Broadband Electrical Spectroscopy To Distinguish Single-Cell Ca2+ Changes Due To Ionomycin Treatment in A Skeletal Muscle Cell Line Part 2

4. Discussion

In Figure 3b, the distribution of the fluorescence related to calcium peaks at a higher ratio and spreads wider after Ionomycin treatment, consistent with an increase of calcium due to treatment. Based on the known action of ionomycin as an electroneutral ionophore, the cells are expected to rapidly transport extracellular calcium inside and then recover normal cytosolic concentrations after the removal of the ionomycin [27,28]. Another common method of monitoring Ca2+ manipulation within cells is nanosecond pulsed electrical fields (nsPES), which deliver a small electrical stimulant to generate pores in the cell plasma membrane, depending on the signal magnitude and polarity [29]. Recent work in this field to look at Ca2+ increase within cells has shown that the presence of sucrose molecules can delay the swelling response typically associated with an increase in intracellular Ca2+; however, this can depend on the external concentration introduced and the voltage-gated channels present on the cell type of study [30,31]. As with our study, these show that while established in creating a gradient to increase cytosolic Ca2+, there are less understood effects on deeper membranes and compartments within the cell after treatment [32]. While the calcium change is the main factor associated with ionomycin treatment, several long-term changes can be noted, including expression of IL-6 [33,34] or CAI [35]; however,  these are unlikely to cause an impact in the short term. Due to the rhythmic nature of Ca2+  function in cells, regulation of levels in the cytosol is carefully controlled by several proteins through storage and release in the sarcoplasmic reticulum. Based on this crucial system, it is anticipated that while the overall distribution increased, some cells returned to fluorescence consistent with their initial values. The rapid peak of fluorescence and accompanying plateau is consistent with previous work observing the rapid flux of cytosolic calcium before slower recovery after treatment with ionomycin [36].

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Similarly, looking at the electrical measurements, the increase is consistent with cytosolic Ca2+ increase and the extracted values are consistent with previous measurements of similar cellular changes. The values from the UNT cells are comparable in scale to the previously published fitting value of 0.22 S/m and 9.49 ε0 [21]. The statistical comparison between these values for the UNT (n = 51) and TRT (n = 20) cells shows that a significant increase exists for conductivity and a significant decrease exists for permittivity. The small magnitude of change is consistent with the expected pattern of recovery after the removal of ionomycin transport complexes. For the maintenance of cell viability, the change in calcium while established would be minimal in a healthy population. The observed change in conductivity and permittivity is consistent with an increase in ions, making a more even distribution of charge throughout the cell cytoplasm. It has been noted earlier that based on the initial Ca2+ imaging data, not every ionomycin-treated cell sustains the increased intracellular Ca2+ concentration, leading to the significant overlap between the UNT and TRT groups. It is also important to note that while this work focuses on the cytosolic changes, the homeostasis of calcium management also occurs in the sarcoplasmic reticulum. However, due to the broadband nature of the reported measurements, changes in other compartments are captured in a wide range of frequencies.

The previous work posits oxidative damage to the mitochondria and flooding of intracellular reactive oxygen species (ROS) and Ca2+ before the start of apoptosis when exposed to chronic oxidative stress [37]. Physiologically, there is a well-established link between Ca2+ levels in skeletal muscle and the ability to maintain the balance of ROS and mitigate the effects of oxidative stress. Not surprisingly, the spectra alteration observed here from intracellular Ca2+ elevation, an increase in ∆S11 in the MHz range followed by a  dip in the GHz range and a less negative value of ∆S21 in the kHz range, is comparable to those from L6 cells exposed to long-term oxidative stress [21]. The previous work identified Ca2+ as a key factor differentiating cells that experienced exposure to oxidative stress, while in this work, the ability to differentiate calcium through modeling shows the milder but noticeable contributions Ca2+ makes to the internal dielectric properties. Alternatively, work using the ratio of impedance at 1 MHz to 300 kHz to characterize individual cell opacity also showed the ability to measure the changes in neutrophils due to calcium ionophore exposure [38]. This work has a higher throughput and therefore, sample size; however, it is limited to blood cells and limited to analysis of size and opacity to characterize the populations studied. By measuring a full spectrum of frequency values rather than relying on a smaller number of frequencies, this work shows the capture of dielectric properties representing complex and multifaceted changes due to elevated Ca2+ levels induced by ionomycin.

The ability to identify cytoplasmic Ca2+ changes has the potential to aid in improving our understanding of many associated skeletal muscle diseases such as DMD, cachexia, and the process of sarcopenia development [2]. Considering that earlier published work also showed that calcium influx is an important factor in the way long-term oxidative stress changes the electrical properties of muscle cells, the results from this study are not meant to differentiate between calcium mismanagement and oxidative stress but rather explain the contribution calcium mismanagement might have towards the oxidative stress responses previously observed. This work is also limited by the selectivity of the electrical spectroscopic method to define specific molecular or ionic contributors with certainty. Due to the complexity of ion management inside cell models, while the treatment is intended to alter intracellular Ca2+ concentration, the effects of other ions or molecules may also contribute to the observed changes in the electrical signal. This limitation of ion sensing selectivity has been reported in an aqueous solution at high frequencies [39,40]. Because electrical measurements are widely non-specific and oxidative diseases have complex and multifaceted effects on muscle cells, the objective is to broaden understanding of how these effects manifest electrically. As ionomycin treatment is known to alter ion concentration inside cells without inducing oxidative stress, this study allows us to focus on the manifestation of calcium imbalance in cellular impedance. Additionally, while this work focuses on skeletal muscle, the potential to monitor neuron resting and altered Ca2+ concentration has implications for many more diseases [41,42]. Typically, measurement of Ca2+ in vivo relies on the inclusion of fluorescent agents such as the Fura-2 in this work to look at cytosolic Ca2+ or Mag-Fluo-4 to look at Ca2+ in the endoplasmic reticulum [20,43]. However, these options require cell labeling and complex treatment processes, both of which are avoided by electrical measurement. The electrical system presented can offer a wider view of the individual cell dielectric properties at multiple frequencies, more rapid measurement, and fewer resources required. There is a need to demonstrate a realistic sensitivity to biological levels of cytoplasm Ca2+ for disease diagnosis or monitoring applications, which will shape our approach in the future. Going forward, the spectral changes seen in this work will be used in a further study of ME/CFS clinical samples looking at how skeletal muscle electrical properties at a variety of frequencies can be correlated with biological changes to further our understanding of this rare disease.

5. Conclusions

Based on the electrical measurements and corresponding extracted parameters, there is a subtle change in MHz and GHz spectral patterns that can be correlated with fluorescent image-based cytoplasmic Ca2+ levels. The electrically measured differences can be further described by changes to the dielectric parameters of cytoplasmic permittivity (εc) and conductivity (σc). In this work, it was found that increased cytoplasmic Ca2+ concentration can be associated with a significant increase in cytoplasmic conductivity and a decrease in cytoplasmic permittivity. This monitoring system improves the depth of information available about intracellular conditions and ion study in the cytoplasm. The work presented here is limited by the lack of comparison with true concentration correlation and selective sensing of the measurement system to particular ions; therefore, further exploration is necessary to develop a true system for disease monitoring. That being said, understanding these Ca2+ levels can help generate understanding and evaluate skeletal muscle disease progression and treatment effectiveness. In addition, by modeling these changes in the context of pre-evaluated oxidative changes, important deductions can be made about how different properties associated with ME/CFS contribute to an overall electrical profile to move toward a unique and rapid diagnostic tool.

Author Contributions: Conceptualization, C.A.F., T.P. and X.C.; methodology, C.A.F., M.F., T.P., and X.C.; software, C.A.F.; validation, C.A.F., M.F., T.P., and X.C.; formal analysis, C.A.F.; investigation, C.A.F., C.S., L.M., T.P., and X.C.; resources, T.P. and X.C.; data curation, C.A.F., C.S., L.M., and T.P.; writing—original draft preparation, C.A.F.; writing—review and editing, M.F., T.P. and X.C.;  visualization, C.A.F.; supervision, M.F., T.P., and X.C.; project administration, T.P., and X.C.; funding acquisition, T.P., and X.C. All authors have read and agreed to the published version of the manuscript.

Funding: C.A.F. and X.C. appreciate support through funding from the National Science Foundation, Division of Electrical, Communications & Cyber Systems Grant 1809623. C.S., L.M. and T.P. are supported through “G. d’Annunzio” University grants.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable. 

Data Availability Statement: Data is available upon request. 

Conflicts of Interest: The authors declare no conflict of interest.

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