The Bioelectric Code: Reprogramming Cancer and Aging From the Interface of Mechanical and Chemical Microenvironments

Cancer is a complex, heterogeneous group of diseases that can develop through many routes. Broad treatments such as chemotherapy destroy healthy cells in addition to cancerous ones, but more refined strategies that target specific pathways are usually only effective for a limited number of cancer types. This is largely due to the multitude of physiological variables that differ between cells and their surroundings. It is therefore important to understand how nature coordinates these variables into concerted regulation of growth at the tissue scale. The cellular microenvironment might then be manipulated to drive cells toward a desired outcome at the tissue level. One unexpected parameter, cellular membrane voltage (Vm), has been documented to exert control over cellular behavior both in culture and in vivo. Manipulating this fundamental cellular property influences a remarkable array of organism-wide patterning events, producing striking outcomes in both tumorigenesis as well as regeneration. These studies suggest that Vm is not only a key intrinsic cellular property, but also an integral part of the microenvironment that acts in both space and time to guide cellular behavior. As a result, there is considerable interest in manipulating Vm both to treat cancer as well as to regenerate organs damaged or deteriorated during aging. However, such manipulations have produced conflicting outcomes experimentally, which poses a substantial barrier to understanding the fundamentals of bioelectrical reprogramming. Here, we summarize these inconsistencies and discuss how the mechanical microenvironment may impact bioelectric regulation.

Membrane voltage (Vm) is defined as the electrical potential difference between the cytoplasm and extracellular space (Levin, 2007). This bioelectric field has been demonstrated to transmit extensive patterning information between cells at tissue-scale. For example, disrupting Vm gradients has been shown to impair regeneration and development, causing the growth of functioning ectopic organs such as eyes in Xenopus and head/brain structures in planaria (Beane et al., 2011; Pai et al., 2012). Excitingly, manipulating Vm has also been shown to induce limb regrowth in non-regenerative species (Tseng and Levin, 2013), prompting much interest in bioelectricity as a future therapeutic tool to restore organs deteriorated during the aging process or accidentally damaged. In addition, manipulating Vm can prevent the formation of tumors (Chernet and Levin, 2014), suggesting promising future cancer treatments. However, it is unclear what Vm manipulation is needed to produce a desired outcome; separate studies report contradicting observations resulting from similar alterations of Vm. In particular, comparable Vm manipulations have been linked to both apoptosis and proliferation, which are seemingly opposite phenotypes (Bortner et al., 1997; Wang et al., 1999; Yu et al., 1999a,b; Thompson et al., 2001). Adding another level of complexity, cellular Vm varies significantly between cell types and with progression of the cell cycle. How then might this broad range of observations surrounding Vm be reconciled into a consistent theory for possible implementation in future medical therapies to combat aging and disease?

Although it is generally accepted that experiments in culture do not recapitulate the complexity of the cellular surroundings in vivo, a number of parameters altered by traditional culture methods are often not accounted for in experimental design and data interpretation. The ability of chemical components of the cellular microenvironment to impact phenotype is a classic topic of study. For example, factors such as hypoxia (Pang et al., 2016) and pH (Damaghi et al., 2013) have been demonstrated to drive cancer progression. However, it is becoming increasingly well recognized that physical signals also contribute to tumorigenesis: substratum stiffness and pressure are two key components of this mechanical microenvironment (Discher et al., 2005; Piotrowski-Daspit et al., 2016). Further studies regarding how mechanical parameters impact Vm are needed to more fully understand the processes that contribute to the emergence of bioelectric field gradients. Additional factors in the microenvironment such as the presence of multiple cell types and microbiota impact the transduction of bioelectric signals (Chernet and Levin, 2014). However, we lack a full understanding of how all of these regulatory cues function together to translate changes in Vm into physiological cellular states.

Not only do variables in the microenvironment change in studies of bioelectricity, but Vm itself may impact several variables within the microenvironment. The conflicting outcomes of previously published experiments then may have resulted from unintentionally altering different regulatory cues simultaneously. In addition, at the cellular level, the output of a given Vm input is often represented as a single biological state, such as growth or death. Apoptosis and proliferation are generally treated as bimodal opposites. However, this interpretation is incomplete at best. Rather than behaving as opposing cellular functions, division and programmed death occur in a coordinated fashion to sculpt growth and form, tuned by the complex web of surrounding microenvironmental signals. This review summarizes the seemingly conflicting reports of bioelectric signaling and discusses two topics that may help explain these inconsistencies: the mechanical microenvironment and the importance of cellular death.

The idea that electricity could contain biological information was first demonstrated experimentally in the late 1700s, when Luigi Galvani electrically stimulated muscle contraction in an amputated frog leg (Verkhratsky et al., 2006). Although initially met with skepticism, the idea of “animal electricity” (Galvani, 1791) eventually led to our current understanding of how the brain and body are connected. Electrical properties are often only associated with excitable cells, such as neuronal tissue. However, all cells possess an electrical potential across the plasma membrane, and thus generate and receive bioelectric signals (Levin, 2010). One of the earliest indications that this electrical potential might serve as a growth-directing field came in 1903, when A.P. Mathews identified an electrical gradient in regenerating hydra (Mathews, 1903).

Modern experiments (Beane et al., 2011, 2013; Pai et al., 2012; Tseng and Levin, 2013; Adams et al., 2016) support the historical hypothesis (Mathews, 1903; Burr and Northrop, 1935) that electrical patterns regulate not only muscle movement and excitable cells as demonstrated by Galvani, but growth and form of the organism as a whole. In the 1970s and 1980s, separate studies measured Vm of different cell types (summarized in Binggeli and Weinstein, 1986). These data showed that cancerous and proliferative tissues were generally more positively charged than non-proliferative cells (Levin, 2012a; Adams and Levin, 2013). Consistent with this observation, it was found that experimentally causing cells to become more negatively charged reversibly blocked cellular proliferation (Cone and Tongier, 1971), which was thought to result from the blockage of ions such as Na+ believed to be involved in DNA synthesis (Binggeli and Weinstein, 1986). When the cytoplasm is more positively charged than the extracellular space, Vm is referred to as “depolarized” (Adams and Levin, 2013). Conversely, when the cytoplasm is more negatively charged than the extracellular space, Vm is considered to be “hyperpolarized” (Figure 1). It was therefore hypothesized that a threshold Vm separates “normal” quiescent or resting cells from proliferative or cancerous tissues (Binggeli and Weinstein, 1986). However, this hypothesis did not account for several factors. First, although Vm was measured directly using patch clamping, these measurements were all conducted under different conditions in separate studies. Some measurements were taken directly in vivo under live dissection conditions (tumors), others were of single cells cultured on glass (fibroblasts), and some involved tissue slices, explanted organs (corneal epithelium), or intact 4–16 cell-stage embryos (Binggeli and Weinstein, 1986).

Notably, it remains unclear how bioelectrical patterns are impacted by age both in cells and whole organisms, adding yet another uncontrolled variable to these data. Even during this early phase of Vm research, it was appreciated that Vm “deteriorates” rapidly under non-physiological conditions: an observation generally not accounted for in reports of that era (Binggeli and Weinstein, 1986). Although cancerous cells were observed to be more depolarized than noncancerous cells (Binggeli and Cameron, 1980), this observation represented an average of the cell population rather than an absolute fixed value.

Today, we recognize that although cellular Vm usually ranges from approximately –90 to –10 millivolts (mV), these values can vary greatly depending on the cell type and physiological state of the cell (Yang and Brackenbury, 2013). Each cell regulates its resting Vm through a variety of mechanisms (Adams and Levin, 2006). ATP-dependent pumps allow the cell to push ions into or out of the cytoplasm, even against their electrochemical gradients. Cells also express a large variety of channels that allow the passage of ions across the plasma membrane. These ion channels may be gated in response to changes in extracellular ion concentrations, Vm, or mechanical stimuli (Coste et al., 2010; Pathak et al., 2014; Wu et al., 2016; Gudipaty et al., 2017). Thus, as might be anticipated, cellular Vm is neither spatially uniform throughout the cell nor static in time. This added level of complexity might carry additional biological signals. For instance, tumor cells confined in narrow microfabricated channels establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, creating a net inflow of water and ions at the leading edge of the cell and an outflow at the trailing edge. This flux enables the migration of metastatic breast cancer cells through narrow channels in culture, independent of actomyosin contractility and integrin signaling (Stroka et al., 2014). Inhibiting the Na+/H+ exchanger involved in this process decreases the velocity of migration. In addition to spatial gradients, temporal variations in intracellular Vm play a significant role in cell cycle progression. Generally, cells are more hyperpolarized during S phase and more depolarized during mitosis, whereas G1 and G2 phases fluctuate partway between these extremes (Barghouth et al., 2015).

This appears to be driven by changes in expression levels of ion channels for K+, Na+, and Cl−, and gating of these channels in response to changes in cell volume or alterations in Vm (Ouadid-Ahidouch et al., 2001; Sundelacruz et al., 2009; Urrego et al., 2014; Barghouth et al., 2015). This cyclical variation in Vm observed during the cell cycle is believed to be required for a successful cell division (Barghouth et al., 2015). The regulation of Vm through time would appear then to be a critical part of bioelectrical signaling.

In addition, the role of mechanical factors in regulating both proliferation and Vm is becoming increasingly evident. During the early days of cell culture, an optimal cell density was known to be necessary for the growth of non-immortalized cells: not only did low cell density inhibit growth (few cell-cell contacts), but cells within an overly confluent monolayer also exhibited reduced proliferation (Todaro and Green, 1963). Intriguingly, it was later found that cells in a confluent monolayer are more hyperpolarized than individual cells (Blennerhassett et al., 1989)—an early indication that mechanical forces may help regulate bioelectric signaling. Despite the growing number of observations that Vm is a dynamic property influenced by features of the microenvironment, it is still generally accepted today that dividing or cancerous cells are more depolarized than non-dividing tissue (Wang, 2004; Fraser et al., 2005; Ouadid-Ahidouch and Ahidouch, 2008; Sundelacruz et al., 2009; Yang and Brackenbury, 2013; Chernet and Levin, 2014) or quiescent cells (Barghouth et al., 2015). This concept is supported by recent experiments employing optogenetic control over ion channels, which have demonstrated that hyperpolarization decreases tumor incidence (Sundelacruz et al., 2009; Levin, 2012b; Chernet and Levin, 2014). Such studies generate excitement that bioelectric control could be implemented in medical strategies to combat cancer. This field also holds promise for restoring organs that are damaged or failing due to aging, potentially improving quality of life or even extending lifespan.

However, will simply hyperpolarizing a tumor (using drug treatments or optogenetic methods) cause cancerous cells to enter a quiescent state or die? Would depolarizing an area of tissue cause a failing organ to regenerate, or would it cause a tumor to form? The observation that mechanical and chemical factors in the microenvironment interact with Vm signals both spatially and temporally has begun to change the bioelectric view of oncogenesis from a cellular switch in the Vm of healthy cells past a “threshold” value to an organism-wide defect in bioelectric patterning (Levin, 2012b).

Despite the growing attention being given to the promise of bioelectricity in medicine, a consistent theory that connects Vm to a desired phenotype remains largely elusive. Experiments aimed at understanding bioelectric regulation at both the cellular level and during global tissue patterning events such as regeneration have yielded conflicting results. Remarkably, at the cellular level, similar Vm manipulations can trigger both growth and death (Bortner et al., 1997; Wang et al., 1999; Yu et al., 1999a,b; Thompson et al., 2001). Specifically, separate studies in which depolarization was induced in culture using drug treatments or by adjusting ion concentration in the cellular medium reported increases in either proliferation or apoptosis (Magnis et al., 1991; Wang et al., 1999; Wang, 2004; Lang et al., 2005; Yang and Brackenbury, 2013; Leanza et al., 2014; Levin, 2014; Table 1). Further, one paper reports that hyperpolarization by potassium channels is responsible for inhibiting apoptosis of murine myeloblastic FDC-P1 cells, suggesting that increased depolarization is necessary for apoptosis to occur via the Mcl-1 pathway in this cell type (Wang et al., 1999). In contrast, another study reports that hyperpolarization is required for inducing apoptosis, which is thought to occur via an efflux of potassium ions, reducing cell size (Lang et al., 2005). Hyperpolarization drives an influx of calcium ions in some cases (Nilius and Wohlrab, 1992; Ouadid-Ahidouch and Ahidouch, 2008), which has notably been reported to contribute either to apoptosis or proliferation (Clapham, 2007).

Although differing levels of calcium ion concentration amplified by Vm changes may seem like an attractive explanation for these disparate observations, it has alternatively been suggested that hyperpolarization does not propel, but inhibits influx of calcium ions by triggering the close of voltage-gated calcium channels (Wang, 2004). It is becoming more widely acknowledged that discrepancies exist regarding what types of ion flow (K+, Ca2+) contribute to either apoptosis or proliferation (Wang, 2004; Lang et al., 2005). Similar debates persist in studies of global patterning events such as regeneration of amputated limbs or reprograming of oncogenic tissues.

A fundamental question in regenerative medicine is how limbs and organs maintain consistent proportions. A study in planaria determined that depolarization by H,K- ATPase is required for proper head and pharynx scaling following amputation (Beane et al., 2013). Disrupting this Vm gradient resulted in shrunken heads and enlarged pharynxes in regenerated worms. It might be expected that such a phenotype would be caused by defects in proliferation compromising regenerative growth. Surprisingly, apoptotic remodeling of tissues, and not proliferation, was required for proper organ size. Disrupting either depolarization or apoptosis resulted in planaria with disproportionate head and pharynx size in response to amputation. These findings illustrate the link between Vm and apoptosis in regeneration. Although increased depolarization is generally correlated with dividing and cancerous cells (Levin, 2007), in this instance it induced apoptosis during regeneration in planaria.

Still, observations that hyperpolarizing treatments inhibit tumor formation (Chernet and Levin, 2014) might seem to suggest that depolarization is a disease phenotype that potential cancer therapies might seek to abolish. However, artificially creating areas of depolarization in bisected planaria did not generate tumors, but caused the formation of ectopic heads (Beane et al., 2011). Further, artificially hyperpolarizing bisected worms after amputation inhibited normal head regeneration (Beane et al., 2011). These studies indicate that depolarization relative to the surrounding tissue is a critical determinant of normal regenerative processes in planaria. Furthermore, depolarization is required for the regrowth of planarian head structures, including brain and eyes. However, separate studies in Xenopus revealed regions of hyperpolarization were critical for eye development (Pai et al., 2012). Expression of hyperpolarizing potassium channels induced ectopic eye formation in regions such as the gut and tail, while depolarization inhibited eye formation (Pai et al., 2012). One experiment that may shed light on these seeming discrepancies involved disrupting bioelectric signals by overexpression of hyperpolarizing channels in the frog embryo: widespread apoptosis or proliferation in the central nervous system of the tadpole was observed depending on which cells of the blastula were hyperpolarized, not simply on a specific Vm value (Pai et al., 2015a). This experiment adds an additional level of complexity to our understanding of bioelectricity, illustrating that Vm behaves like a morphogen field (Levin, 2010) rather than a cellular switch. Vm orchestrates behavior at both the cellular (Levin, 2010, 2012b, 2014) and tissue levels (Sundelacruz et al., 2009; Levin, 2010; Adams and Levin, 2013; Chernet and Levin, 2014) in the form of a bioelectric field gradient (Figure 1).

In this way, Vm comprises a key component of the external cellular microenvironment. However, in contrast to a traditional chemical morphogen, Vm functions at the interface of chemical and mechanical signals by impacting the flow of biochemically important ions such as Ca2+ by creating an electrical gradient across cells. In turn, this electrochemical gradient gates voltage-sensitive ion channels, thus creating a tightly connected communication pathway between the cell and its microenvironment (Clapham, 2007; Ohkubo and Yamazaki, 2012; Rothberg and Rothberg, 2012; Martinac, 2014). In this way, cells can be described as charged points, creating an electrical field across the tissue, termed bioelectricity (Levin, 2007). This bioelectric field is formed by spatial differences in Vm both within and across individual cells. Bioelectricity also carries information temporally, by changing at the same time as growth or injury of the tissue (Levin, 2007, 2010, 2012b, 2014; Tseng and Levin, 2013; Barghouth et al., 2015). For instance, cells located at the edge of a wound become depolarized, then migrate and proliferate to close the lesion (Chifflet et al., 2005). Wounds can thus be described as breaks in the tissue-wide bioelectric circuit (Levin, 2007; Figure 1).

Not only have disparate outcomes been documented in response to bioelectric manipulations, but the postulated mechanism of Vm transduction also differs between studies. Both the planarian and Xenopus studies found that calcium signaling was critical for alterations in Vm to be transduced into a morphogenetic output. In planaria, depolarization proceeds via activation of L-type calcium channels, thus increasing the concentration of Ca2+ ions in the anterior region of the animal (Beane et al., 2011). Ca2+ is then thought to drive anterior gene expression through the activation of factors such as cAMP response element-binding protein (CREB). In Xenopus, hyperpolarization-induced ectopic eye formation is also calcium-dependent, as inhibiting voltage-gated calcium (Cav) channels represses this phenotype. In vivo, cells reside amongst different cell types as well as in the presence of a complex microbial network. Remarkably, bacteria can participate in propagating Vm signals (Chernet and Levin, 2014). This was discovered in experiments exploring the ability of hyperpolarization to inhibit tumor formation in Xenopus. Transduction of this bioelectrical signal into tumor repression proceeds via Vm-modulated transport of histone deacetylase 1 (HDAC1), a factor involved in control of the cell cycle, apoptosis, and differentiation (Chernet and Levin, 2014). Further, HDAC1 was found to be inhibited by butyrate, a by-product of native bacteria in Xenopus. In this case, the inhibition of tumor-like structures depends on a balance of both bioelectric cues and microbial (HDAC inhibition) signals, not calcium channels. Therefore, not only do cells internally transduce Vm signals into phenotypic changes, the surrounding microenvironment also plays a substantial role in the physiological outcome of a given Vm input.

The key to resolving the differing observations associated with bioelectric manipulation may lie in the consideration of Vm not just as one morphogenetic property, but as a key parameter defined within a network of additional microenvironmental signals.

There are many examples in which disrupting the endogenous bioelectric field, by treating with ion channel inhibitors or overexpressing certain ion channels, has been demonstrated to control organ identity and placement in developing or regenerating organisms (Adams et al., 2007; Levin, 2007, 2010, 2012b; Tseng et al., 2007; Morokuma et al., 2008; Chernet and Levin, 2013; Tseng and Levin, 2013; Barghouth et al., 2015; Neuhof et al., 2016). For instance, depolarization was found to be a key step in the regeneration of the planarian head (Beane et al., 2011). Intriguingly, manipulation of the bioelectric gradient induces regrowth of amputated appendages in species such as Xenopus (Tseng and Levin, 2012, 2013), which lose regenerative capability with increasing age. Optogenetic hyperpolarization of amputated tail stumps in Xenopus tadpoles was found to induce regeneration of a complete tail structure containing a functioning spinal cord (Adams et al., 2013).

Because manipulating Vm or ion flux can alter phenotypes such as regeneration in aging organisms and correct tissue homeostasis defects such as cancer, much effort is currently being devoted to understanding the gene expression changes through which the bioelectric field regulates regeneration and tissue homeostasis. Specifically, microarray analysis has revealed conserved gene networks regulated by Vm depolarization (Pai et al., 2015b) across three different processes and species (embryogenesis in Xenopus; spinal cord regeneration in axolotl; and human cells in culture). Common regulatory processes include cell cycle, cell death, and differentiation, as well as factors associated with cytoskeletal organization, cell interactions, and cell movement. However, these results represent a single time point: further analysis is needed to examine the genetic changes accompanying temporal fluctuations in Vm. An additional study in Xenopus revealed that genes associated with hyperpolarization varied temporally in expression (Langlois and Martyniuk, 2013). Specifically, voltage-gated potassium channels decreased at the earliest stages of embryogenesis, but increased in expression during later stages of development. Changes in the levels of these channels could contribute to changes in Vm during growth. This supports the observation that Xenopus embryogenesis is accompanied by bioelectric gradients that direct anatomical form (Vandenberg et al., 2011; Adams et al., 2016; Sullivan et al., 2016). Although this information provides valuable insight and confirms that Vm regulation is evolutionarily conserved, it is still unclear how an individual cell translates a given Vm into a fate decision.

Furthermore, how is this information coordinated across the cells of a tissue to form the complex structure of an organ? The answers to such questions are collectively referred to as the “bioelectric code” (Tseng and Levin, 2013). Analogous to the way cracking the genetic code provided us with a deeper understanding of heritable illness, understanding the workings of bioelectricity is expected to provide exciting alternative therapies for both cancer and regeneration of organs lost to accidents or deteriorated due to aging, by allowing us to reprogram growth patterns (Levin, 2012b, 2014; Adams and Levin, 2013).

There is a strong connection between age and regenerative capacity. For instance, there is an age-dependent decline in the ability of mice to regrow tissues including lung (Paxson et al., 2011) and muscle (Conboy and Rando, 2005). In addition, the proliferative ability of cell populations including β-cells (Tschen et al., 2009) and T-cells (Mackall and Gress, 1997) decreases with age. This decline in regenerative capacity is a large factor in onset of the disability and frailty often associated with aging, due to loss of muscle mass and inability to heal muscle tissue after injury. This is believed to be due to signaling downstream of members of the transforming growth factor beta (TGFβ) family, such as myostatin and growth differentiation factor 11 (GDF11) (Egerman et al., 2015), or Notch (Conboy et al., 2003). However, the mechanism of age-related muscle loss remains unclear. Much effort has focused on identifying molecular components that are impacted by aging: the idea being that artificially returning altered levels of signaling molecules in aged organisms to levels observed in youth might reverse the deleterious effects of aging (Egerman et al., 2015). However, conflicting results have been obtained regarding whether molecules, such as GDF11, are up- or down-regulated as a consequence of aging (Egerman et al., 2015). In addition, simply increasing/decreasing levels of one factor may not be an optimal strategy, as many other processes may be impacted. Tuning the activity of one molecule or pathway without a thorough understanding of all processes that might be impacted could produce unwanted effects. A simple example is that blocking cell death does not provide us with immortality, but instead potentiates the development of cancer (Reed et al., 1996).

Bioelectric signaling appears to function as a global regulatory mechanism providing us with the capability of inducing the formation of an entire planarian head without a full understanding of every pathway that is involved in this complex regenerative process (Beane et al., 2011). The ability to regulate regeneration of muscle or deteriorated organs without harming other processes may thus be a potential use of Vm.

Whether changes in bioelectric gradients are involved in the age-related decline of regenerative ability is an open question. More research is needed to determine this. However, Vm may play a role in age-related ailments of neural tissue. In particular, Vm is involved in regulating intracellular Ca2+ levels (Clapham, 2007). Ca2+ influx occurs through ion channels such as Cav (Catterall, 2011) and transient receptor potential (TRP) channels (Clapham, 2003), while outflux primarily proceeds via uptake into mitochondria and endoplasmic reticulum (ER) (Bezprozvanny and Mattson, 2008). Perturbing this delicate balance can lead to diseases such as Alzheimer's. For example, blocking K+ channels might cause a neuronal cell to become more hyperpolarized; this imparts a more overall negative charge to the cell, creating a more favorable electrochemical gradient for entry of positively charged ions (Clapham, 2007; Bezprozvanny and Mattson, 2008) such as Ca2+. Elevated intracellular Ca2+ levels can lead to increased excitotoxicity and apoptosis (Bezprozvanny and Mattson, 2008), aiding the progression of neurodegenerative disease. It has long been known that a connection exists between Alzheimer's and age (Hardy, 2006). Age is also associated with increased neuronal intracellular Ca2+ levels (Thibault and Landfield, 1996). Although Vm and voltage-gated calcium channels are implicated in this process (Thibault and Landfield, 1996), many additional components are involved in heightened Ca2+ entry, such as the pores formed by amyloid β-peptide (Bezprozvanny and Mattson, 2008). More research is needed to determine how age impacts bioelectric gradients specifically in both neural and non-neural tissues.

It might be expected that animals with high regenerative capacity are more prone to cancer, because their cells are presumably highly prolific. However, the opposite is true, suggesting that highly regenerative species such as planaria and axolotls have tight regulation of morphogenetic patterning in both space and time (Levin, 2012b). The strong patterning regulation that enables organisms with high regenerative ability not only to maintain their form following injury but to ward off cancerous growth draws a close relationship between cancer and regeneration (Levin, 2012b). Intriguingly, an inverse relationship between the diagnosis of age-related diseases such as Alzheimer's and risk of developing cancer has been observed (Driver et al., 2012). This suggests that cancer, injury, and aging are not necessarily different ailments requiring specialized treatment strategies, but variations of a common morphogenetic patterning defect.

Although there are conflicting results and proposed mechanisms regarding bioelectric signaling, the studies discussed so far appear to agree that Vm transfers some form of information to the cell that is propagated as a stable physiological state (Neuhof et al., 2016). Logically, a change in Vm that triggers division, differentiation, or gene expression in one cell could impact surrounding cells, and thus propagate throughout the tissue. However, changes in Vm can also induce apoptosis. Can a dead cell communicate information to its surrounding tissue? Although counterintuitive, it has become clear that apoptosis is required for many growth and tissue maintenance processes including embryonic development (Haanen and Vermes, 1996), tumor prevention (Hanahan and Weinberg, 2000), and healthy maintenance of the epithelial cell barrier (Rosenblatt et al., 2001; Andrade and Rosenblatt, 2011; Gu et al., 2011; Slattum and Rosenblatt, 2014; Eisenhoffer et al., 2015). The expression of gene networks associated not only with proliferation but also with apoptosis is increased in the first stages of embryogenesis (Langlois and Martyniuk, 2013).

One of the earliest indications that cellular death is necessary for life processes came in 1842, when examinations of amphibian development revealed that both cellular proliferation and death occurred during embryonic growth (Vogt, 1842; Jacobson et al., 1997). Later on, it was recognized that apoptosis is an integral part of many processes during embryogenesis including the formation of the early structure of the brain, which is critical for proper brain function (Oppenheim, 1991; Kuida et al., 1996). Apoptosis plays key roles in sculpting appendages (Saunders et al., 1962; Milligan et al., 1995; Jacobsen et al., 1996), forming tubes and lumina (Glucksmann, 1951), metamorphosis (Lockshin, 1981), controlling cell numbers (Rosenblatt et al., 2001; Andrade and Rosenblatt, 2011), and deleting unwanted structures (Jacobson et al., 1997). Blocking apoptosis in the embryo was found to have deleterious or even fatal outcomes (Kuida et al., 1996). Apoptosis is not only necessary for embryonic development, but is required for regeneration in some species. For example, apoptosis controls the tissue remodeling essential for correct size ratios and cell lineage specification during planarian regeneration (Beane et al., 2013). In addition, studies in Hydra revealed that apoptosis was essential for head regeneration following amputation (Chera et al., 2009). Remarkably, a layer of apoptotic cells near the amputation site provided an increased source of Wnt3, functioning in the synchronized division of nearby stem cells. In this way, the dying cells propagated patterning information to the proliferating cells. Not only do apoptotic cells modify the chemical microenvironment of neighboring cells, but their elimination may change the geometry and density of a tissue.

Disruption of the homeostatic balance between proliferation and death is proposed to be a primary driving force for both tumorigenesis (proliferation favored over apoptosis) and organ deterioration (apoptosis favored over proliferation) (Andrade and Rosenblatt, 2011; Slattum and Rosenblatt, 2014). The push to understand the disruption of this homeostasis may be behind the unfortunate categorization of apoptosis and proliferation into two opposing phenotypes. However, it is becoming increasingly accepted that apoptosis and proliferation work together to orchestrate growth, development, and maintenance of tissues. Furthermore, the cellular transition from growth to death does not appear to occur as the bimodal switch we often envision. Cells are capable of recovering from apoptosis even after apparently late stages, including caspase activation and DNA damage, a process termed “anastasis” (Tang et al., 2012). During early stages of apoptosis recovery, genes associated with proliferation and cell cycle are enriched. As anastasis progresses, cells take on a migratory phenotype and upregulate genes associated with focal adhesions and regulation of the actin cytoskeleton (Sun et al., 2017). Remarkably, scratch wounds of cell monolayers close faster when induced to undergo anastasis by treatment with and subsequent removal of ethanol, which induces apoptosis (Sun et al., 2017). It is therefore unclear at what point a cell can be considered fully dead, and recovery from apoptosis can even lead to enhanced healing and expression of proliferation-associated genes.

Current strategies for studying bioelectricity often involve manipulating Vm in a given tissue region then observing a particular phenotype, which functions as the output of the Vm input. However, proliferative and apoptotic phenotypes appear to have a complex co-dependence in many situations, and perhaps even cannot be absolutely characterized. The interconnection between apoptosis and proliferation as well as the disparities among bioelectric manipulation experiments imply that the difference between a regenerative signal and a tumor-initiating cue may be very subtle. A more comprehensive understanding of all the factors that contribute to bioelectrical signaling is needed to determine whether a Vm alteration will result in a beneficial or deleterious program.

Although much attention has traditionally been devoted to understanding how cell-intrinsic parameters (such as genetic alterations and changes in protein expression) drive phenotypes related to aging and cancer, it has become increasingly well recognized that the cellular microenvironment also plays a large role in cancer-related cellular behaviors as well as growth and form at the tissue-scale. The microenvironment functions not only to guide the cell in spatial dimensions, but directs tissue-scale growth through time (Figure 2). In addition, not only is Vm closely related to the microenvironment, the microenvironment is part of the Vm definition. Vm is the difference between electrical charge within the cytoplasm and the external medium (Levin, 2010). Notably, even if the cell did not have any way of controlling its internal charge via ion channels or pumps, Vm could still be changed by altering the charge of the extracellular medium alone.