Muscle Cell Death Research Articles

CARD9 mediates necrotic smooth muscle cell-induced inflammation in macrophages contributing to neointima formation of vein grafts.

Inflammation plays an important role in the neointima formation of grafted veins. However, the initiation of inflammation in grafted veins is still unclear. Here, we investigated the role and underlying mechanism of an innate immunity signalling protein, caspase-associated recruitment domain 9 (CARD9) in vein grafts in mice. In early murine vein grafts, we observed robust death of smooth muscle cells (SMCs), which was accompanied by infiltration of macrophages and expression of pro-inflammatory cytokines. Meanwhile, SMC necrosis was associated with the expression of pro-inflammatory cytokines in macrophages in vitro. To explore the mediators of necrotic SMC-induced inflammation in grafted veins from mice, we examined the expression of CARD family proteins and found CARD9 highly expressed in infiltrated macrophages of grafted veins. CARD9-knockout (KO) inhibited necrotic SMC-induced pro-inflammatory cytokine expression and NF-κB activation. Furthermore, CARD9-KO suppressed necrotic SMC-induced expression of VEGF in macrophages. Finally, CARD9-KO decreased neointima formation of grafted veins in mice. The innate immune protein CARD9 in macrophages may mediate necrotic SMC-induced inflammation by activating NF-κB and contributed to neointima formation in the vein grafts.

Biomechanical and pathological changes of taut bands in rats after repeated low-frequency electrical stimulation

Objective To investigate the biomechanical and pathological changes in vivo in the taut bands (TB) of biceps femoris in rats after repeated low-frequency electrical stimulation. Methods Twenty-eight Wistar rats were randomly divided into a control group, an electrical intensity-dependent fatigue group, which were subject to electric intensity-dependent fatigue test, and an electrical frequency-dependent fatigue group, which were subject to electrical frequency-dependent fatigue test. After fatigue tests, the taut band of the biceps femoris and the non-taut band of the contralateral biceps femoris were harvested for pathological observation. The maximum contraction force (MCF), electrical intensity- and frequency-dependent fatigue characteristics and any pathological changes in the TBs were assessed and compared to the non-taut band region of the other biceps femoris. Results The MCF at the 15th and 20th stimulation (1.42±0.28 g and 0.93±0.54 g respectively) were significantly lower than that at the 1st and 5th stimulation of the TBs. High stimulation intensity (HSI) at the 15th and 20th stimulation (3.76±0.71 V and 3.44±0.97 V) were also significantly lower than at the 1st TB stimulation. At the 10th, 15th and 20th stimulation of the TBs, MCF and HSI were both significantly lower than in the bands which were not tight. In the frequency-dependent fatigue stimulation tests, the frequency which generated the MCF of the TBs was significantly lower than in the bands which were not tight, while the MCF of the TBs was significantly higher than that of non-TBs. After either intensity or frequency fatigue testing, more severe edema, uneven cytoplasmic death and degeneration of muscle fibers were observed in sections from TBs than from the bands which were not tight. Conclusions Taut muscle bands are significantly less fatigue-resistant than normal muscle fibers. Taut bands may contribute to the fatigue of myofascial pain syndromes. Key words: Myofascial pain; Taut bands; Fatigability; Muscles; Electical stimulation

Tissue transglutaminase-catalysed cross-linking induces Apolipoprotein E multimers inhibiting Apolipoprotein E's protective effects towards amyloid-beta-induced toxicity.

Cerebral amyloid angiopathy (CAA) is a pathological hallmark of Alzheimer's disease (AD) and characterized by deposition of amyloid-β (Aβ) protein and smooth muscle cell (SMC) death in cerebral vessel walls. Apolipoprotein E (ApoE) is of importance in both Aβ accumulation and Aβ-mediated toxicity towards SMCs in the cerebral vessel wall, although its exact role in CAA pathogenesis remains unclear. Tissue transglutaminase (tTG) is an enzyme capable of inducing both protein complexes and altered protein bioactivity via post-translational cross-linking. In CAA, tTG and its catalytic activity are associated with deposited Aβ. Furthermore, several apolipoproteins are known substrates of tTG. We therefore investigated whether ApoE is a substrate for tTG and if this affects ApoE's bioactivity. We found strong binding of different ApoE isoforms with tTG and demonstrated tTG-catalysed ApoE multimers. In post-mortem human AD cases, ApoE colocalized with insitu active tTG in CAA. Moreover, human brain SMCs treated with Aβ demonstrated enhanced secretion of both ApoE and tTG, and of TG cross-links in the extracellular matrix. Interestingly, tTG-catalysed cross-linked ApoE failed to protect SMCs against Aβ-mediated cytotoxicity. Together, our data demonstrate a novel tTG-driven post-translational modification of ApoE that might play an important role in CAA. Cerebral amyloid angiopathy (CAA) is a pathological hallmark of Alzheimer's disease (AD) and characterized by amyloid-β (Aβ) protein deposition and cerebral smooth muscle cell (SMC) death. We found that, in contrast to normal vessels, in CAA apolipoprotein E (ApoE) is cross-linked by tissue transglutaminase (tTG) resulting in stable ApoE complexes. These complexes no longer protect cerebral SMC from Aβ-mediated toxicity. Our findings demonstrate a novel mechanism explaining the Aβ-mediated cerebral SMC cell death characteristic of CAA in AD cases.

Abstract 353: Elastase-Activated Stress Response of Vascular Cells

Introduction: Rupture of atherosclerotic plaques is the most abundant cause for stroke. The serine protease elastase plays an important role as it induces death of endothelial cells (ECs) and smooth muscle cells (SMCs), and breaks down the fibrous cap of atherosclerotic plaques. Increased elastase concentrations were found in patients with symptomatic stenosis. We previously showed that elastase activates the endoplasmic reticulum (ER) stress signaling pathway unfolded protein response (UPR) in rupture-prone plaques of human carotid artery. However, signaling pathways elicited by elastase in vascular cells were largely unknown. We hypothesized that elastase induces cell-type dependent responses in ECs, SMCs and macrophages (M[[Unable to Display Character: Ф]]). Methods and Results: Different forms of cell death and UPR activation were analyzed in primary and immortalized endothelial cells, coronary artery smooth muscle cells (HCASMCs) and M[[Unable to Display Character: Ф]] after treatment with human neutrophil elastase. To discriminate between the involved cell death types, three independent assays were performed. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assay by confocal microscopy (p < .01), caspase3/7 activity by chemiluminescence-assays (p < .01) and cell-cycle analysis by flow cytometry revealed that an autophagic/apoptotic cell death was induced upon elastase treatment. This appeared specific for ECs, as it was absent in M[[Unable to Display Character: Ф]]. Necrosis (as determined by chemiluminescent lactate dehydrogenase-release assay) and necroptosis (assessed by flow cytometry) played only minor roles. The involvement of the UPR was investigated on protein and / or gene expression level. The high levels of GRP78, phospho-PERK, phospho-eIF2α, spliced XBP1 and CHOP indicate a strongly activated UPR that may give rise to the subsequent induced autophagic/apoptotic cell death. Conclusion: Elastase plays a significant role in plaque stability and cell survival likely through activation of a UPR/autophagic type of endothelial cell death. This may explain underlying molecular links how elastase destabilizes atherosclerotic plaques.

Saturated Fatty Acids Differentially Affects Adipocytes and Muscle Cells

Obesity and obesity related type 2 diabetes have become serious health issues in the US and worldwide. It is certain that the human genetic coding cannot been changed so dramatically by evolution during such a short period of time. Environmental factors including diets may play an important role in developing obesity/type 2 diabetes. Since adipose and muscle tissues are significantly affected in individuals with obesity, adipocytes and muscle cells were cultured in the conditions to mimic hyperlipidemia situations that happen during the development of obesity. We have observed that saturated fatty acids at high concentrations differentially affects adipocytes and muscle cells. The saturated fatty acid palmitic acid damages muscle cells and induces muscle cell death while causing more lipid accumulation in adipocytes. Polyunsaturated fatty acid linoleic acid also causes more lipid accumulation in adipocytes but it does not damage muscle cells. A few new candidate genes that are regulated by a saturated fatty acid in muscle cells have been identified by cDNA microarray. Further studies will be conducted to explore the mechanisms at the molecular level.

PPARδ modulates oxLDL-induced apoptosis of vascular smooth muscle cells through a TGF-β/FAK signaling axis

The peroxisome proliferator-activated receptor delta (PPARδ) has been implicated in the modulation of vascular homeostasis. However, its roles in the apoptotic cell death of vascular smooth muscle cells (VSMCs) are poorly understood. Here, we demonstrate that PPARδ modulates oxidized low-density lipoprotein (oxLDL)-induced apoptosis of VSMCs through the transforming growth factor-β (TGF-β) and focal adhesion kinase (FAK) signaling pathways. Activation of PPARδ by GW501516, which is a specific ligand, significantly inhibited oxLDL-induced cell death and generation of reactive oxygen species in VSMCs. These inhibitory effects were significantly reversed in the presence of small interfering (si)RNA against PPARδ, or by blockade of the TGF-β or FAK signaling pathways. Furthermore, PPARδ-mediated recovery of FAK phosphorylation suppressed by oxLDL was reversed by SB431542, a specific ALK5 receptor inhibitor, indicating that a TGF-β/FAK signaling axis is involved in the action of PPARδ. Among the protein kinases activated by oxLDL, p38 mitogen-activated protein kinase was suppressed by ligand-activated PPARδ. In addition, oxLDL-induced expression and translocation of pro-apoptotic or anti-apoptotic factors were markedly affected in the presence of GW501516. Those effects were reversed by PPARδ siRNA, or inhibitors of TGF-β or FAK, which also suggests that PPARδ exerts its anti-apoptotic effect via a TGF-β/FAK signaling axis. Taken together, these findings indicate that PPARδ plays an important role in the pathophysiology of disease associated with apoptosis of VSMC, such as atherosclerosis and restanosis.

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmdmdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca2+ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.

Expression and Function of Inositol 1,4,5-Trisphosphate Receptors in the Heart

Calcium is a second messenger that is essential for the control of a variety of cellular functions. In the heart, calcium plays an integral role in many cellular processes including muscle contraction, gene expression, and cell death. The inositol 1,4,5-trisphosphate receptor (IP3R) is a calcium channel that is ubiquitously expressed throughout the body. There are three IP3R isoforms encoded by separate genes. All three IP3R isoforms are expressed in the heart, with IP3R-2 reported to being most predominant with regards to both expression levels and functional significance. The role of IP3R signaling in the heart has been controversial in part due to its low expression level compared to other calcium channels. However, several groups have shown that IP3R-2 may play a key role in the progression of hypertrophy and apoptosis. The functional roles of IP3R-1 and IP3R-3 in the heart are essentially unexplored despite measureable expression levels. We hypothesize that all three IP3R isoforms can contribute to calcium transients and cardiac stress induced by endothelin-1 (ET-1). As our models we used embryonic mouse ventricular cardiomyocytes and the H9C2 cell line. Contrary to previous reports, we found that there is similar level of expression of all three IP3Rs in mouse ventricular cardiomyocytes and H9C2 cells. We next treated H9C2 cells with ET-1 for up to 96 hours and compared RNA and protein levels to controls. We found that was similar protein and RNA levels of all three IP3R isoforms in H9C2 cells after 48, 72 and 96 hours treatment with ET-1. These results demonstrate that all three IP3R isoforms are expressed in the heart at similar levels. Our results also suggest that ET-1 does not significantly affect IP3R expression levels, with possible significance for cardiac hypertrophy.

Titanium implant impairment and surrounding muscle cell death following neuro-myoelectrostimulation: An in vivo study.

Electrical currents have deleterious effects on biomedical metallic implants. However, following arthroplasty, neuro-myoelectrostimulation (NMES) is often used in patient rehabilitation. Such a rehabilitation technique could compromise patient recovery through deleterious effects on metallic alloys and biological tissues. The purpose of our study was to assess the effects of NMES on a Ti6Al4V implant placed in a rat tibial crest and the surrounding muscle tissues. This in vivo study allowed to bring to the fore the prosthesis behavior under mechanical and electromagnetic loads induced by NEMS stimulation. After 3 weeks, implant-to-bone adhesion significantly decreased in stimulated animals compared with nonstimulated animals. Surface mapping indicated titanium implant degradation after NMES. Furthermore, NMES alone did not induce muscle damage contrary to that found in implanted animals. The muscle damage rate was significantly higher in implanted and stimulated animals compared with implanted-only animals. It seems obvious that rehabilitation programs using the NMES technique could induce early deterioration of biomaterial employed for surgical implants. Clinicians should reconsider the use of NMES as a rehabilitation technique for patients with titanium prostheses.

Abstract 13514: Apoptogenic Protein is a Novel Phenotypic Marker of the Contractile Vascular Smooth Muscle Cells

Introduction: Apoptogenic protein (Apop) is a mitochondrial protein originally identified in the atherosclerotic cells cultured from thoracic aortas of ApoE-deficient mice. Functional analysis revealed that the expression of Apop using an expression vector induces apoptotic death of smooth muscle cells (SMC) in culture through the release of cytochrome c from mitochondria, followed by the activation of the caspase cascade. Here, we show that Apop is expressed in the contractile SMC, and can be a novel phenotypic marker of the SMC. Methods and Results: Human aortic smooth muscle cells (HASMC) and human coronary atery smooth muscle cells (HCASMC) were cultured in HuMedia-SB2 supplanted with hEGF, hFGF, insulin and 5% FBS. Differentiated HASMC were cultured on laminin-coated dishes in HuMedia-SD2 supplemented with heparin and 1% FBS. Gene expression level was analyzed by real time PCR. SM2 and SMemb were used as differentiation marker and synthetic SMC marker, respectively. The differentiated SMC showed a higher level of expression of SM2, and the ratio of SM2/SMemb was higher compared to the synthetic SMC. Apop gene expression was higher in differentiated HASMC compared to the synthetic phase cells. Similarly, HCASMC showed a higher level of Apop and SM2 expression and SM2/SMemb expression ratio in differentiated cells compared to the cells in synthetic phase. These results indicate that Apop is expressed in differentiated SMC at a higher level than synthetic SMC. CArG box, which is often present in phenotypic marker genes expressed in differentiated SMC, exists in the intron1 region of Apop gene. Serum response factor (SRF) binds to CArG box, and regulates the expression of the genes expressed in differentiated SMC. The silencing of SRF using siRNA reduced the expression of Apop in differentiaed HASMC, indicating that SRF regulates the expression of Apop in differentiated cells. Moreover, immunostaining using human atherosclerotic coronary artery specimens revealed that Apop gene is co-expressed with the differentiation marker, SM2 in the coronary artery SMC. Conclusion: These results indicate that Apop is a novel phenotypic marker of the differentiated SMC, and may be a novel therapeutic target for the prevention of the development of atherosclerosis.

Olmesartan inhibits cultured rat aortic smooth muscle cell death induced by cyclic mechanical stretch through the inhibition of the c-Jun N-terminal kinase and p38 signaling pathways

Acute aortic dissection (AAD) is a life-threating disease; however, there is almost no effective pharmacotherapy for it. An increase in c-Jun N-terminal kinase (JNK) phosphorylation and smooth muscle cell (SMC) apoptosis is observed tissues in patients with AAD. Therefore, we hypothesized that an acute rise in blood pressure leads to SMC death through phosphorylation of JNK or p38, which may cause AAD. We investigated the influence of cyclic mechanical stretch, which mimics an acute increase in blood pressure, on cultured rat aortic SMCs (RASMCs) and examined the changes in JNK and p38 phosphorylation. Further, we investigated the effect of olmesartan, an angiotensin II receptor blocker, on stretch-induced RASMC death. We found that mechanical stretch-induced RASMC death in a time-dependent manner, which correlated with the phosphorylation of JNK and p38. Olmesartan inhibited RASMC death and the phosphorylation of JNK and p38. JNK and p38 inhibitors reversed stretch-induced RASMC death. These results suggest that acute mechanical stretch causes JNK and p38 phosphorylation, which may result in SMC death leading to aortic dissection. Olmesartan may be used for pharmacotherapy to prevent aortic dissection, independent of its blood pressure-lowering effect, through its inhibition of JNK and p38 phosphorylation.

Biomarkers of plaque instability.

Atherosclerosis is the proximate cause of arterial thrombosis, leading to acute occlusive cardiovascular syndromes. Thrombosis in atherosclerosis usually results from rupture of the fibrous cap of atherosclerotic plaques with a smaller proportion resulting from superficial endothelial erosion. Ruptured plaques are often associated with intimal and adventitial inflammation, increased size of lipid-rich necrotic core with thinned out collagen-depleted fibrous cap, outward remodeling, increased plaque neovascularity, intraplaque hemorrhage, and microcalcification. By inference, non-ruptured plaques with similar compositional features are considered to be at risk for rupture and hence are labeled vulnerable plaques or high-risk plaques. Identification of vulnerable plaques may help in predicting the risk of acute occlusive syndromes and may also allow targeting for aggressive systemic and possibly local therapies. Plaque rupture is believed to result from extracellular matrix (which comprises the protective fibrous cap) dysregulation due to excessive proteolysis in the context of diminished matrix synthesis. Inflammation is believed to play a key role by providing matrix-degrading metalloproteinases and also by inducing death of matrix-synthesizing smooth muscle cells. Systemic markers of inflammation are thus the most logical forms of potential biomarkers which may predict the presence of vulnerable or high-risk plaques. Several studies have suggested the potential prognostic value of a variety of systemic markers, but regrettably, their overall clinical predictive value is modestly incremental at best, especially for individual subjects compared to groups of patients. Nevertheless, continued investigation of reliable, cost-effective biomarkers that predict the presence of a high-risk plaque and future athero-thrombotic cardiovascular events with greater sensitivity and specificity is warranted.

Low-frequency low energy ultrasound combined with microbubbles induces distinct apoptosis of A7r5 cells.

The present study aimed to investigate whether low frequency low energy ultrasound combined with microbubbles induces apoptotic cell death of A7r5 rat aortic vascular smooth muscle cells, and to identify the possible mechanisms underlying this effect. Ultrasonic waves (45 kHz with 0.3 Wcm2 of intensity for 0, 10, 20 and 30 sec) were used together with different dosages of SonoVue™ microbubbles (0, 14, 28, 42 and 56 µl), respectively. The cell viability and apoptotic rate were determined by trypan blue staining immediately following treatment and flow cytometry 24 h thereafter. The treatment conditions resulting in the lowest amount of necrosis, highest apoptotic rate and lowest microbubble dosage was selected for the US+MB group, which was treated with ultrasound combined with microbubbles. The cell proliferation 24 h following treatment was determined and western blot analysis was applied to examine the expression of apoptosis‑associated proteins, B-cell lymphoma 2 (Bcl‑2) and Bcl-2-associated X (Bax). The harmonic acoustic pressure amplitude was measured to obtain the cavitation intensity. The combination of 20 sec ultrasound irradiation and 14 µl SonoVue™ was selected as the treatment conditions for the US+MB group. The results demonstrated that both ultrasound alone (the US group) and in combination with microbubbles significantly inhibited the proliferation of A7r5 cells compared with that of the control (P<0.01), and the suppression in the US+MB group was significantly greater (P<0.01). The apoptotic rate in A7r5 cells induced by this combination treatment (16.62±0.93%) was significantly higher than that in the control (3.93±0.39%; P<0.01) and US (6.88±1.87%; P<0.01) groups. Treatment with ultrasound combined with microbubbles increased the expression of Bax and decreased the ratio of Bcl‑2/Bax compared with those in the control and US groups. The cavitation induced by ultrasound combined with microbubble treatment was more intense than that by ultrasound alone. The results demonstrated that the cell death and apoptosis of A7r5 cells were associated with ultrasound duration and microbubble dosage. Low frequency ultrasound combined with microbubbles induced apoptosis in A7r5 cells through the upregulation of Bax and the downregulation of the Bcl‑2/Bax ratio, where the cavitation effect may have an important role.

The effect of high expression of myogenic differentiation factor D in muscle satellite cells on prevention of denervation muscle atrophy: an experimental study in rats

Objeetive To study the effect of implantation of muscle satellite cells (MSCs) transtected with myogenic differentiation factor D (MyoD) high expression plasmids on denervated gastrocnemius muscle atrophy in rats.Methods Plasmid of high expression of MyoD was constructed and used to transfect MSCs derived from Sprague-Dawley rats.These transfected MSCs were transplanted into the denervated gastrocnemius muscles in a rat sciatic nerve transection model.At 2 and 4 weeks postoperatively,gastrocnemius muscles were harvested for measurement of wet muscle weight and muscle fiber cross sectional area from H&E stained tissue sections.The expression of MyoD was quantified with real-time PCR.Results Confocal microscopy detected MyoD expression in the transfected MSCs with a transfection efficiency of 43 ％.Gastrocnemius muscle wet weight of the MyoD transfected MSC group was higher than those of the other groups (P ＜ 0.05).The level of MyoD gene expression was higher in the MyoD transfected MSC group than in the other groups (P ＜ 0.05).Muscle cell degeneration and cell death was noted in the control group at 2 and 4 weeks postoperatively.Vacuole formation and myofiber rupture were also observed.Myofiber regeneration was present in the MSC group and MyoD transfected MSC group.Muscle fiber cross sectional area in the MyoD transfected MSC group was higher than the other groups at both 2 and 4 week time points.The differences were statistically significant (P ＜ 0.05).Conclusion Transplantation of high MyoD expression MSCs can delay denervation atrophy of gastrocnemius muscle in rats. Key words: Muscular atrophy; Skeletal muscle satellite cells; Myogenic differentiation factor D; Denervated muscle atrophy; Gene therapy

G.P.11: SMCHD1 mutations cause FSHD type 2 and act as modifiers of disease severity

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder of wide clinical variability. Contractions of the D4Z4 macro-satellite repeat on the permissive chromosome 4qA haplotype are the molecular basis of the disease. D4Z4 repeats with less than 11 copies are associated with chromatin decondensation and hypomethylation allowing for expression of the flanking DUX4 gene which is highly cytotoxic and causes skeletal muscle cell death. Recently, mutations in the SMCHD1 gene have been identified in a subset of patients without D4Z4 repeat contractions (FSHD2). Haploinsufficiency of the SMCHD1 protein results in hypomethylation of D4Z4 and a more open chromatin structure permitting activation of DUX4. We have screened phenotypic FSHD patients from 95 unrelated families for mutations in SMCHD1 by next generation sequencing (NGS). We identified mutations (deletions, missense, splice site and nonsense) in nine index patients without D4Z4 contractions. We developed a pyrosequencing assay to determine the methylation status of the D4Z4 repeat array and found significantly lower methylation levels for FSHD2 patients than for healthy controls and FSHD1 patients. SMCHD1 mutations were also found in two patients with moderately contracted D4Z4 alleles thus suffering from FSHD1 and 2. Comparing the phenotype of patients, all FSHD2 patients were relatively mildly affected while patients with FSHD1+2 were much more severely affected than expected from their D4Z4 copy number. Our findings confirm the digenic inheritance of FSHD2 with mutations in SMCHD1 and hypomethylation of D4Z4. In cases of “double trouble” with FSHD1 and 2, SMCHD1 mutations seem to act as a modifier of disease severity. With SMCHD1 mutations found in 16.4% of phenotypic FSHD patients, FSHD2 cannot be considered to be excessively rare. Hence, we suggest including sequencing of SMCHD1, haplotyping and methylation analysis in the workflow of molecular FSHD diagnostics.

Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy.

Disruption of the dystrophin complex causes muscle injury, dysfunction, cell death and fibrosis. Excess transforming growth factor (TGF) β signaling has been described in human muscular dystrophy and animal models, where it is thought to relate to the progressive fibrosis that characterizes dystrophic muscle. We now found that canonical TGFβ signaling acutely increases when dystrophic muscle is stimulated to contract. Muscle lacking the dystrophin-associated protein γ-sarcoglycan (Sgcg null) was subjected to a lengthening protocol to produce maximal muscle injury, which produced rapid accumulation of nuclear phosphorylated SMAD2/3. To test whether reducing SMAD signaling improves muscular dystrophy in mice, we introduced a heterozygous mutation of SMAD4 (S4) into Sgcg mice to reduce but not ablate SMAD4. Sgcg/S4 mice had improved body mass compared with Sgcg mice, which normally show a wasting phenotype similar to human muscular dystrophy patients. Sgcg/S4 mice had improved cardiac function as well as improved twitch and tetanic force in skeletal muscle. Functional enhancement in Sgcg/S4 muscle occurred without a reduction in fibrosis, suggesting that intracellular SMAD4 targets may be important. An assessment of genes differentially expressed in Sgcg muscle focused on those encoding calcium-handling proteins and responsive to TGFβ since this pathway is a target for mediating improvement in muscular dystrophy. These data demonstrate that excessive TGFβ signaling alters cardiac and muscle performance through the intracellular SMAD pathway.

Azelnidipine inhibits cultured rat aortic smooth muscle cell death induced by cyclic mechanical stretch.

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. Clarifying the detailed mechanism for aortic dissection is of great significance for establishing effective pharmacotherapy for this high mortality disease. In the present study, we evaluated the influence of biomechanical stretch, which mimics an acute rise in blood pressure using an experimental apparatus of stretching loads in vitro, on rat aortic smooth muscle cell (RASMC) death. Then, we examined the effects of azelnidipine and mitogen-activated protein kinase inhibitors on mechanical stretch-induced RASMC death. The major findings of the present study are as follows: (1) cyclic mechanical stretch on RASMC caused cell death in a time-dependent manner up to 4 h; (2) cyclic mechanical stretch on RASMC induced c-Jun N-terminal kinase (JNK) and p38 activation with peaks at 10 min; (3) azelnidipine inhibited RASMC death in a concentration-dependent manner as well as inhibited JNK and p38 activation by mechanical stretch; and (4) SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) protected against stretch-induced RASMC death; (5) Antioxidants, diphenylene iodonium and tempol failed to inhibit stretch-induced RASMC death. On the basis of the above findings, we propose a possible mechanism where an acute rise in blood pressure increases biomechanical stress on the arterial walls, which induces RASMC death, and thus, may lead to aortic dissection. Azelnidipine may be used as a pharmacotherapeutic agent for prevention of aortic dissection independent of its blood pressure lowering effect.

Cross talk between vascular smooth muscle cells and monocytes through interleukin-1β/interleukin-18 signaling promotes vein graft thickening.

Interleukin (IL)-1β and IL-18 are key proinflammatory cytokines that play important roles in the pathophysiology of vein graft remodeling. However, the mechanism of IL-1β/IL-18 production and its role in the development of graft remodeling remain unclear. IL-1β/IL-18 were rapidly expressed in venous interposition grafts. Vascular smooth muscle cell (VSMC) death and monocytic inflammasome activation occurred in grafted veins. Necrotic VSMCs induced the expression of IL-1β, IL-18, and other inflammasome-associated proteins in monocytes, which was partially inhibited by their antagonist, recombinant IL-1ra-Fc-IL-18bp. Activated monocytes stimulated proliferation of VSMCs by activating cell growth-related signaling molecules (AKT, STAT3, ERK1/2, and mTOR [AKT/protein kinase B, signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, mammalian target of rapamycin]) and increasing production of platelet-derived growth factor-bb; these effects were suppressed by IL-1ra-Fc-IL-18bp. Activated monocytes also promoted migration of VSMCs, which was independent of IL-1β/IL-18 signaling. Importantly, administration of IL-1ra-Fc-IL-18bp inhibited activation of cell growth-related signaling molecules, VSMC proliferation, and vein graft thickening in vivo. Our work identified an interaction among necrotic VSMCs, monocytes, and viable VSMCs through IL-1β/IL-18 signaling, which might be exploited as a therapeutic target in vein graft remodeling.

Embryonic Stem Cell–Derived Cardiac Myocytes Are Not Ready for Human Trials

Recently, a preclinical report assessed the efficacy, safety, and feasibility of using human embryonic stem cells to regenerate infarcted myocardium in a nonhuman primate model. This commentary evaluates that study by pointing out key weaknesses, offering an alternative perspective, and summarizing major unresolved issues. Our conclusion is that significant challenges remain before human embryonic stem cells are ready for use in clinical trials. Ischemic heart disease (IHD) affects millions of patients. The cardiac dysfunction that results from IHD is secondary to the death of cardiac muscle cells. Current IHD treatments are inadequate, and there is a need to develop novel therapies to improve the lives of affected patients. New cellular therapies are being developed for repairing damaged hearts via induction of cardiac myocyte regeneration. In our view, approaches that can regenerate new, functional myocardium need to be tested first in small animal models and then in appropriate large animal models. Those approaches that are shown to be safe and to improve cardiac function in preclinical studies should then be considered for clinical application. In a recent report in Nature , Chong et al1 describe experiments designed to test the potential of human embryonic stem cell–derived cardiac myocytes (hESC-CMs) to regenerate infarcted myocardium in a nonhuman primate model. This study was proposed by the authors as proof of concept for trials of these cells in patients. Our commentary looks at this report with an eye toward safety and efficacy of hESC-CMs. A major advance in the study of Chong et al1 is that an approach has been developed to produce large quantities of hESC-CMs that seem to be sufficient for future use in humans with IHD. The authors were able to amplify 1 billion hESC-CMs, a number purported sufficient to regenerate an adult human infarcted heart. This approach addresses …

FRI0521 Cd28null T Cells Kill Autologous Muscle Cells from Polymyositis Patients in Vitro by Perforin-Dependent Mechanisms

BackgroundInflammatory infiltrates of muscles in polymyositis are dominated by CD4+CD28null and CD8+CD28null T cells1. In contrast to conventional CD28+ T cells, these cells have a restricted T cell-receptor repertoire, rapidly...