Evaluation of voltage gated calcium channel activity as a mechanism for ELF-EMF induced tendon injury susceptibility

1. Overview

Electrical and electromagnetic stimulation has been shown to modulate anabolic and catabolic gene and protein expression in bone and cartilage through voltage gated calcium channels^1,2. However, the voltage gated calcium channel activity in tendons in response to chronic EMF exposure and the potential contribution to injury susceptibility has remained untested.

This proposal aims to test how voltage gated calcium channel activity in tenocytes may be affected by long term ELF-EMF exposure in an ex vivo model. Using previously validated electromagnetic stimulation devices³ and specific ion channel blockers⁴, we will probe mechanisms through which ELF-EMFs may contribute to tendon extracellular matrix structure and mechanics.

2. Introduction

Tenocytes within the tendons are responsible for the maintenance and functionality of the highly organized and elastic extracellular matrix (ECM), giving the tendons the ability to withstand repeated stretching without failure. The ECM in tendons is chiefly comprised of collagen and proteoglycans that provide the tissue with high tensile strength and elasticity to prevent failure during everyday movement. The ECM molecules also bind to the tenocytes and couple the mechanical deformation of the tissue to the deformation of the cell. This mechanical signaling can drive downstream signaling cascades and transcriptional changes that contribute to cell proliferation and apoptosis as well as ECM production and degradation⁵. The process by which mechanical cues are translated to biochemical cues that alter gene expression and activity is known as mechanotransduction.

Calcium signaling is a common mechanotransduction modality where membrane bound ion channels respond to mechanical deformation and fluid flow by opening and creating an increase in cytosolic calcium concentration. This can trigger downstream pathways which contribute to cell proliferation and ECM production and degradation ⁶. Voltage gated calcium channels (VGCCs) are activated when an electric potential is induced across the membrane during, typically during osmotic pressure changes and mechanical deformation⁴, but they can also be activated by exogenous electrical or electromagnetic fields¹ . While it has been shown that modulations in ion channel activity can increase proliferation and matrix deposition in chondrocyte and mesenchymal stem cells ^7,8, the effect of EMF on tenocyte calcium channel activity and subsequent matrix remodeling has yet to be explored.

In the proposed study, bovine tendon explants will be cultured with and without VGCC inhibitors and stimulated with ELF-EMF. The tendon explant model will allow for more realistic loading environment for delivery of EMFs without requiring time-intensive and costly in vivo animal models. An ex vivo environment will allow us to systematically test the effects of controlled EMF exposure and VGCC activity over many time points with the same biological replicate.

Central hypothesis: ELF-EMF stimulation will result in upregulation of matrix degrading genes while reducing collagen and proteoglycan content and ultimate tensile strength compared to unstimulated controls, and that VGCC inhibition with verapamil will mitigate these EMF-induced effects.

3. Methods

Participants/Sample

This study will be completed under the supervision of Dr. Deva Chan (PI) in the Chan Musculoskeletal Research and Innovation Lab at Purdue University. With the strong foundation of mechanobiology and electrical stimulation models within the lab, the project will reasonably be completed within a 6-month time span. Results from this will be published in a peer-reviewed journal manuscript and an open access preprint on ResearchHub to enable timely insights into the connection between chronic ELF-EMF exposure and tendon injury susceptibility.

Experimental samples will consist of bovine tendon explants obtained from a local abattoir with n = 6 biological replicates per experimental group to account for biological variation. The explant model will enable close approximation to in vivo conditions for cell-ECM interactions as well as heterogeneous ELF-EMF delivered to the tenocytes through the dense ECM. This explant model will also allow for the collection of multiple samples from the same biological replicate over different time points to increase statistical power.

Materials and Procedures

Bovine metacarpophalangeal joints will be obtained from a local abattoir and dissected under sterile conditions. Tendon explants will be taken from tensional sections of deep digital flexor tendons to avoid variations in mechanotransduction between tensional and compressed regions of the tendon⁹. Explants will be cultured in Dulbecco’s modified eagle serum (DMEM) supplemented with 1% antibiotic/antimycotic and 10% fetal bovine serum. A preculture period of 2 days will be implemented before stimulation to allow for stabilization, and cell viability will be measured with live/dead cell viability assay periodically throughout the loading period.

An IGEA I-One therapy device will be used to deliver electromagnetic field stimulation to explant cultures³. The explants will be stimulated with 50 mG electromagnetic field at 60 Hz continuously for up to 2 months to simulate chronic EMF exposure while maintaining ex vivo

To evaluate the effects of voltage gated calcium channel activity, VGCCs will be blocked using specific inhibitor verapamil¹.

Key outcomes:

Gene expression: mRNA expression of matrix remodeling genes (COL1A1, COL1A2, ELN, MMP2, MMP9, ASPN) and tenocyte differentiation markers (SCX, TNMD) will be measured using RT-qPCR.
ECM Composition: Collagen I and proteoglycan content will be assessed using bovine collagen I ELISA kits and SDS-PAGE, respectively
Tissue mechanics: Young’s modulus and ultimate tensile strength will be measured with tensile testing with a standard load frame to assess injury susceptibility at each time point.

Planned Analyses

For multiple time points up to 2 months, explants will be split into 3 groups: EMF stimulated, EMF stimulated + VGCC blocker, and control (no EMF, no VGCC blocker)

Matrix remodeling gene expression, ECM content, and mechanics can be compared with ANOVA and post hoc Tukey.

We hypothesize that EMF stimulation will result in upregulation of matrix degrading genes while reducing collagen and proteoglycan content and ultimate tensile strength compared to unstimulated controls, and that VGCC inhibition with verapamil will mitigate these EMF-induced effects.

Contingency plan:

If EMF stimulated samples at each time point do not show an increase matrix degradation gene expression, then further studies will be completed to examine the integration of non-VGCC, mechanosensitive ion channels in response to EMF as deformation of piezoelectric collagen within the ECM may also contribute to tenocyte response.

If control samples show tenocyte dedifferentiation, then a tensioning method will be employed to maintain physiological levels of mechanical stress within cultures.

If VGCC inhibition does decrease degenerative effects of EMF, then further investigation into downstream signaling cascades (MAPK/ERK, calmodulin/calcineurin) will be executed to further elucidate mechanisms of action.

Ethics and Data Management

No IACUC or PACUC protocols will be needed for this proposal as tissues will be collected from local abattoir.

Finalized protocols will be preprinted before experiments on ResearchHub and analysis tools and datasets will be publicly available through open access data repositories (ie Github, Dataverse).

5. Budget

The anticipated costs for this project include the following:

Tissue dissection and culture reagents

Collagen and proteoglycan content assays (ELISAs, SDS-PAGE)

RNA isolation and RT-qPCR reagents

EMF stimulation device

Personnel and facility costs

6. References

1. Xu, J., Wang, W., Clark, C. C. & Brighton, C. T. Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels. Osteoarthritis Cartilage 17, 397–405 (2009).

2. Ermakov, Y. A., Kamaraju, K., Sengupta, K. & Sukharev, S. Gadolinium Ions Block Mechanosensitive Channels by Altering the Packing and Lateral Pressure of Anionic Lipids. Biophys. J. 98, 1018–1027 (2010).

3. HOCHSTENBACH, F. System and method for applying a low frequency magnetic field to biological tissues. (2018).

4. Lv, M. et al. Calcium Signaling of in situ Chondrocytes in Articular Cartilage under Compressive Loading: Roles of Calcium Sources and Cell Membrane Ion Channels. J. Orthop. Res. Off. Publ. Orthop. Res. Soc. 36, 730–738 (2018).

5. Subramanian, A., Kanzaki, L. F., Galloway, J. L. & Schilling, T. F. Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling. eLife 7, e38069 (2018).

6. Fitzgerald, J. B. et al. Mechanical Compression of Cartilage Explants Induces Multiple Time-dependent Gene Expression Patterns and Involves Intracellular Calcium and Cyclic AMP. J. Biol. Chem. 279, 19502–19511 (2004).

7. Zerillo, L. et al. Extremely low frequency–electromagnetic fields promote chondrogenic differentiation of adipose-derived mesenchymal stem cells through a conventional genetic program. Sci. Rep. 14, 10182 (2024).

8. Lv, M. et al. Calcium signaling of in situ chondrocytes in articular cartilage under compressive loading: Roles of calcium sources and cell membrane ion channels. J. Orthop. Res. 36, 730–738 (2018).

9. Rees, S. G., Waggett, A. D., Dent, C. M. & Caterson, B. Inhibition of aggrecan turnover in short-term explant cultures of bovine tendon. Matrix Biol. 26, 280–290 (2007).

Evaluation of voltage gated calcium channel activity as a mechanism for ELF-EMF induced tendon injury susceptibility

Funding Progress

Nonprofit Recipient

Funders

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Funding Progress

Nonprofit Recipient

Funders

Topics

DOI

Evaluation of voltage gated calcium channel activity as a mechanism for ELF-EMF induced tendon injury susceptibility

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI