ReviewNovel insights into cardiomyocytes provided by atomic force microscopy
Introduction
In the late 1980s, the atomic force microscope (AFM) was developed by Binnig et al. [1] to investigate surfaces of insulators at the atomic scale, while avoiding damages to the specimens. AFM is a scanning probe technique, which exploits the deflections of a cantilevered spring to image and probe different kind of samples. Fundamental components and working principles of the AFM are shown in Fig. 1.
The atomic force microscope has proven to be a versatile tool of investigation, since it can either acquire a sample topography or measure various mechanical properties, such as stiffness, adhesive and viscoelastic behaviour; in the former case, the AFM works as an imaging machine, whereas in the latter it can be considered a mechanical instrument [2]. In addition, as the AFM can work in aqueous environments, it was forthwith thought to be used for studying biological specimens either close to or under physiological conditions.
Cardiovascular diseases are still the leading cause of death worldwide. Particularly, according to Gillespie et al. [3], heart disease and stroke are the first and fourth causes of death, respectively, in the United States. The proper functioning of the heart is pivotal for living organisms, and cardiology research is mainly focused on the thorough comprehension of bio-chemo-mechanical principles underlying the physiological behaviour of the heart. Yet, the effects of inherited or incidental cardiac dysfunctions are under investigation, likewise any possible therapy, such as drug administration and tissue regeneration/transplantation. The study of aetiology, hallmarks and progress of a cardiac pathology can be considered a multidisciplinary task, since it involves physicians, biologists, and more recently biophysicists and bioengineers. Altogether, they investigate cardiac behaviour both at the macroscopic, e.g. via echocardiography or ventriculography, and microscopic level.
In recent years, more and more researchers focus on the basic constituent of cardiac tissue, mainly on cardiomyocytes (CMs), which are the core unit of the heart, generating and transmitting the contractile force. In this scenario, AFM can play a key role, due to its high resolution, both in topographic and force terms. The present work aims to review the studies that involved AFM technology to investigate the structure and function of CMs. In addition, original data are presented as further characterization of cardiomyocytes in terms of plasticity index and stress relaxation behaviour.
Section snippets
AFM as imaging machine applied to cardiomyocytes
Over the years, various biological samples have been characterized for their topography by AFM, as thoroughly reviewed in [4], [5]. Regarding the aim of the present work, in 1999, Perez-Terzic et al. [6] imaged sarcolemma-stripped CMs from neonatal rats, focusing on the nuclear pore complexes (NPCs). Data showed that both Ca2+ and ATP/GTP depletion induced a significant decrease in the percentage of open NPCs compared to untreated CMs, the second treatment resulting also in more relaxed pores
Assessment of elastic behaviour of cardiomyocytes
Early after its invention, the atomic force microscope was used to assess the mechanical properties, namely hardness, elastic and plastic behaviour, by nano-indentation of the investigated sample [14]. The mechanical analysis by AFM at the atomic level has raised great interest in the field of mechanotransduction, which is the study of cellular and molecular processes that convert mechanical cues into biochemical signals [15].
A large number of studies on the elastic properties of living cells,
Other applications of AFM on cardiomyocytes
Beyond the already discussed assessment of contractile activity, AFM was employed to induce contraction in living cells, activating their mechano-sensitive ion channels. This was exploited by He et al. [62] to demonstrate the formation of membrane nanotubes as a transport mechanism between NRVMs and rat fibroblasts that are not strictly connected by gap junctions. Using simultaneous fluorescence imaging, authors showed that the Ca2+ signal from induced contraction propagated through the
Conclusion
The interest in cardiovascular research is constantly fostered by the striking amount of deaths caused by cardiovascular diseases, therefore new methods and approaches are being pursued to better understand the pathophysiological conditions of the heart, both at the macro- and microscopic level.
The present work has been focused mainly on studies performed on single cells and cellular clusters, and reviewed most of the literature about cardiomyocytes investigation via AFM, ranging from the first
Conflict of interest statement
The authors have no conflict of interest to declare.
Fundings
This work was supported by the “Fondation Leducq”, Transatlantic Network of Excellence (grant number 14-CVD 03).
Acknowledgements
Authors gratefully thank Thomas Lanzicher for his contribution with the plasticity index data on NRVMs and Dr. Valentina Martinelli for providing the NRVMs used in this study.
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