In vitro cell stretching technology (IsoStretcher) as an approach to unravel Piezo1-mediated cardiac mechanotransduction

https://doi.org/10.1016/j.pbiomolbio.2020.07.003Get rights and content

Abstract

The transformation of electrical signals into mechanical action of the heart underlying blood circulation results in mechanical stimuli during active contraction or passive filling distention, which conversely modulate electrical signals. This feedback mechanism is known as cardiac mechano-electric coupling (MEC). The cardiac MEC involves complex activation of mechanical biosensors initiating short-term and long-term effects through Ca2+ signals in cardiomyocytes in acute and chronic pressure overload scenarios (e.g. cardiac hypertrophy). Although it is largely still unknown how mechanical forces alter cardiac function at the molecular level, mechanosensitive channels, including the recently discovered family of Piezo channels, have been thought to play a major role in the cardiac MEC and are also suspected to contribute to development of cardiac hypertrophy and heart failure. The earliest reports of mechanosensitive channel activity recognized that their gating could be controlled by membrane stretch. In this article, we provide an overview of the stretch devices, which have been employed for studies of the effects of mechanical stimuli on muscle and heart cells. We also describe novel experiments examining the activity of Piezo1 channels under multiaxial stretch applied using polydimethylsiloxane (PDMS) stretch chambers and IsoStretcher technology to achieve isotropic stretching stimulation to cultured HL-1 cardiac muscle cells which express an appreciable amount of Piezo1.

Introduction

Mechanosensory transduction is an ancient sensing mechanism involving mechanosensitive (MS) ion channels. These highly efficient biological force-sensing macromolecules are tightly coupled to the mechanics of biological cell membranes. Together with the cytoskeleton, integrins, G-protein-coupled receptors and membrane-bound enzymes, they represent major molecular mechanosensors tasked with transducing mechanical stimuli, exerted on the cell membrane, into electrochemical intracellular signals (Hamill and Martinac, 2001; Ingber, 2006). By operating on a millisecond time scale these channels play a central role in the physiology of touch, hearing and blood pressure regulation, for example (Martinac, 2004). Force-induced changes in these channels result in electrical signal transduction mediated via ion fluxes defined by channel conductive properties (conductance, ion selectivity) and electrochemical ion gradients. Studies of MS ion channels have been conducted for more than 30 years, including the first report of MS channels in bacteria (Martinac et al., 1987), which have greatly contributed to our understanding of the basic biophysical principles underlying the physiology of mechanosensory transduction in higher eukaryotes, including animals and humans (Cox et al., 2019; Martinac and Cox, 2017). However, a complete understanding of the molecular force-sensing mechanisms at play remains elusive.

The recent exciting discovery of the Piezo mechanically gated ion channel family (Coste et al., 2010) and their role in eukaryotes (Kim et al., 2012) has opened the possibility of closing the gap in our knowledge of the physiology and pathology of mechanotransduction processes. However, as we will point out, biological assessment of such MS entities requires availability of novel, organ-mimicking stretch technologies, which we will review alongside with our recently bioengineered IsoStretcher system technology that was here applied to study Piezo1 channel properties in murine atrial HL-1 cells.

Section snippets

Mechanosensitivity of Piezo1 channels

The Piezo channel family includes two isoforms: Piezo1 and Piezo2. Acting as Ca2+-permeable non-selective cation channels, they were initially indicated in 2010 to play important roles in mechanotransduction processes in various species including animal, plant and other eukaryotic species, throughout most tissues and cells (Coste et al., 2010). The two isoforms of Piezo encoded as PIEZO1 and PIEZO2 genes are sharing approximate 50% gene identity with each other and were considered to have some

Overview of stretching technologies

As the questions around mechanosensitive ion channels, such as Piezo1, have become increasingly important, many devices have been built to exert mechanical strain on cells. Various approaches emerged in the field of cell stretching, a few of which are presented and discussed here before introducing the IsoStretcher, an isotropic cell stretching device.

Furthermore, other groups have concentrated on alternative stimulation methods, like shear-stress, indentation and pressure. These methods were

Conclusion

MS channel Piezo1 is involved in a broad range of physiological and pathological processes in the body. In the cardiovascular system, the activities of Piezo1 have been mainly examined in ECs or RBCs. Importantly, Piezo1 is also thought to be able to respond to mechanical forces such as stretch due to cardiac contraction and to act as a primary force sensor in the heart, although the exact role of Piezo1 in cardiomyocytes still needs to be unravelled through further studies using different

Adult mouse cardiac fibroblast isolation and primary culture

Three 12-week-old male C57BL/6J mice with an average body weight of 29.53 g (SD = 0.81) were euthanized according to guidelines of the Animal Research Act 1985 No 123 (New South Wales, Australia). The isolation and primary culture of mouse CFs were following a published protocol by Zeigler et al. (2016).

Quantitative real-time polymerase chain reaction (RT-PCR)

RNA was extracted from adult mouse CFs and cultured HL-1 cells with the RNeasy Fibrous Tissue Mini Kit (QIAGEN), following the manufacturer’s protocol. cDNA was synthesized using the SuperScript

Author contributions

Yang Guo: Conceptualization, Data curation, Formal analysis, Methodology, Visualization, Writing - original draft, Writing - review & editing.

Anna-Lena Merten: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing - original draft, Writing - review & editing.

Ulrike Schöler: Data curation, Formal analysis, Writing - review & editing.

Ze-Yan Yu: Supervision, Validation, Writing - review & editing

Jasmina Cvetkovska: Data curation.

Diane Fatkin: Data

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors are grateful for the support and advice of Dr. Charles D Cox in the HL-1 cell stretching experiments, as well as Dr. Louise Dunn and Professor Roland Stocker for their help in designing the mouse Piezo1 RT-PCR primers.

The authors gratefully acknowledge funding of the German Federal Ministry for Economy and Energy due to a resolution by the German Bundestag (ZIM and BMWi, #ZF4134304CR6) and funding of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the German

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