Cardiomyocyte internal structure of a Duchenne muscular dystrophy murine model by second harmonic generation multiphoton microscopy

International audienceDuchenne muscular dystrophy (DMD) is a severe and lethal disease that affects ~ 1/3,500 boys per year. It is linked to mutations in the dystrophin gene on the X chromosome coding for a major and large cytoskeletal protein. By using a DMD mouse model (mdx), we have previously shown the development of dilated cardiomyopathy with aging is associated with a dysfunction of the contractile properties of the ventricular cardiomyocytes (Fauconnier et al PNAS). However, the link between the dystrophin deficiency, the internal structure and the contractile dysfunction of the cardiomyocytes is unclear.In this work, second harmonic microscopy (SHG) was used to acquire images on mdx and control alive cardiomyocytes at different ages and in different experimental conditions to study the cellular cytoskeleton and contractile apparatus organization in absence of artefacts generated by histologic fixation and use of antibody. SHG is a technique based on second harmonic response of biological organized systems harboring non-centrosymmetry or chromophores. SHG makes functional imaging possible on muscle cells in absence of fluorescent staining [1,2]. Indeed, by measuring the SHG anisotropy important results are extracted on the structural state of actomyosin motors with an impressive sensitivity of myosin filament conformation2. The multiphotonic microscopy allows following dynamic formation of myosin filaments 1 and the organization of key sarcomeric cardiac proteins. Accurate image analysis tools should allow the analysis of several regional properties of the cardiomyocytes obtained from the SHG acquisition, such as the scattering, variance and organization of the myofibril signal, the sarcomere length.[1] H. Liu, Y. Shao, W. Qin, R.B. Runyan, M. Xu, Z. Ma, T.K. Borg, R.Markwald, and B. Z. Gao. Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy, Cardiovascular Research (2013) 97, 262–270[2] V. Nucciottia, C. Stringarib, L. Sacconib, F. Vanzib, L. Fusia, M. Linaria, G. Piazzesia, V. Lombardia, and F. S. Pavone. Probing myosin structural conformation in vivo by second-harmonic generation microscopy, PNAS (2010) doi/10.1073/pnas.0914782107

Mammary SLAMF3 Regulates Store-Operated Ca2+ Entry and Migration Through STIM1 in Breast Cancer Cells and Cell Lines

International audienceStore Operated Calcium Entry (SOCE) is the main route for calcium entry in breast cells. After it’s activation by STromal Interaction Molecule (STIM) during endoplasmic reticulum store depletion, membrane channels ORAI are the main actors of this cell calcium entry. STIM, ORAI and SOCE alterations might contribute to Breast Cancer (BC) carcinogenesis. Recently, we reported the tumor suppressor role of Signaling Lymphocytic Activation Molecule Family member 3 (SLAMF3) on HepatoCellular Carcinoma (HCC) progression. SLAMF3 has been shown to regulate the activity of immune cells by modulating the calcium influx. In this report, we aimed at exploring the role of SLAMF3 in regulating SOCE and migration of BC cells. We quantified and compared the expression of SLAMF3 and STIM1 by quantitative RT-PCR in tumor and healthy resections of 14 patients followed at the University Hospital of Amiens. The expressions of SLAMF3 and STIM1 were also quantified and compared in non-invasive T47D and invasive MDA-MB-231 cell lines by quantitative RT-PCR, Western blot and flow cytometry. We determined the Ca2+ basal entry as well as SOCE by Mn2+ quenching and calcium imaging, respectively, in T47D and MDA-MB-231 cells overexpressing SLAMF3 ectopically. The cell proliferation and migration/invasion were investigated by MTT, wound healing assay and Boyden chambers tests, respectively. First, we report the expression of SLAMF3 in mammary epithelial cells. We highlight the complete loss of SLAMF3 expression in invasive BC cell lines compared to non-invasive cells. In addition, we show that the forced expression of SLAMF3 in invasive cells down-regulate specifically the STIM1 expression in invasive compared to non-invasive mammary cell lines. Interestingly, an inverse correlation is observed between the low expression of SLAMF3 and the high expression of STIM1 in primary human BC tissues. Our results indicate that SLAMF3 reduces SOCE and therefore restricts BC cell migration by decreasing STIM1 expression. Therefore, SLAMF3 might be used as a predictive marker of BC evolution and aggressiveness