiogenic phenotype of HMEC-1 cells. More importantly, when delivered in vivo, pHRE-HO-1 significantly improved the post-ischaemic foot BF in mice subjected to femoral artery ligation. These effects were associated with reduced levels of pro-inflammatory cytokines (IL-6 and CXCL1) and lower numbers of transferase-mediated dUTP nick-end labelling-positive cells. Moreover, HO-1 delivered into mouse skeletal muscles seems to influence the regenerative potential of myocytes as it significantly changed the expression of transcriptional (Pax7, MyoD, myogenin) and post-transcriptional (miR-146a, miR-206) regulators of skeletal muscle regeneration.ConclusionOur results suggest the therapeutic potential of HO-1 for prevention of adverse effects in critical limb ischaemia.

Ischemic Injury Following EUS Guided Celiac Plexus Neurolysis

Case Presentation: A 51 yo M with metastatic pancreatic adenocarcinoma with vascular invasion presents with abdominal pain. The patient had an ERCP with CBD stent exchange and EUS with celiac plexus neurolysis (CPN). During the EUS CPN, a total of 10 cc of .25% bupivicaine and 10 cc of dehydrated Etoh were injected. One day following the procedure he had melena and subsequent coffee ground emesis with a 4 gram drop in hemoglobin from admission. An EGD showed extensive ulceration and inflammation along the lesser curvature, which was suggestive of ischemic injury. Discussion: Treatment of pain in pancreatic cancer is an important objective in palliative care. Approximately 80-85% of patients have pain associated with advanced pancreatic cancer. Options for pain control include pharmacotherapy as well as CPN, which can be performed percutaneously, surgically or under the guidance of EUS. EUS CPN has been shown to be effective in achieving pain control. In one meta-analysis of 119 patients, pain was alleviated in 73% of patients. CPN is overall felt to be safe but practitioners should be aware of the possible complications. Side effects include local pain, unopposed parasympathetic activity, which can result in diarrhea (44%) and hypotension (38%). Serious complications include retroperitoneal hemorrhage and peripancreatic abscess. In a series of 189 EUS guided celiac plexus blocks and 31 CPNs, the following 4 complications were reported: hypotension, retroperitoneal abscess, and 2 with post-procedural pain. One case report notes ischemic complications following CPN, including gastric ischemia/necrosis, splenic infarction, and pancreatic necrosis in the distribution of the celiac artery. Similarly, our patient likely developed ischemic injury following CPN. Ethanol can cause vasospasm and severe cases of vasoconstriction can lead to vascular and smooth muscle cell death. Conclusion: CPN is an effective modality for treatment of pain in advanced pancreatic cancer. However, physicians should also be aware of the complications. Patients who develop signs of GI bleeding after CPN should be evaluated for ischemic injury.Figure: No Caption available.

G.P.34 Gene expression profiling in tibial muscular dystrophy reveals new involved molecular pathways

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. To identify these components we used gene expression profiling from muscle biopsies samples of TMD patients, healthy controls and from phenotypically overlapping Welander distal myopathy (WDM) patients. The profiling results were confirmed through RT-PCR and protein level analysis and we identified an activation of the unfolded protein response (UPR). UPR was then confirmed through elevation of marker genes HSPA5 (BIP) and XBP1 and the presence of ER-stress specific XBP1 splicing events. However, UPR activation appears to be insufficient, leading to build-up of ubiquitinated proteins which in turn cause activation of the autophagic system. Massive accumulation of LC3b positive autophagosomes within the rimmed vacuolated regions of degenerated muscle fibers suggests that this apparently compensatory mechanism is not capable of restoring equilibrium since these fibers degenerate further and disappear in the end stage of the pathology cascade.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue-specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR-transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3-the more active form of TH. D2 mRNA was induced in the TR-overexpressing cells by low levels of TH. D2 promoter contains a TH-response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cell's ability to respond to low levels of T4.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Inhibition of cell surface expression of endothelial adhesion molecules by ursolic acid prevents intimal hyperplasia of venous bypass grafts in rats

Despite rapid progress in surgical techniques, there is still a significant lack of surgery-supportive pharmacological treatments. The aim of this study was to test the hypothesis that ursolic acid (UA) may prevent intimal hyperplasia of venous bypass grafts. The hypothesis was tested by means of primary cell isolation and culture followed by real-time polymerase chain reaction, western blotting, fluorescence microscopy and fluorescence-activated cell sorting analyses, as well as an in vivo rat model for intimal hyperplasia of venous bypass grafts and immunohistochemistry and histochemistry. The local application of UA significantly inhibited intimal hyperplasia in vivo (intimal thickness control: 25 µm, UA group: 18 µM-8 weeks after surgery). The UA treatment of grafts significantly resulted in reduced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression, reduced infiltration of the grafts vessel wall by CD45-positive cells and increased smooth muscle cell (SMC) death. In in vitro condition, it could be shown that UA inhibits VCAM-1 expression downstream of NFκB and is likely to interfere with VCAM-1 protein synthesis in endothelial cells. Quantification of cell death in vascular smooth muscle cells treated with UA indicated that UA is a potent inducer of SMC apoptosis. Our results suggest that UA-mediated inhibition of endothelial VCAM-1 expression reduces the infiltration of venous bypass grafts by CD45-positive cells and inhibits intimal hyperplasia. Apoptosis induction in SMCs may be another method in which UA reduces intimal thickening. UA may constitute a surgery-supportive pharmacon that reduces intimal hyperplasia of vein grafts.

PET Imaging of Denervation-Induced Muscle Cell Death with 18F-ML-10, a Novel Apoptosis Tracer (P07.198)

Objective: Molecular imaging of denervation-induced myocyte apoptosis by 18 F-ML-10 PET. Background Muscle denervation is common in numerous neurological disorders, ranging from injuries to peripheral nerves to motor neuron disease. Currently, denervation is diagnosed by EMG and/or muscle biopsy, both invasive and limited in spatial information. Molecular imaging tool for denervation injury is therefore much needed. Since apoptosis has been shown to be an important mechanism in denervation-induced myocyte cell-death, we hypothesized that 18 F-ML-10, a PET tracer of apoptosis, may be useful for the respective clinical indications. Design/Methods: In a proof-of-concept study, denervation injury was induced by transection of the left sciatic nerve in rats. Animals were subjected to 18 F-ML-10 PET/CT scans at 24h, 3 and 7 days post-transection. Tracer uptake in muscles innervated by the sciatic nerve was compared to the uptake in contralateral muscles. Neurological deficits were followed from time of nerve transection to last 18 F-ML-10 scan, and assessed for correlation with tracer uptake in the target muscles. Results: Unilateral increase in 18 F-ML-10 uptake were observed specifically in the left hamstring muscles in all rats during the assessed time window, with generation of respective “hot spots” in the affected muscles. Substantially lower activity was measured in the muscles not exclusively innervated by the transected sciatic nerve. Uptake was highest at 24h after denervation, concurrent with the apoptotic peak, and decreased by 7 days post-surgery. Neurological deficits were well-correlated with the increased tracer uptake. Conclusions: This study provides the first application of apoptosis imaging with PET for a neuromuscular disorder. This study revealed “hot spots” generated by 18 F-ML-10, selectively at the site of the denervated muscles. Striving to advance diagnosis of motor neuron disease, and pending on further research, molecular imaging with 18 F-ML-10 may serve as a useful tool for “virtual biopsy”, i.e., non-invasive, integrative detection of muscle denervative processes. Supported by: Financially supported by Aposense Ltd. Disclosure: Dr. Reshef has received personal compensation for activities with Aposense Ltd as an employee. Dr. Ben-Ami has received personal compensation for activities with Aposense Ltd as an employee. Dr. Freedman has nothing to disclose. Dr. Davidson has received personal compensation for activities with Aposense Ltd as an employee. Dr. Mishani has nothing to disclose. Dr. Ziv has received personal compensation for activities with Aposense Ltd as an employee.

ATP release is altered in a mouse model for Duchenne muscular dystrophy and signals for proteins that promote cell death

Several reports have described changes in ATP signaling in models for muscular dystrophy; nevertheless, none of them has been directly related with the dystrophy pathology. To study this pathway we used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. ATP was measured using luciferase, protein expression by Western blots and mRNA by qPCR. The basal ATP release occurs in bursts peaking every 5 minutes in fibers from both mice, being several fold more important in mdx fibers. We also studied the expression of different components of this pathway in isolated triads. We found that P2Y2 receptor and pannexin‐1 are increased and P2Y1 receptor is decreased in mdx fibers. Moreover, ‘selective’ dye uptake suggests that the activity of pannexin channels is elevated in dystrophic fibers. Upon stimulation with extracellular ATP, we found that a number of genes are differentially expressed between normal and dystrophic fibers. Among these genes, over expression of proapoptotic genes as Bax, Bim and PUMA in mdx fibers contrast to the repression of the same genes seen in normal fibers. This study suggests that ATP signaling is altered in mdx fibers and this pathway appears to be involved in the activation of a mechanism related with muscle cell death in muscular dystrophy.FONDAP‐15010006, AFM‐14562, FONDECYT‐1110467, CONICYT‐PhD fellowship (DV)

Structural Adaptation of Microvessels in Disease States

Hypertension, diabetes, and obesity are often associated with impaired microvascular function and structural adaptation. A.R. Pries and T.W. Secomb have developed a mathematical model of structural adaptation based on known physiological responses to shear stress, circumferential stress, and metabolic demand under healthy conditions. While this model captures key aspects of microvascular remodeling, it does not explicitly incorporate signaling pathways. As altered signaling pathways are a prominent feature of many disease states, in its current state this model cannot be used to predict the effects of diseases on vessel remodeling. Using the Pries and Secomb model as a framework, we have developed a model that incorporates relevant signaling pathways. In our model, diameter changes with nitric oxide, a vasodilator, and endothelin‐1, a vasoconstrictor, which are both functions of shear stress. Wall area changes with circumferential stress as well as the growth and death of vascular smooth muscle cells. Currently our model reflects the steady state trends in vessel geometry under normal conditions. Work is ongoing to further validate the model and examine vascular remodeling in disease states. Our long term goal is to improve the understanding of vascular adaptation in disease states and to create modeling tools which provide biologically testable hypotheses for experimentalists and clinicians.

Wild‐type amyloid beta 1‐40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity

Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-β (Aβ) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aβ-peptide deposits [composed mainly of wild-type (WT) Aβ(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aβ(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aβ(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aβ(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aβ(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA.

Urocortins Improve Dystrophic Skeletal Muscle Structure and Function through Both PKA- and Epac-Dependent Pathways

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Age-related loss of muscle fibres is highly variable amongst mouse skeletal muscles

Sarcopenia is the age-related loss of skeletal muscle mass and strength, attributable in part to muscle fibre loss. We are currently unable to prevent fibre loss because we do not know what causes it. To provide a platform from which to better understand the causes of muscle fibre death we have quantified fibre loss in several muscles of aged C57Bl/6J mice. Comparison of muscle fibre numbers on dystrophin-immunostained transverse tissue sections at 6 months of age with those at 24 months shows a significant fibre loss in extensor digitorum longus and soleus, but not in sternomastoid or cleidomastoid muscles. The muscles of the elderly mice were mostly lighter than their younger counterparts, but fibres in the elderly muscles were of about the same cross-sectional area. This study shows that the contribution of fibre death to sarcopenia is highly variable and that there is no consistent pattern of age-related fibre loss between skeletal muscles.

An update on pathogenic mechanisms of inflammatory myopathies

Our understanding of the pathogenesis of the inflammatory myopathies suggests an interplay between adaptive, innate immune, and nonimmune mechanisms in the damage and dysfunction that occur in myopathic muscle tissue. This review gives an update on the recent findings concerning some of these mechanisms and their relevance to disease diagnosis, prognosis, and therapy. The presence of several additional immune cell types (CD-28 null T cells, regulatory T cells, plasmacytoid dendritic cells, plasma cells) and their roles in the various subsets of myositis are discussed. Likewise several new autoantibodies (e.g. 3-hydroxy-3 methylglutaryl-coenzyme-A reductase and melanoma differentiation-associated gene 5) and their association with disease phenotype are described. The review also discusses emerging evidence that cytokines (type 1 interferon) and Toll-like receptor signaling influence the local immune cell activation and response. The mechanisms involved in muscle degeneration are not clearly defined, but recent studies point to a role for nonimmune mechanisms such as endoplasmic reticulum stress and autophagy in skeletal muscle cell death and dysfunction in myositis. The muscle microenvironment in inflammatory myopathy is complex. Multiple players such as adaptive and innate immune cells, cytokines, and chemokines as well as nonimmune mechanisms are involved. Understanding the nature of the relevant cell types and the molecular pathways underlying particular disease phenotypes should help to define therapeutic targets for myositis.