Performance of Repetitive Tasks Induces Decreased Grip Strength and Increased Fibrogenic Proteins in Skeletal Muscle: Role of Force and Inflammation

Background This study elucidates exposure-response relationships between performance of repetitive tasks, grip strength declines, and fibrogenic-related protein changes in muscles, and their link to inflammation. Specifically, we examined forearm flexor digitorum muscles for changes in connective tissue growth factor (CTGF; a matrix protein associated with fibrosis), collagen type I (Col1; a matrix component), and transforming growth factor beta 1 (TGFB1; an upstream modulator of CTGF and collagen), in rats performing one of two repetitive tasks, with or without anti-inflammatory drugs. Methodology/Results To examine the roles of force versus repetition, rats performed either a high repetition negligible force food retrieval task (HRNF), or a high repetition high force handle-pulling task (HRHF), for up to 9 weeks, with results compared to trained only (TR-NF or TR-HF) and normal control rats. Grip strength declined with both tasks, with the greatest declines in 9-week HRHF rats. Quantitative PCR (qPCR) analyses of HRNF muscles showed increased expression of Col1 in weeks 3–9, and CTGF in weeks 6 and 9. Immunohistochemistry confirmed PCR results, and also showed greater increases of CTGF and collagen matrix in 9-week HRHF rats than 9-week HRNF rats. ELISA, and immunohistochemistry revealed greater increases of TGFB1 in TR-HF and 6-week HRHF, compared to 6-week HRNF rats. To examine the role of inflammation, results from 6-week HRHF rats were compared to rats receiving ibuprofen or anti-TNF-α treatment in HRHF weeks 4–6. Both treatments attenuated HRHF-induced increases in CTGF and fibrosis by 6 weeks of task performance. Ibuprofen attenuated TGFB1 increases and grip strength declines, matching our prior results with anti-TNFα. Conclusions/Significance Performance of highly repetitive tasks was associated with force-dependent declines in grip strength and increased fibrogenic-related proteins in flexor digitorum muscles. These changes were attenuated, at least short-term, by anti-inflammatory treatments

Quality Assessment of Published Systematic Reviews in High Impact Cardiology Journals: Revisiting the Evidence Pyramid

Objective: Systematic reviews are increasingly used as sources of evidence in clinical cardiology guidelines. In the present study, we aimed to assess the quality of published systematic reviews in high impact cardiology journals. Methods: We searched PubMed for systematic reviews published between 2010 and 2019 in five general cardiology journals with the highest impact factor (according to Clarivate Analytics 2019). We extracted data on eligibility criteria, methodological characteristics, bias assessments, and sources of funding. Further, we assessed the quality of retrieved reviews using the AMSTAR tool. Results: A total of 352 systematic reviews were assessed. The AMSTAR quality score was low or critically low in 71% (95% CI: 65.7–75.4) of the assessed reviews. Sixty-four reviews (18.2%, 95% CI: 14.5–22.6) registered/published their protocol. Only 221 reviews (62.8%, 95% CI: 57.6–67.7) reported adherence to the EQUATOR checklists, 208 reviews (58.4%, 95% CI: 53.9–64.1) assessed the risk of bias in the included studies, and 177 reviews (52.3%, 95% CI: 45.1–55.5) assessed the risk of publication bias in their primary outcome analysis. The primary outcome was statistically significant in 274 (79.6%, 95% CI: 75.1–83.6) and had statistical heterogeneity in 167 (48.5%, 95% CI: 43.3–53.8) reviews. The use and sources of external funding was not disclosed in 87 reviews (24.7%, 95% CI: 20.5–29.5). Data analysis showed that the existence of publication bias was significantly associated with statistical heterogeneity of the primary outcome and that complex design, larger sample size, and higher AMSTAR quality score were associated with higher citation metrics. Conclusion: Our analysis uncovered widespread gaps in conducting and reporting systematic reviews in cardiology. These findings highlight the importance of rigorous editorial and peer review policies in systematic review publishing, as well as education of the investigators and clinicians on the synthesis and interpretation of evidence

Mutation in Osteoactivin Decreases Bone Formation in Vivo and Osteoblast Differentiation in Vitro

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb+/SjJ (D2J/Gpnmb+)]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb+ mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb+ osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation

Quality Assessment of Published Systematic Reviews in High Impact Cardiology Journals: Revisiting the Evidence Pyramid

Objective: Systematic reviews are increasingly used as sources of evidence in clinical cardiology guidelines. In the present study, we aimed to assess the quality of published systematic reviews in high impact cardiology journals.Methods: We searched PubMed for systematic reviews published between 2010 and 2019 in five general cardiology journals with the highest impact factor (according to Clarivate Analytics 2019). We extracted data on eligibility criteria, methodological characteristics, bias assessments, and sources of funding. Further, we assessed the quality of retrieved reviews using the AMSTAR tool.Results: A total of 352 systematic reviews were assessed. The AMSTAR quality score was low or critically low in 71% (95% CI: 65.7–75.4) of the assessed reviews. Sixty-four reviews (18.2%, 95% CI: 14.5–22.6) registered/published their protocol. Only 221 reviews (62.8%, 95% CI: 57.6–67.7) reported adherence to the EQUATOR checklists, 208 reviews (58.4%, 95% CI: 53.9–64.1) assessed the risk of bias in the included studies, and 177 reviews (52.3%, 95% CI: 45.1–55.5) assessed the risk of publication bias in their primary outcome analysis. The primary outcome was statistically significant in 274 (79.6%, 95% CI: 75.1–83.6) and had statistical heterogeneity in 167 (48.5%, 95% CI: 43.3–53.8) reviews. The use and sources of external funding was not disclosed in 87 reviews (24.7%, 95% CI: 20.5–29.5). Data analysis showed that the existence of publication bias was significantly associated with statistical heterogeneity of the primary outcome and that complex design, larger sample size, and higher AMSTAR quality score were associated with higher citation metrics.Conclusion: Our analysis uncovered widespread gaps in conducting and reporting systematic reviews in cardiology. These findings highlight the importance of rigorous editorial and peer review policies in systematic review publishing, as well as education of the investigators and clinicians on the synthesis and interpretation of evidence

Molecular, Phenotypic Aspects and Therapeutic Horizons of Rare Genetic Bone Disorders

A rare disease afflicts less than 200,000 individuals, according to the National Organization for Rare Diseases (NORD) of the United States. Over 6,000 rare disorders affect approximately 1 in 10 Americans. Rare genetic bone disorders remain the major causes of disability in US patients. These rare bone disorders also represent a therapeutic challenge for clinicians, due to lack of understanding of underlying mechanisms. This systematic review explored current literature on therapeutic directions for the following rare genetic bone disorders: fibrous dysplasia, Gorham-Stout syndrome, fibrodysplasia ossificans progressiva, melorheostosis, multiple hereditary exostosis, osteogenesis imperfecta, craniometaphyseal dysplasia, achondroplasia, and hypophosphatasia. The disease mechanisms of Gorham-Stout disease, melorheostosis, and multiple hereditary exostosis are not fully elucidated. Inhibitors of the ACVR1/ALK2 pathway may serve as possible therapeutic intervention for FOP. The use of bisphosphonates and IL-6 inhibitors has been explored to be useful in the treatment of fibrous dysplasia, but more research is warranted. Cell therapy, bisphosphonate polytherapy, and human growth hormone may avert the pathology in osteogenesis imperfecta, but further studies are needed. There are still no current effective treatments for these bone disorders; however, significant promising advances in therapeutic modalities were developed that will limit patient suffering and treat their skeletal disabilities

Increased CTGF and collagen staining decreases after anti-inflammatory drug treatments.

<p>CTGF immunohistochemistry (panels A–F) and Masson's trichrome staining (panels G,H) increased in flexor digitorum muscles (M) and lumbricals in TR-HF and 6-week HRHF task rats, and decrease with anti-inflammatory drugs. Panels A–F show CTGF immunostaining (red) of flexor digitorum muscles from: <i>(A)</i> TR-HF, <i>(B)</i> TR-HF treated with anti-TNF-α, <i>(C)</i> untreated 6-week HRHF (showing CTGF-immunostained cells at edges of myofibers), <i>(D)</i> 6-week HRHF treated with anti-TNF-α (inset shows small CTGF-immunostained cells in endomyseum), <i>(E)</i> 6-week HRHF treated with ibuprofen, and <i>(F)</i> untreated 6-week HRHF (showing increased CTGF-immunostained cells in endomyseum (ct)). <i>(G)</i> Quantification of percent area with CTGF immunostaining. **p<0.01 compared to NC; ^## p<0.01 compared to TR-HF; ^&&p<0.01 compared to untreated 6-week HRHF; n = 4–12/gp. <i>(F)</i> Lumbricals from Region B stained with Masson's Trichrome showing: <i>(H)</i> increased blue stained collagen matrix in an untreated 6-week HRHF rat, and <i>(I)</i> decreased blue staining in a 6-week HRHF treated with anti-TNF-α. Scale bars = 50 µm.</p

Connective tissue growth factor immunostaining increased more in HRHF muscles than in HRNF.

<p>Cross-sections (panels A,B,D), and longitudinal sections (panel C) from region A of flexor digitorum muscles (M) are shown. <i>(A)</i> NC muscle showing only low levels of CTGF immunostaining. <i>(B)</i> 9-week HRNF muscle showing a small increase in CTGF-immunostained cells at peripheral edges of myofibers (arrows). <i>(C)</i> 9-week HRHF muscle showing increased CTGF in larger mast-like cells (arrowheads) near a blood vessel (bv). <i>(D)</i> 9-week HRHF muscle also contained smaller CTGF-immunostained cells (arrows) at the periphery of myofibers. Inset shows higher power of the CTGF-immunostained cells at edges of myofibers. <i>(E)</i> Quantification of CTGF immunostaining in flexor digitorum muscles. <i>(F)</i> A lane from a representative Western blot showing that the CTGF antibody used for the immunohistochemistry detects a band at 38 kDa in the flexor digitorum muscles, the expected molecular weight of CTGF. The whitish band below the 38 kDa band indicates the site of GAPDH. The gel was first stained with anti-GAPDH and then stripped prior to staining with anti-CTGF. ***p<0.001 and ^###p<0.001, compared to NC and TR-HF, respectively (n = 3–10/gp). Scale bars = 50 µm.</p

TGFB1 increases in TR-HF and 6-week HRHF rats and is attenuated by ibuprofen.

<p><i>(</i><b><i>A</i></b><i>)</i> NC muscle cut in cross-section showing no TGFB1 immunoreactive cells. <i>(B)</i> 6-week HRHF muscle showing TGFB1-immunostained cells at edges of myofibers (arrows). <i>(C)</i> Muscle from a 6-week HRHF rat treated with ibuprofen (6-wk HRHF+IBU) showing fewer TGFB1-immunostained cells than in panel B. <i>(D)</i> Muscle from a 6-week HRHF rat treated with anti-TNF-α (6-wk HRHF+anti-TNF) showing reduced TGFB1 immunostaining. <i>(E)</i> ELISA results for TGFB1 in muscles from NC, 6-week HRNF, TR-HF, and 6-week HRHF rats; n = 3–8/gp. <i>(F)</i> Quantification of percent area of muscle with TGFB1 immunostaining. *p<0.05 compared to NC; ^&p<0.05 compared to untreated 6-week HRHF; n = 4–10/gp. Scale bars = 50 µm.</p

Collagen immunostaining increases in flexor digitorum muscles with HRNF and HRHF tasks.

<p>Cross-sections (panels A,B,D), and longitudinal sections (panel C) from region A of flexor digitorum muscle (M) are shown. <i>(A)</i> NC muscle (M) showing no immunostaining for collagen type 1 in or around individual myofibers. <i>(B)</i> 9-week HRNF muscle showing increased collagen type I staining around a few myofibers. Arrows indicate collagen immunoreactive staining extending around individual myofibers. Inset shown higher power photo of area in panel B indicated with an asterisk. <i>(C,D)</i> 9-week HRHF muscle showing increased collagen type I staining in the endomyseum (panel C), and in small cells at the edges of some myofibers (arrows; panel D), as well as within some myofibers (panel D).The T in panel C indicates a small tendon slip within the muscle mass region. Similar results were observed in n = 3/gp. <i>(E)</i> Lanes from a representative Western blot of flexor digitorum muscles from HRHF rats probed with the collagen type 1 antibody used for the immunohistochemistry. Lane 1 shows the standards; Lane 2 shows bands at the expected molecular weights of procollagen type I (approximately 140 kDa; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038359#pone.0038359-MartinezSalgado1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038359#pone.0038359-Iwasaki1" target="_blank">[46]</a>) and mature collagen (75 Kda here; known to be between 70 and 90 kDa; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038359#pone.0038359-MartinezSalgado1" target="_blank">[45]</a>); Lane 3 from a different muscle sample showing mainly detection of a band at the molecular weight of mature collagen. Scale bars = 50 µm.</p