Array atomic force microscopy for real-time multiparametric analysis.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale biological and physical systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. Conventional AFMs only permit sequential single-point analysis; widespread adoption of array AFMs for simultaneous multipoint study is challenging owing to the intrinsic limitations of existing technological approaches. Here, we describe a prototype dispersive optics-based array AFM capable of simultaneously monitoring multiple probe-sample interactions. A single supercontinuum laser beam is utilized to spatially and spectrally map multiple cantilevers, to isolate and record beam deflection from individual cantilevers using distinct wavelength selection. This design provides a remarkably simplified yet effective solution to overcome the optical cross-talk while maintaining subnanometer sensitivity and compatibility with probe-based sensors. We demonstrate the versatility and robustness of our system on parallel multiparametric imaging at multiscale levels ranging from surface morphology to hydrophobicity and electric potential mapping in both air and liquid, mechanical wave propagation in polymeric films, and the dynamics of living cells. This multiparametric, multiscale approach provides opportunities for studying the emergent properties of atomic-scale mechanical and physicochemical interactions in a wide range of physical and biological networks

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor β receptor I (TGFβRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a "resting" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation

Mechanical regulation of cardiac fibroblast profibrotic phenotypes.

Cardiac fibrosis is a serious condition currently lacking effective treatments. It occurs as a result of cardiac fibroblast (CFB) activation and differentiation into myofibroblasts, characterized by proliferation, extracellular matrix (ECM) production and stiffening, and contraction due to the expression of smooth muscle α-actin. The mechanical properties of myocardium change regionally and over time after myocardial infarction (MI). Although mechanical cues are known to activate CFBs, it is unclear which specific mechanical stimuli regulate which specific phenotypic trait; thus we investigated these relationships using three in vitro models of CFB mechanical activation and found that 1) paracrine signaling from stretched cardiomyocytes induces CFB proliferation under mechanical conditions similar to those of the infarct border region; 2) direct stretch of CFBs mimicking the mechanical environment of the infarct region induces a synthetic phenotype with elevated ECM production; and 3) progressive matrix stiffening, modeling the mechanical effects of infarct scar maturation, causes smooth muscle α-actin fiber formation, up-regulation of collagen I, and down-regulation of collagen III. These findings suggest that myocyte stretch, fibroblast stretch, and matrix stiffening following MI may separately regulate different profibrotic traits of activated CFBs

Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin

Background: Pressure overload of the heart occurs in patients with hypertension or valvular stenosis and induces cardiac fibrosis because of excessive production of extracellular matrix by activated cardiac fibroblasts. This initially provides essential mechanical support to the heart, but eventually compromises function. Osteopontin is associated with fibrosis; however, the underlying signaling mechanisms are not well understood. Herein, we examine the effect of thrombin-cleaved osteopontin on fibrosis in the heart and explore the role of syndecan-4 in regulating cleavage of osteopontin. Methods and Results: Osteopontin was upregulated and cleaved by thrombin in the pressure-overloaded heart of mice subjected to aortic banding. Cleaved osteopontin was higher in plasma from patients with aortic stenosis receiving crystalloid compared with blood cardioplegia, likely because of less heparin-induced inhibition of thrombin. Cleaved osteopontin and the specific osteopontin peptide sequence RGDSLAYGLR that is exposed after thrombin cleavage both induced collagen production in cardiac fibroblasts. Like osteopontin, the heparan sulfate proteoglycan syndecan-4 was upregulated after aortic banding. Consistent with a heparan sulfate binding domain in the osteopontin cleavage site, syndecan-4 was found to bind to osteopontin in left ventricles and cardiac fibroblasts and protected osteopontin from cleavage by thrombin. Shedding of the extracellular part of syndecan-4 was more prominent at later remodeling phases, at which time levels of cleaved osteopontin were increased. Conclusions: Thrombin-cleaved osteopontin induces collagen production by cardiac fibroblasts. Syndecan-4 protects osteopontin from cleavage by thrombin, but this protection is lost when syndecan-4 is shed in later phases of remodeling, contributing to progression of cardiac fibrosis

Glypican-6 expression is increased in the failing mouse heart.

<p>Schematic of the aortic banding (AB) heart failure model with heart regions indicated (A). Chronic pressure overload was induced in adult mice by banding of the ascending aorta. RA: right atrium, RV: right ventricle, LA: left atrium, LV: left ventricle. Relative LV mRNA levels of GPC1 (B), GPC2 (C), GPC3 (D), GPC4 (E) and GPC6 (F) after 24h, 1, 3, 16 and 18 weeks of AB or sham-operation in male mice (n = 3–10). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.t001" target="_blank">Table 1</a> for animal characteristics. mRNA expression was normalized to ribosomal protein L32 (RPL32) expression. Representative immunoblots and quantitative data of full length GPC6 (GPC6FL; Mw ≈62kDa) in LV protein lysates from AB- and sham-operated control mice analyzed under reducing conditions (+ dithiothreitol). For immunoblotting of heparan sulfate (HS) proteoglycans in tissue, proteoglycans were methanol (MetOH) precipitated prior to digestion with heparan sulfate degrading enzymes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.ref008" target="_blank">8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.ref036" target="_blank">36</a>](G and H; n = 3 at all time-points). Recombinant human GPC6 produced in <i>E</i>.<i>coli</i> was used as positive control (Rec.hGPC6). Data are presented as mean ± S.E.M. Unpaired Student’s <i>t-test</i> was used for statistical testing vs. controls at respective time-points. *<i>P<</i>0.05; **<i>P<</i>0.01; ***<i>P<</i>0.001. Pearson correlations of LV GPC6/RPL32 mRNA vs. LV weight/tibia length (TL)(I), ACTA1/RPL32 mRNA (J), lung weight/TL (K) and NPPA/RPL32 mRNA (L) in AB- and sham-operated mice (24h-18 weeks).</p

Glypican-6 enhances ERK1/2 signaling and hypertrophic responses in cultured cardiomyocytes.

<p>Representative immunoblots and quantification of phospho-extracellular signal-regulated kinase (pERK)1 (pERK 44) relative to total ERK1 (totERK 44; Mw ≈44 kDa) and phospho-ERK2 (pERK 42) relative to total ERK2 (totERK 42; Mw ≈42 kDa) in neonatal rat cardiomyocytes (NCM) transduced with an adenovirus encoding human GPC6 (AdhGPC6) or empty vector (AdNull; A-C), n = 5. Vinculin was used as loading control. Immunoblots in A were run under reducing conditions (+dithiothreitol) revealing the N-terminal domain of GPC6 (GPC6N; ≈35kDa). The full length GPC6 (GPC6FL; Mw ≈62kDa) band represents non-glycanated GPC6 where N- and C-terminal domains are held together by disulfide bonds. Relative mRNA levels of atrial and brain natriuretic peptides (NPPA and NPPB, respectively, D and E) normalized to ribosomal protein L4 (RPL4) in NCM transduced with AdhGPC6 or empty vector, and treated with the dual specificity kinase (MEK1/2) inhibitor U0126 or vehicle control, n = 9–18 from three separate cell cultures. [³H] leucine incorporation in NCM transduced with AdhGPC6 or empty vector and treated with U0126 or vehicle control (F), relative to AdNull, non-treated control, n = 6–12. Serum was used as a positive control. Relative mRNA levels of α-skeletal actin (ACTA1, G) normalized to ribosomal protein L4 (RPL4) in NCM transduced with AdhGPC6 or empty vector, and treated with U0126 or vehicle control, n = 8–18 from three separate cell cultures (<i>t-test</i> AdNull vs. AdhGPC6, p = 0.0063). Data are presented as mean ± S.E.M. Unpaired Student’s <i>t-test</i> (B and C) and one-way ANOVA with Bonferroni post-hoc test (D-G) were used to test for statistical significance. *<i>P<</i>0.05; **<i>P<</i>0.01; ***<i>P<</i>0.001; AdhGPC6-transduced NCM significantly different from empty vector control or U0126-treated groups.</p