Within approximately the last 10 years, experimental results and clinical data have established that both fibroblast and myofibroblast cell populations in the mammalian heart (including the adult human) can play very important functional roles in physiological and pathophysiological settings, respectively (Nattel, 2002; Baudino et al., 2006; Munoz et al., 2008; Porter and Turner, 2009; Baum and Duffy, 2011; Biernacka and Frangogiannis, 2011). The fibroblast population, when judged in terms of cell numbers, is predominant in all adult mammalian hearts (cf. Baudino et al., 2006). Until quite recently, these fibroblasts were considered to be passive. That is, they were thought to have few, if any primary electrophysiological roles. Rather, both fibroblasts and myofibroblasts were believed to function predominantly in the synthesis, and degradation (homeostasis) of the extracellular matrix, largely as a consequence of their roles in collagen production/breakdown (Weber et al., 1993). However, this view changed after publication of data demonstrating that both fibroblasts and myofibroblasts have the capability of communicating (cell-to-cell) electrically and metabolically through conventional connexins within gap junctions. This connexin-mediated electrotonic communication may also include functional interactions of these fibroblasts/myofibroblasts with adjacent myocytes (Gaudesius et al., 2003; Chilton et al., 2007; Zlochiver et al., 2008; Kakkar and Lee, 2010). It is now apparent that both the fibroblast and the myofibroblast cell populations can serve as a significant source of paracrine substances that, when secreted, act on adjacent cells (Powell et al., 1999; Pedrotty et al., 2009; Rohr, 2011). Both fibroblasts and myofibroblasts can also be regulated by neurotransmitters, co-transmitters, peptides, and circulating hormones (Brilla et al., 1995; Rose et al., 2007; cf. Rose and Giles, 2008). Important early work on isolated fibroblasts also drew attention to the fact that the electrophysiological properties of these cells can be modulated significantly by mechanical perturbations, including both stretch and shear forces. Abramochkin et al. (2014) have recently reviewed this data.

Experimental work, based mainly on co-culture methods, combined with simulations using mathematical models of, e.g., human atrial myocytes and fibroblasts, have drawn attention to the possibility that the fibroblast cell population could significantly modulate the electrophysiological substrate in a number of cardiac tissues. Papers that deal specifically with these interactions in the atria include: (Maleckar et al., 2009a; Ashihara et al., 2012; McDowell et al., 2013) and ventricles (MacCannell et al., 2007; Sachse et al., 2008; McDowell et al., 2011) of the human heart. However, in all cases, these interactions have been assumed to be passive; that is, to not involve any regenerative or propagated responses that originate in the fibroblast/myofibroblast cell population (Gaudesius et al., 2003; Maleckar et al., 2009a; McDowell et al., 2011, 2013).

Important early work on atrial rhythm disturbances by Spach and his colleagues (Spach and Boineau, 1997) drew attention to the ability of “microfibrosis” to alter the electrical substrate in the atrium and thus promote arrhythmias. Spach et al. (2007) also recognized that in the human atrium an important consequence of aging was an increased incidence of supraventricular rhythm disturbances with some fibrosis. Enhanced fibrosis in the setting of healthy aging is now a well-known contributor to atrial flutter/fibrillation (Nattel, 2002; Biernacka and Frangogiannis, 2011; Diness et al., 2011; Guzadhur et al., 2012).

Recently, it has been reported that myofibroblasts derived from explanted human right atrial tissue (the right atrial appendage) can express measurable (and potentially functionally important) Na⁺currents. Specifically, Chatelier et al. (2012) have reported that approximately 75% of the myofibroblasts that are isolated from the human right atrial appendage express Na⁺ current after being maintained in a primary 2-D cell culture 8–12 days. They observed that 100% of these cells expressed Na⁺ current after 13–17 days in culture. These investigators have also provided evidence that this Na⁺ current is generated by the same Na⁺ channel α subunit, Na_v 1.5 that produces the Na⁺ current in the atria and ventricles of the adult human heart.

After the properties of this Na⁺-selective integral membrane protein in fibroblasts/myofibroblasts are understood more completely, a paradigm shift concerning the functional properties of human cardiac syncytium in both health and disease may be required. As an example, the expression of Na_v 1.5 in this cell population raises the possibility that the myofibroblast cell population could be the source of ectopic foci. That is, this Na⁺ current could provide a significant source of regenerative inward current. In principle, the resulting depolarization could serve both as the trigger and the substrate for supraventricular rhythm disturbances originating in the fibroblast/myofibroblast cell population.

The main focus of our study was to complement and extend the Chatelier et al. (2012) findings by addressing four separate, but related, objectives:

Here, we report our preliminary findings based on conventional whole-cell voltage-clamp measurements of the electrophysiological properties of single myofibroblasts that were isolated from the human right atrial appendage and then placed in conventional 2-D tissue culture for approximately 6 days (2–4 passages). PCR analyses of the expression of a number of α and β subunits of mammalian Na⁺ channels are also presented. Our results are compared and contrasted with those in the original paper on this topic (Chatelier et al., 2012). Mathematical models of the human atrial myocyte and/or the fibroblast/myofibroblast are used to explore and illustrate plausible functional consequences of Na⁺ current expression in the myofibroblast population.

Taken together, this experimental and theoretical work contributes to ongoing discussions regarding possible functional roles for Na⁺ current in human atrial myofibroblasts. These findings also raise interesting possibilities and questions concerning novel molecular targets in the compromised human atrium, e.g., during persistent atrial fibrillation (Benito et al., 2008; Biernacka and Frangogiannis, 2011; Aguilar-Shardonofsky et al., 2012). This class of rhythm disturbance is of importance due to its increased incidence during the healthy aging process (Benito et al., 2008; Aguilar-Shardonofsky et al., 2012; Guzadhur et al., 2012); and because persistent atrial fibrillation can increase the incidence of transient ischemic attack and/or stroke (cf. Bersohn et al., 2012).

A conventional whole-cell patch clamp method was used to characterize the predominant transmembrane ionic currents in these single isolated human atrial myofibroblasts. Representative families of membrane currents are shown in Figure 1. In this myofibroblast and in the majority of others that were studied, voltage clamp depolarizations resulted in rapid activation of a small outward current. Larger depolarizations (in the +60 to +100 mV range) consistently gave rise to large “noisy” transmembrane records, that are characteristic of Ca²⁺-activated K⁺ currents. In contrast, hyperpolarizing clamp steps from the holding potential produced much smaller changes in transmembrane current, similar to the pattern that has been reported in rat ventricular fibroblasts/myofibroblasts (Chilton et al., 2005).

A main focus of this study was to determine whether transient inward Na⁺ currents could be detected in these myofibroblasts. For this reason, families of transmembrane currents were generated from two different holding potentials. After each cell was voltage clamped near the resting potential, the holding potential was set to −50 mV, and step depolarizations lasting 100 ms were applied in 10 mV increments covering the range of membrane potentials −120 mV to +100 mV. As shown in the bottom row of Figure 1, this protocol produced small but measurable transient inward currents (indicated by the red traces). To enhance the likelihood of detecting Na⁺ current, and to attempt to record the maximum transient inward Na⁺ current that could be generated by each individual myofibroblast, the holding potential was then set to −120 mV. Transmembrane currents, in response to the same levels of depolarization, were elicited. These families of currents are shown in the top row of Figure 1. As expected (as a consequence of the more hyperpolarized holding potential removing inactivation), this maneuver resulted in significantly larger transient inward currents (see red traces). This type of protocol was applied to 40 myofibroblasts in total: the data indicate that approximately 50% of these isolated myofibroblasts express a measurable transient inward current, when the holding potential was −120 mV (see Discussion).

In this series of experiments, it became apparent that there was considerable variability in the expression levels of these Na⁺ currents. The scatterplots in Figure 2 illustrate and summarize this variability. Note that when the transient inward Na⁺ current is measured, either in terms of current density (left) in response to a depolarization to 0 mV or plotted as peak current, (right) there is very significant heterogeneity or scatter. The average current density at 0 mV was approximately 3 pA/pF when the holding potential was −120 mV.

The raw data (left) and current-voltage relationship (right) in Figure 3 provide useful information concerning the peak size, as well as the activation and inactivation kinetics, and the voltage-dependence of this peak inward current in the human atrial myofibroblast, respectively. The family of currents shown in the left panel of Figure 3 was obtained by applying rectangular voltage clamp depolarizations from a holding potential of −120 mV, as illustrated in the inset. The peak current in response to this step-depolarization to 0 mV is highlighted in red. This current trace provides a point of reference for the summarized data described in Figure 2.

A representative peak inward current-voltage relationship is shown in the right panel of Figure 3. Note that this inward current first activates at a membrane potential near −50 mV, and that it is maximal near 0 mV. The positive limb of this I–V curve could not be obtained in its entirety due to the rapid activation of a substantial and noisy outward current (see Figure 1) at membrane potentials positive to approximately +50 mV. Overall, this pattern of current changes closely resembles families of currents and peak current-voltage relationships that have been published for Na⁺ current in cardiac myocytes. This includes myocytes from human atrium (Baba et al., 2006), and data based on heterologous expression, when the α subunit of the cardiac Na⁺ channel, Na_v 1.5 is overexpressed in mammalian cell lines, and then studied with conventional patch clamp methods.

The raw data and corresponding analysis shown in Figure 4 summarize our experimental measurements of the steady-state inactivation characteristics of the Na⁺ current in these human atrial myofibroblasts. A conventional two-step voltage clamp protocol was used to progressively inactivate this Na⁺ current. From a holding potential of −120 mV, a relatively long “inactivating” prepulse (500 ms) was applied, and this was followed immediately by a second step-depolarization to −10 mV. As expected and as illustrated in the superimposed currents in the left panel, when the prepulse voltage depolarized the cell beyond approximately −40 mV, peak Na⁺ current decreased progressively. This voltage-dependent inactivation was characterized over the entire physiological range of membrane potentials, as shown in the plot in the right panel of this Figure. The best-fit parameters for this steady-state inactivation relationship were: membrane potential for half an activation or V_h −65 mV, and maximum slope factor 8.4 mV. In this panel of Figure 4, the sigmoid relationship shown in red is a derived relationship depicting voltage-dependent activation for the Na⁺ currents shown in Figure 3 (right).

These electrophysiological findings are somewhat similar to those in the original paper on this topic (Chatelier et al., 2012). Certainly, both data sets reveal the functional expression of Na⁺ channels in tissue cultured human atrial myofibroblasts.

Additional molecular evidence for the expression of Na⁺ channels in these human atrial myofibroblasts is presented in Figure 5. These histograms are based on our PCR analyses of the relative expression levels of a range of human Na⁺ channel (SCN-5) α and β subunits. From panel A note the two strongest α subunit signals are for the “nerve” Na⁺ channels SCN9A and SCN2A, respectively. However, the predominant (TTX-insensitive) mammalian heart α subunit was also be detected. Figure 5B presents analogous PCR data directed toward Na⁺ channel β subunits. Here, the β₁ subunit represents the predominant signal (see Discussion).

Our data (Figures 1–5) and the Chatelier et al. publication (2012) that first reported functional expression of Na⁺ current in human atrial myofibroblasts suggest the possibility that these cells may be able to exhibit regenerative, action potential-like responses when appropriate depolarizing stimuli are applied. Figures 6–8 illustrate the results of in silico tests of this possibility.

For these simulations, two quite different starting conditions were chosen: (i) the myofibroblast was assumed to have a resting membrane potential E_m of −35 mV, and (ii) the myofibroblast E_m values were set at −60 mV. This was done in recognition of the uncertainty of the fibroblast resting potential in situ (see Discussion), and because the Na⁺ current is inactivated almost completely at −35 mV. In both cases, selected 16 ms de- and hyperpolarizing stimuli were applied intracellularly, as diagrammed in Figure 6A.

The results in Figures 6B,C illustrate the responses when the myofibroblast was stimulated from a steady membrane potential of −35 mV, and no I_Na was introduced/expressed. As expected, the myofibroblast membrane potential responds to small rectangular stimuli with a quasi-exponential electrotonic de- or hyperpolarization, having characteristic time-courses governed approximately by the product of the mean input resistance (3–5 Gigohms) and single cell capacitance (6–10 pF). Note that the original mathematical model of the myofibroblast employed in these simulations (MacCannell et al., 2007) includes a rapidly activating delayed rectifier K⁺ current, in accordance with experimental data from rat ventricular myofibroblasts. This K⁺ current is activated when E_m depolarizes positive to approximately −30 mV. Indeed, the myofibroblast exhibits a partial repolarization (top trace, panel B) at positive membrane voltages (approximately 0 mV). In addition, a post-stimulus hyperpolarization due to the subsequent deactivation of this K⁺ current was observed. The results in Figure 6D,E were obtained using the same 4 constant current stimuli (panel A) after the Na⁺ current (identified in our study; Figures 1–4) was introduced. Note (panel E) that there is a tendency for regenerative depolarization in the voltage responses corresponding to all 3 depolarizing stimuli when E_m is set at −65 mV. The data in panels F and G is analogous to that in panels D and E, except that in these simulations I_Na equations that replicate the Na⁺ current described by Chatelier et al. (2012) have been “introduced” into the myofibroblast.

In the next two sets of computational studies, a mathematical model of the human atrial myocyte was combined with the modified mammalian myofibroblast model that is described in Methods. Three sets of computations were done: (i) a fixed number of in silico myofibroblasts were coupled to a single human atrial myocyte through a linear resistance corresponding to a conductance of 0.5 nS. These results are shown in the left column, panels B,E,H of Figure 7. (ii) In the second set of computations, Na⁺ current consistent with the biophysical properties depicted in Figures 1–4 in this paper was introduced into the myofibroblast cell population. These results are shown in the middle column, or panels C, F, and I of Figure 7. (iii) Finally, an analogous set of computations was done, this time using the Na⁺ current having the biophysical properties reported by Chatelier et al. (2012). In Figure 7, these computations are shown in the third column, or panels D,G,J. For a reference and starting point, panel A shows the atrial myocyte resting potential and action potential waveform (red); and also illustrates the assigned myofibroblast resting potential when these two types of cells are not coupled. Overall, this pattern of responses was not particularly revealing with respect to Na⁺-current dependent patterns of action potential waveforms or the electrotonic responses in the myofibroblast population. Specifically, the expression of Na⁺ current in the myofibroblast appeared to have no significant effects on the electrotonic waveform.

Note, however, that an important variable was that the myofibroblast resting membrane potential was initially set to that reported for these isolated cells, namely approximately −35 mV. After coupling in the 1:1 (panels B–D) and 1:3 (panels E–G) cases the fibroblast hyperpolarized and experienced a significant electrotonic depolarization and repolarization driven by the myocyte action potential. In the case of 1:9 ratio coupling (panels H–J) the atrial myocyte depolarized significantly and exhibited only a weak regenerative response, which resulted in a small electrotonic depolarization in the myofibroblast(s). We note that when the resting potential in either the myocyte or the fibroblasts is positive to approximately −60 mV, I_Na would be strongly inactivated (see Figure 4), and thus would not contribute to the membrane voltage profile in the myofibroblast or the immediately adjacent myocyte.

After recognizing that the resting membrane potential of the myofibroblast was a critically important determinant of the size of the peak inward Na⁺ current, a second set of computations was carried out with the membrane potential of the myofibroblast set to approximately −65 mV, rather than −35 mV. In Figure 8, the presentation of the data is the same as that of data Figure 7. Thus, the panels in column 1 (B,D,E,H) were based on computations done with no Na⁺ current in the myofibroblast cell population, and with coupling ratios of myocyte to myofibroblasts of 1:1, 1:3, and 1:9, respectively. As expected from our previous papers (MacCannell et al., 2007; Maleckar et al., 2009a), the myocyte membrane potential dominates that of the myofibroblast at low myofibroblast/myocyte ratios. In distinction, to when the myofibroblast resting membrane potential is much more depolarized (−35 mV), when the coupling ratio is 1:3 or 1:9, both the myofibroblasts and the myocytes have well-polarized resting membrane potentials. Partly for this reason, the waveform of the voltage change in the myofibroblast also differs substantially. This arises from the fact that more hyperpolarized resting membrane potential removes inactivation from the Na⁺ channels. This effect would be expected to be strongly manifested in the computations shown in panels C,F,I—as well a D,G,I, since the voltage dependence for inactivation of the Na⁺ current falls well within the range of the resting membrane potential of the myofibroblast.

Careful inspection of some of the computational results in Figures 7, 8 may suggest that activation of I_Na in the myofibroblast influences the overall “substrate” electrophysiological profile. Thus, in some cases, the membrane potential in the myofibroblast(s) depolarizes, when the atrial myocyte is repolarizing.

The computational results shown in Figure 9 provide some further insight into this intriguing possibility. All of these computations were done using a 1 myocyte to 1 myofibroblast ratio. Two different values of coupling (intercellular) resistance, either 0.5 nS (top row) or 8 nS (bottom row) were selected for study. As expected, when intercellular resistance is very small, the myocyte action potential produces an electrotonic response in the myofibroblast that is essentially unattenuated.

Note that at both these values of intercellular resistance (panels C,D), the current flowing between the “cells” is dominated by the net inward (depolarizing) and outward (repolarizing) currents generated by the atrial myocyte. Panels E and F show the net transmembrane current in the myofibroblast population. Again, the inward, depolarizing current appears to be contributed mainly by the activation of I_Na in the atrial myocyte(s), as opposed to the myofibroblast(s) (see Discussion).

Doctors Robert Clark and Darrell Belke were responsible for the experimental work shown in Figures 1–4 and 5, respectively. Doctors Jussi Koivumäki and Mary Maleckar produced the simulation results in Figures 6–9. Doctor Wayne Giles planned and wrote draft #1 of this manuscript. All authors read and provided comments on the original and revised manuscripts.