Atomic force microscopy-single-molecule force spectroscopy unveils GPCR cell surface architecture.

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.

Compromised Biomechanical Properties, Cell-Cell Adhesion and Nanotubes Communication in Cardiac Fibroblasts Carrying the Lamin A/C D192G Mutation.

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.

Biophysical properties of cardiomyocyte surface explored by multiparametric AFM.

PeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) multiparametric AFM mode was adapted to qualitative and quantitative study of the lateral membrane of cardiomyocytes (CMs), extending this powerful mode to the study of soft cells. On living CM, PeakForce QNM depicted the crests and hollows periodic alternation of cell surface architecture previously described using AFM Force Volume (FV) mode. PeakForce QNM analysis provided better resolution in terms of pixel number compared to FV mode and reduced acquisition time, thus limiting the consequences of spontaneous living adult CM dedifferentiation once isolated from the cardiac tissue. PeakForce QNM mode on fixed CMs clearly visualized subsarcolemmal mitochondria (SSM) and their loss following formamide treatment, concomitant with the interfibrillar mitochondria climbing up and forming heaps at the cell surface. Interestingly, formamide-promoted SSM loss allowed visualization of the sarcomeric apparatus ultrastructure below the plasma membrane. High PeakForce QNM resolution led to better contrasted mechanical maps than FV mode and provided correlation between adhesion, dissipation, mechanical and topographical maps. Modified hydrophobic AFM tip enhanced contrast on adhesion and dissipation maps and suggested that CM surface crests and hollows exhibit distinct chemical properties. Finally, two-dimensional Fast Fourier Transform to objectively quantify AFM maps allowed characterization of periodicity of both sarcomeric Z-line and M-band. Overall, this study validated PeakForce QNM as a valuable and innovative mode for the exploration of living and fixed CMs. In the future, it could be applied to depict cell membrane architectural, mechanical and chemical defects as well as sarcomeric abnormalities associated with cardiac diseases.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.