MAB1691 Sigma-AldrichAnti-Troponin I Antibody, a.a. 186-192, clone C5

In addition to the primary symptoms arising from inflamed joints, muscle weakness is prominent and frequent in patients with rheumatoid arthritis (RA). Here, we investigated the mechanisms of arthritis-induced muscle dysfunction in rats with adjuvant-induced arthritis (AIA).AIA was induced in the knees of rats by injection of complete Freund's adjuvant and was allowed to develop for 21 days. Muscle contractile function was assessed in isolated extensor digitorum longus (EDL) muscles. To assess mechanisms underlying contractile dysfunction, we measured redox modifications, redox enzymes and inflammatory mediators, and activity of actomyosin ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase.EDL muscles from AIA rats showed decreased tetanic force per cross-sectional area and slowed twitch contraction and relaxation. These contractile dysfunctions in AIA muscles were accompanied by marked decreases in actomyosin ATPase and SR Ca(2+)-ATPase activities. Actin aggregates were observed in AIA muscles, and these contained high levels of 3-nitrotyrosine and malondialdehyde-protein adducts. AIA muscles showed increased protein expression of NADPH oxidase 2/gp91(phox), neuronal nitric oxide synthase, tumor necrosis factor α (TNF-α), and high-mobility group box 1 (HMGB1). Treatment of AIA rats with EUK-134 (3 mg/kg/day), a superoxide dismutase/catalase mimetic, prevented both the decrease in tetanic force and the formation of actin aggregates in EDL muscles without having any beneficial effect on the arthritis development.Antioxidant treatment prevented the development of oxidant-induced actin aggregates and contractile dysfunction in the skeletal muscle of AIA rats. This implies that antioxidant treatment can be used to effectively counteract muscle weakness in inflammatory conditions.

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro-generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG-fibrinogen hydrogel. Once engineered to express placental-derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG-fibrinogen-embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient-specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.

A K141N missense mutation in heat shock protein (HSP) B8, which belongs to the small HSP family, causes distal hereditary motor neuropathy, which is characterized by the formation of inclusion bodies in cells. Although the HSPB8 gene causes hereditary motor neuropathy, obvious expression of HSPB8 is also observed in other tissues, such as the heart. The effects of a single mutation in HSPB8 upon the heart were analyzed using rat neonatal cardiomyocytes. Expression of HSPB8 K141N by adenoviral infection resulted in increased HSPB8-positive aggregates around nuclei, whereas no aggregates were observed in myocytes expressing wild-type HSPB8. HSPB8-positive aggresomes contained amyloid oligomer intermediates that were detected by a specific anti-oligomer antibody (A11). Expression of HSPB8 K141N induced slight cellular toxicity. Recombinant HSPB8 K141N protein showed reactivity against the anti-oligomer antibody, and reactivity of the mutant HSPB8 protein was much higher than that of wild-type HSPB8 protein. To extend our in vitro study, cardiac-specific HSPB8 K141N transgenic (TG) mice were generated. Echocardiography revealed that the HSPB8 K141N TG mice exhibited mild hypertrophy and apical fibrosis as well as slightly reduced cardiac function, although no phenotype was detected in wild-type HSPB8 TG mice. A single point mutation of HSPB8, such as K141N, can cause cardiac disease.

Activation of ErbB2/4 receptor tyrosine kinases in cardiomyocytes by neuregulin treatment is associated with improvement in cardiac function, supporting its use in human patients with heart failure despite the lack of a specific mechanism. Neuregulin infusion in rodents increases cardiac myosin light chain kinase (cMLCK) expression and cardiac myosin regulatory light chain (RLC) phosphorylation which may improve actin-myosin interactions for contraction. We generated a cMLCK knockout mouse to test the hypothesis that cMLCK is necessary for neuregulin-induced improvement in cardiac function by increasing RLC phosphorylation.The cMLCK knockout mice have attenuated RLC phosphorylation and decreased cardiac performance measured as fractional shortening. Neuregulin infusion for seven days in wildtype mice increased cardiac cMLCK protein expression and RLC phosphorylation while increasing Akt phosphorylation and decreasing phospholamban phosphorylation. There was no change in fractional shortening. In contrast, neuregulin infusion in cMLCK knockout animals increased cardiac performance in the absence of cMLCK without increasing RLC phosphorylation. In addition, CaMKII signaling appeared to be enhanced in neuregulin-treated knockout mice.Thus, Neuregulin may improve cardiac performance in the failing heart without increasing cMLCK and RLC phosphorylation by activating other signaling pathways.

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca(2+) sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca(2+) transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

Although clinical benefit can be achieved after cardiac transplantation of adult c-kit+ or cardiosphere-derived cells for myocardial repair, these stem cells lack the regenerative capacity unique to neonatal cardiovascular stem cells. Unraveling the molecular basis for this age-related discrepancy in function could potentially transform cardiovascular stem cell transplantation. In this report, clonal populations of human neonatal and adult cardiovascular progenitor cells were isolated and characterized, revealing the existence of a novel subpopulation of endogenous cardiovascular stem cells that persist throughout life and co-express both c-kit and isl1. Epigenetic profiling identified 41 microRNAs whose expression was significantly altered with age in phenotypically-matched clones. These differences were correlated with reduced proliferation and a limited capacity to invade in response to growth factor stimulation, despite high levels of growth factor receptor on progenitors isolated from adults. Further understanding of these differences may provide novel therapeutic targets to enhance cardiovascular regenerative capacity.

Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening.

Heart regeneration through in vivo cardiac reprogramming has been demonstrated as a possible regenerative strategy. While it has been reported that cardiac reprogramming in vivo is more efficient than in vitro, the influence of the extracellular microenvironment on cardiac reprogramming remains incompletely understood. This understanding is necessary to improve the efficiency of cardiac reprogramming in order to implement this strategy successfully. Here we have identified matrix identity and cell-generated tractional forces as key determinants of the dedifferentiation and differentiation stages during reprogramming. Cell proliferation, matrix mechanics, and matrix microstructure are also important, but play lesser roles. Our results suggest that the extracellular microenvironment can be optimized to enhance cardiac reprogramming.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. DMD patients lack dystrophin protein and develop skeletal muscle pathology and dilated cardiomyopathy (DCM). Approximately 20% succumb to cardiac involvement. We hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent DCM in animal models of DMD. ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and α-actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn(-/-) mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin(+) cardiac stem cells were detected in ADM-injected heart. Nestin(+) striated cells were also detected in four of five mdx/utrn(-/-) hearts injected with ADMs. In contrast, when ADMs were injected into the heart of aged mdx mice with advanced fibrosis, no functional improvement was detected by echocardiography. Instead, ADMs exacerbated some features of DCM. No dystrophin protein, increase in CD31 expression, or increase in Nestin(+) cell proliferation was detected following ADM injection in aged mdx heart. Dystrophin was observed following transplantation of ADMs into the hearts of young mdx mice, however, suggesting that pathology in aged mdx heart may alter the fate of donor stem cells. In summary, ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.