Development of Magnetic Torque Stimulation (MTS) Utilizing Rotating Uniform Magnetic Field for Mechanical Activation of Cardiac Cells

Abstract

Regulation of cell signaling through physical stimulation is an emerging topic in biomedicine.

Background: While recent advances in biophysical technologies show capabilities for spatiotemporal stimulation, interfacing those tools with biological systems for intact signal transfer and noncontact stimulation remains challenging. Here, we describe the use of a magnetic torque stimulation (MTS) system combined with engineered magnetic particles to apply forces on the surface of individual cells. MTS utilizes an externally rotating magnetic field to induce a spin on magnetic particles and generate torsional force to stimulate mechanotransduction pathways in two types of human heart cells-cardiomyocytes and cardiac fibroblasts.

Methods: The MTS system operates in a noncontact mode with two magnets separated (60 mm) from each other and generates a torque of up to 15 pN µm across the entire area of a 35-mm cell culture dish. The MTS system can mechanically stimulate both types of human heart cells, inducing maturation and hypertrophy.

Results: Our findings show that application of the MTS system under hypoxic conditions induces not only nuclear localization of mechanoresponsive YAP proteins in human heart cells but also overexpression of hypertrophy markers, including β-myosin heavy chain (βMHC), cardiotrophin-1 (CT-1), microRNA-21 (miR-21), and transforming growth factor beta-1 (TGFβ-1).

Conclusions: These results have important implications for the applicability of the MTS system to diverse in vitro studies that require remote and noninvasive mechanical regulation.

Keywords: cardiac cells; hypoxia; magnetogenetics; mechanotransduction; torsional magnetic stimulation.

Figure 1

Schematics of magnetic torque stimulation (MTS) system (left) and its application for mechanostimulation of cardiac cells (right).

Figure 2

Numerical simulation of MTS system for mechanical stimulation of cells. Configuration of MTS with induced normalized magnetic flux density distribution and the stream line plots (A). Cross line plots in x- and y-directions, respectively (B). Normalized magnetic flux density field from the simulation result and the torque induced by the magnetic beads using the Equation (1) with the proposed coefficient (C).

Figure 3

Effects of forces generated by the MTS system on protein localization and gene expression in AC16 cells. Representative fluorescence images of AC16 cells in the groups: MPs only (control), MF only, hypoxia, and hypoxia with MF-MPs for 24 h (scale bars: 100 µm) (A). Immunofluorescence images of staining for YAP (green) and the nuclear marker DAPI (blue) showed that YAP translocated from the cytosol to the nucleus. Quantification of YAP expression in the cytosol and nucleus of cultured AC16 cells of each group (B). Quantitative mRNA expression data for genes related to cardiac hypertrophy in cultured AC16 cells of each group analyzed by RT-PCR (C). A t-test was used to compare the control and MF-MPs with Hyp groups (****, p < 0.0001; ***, p < 0.001; **, p < 0.01). There were two independent experiments, and RT-PCR analysis was duplicated.

Figure 4

Effects of forces generated by the MTS system on protein localization and gene expression in hCF cells. Representative fluorescence images of YAP (green) and DAPI (blue) staining in hCF cells in the group of MPs only (control), MF only, hypoxia, and hypoxia with MF-MPs for 24 h (scale bars: 100 µm) (A). Quantitative analysis and comparison of YAP expression in the cytosol and nucleus of cultured hCF cells of each group (B). Quantitative mRNA expression data for cultured hCF cells in each group, as assessed by RT-PCR (C). Statistical analysis was conducted with a t-test to compare the control and MF-MPs with Hyp groups (****, p < 0.0001; **, p < 0.01; *, p < 0.05). There were two independent experiments, and RT-PCR analysis was duplicated.