Atomic Force Microscopy: an innovative technology to explore cardiomyocyte cell surface in cardiac physio/pathophysiology

International audienceAtomic Force Microscopy (AFM) has emerged these recent years as a multifunctional toolbox for studying biological samples in physiological conditions. Although its use has spread among biologists community, cardiology remains a scientific field where not been extensively used yet. Heart diseases are nowadays a major human threat, and cause the death of millions of people each year. A convergent point to all heart diseases seems to be related to the defect of the cardiomyocyte, the contractile unit of the he reason, many scientists got interested in this cell type. However, very few studies use a technology such as AFM and its derivatives (force spectroscopy, multiparametric imaging) to explore this cell. The aim of this review is thus to give a comprehensive an interest of the biophysical approach made possible by AFM studies. We will show how AFM has been and can be used to study fix living cardiomyocytes, and, how combined with other types of microscopy, it can help getting a better understanding o pathologies or drugs. This review is the first dedicated to the use of AFM technics in cardiology, and gives new insights in fundamental questions surrounding cardiomyocytes, that can be answered using such a technology

Atomic Force Microscopy: an innovative technology to explore cardiomyocyte cell surface in cardiac physio/pathophysiology

International audienceAtomic Force Microscopy (AFM) has emerged these recent years as a multifunctional toolbox for studying biological samples in physiological conditions. Although its use has spread among biologists community, cardiology remains a scientific field where not been extensively used yet. Heart diseases are nowadays a major human threat, and cause the death of millions of people each year. A convergent point to all heart diseases seems to be related to the defect of the cardiomyocyte, the contractile unit of the he reason, many scientists got interested in this cell type. However, very few studies use a technology such as AFM and its derivatives (force spectroscopy, multiparametric imaging) to explore this cell. The aim of this review is thus to give a comprehensive an interest of the biophysical approach made possible by AFM studies. We will show how AFM has been and can be used to study fix living cardiomyocytes, and, how combined with other types of microscopy, it can help getting a better understanding o pathologies or drugs. This review is the first dedicated to the use of AFM technics in cardiology, and gives new insights in fundamental questions surrounding cardiomyocytes, that can be answered using such a technology

TNFα production or expression by macrophages (isolated from uninfected immunocompetent mice) upon interaction with resting conidia of parental (<i>ku80</i>) and <i>ags</i>Δ_<i>5T</i> strains or <i>ags</i>Δ_<i>5T</i> conidial NaCl extract (3.2 µg proteins) respectively.

<p>(A) TNFα was quantified after 5 h incubation of the conidia with macrophages; (B) Relative expression of TNFα assessed by real time RT-PCR in total RNA from macrophages after 5 h incubation of the <i>ags</i>Δ_<i>5T</i> conidial NaCl extract with macrophages. NaCl supernatant from <i>ku80</i> resting conidia incubated for 2 h in 0.5M NaCl was used as a control. NS: Non-stimulated. *, P<0.05.</p

Surface analysis of resting conidia of <i>ags</i>Δ_<i>5T</i> mutant and parental (<i>ku80</i>) strains.

<p>(A): height images (z-range = 1 µm; recorded in water with silicon nitride tips). Atomic Force Microscopy (AFM) images showing the amorphous surface without the rodlet layer on the triple <i>ags</i>Δ_<i>5T</i> mutant conidia whereas the rodlet are observed on the parental strain conidial surface. (B): TEM observations. Note the presence of an extracellular material on the surface of the <i>ags</i>Δ_<i>5T</i> conidia (arrow); CW: cell wall. (C): SDS-PAGE (15% gel) of Hydrofluoric acid (HF) extracts of rodlets from resting conidia showing the two bands, 16 kDa and 14.5 kDa of RodAp classically seen from HF treatment of the conidia <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003716#ppat.1003716-Aimanianda1" target="_blank">[10]</a>. Data are representative of at least three independent experiments.</p

Conidiocidal activity of macrophages isolated from uninfected p47<i>^phox</i>^−/− mice against resting conidia of <i>ags</i>Δ_<i>5T</i> and parental (<i>ku80</i>) strains.

<p>Conidiocidal activity is expressed in percentage of CFU inhibition after 2 and 6 h incubation of the conidia with macrophages. Data are representative from at least three independent experiments. *, P<0.05.</p

Cyclophosphamide immunosuppressed mice and anti-Ly6G treated neutropenic mice infected with resting conidia of <i>ags</i>Δ_<i>5T</i> and parental (<i>ku80</i>) strains.

<p>(A–C) Cyclophosphamide immunosuppressed mice; (D–E) anti-Ly6G treated neutropenic mice; (A) Survival (%) and (B) fungal growth estimated as CFUs in lung. (C and E) lung histology (periodic acid-Schiff-staining). Note the polymorphonuclear cells and mononuclear infiltrates surrounding the bronchi in <i>ku80</i> infected lung. (D) Histological appearance of lungs of anti-Ly6G neutropenic mice infected with conidia of <i>ags</i>Δ_<i>5T</i> and <i>ku80</i> (Gomori's methanamine silver-staining). Note the absence of mycelial development of <i>ags</i>Δ_<i>5T</i> conidia in neutropenic mice. Data are representative of at least three independent experiments. *: p<0.05.</p

Phagocytosis activity by isolated macrophages from uninfected mice against resting conidia of <i>ags</i>Δ_<i>5T</i> and parental (<i>ku80</i>) strains.

<p>Index of phagocytosis is expressed in number of conidia per alveolar macrophage after 1*, P<0.05.</p

Imaging and adhesive properties of <i>A. fumigatus</i> resting conidia of the parental strain and <i>ags</i>Δ_<i>5T</i> mutant.

<p>Structural changes of <i>ags</i>Δ_<i>5T</i> correlate with a loss of cell surface adhesive properties. (A–C) parental strain; (D–F) <i>ags</i>Δ_<i>5T</i> mutant; (A, D) height images (z-range = 1 µm; recorded in water with silicon nitride tips); (B, E) adhesion force maps (z-range: 5 nN) corresponding to the height image; (C, F) Representative force-distance curves and adhesion force histograms (n = 1024) recorded on the surface of parental strain (C) and <i>ags</i>Δ_<i>5T</i> (F).</p

NaCl extracted proteins from the surface of <i>ags</i>Δ_<i>5T</i> resting conidia.

<p>SDS-PAGE (10% gel) of proteins extracted after 2 h incubation of <i>ags</i>Δ_<i>5T</i> and <i>ku80</i> resting conidia in 0.5 M NaCl.</p

Proteins identified in the NaCl extract of <i>ags</i>Δ_<i>5T</i> and <i>ags</i>Δ_<i>n8</i> conidia.

<p>Identification was done by MS/MS and MS with a mascot score above a threshold of 54. Details are showed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003716#ppat.1003716.s009" target="_blank">Table S1</a>.</p