Hydrogen sulphide suppresses human atrial fibroblast proliferation and transformation to myofibroblasts

Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca2+-activated K+ current (BKCa), transient outward K+ current (Ito) and inwardly rectifying K+ current (IKir) were found in human atrial fibroblasts. Current density of BKCa (IC50 = 69.4 μM; n = 6), Ito (IC50 = 55.1 μM; n = 6) and IKir (IC50 = 78.9 μM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100–500 μM) inhibited fibroblast proliferation induced by transforming growth factor-β1 (TGF-β1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre-conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode Ito), but not KCa1.1 (encode BKCa) and Kir2.1 (encode IKir). Furthermore, H2S significantly attenuated TGF-β1–stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-β–induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K+ channel activity and moderates their differentiation towards myofibroblasts.


Introduction
Cardiac fibroblasts are fundamentally involved in cardiac remodelling in normal ageing heart [1] and in damaged myocardium [2]. Aberrant proliferation of fibroblasts and their transformation to myofibroblasts is a hallmark of cardiac fibrosis, which is characterized by excessive extracellular matrix built-up leading to loss of tissue compliance [3,4]. Because of their wide-ranging participation in myocardial pathophysiology, cardiac fibroblasts represent an attractive target in managing cardiac disorders, including cardiac hypertrophy, heart failure and arrhythmias [5]. Indeed, atrial fibrosis has been closely associated with atrial fibrillation [6,7] and sinus node dysfunction [8].
Hydrogen sulphide (H 2 S) is an endogenously generated gaseous transmitter that has been reported to attenuate cardiac fibrosis [9]. It is known to mediate its effects by modulating ion channel activity in many cellular systems [10]. Hydrogen sulphide was the first opener of K ATP channel identified in vascular smooth muscle cells [11]. Through activation of K ATP channels, H 2 S lowers blood pressure, protects heart from ischaemia and reperfusion injury [12,13]. We have recently reported that H 2 S inhibited delayed rectifier potassium channels in human iPS-derived cardiomyocytes [14]. Yet, effect of H 2 S on cardiac fibroblasts remains poorly understood. We hypothesized that H 2 S inhibits proliferation of atrial fibroblasts by inhibiting functioning of potassium channels. We present supporting data that H 2 S may potentially modulate cardiac fibrosis by inhibiting BK Ca , I to and IK ir , independent of K ATP channels, leading to decreased proliferation and suppression of transforming growth factor-b1 (TGF-b1)-induced myofibroblast transformation of atrial fibroblasts.

Fibroblast isolation
Patients undergoing mitral valve repair and coronary bypass surgery (n = 10) were recruited after informed consent in protocol approved by institutional review board of Singapore General Hospital that conformed to the Declaration of Helsinki. Atrial appendages were collected as surgical by-product. Human atrial fibroblasts were isolated by mincing the appendages to less than 1 mm 3 and followed by 0.1% trypsin digestion for 20 min. before plating onto tissue culture-treated 60-mm dishes to produce fibroblastic outgrowth from minced tissue pieces. The isolated fibroblasts were confirmed with expression of collagen I (1/20; Southern Biotech, Birmingham, AL, USA) and anti-human fibroblast (1/1000; Sigma-Aldrich, St. Louis, MO, USA) antibodies (Fig. S1). Atrial fibroblasts were passaged as monolayer in 10% foetal bovine serum-supplemented DMEM. Fibroblasts between passage 1 and 3 were used for subsequent experiments.

Electrophysiological recordings
Cell were placed on the stage of a Nikon Diaphot inverted microscope and superfused continuously at 36 AE 1°C with Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 HEPES and 10 Glucose (pH adjusted to 7.4 with NaOH). The patch-clamped cell was superfused by means of a temperature-controlled micro-superfusor (TC-324B, Warner Instruments, Hamden, CT, USA). Patch pipettes were made from borosilicate glass shanks (Sutter Instrument, Novato, CA, USA) and pulled with a Brown-Flaming puller (Model P-97; Sutter Instrument Co), and had tip resistances of 2-3 MΩ when filled with pipette solution. Pipette tips were polished (Microforge MF830; Narishige, Tokyo, Japan). These patch pipettes were filled with a standard solution containing (in mM) 140 KCl, 1.2 MgCl 2 , 0.05 EGTA, 10 HE-PES, 0.1 GTP and 5.0 Mg ATP (pH adjusted to 7.2 with KOH). For Na + current recording, the patch pipettes were filled with (in mM) 35 NaCl, 105 CsF, 0.1 EGTA and 10 HEPES (pH adjusted to 7.4 with CsOH). After a gigaohm seal was obtained by negative pressure suction, the cell membrane was ruptured by a gentle suction to establish whole-cell configuration with a seal resistance >800 MΩ. The cell membrane capacitance (40.27 AE 8.2 pF) was electrically compensated with the pulse software. The series resistance (Rs, 3-5 MΩ) was compensated by 50-70% to minimize voltage errors. Currents were elicited with voltage protocols as described in the following results section for different individual current recordings. Whole-cell voltageclamp experiments were performed with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) interfaced to a Digidata 1322A data acquisition system controlled by Clampex version 8.1 software (Axon Instruments). Data were analysed with pCLAMP software (Version 10.0; Axon Instrument) and Origin 8.0 (OriginLab, Northampton, MA, USA).

Cell proliferation and apoptosis assay
Cell proliferation assay was performed with BrdU kit (Roche, Basel, Switzerland). Briefly, cells were plated on 96-well plate at a density of 3000/well and cultured for 24 hrs. After 4 hrs of serum starvation, cells were incubated for 24 hrs with medium containing ion channel blockers, NaHS or growth factors. BrdU labelling solution (100 lM) diluted 10 times in DMEM (0.1% FBS) was added to each well and the plates were incubated at 37°C for an additional 2 hrs. Incorporated BrdU was detected by an anti-BrdU antibody for 90 min. and colorimetric development proceeded for 15 min. before analysis by ELISA plate reader (SpectraMax, Molecular Device, Sunnyvale, CA, USA). Cellular apoptosis assay was performed with Caspase-3 Fluorescence Assay kit as instructed (Cayman Chemical, Ann Arbor, MI, USA). Briefly, cells were plated on 96-well plate at a density of 10 4 /well and cultured for 24 hrs. After 4 hrs of serum starvation, cells were incubated for 24 hrs with medium containing NaHS. Fluorescent intensity was obtained with ELISA plate reader (SpectraMax, Molecular Device) at 485 nm excitation and 535 nm emission wavelengths.

Statistical analysis
Data were expressed as mean AE SE. Statistical significance of the difference between groups was determined with Student's t-test. A value of P < 0.05 was considered statistically significant.

Hydrogen sulphide suppresses ion currents in human atrial fibroblasts
Multiple ionic channels are reported to be expressed in human cardiac ventricular fibroblasts [15], ionic channels in our atrial fibroblasts were activated by depolarization voltage between À70 and +60 mV from a holding potential of À80 mV (0.2 Hz) to elicit total outward K + currents. Activated currents that were sensitive to paxilline (1 mM), a specific BK Ca inhibitor, were significantly suppressed at +60 mV, confirming the presence of BK Ca current (52%; 163/309 cells) in human atrial fibroblasts (Fig. 1A). Under identical voltage-clamp condition, exposure to 100 lM NaHS (as a donor of H 2 S) similarly reduced the peak current density of BK Ca (Fig. 1B). The inhibitory effects observed could not be washed out (Fig. 1C). The presence of NaHS resulted in a voltage-dependent suppression of the I-V curve from 10.5 AE 1.2 pA/pF to 6.8 AE 0.9 pA/pF at +40 mV (P < 0.01; n = 6) (Fig. 1D) and a dose-dependent inhibition of BK Ca peak current density with an IC 50 of 69.4 lM (Fig. 1E).
To verify the specificity of H 2 S inhibition on BK Ca , we assessed its effect in the presence of naringenin (10 lM), a specific opener of BK Ca [16]. BK Ca currents were elicited with clamp pulses at +40 mV from a holding potential of À80 mV under control condition (Fig. 1F). Compared with baseline (9.9 AE 0.8 pA/pF), naringenin increased BK C a current significantly (14.1 AE 0.5 pA/pF; P < 0.01; n = 6), but addition of NaHS returned naringenin-induced current to baseline (9.2 AE 0.4 pA/pF; P < 0.05; n = 6) (Fig. 1G). The rising phase of the BK Ca currents at 50 mV with activation s (s act ) at baseline (15.4 AE 0.1 ms) was lowered significantly by naringenin (8.6 AE 0.2 ms; P < 0.01; n = 6), but reversed to baseline after addition of NaHS (15.4 AE 0.2 ms; P < 0.05; n = 6), which confirmed its modulation of BK Ca channel kinetics (Fig. 1H).
Similarly, under conditions to elicit total outward K + currents, a 4aminopyridine (4-AP; 0.5 mM)-sensitive current was detected, indicating the presence of transient outward currents, I to (34%; 104/309 cells) in the atrial fibroblasts ( Fig. 2A). Under identical voltage-clamp condition, exposure of fibroblasts to 100 lM NaHS reduced the peak (G) Summarized data for peak BK Ca currents at +40 mV at baseline, in the presence of Naringenin (10 lM), and in the presence of NaHS (100 lM) (*P < 0.05 versus basal levels; # P < 0.05 versus Naringenin alone; n = 6). (H) Plot of the activation s (s act ) as a function of membrane potential in the presence of Naringenin (10 lM) and Naringenin together with NaHS (100 lM) (**P < 0.01 versus basal levels; # P < 0.05 versus Naringenin alone; n = 6). current density of I to (Fig. 2B). The inhibitory effects occurred within 1 min., reached saturation by 10 min. and could not be washed out (Fig. 2C). Addition of NaHS showed a voltage-dependent suppression of the I to current in the I-V curve from 18.2 AE 1.5 pA/pF to 12.7 AE 1.7 pA/pF at +40 mV (P < 0.05; n = 6) (Fig. 2D) and demonstrated a dose-dependent inhibition of peak current density with an IC 50 of 55.1 lM (Fig. 2E). Steady-state activation of I to was unaffected by NaHS (Fig. 2F). [The curves were fitted by the Boltzman equation: , where G/G max represents a ratio of conductance to the maximum conductance, and V T represents the values of the depolarizing pulses]. The half-maximum activation voltage (V 1/2 ) and slope factor under control condition were 17.2 AE1.5 mV and 19.3 AE 1.3, respectively, which were not significantly different from those in the presence of NaHS (V 1/2 : 18.3 AE 1.2 mV, slope factor 20.2 AE 1.2) (P = NS; n = 6). In contrast, NaHS significantly influenced the steady-state inactivation of I to (Fig. 2G). When fitted to Boltzman function, I/I max =1/[1 + exp(V T À V 1/2 /j)], the half-maximum inactivation voltage (V 1/2-inact ) and slope factor under control condition were À53.6 AE 1.2 mV and 9.08 AE 1.1, respectively, which were significantly different from those in the presence of NaHS (V 1/2-inact : À71.1 AE 3.1 mV, slope factor 14.7 AE 2.4) (P < 0.05; n = 6). Furthermore, recovery of I to from inactivation was analysed by delivering two identical 500 ms depolarizing pulses from À80 to +60 mV and varying the interpulse from 50 to 3500 ms. Addition of NaHS shifted the curve right and increased the half-recovery time of I to from of 461.7 AE 57 to 1218.2 AE 49 ms. (P < 0.01; n = 6) (Fig. 2H), confirming inhibition of NaHS on the kinetic property of I to channel recovery. Furthermore, these properties of I to were similar to those reported in human ventricular fibroblasts [15].
The inhibitory effect of NaHS on I to was further confirmed in the presence of NS5806 (10 lM), a specific opener of I to [17] (Fig. 2I). The I to currents were elicited with clamp pulses at +40 mV from a holding potential of À80 mV. Compared with baseline (18.8 AE 0.85 pA/pF), peak current density significantly increased (24.9 AE 1.5 pA/pF; P < 0.05; n = 6) after the addition of NS5806, but additional presence of NaHS (100 lM) returned the NS5806-stimulated currents to baseline levels (18.6 AE 0.6 pA/pF; P < 0.01; n = 6) (Fig. 2J). After exposure to NS5806 (10 lM), inactivation of I to was significantly subdued, as reflected by an expansion in time constant (s, from 8.6 AE 0.2 to 13.6 AE 0.7 ms at +30 mV, P < 0.05; n = 6). However, addition of 100 lM NaHS returned the time constant to 10.1 AE 0.9 ms at +30 mV in the presence of 10 lM NS5806 (Fig. 2K), confirming inhibition of H 2 S on I to current.
Besides BK Ca and I to currents, an inward rectifier current activated by hyperpolarization voltage steps on a holding potential of À40 mV that was sensitive to Ba 2+ (0.5 mM) was found, indicating the presence of IK ir inward current (28%; 28/309 cells) in the atrial fibroblasts (Fig. 3A). Exposure of atrial fibroblasts to 100 lM NaHS reduced the peak current density of IK ir (Fig. 3B). The inhibitory effects occurred within 1 min., reached saturation at 10 min. and could not be washed out (Fig. 3C). NaHS showed a voltage-dependent suppression of the IK ir current on the I-V curve from À4.4 AE 0.1 pA/pF to À3.0 AE 0.1 pA/pF at À110 mV (P < 0.05; n = 6) (Fig. 3D) and a dosedependent inhibition of peak current density with an IC 50 of 78.9 lM (Fig. 3E).
A minority of the atrial fibroblasts (1%; 1/54 cells) were found to exhibit inward currents with 50 ms voltage steps between À60 and +70 mV from À80 mV holding potential in 10 mV increments that resembled sodium current, indicating that K + currents represent the major ionic species in human atrial fibroblasts.
K ATP channel has been reported to affect cellular proliferation [19]. However, modulation of K ATP channel (30%; 22/73 cells) ( Fig. 5A and B) and Kir6.2 (responsible for K ATP ) gene expression (Fig. 5C) by H 2 S while confirming its role in enhancing current density, failed to show any appreciable effect on proliferation of our atrial fibroblasts. The K ATP currents were elicited from voltage-clamped at the holding potential of À40 mV, voltage ramps were applied every 9 sec. from À120 mV to +60 mV at 20 mV/sec. and subsequently ramps to À40 mV at À100 mV/sec. Consistently, activation of the K ATP channel by 30 lM pinacidil (specific channel enhancer) or its inhibition by 100 lM glibenclamide (specific channel inhibitor) did not significantly affect cellular proliferation despite the observed drastic modulation of current density ( Fig. 5D and E). Down-regulation of fibroblast growth was observed only in the additional presence of NaHS with glibenclamide (19.9 AE 2.9% reduction versus control; P < 0.01; n = 4) or NaHS with pinacidil (22.5 AE 4.2% reduction versus control; P < 0.05; n = 4), suggesting that H 2 S inhibition of proliferation was independent of its modulating role of K ATP channel in atrial fibroblasts. Gene expression showed that H 2 S reduced the mRNA level of KCa1.1 (responsible for BK Ca ), Kv4.3 (responsible for I to ), Kir2.1 (responsible for IK ir ) in TGF-b1-stimulated fibroblasts by 42.3 AE 5.1% (P < 0.05; n = 4), 76.9 AE 3.5% (P < 0.01; n = 4), 90.8 AE 4.7% (P < 0.01; n = 4), respectively, at 12 hrs after addition of NaHS (Fig. 6A-D). Furthermore, pre-treatment with NaHS decreased mRNA level of Kv4.3 by 21.6 AE 2.2% (n = 4; P < 0.05 versus basal levels), but not that of KCa1.1 and Kir2.1. Furthermore, NaHS enhanced production of endogenous H 2 S by enhancing cystathionine c-lyase (CSE) mRNA levels and maintaining its expression even in the presence of D,L-propargylgylcine (PPG), a potent inhibitor of CSE ( Fig. 6E and F). These results indicated that H 2 S inhibited fibroblast proliferation by regulating Kv4.3 mRNA expression and inhibiting I to current, possibly via an autocrine feedback mechanism.

Discussion
Multiple potassium channels are known to express in cardiac ventricular fibroblasts [15] and inhibition of BK Ca current resulted in suppression of fibroblast proliferation [18]. Transient outward K + current, I to , is present in neonatal rat cardiac fibroblasts (encoded by Kv1.4) [22] and human ventricular fibroblasts (encoded by Kv4.3) [15]. Similarly, Ba 2+ -sensitive inward rectifier K + current (encoded by Kir2.1/ Kir2.3) is present in human ventricular fibroblasts [15] and rat ventricular fibroblasts [23] whereby its modulation may have major significance in cardiac fibrosis. However, their roles in atrial fibroblasts which are more actively participating in cardiac fibrosis [24], are relatively not well understood.
We demonstrated that H 2 S dose-dependently inhibited BK Ca , I to and IK ir in human atrial fibroblasts within minutes, suggesting an acute modulation of H 2 S on such channels. The inhibitory effect of H 2 S on BK Ca , I to and IK ir was observed at 25-400 lM. The physiological levels of plasma H 2 S have been reported to be 50-160 lM in human brain [25] and 50-100 lM in human serum [26]. As NaHS dissolved in saline, one-third of the H 2 S exists as an undissociated gas, and the remaining two-third as the HS À anion [27]. Therefore, the physiologically relevant concentration of H 2 S (25-400 lM) used in this study, which effectively blocked BK Ca , I to and IK ir in vitro, is likely to be attainable in vivo.
We found that NaHS attenuated naringenin-induced BK Ca activation and decelerated the transition from closed to open state of the channel, suggesting a role for H 2 S in regulating BK Ca channel kinetic and voltage sensitivity. However, NaHS had no effect on the half-maximum voltage activation, but shifted the steady-state inactivation curve to the left, indicating that the voltage-dependent steady-state inactivation kinetics of I to channel were altered. Furthermore, NaHS markedly shifted the recovery curve of I to to the right, indicating that H 2 S attenuated the recovery of I to from inactivation. These results indicated that H 2 S inhibited I to through facilitation of steady-state inactivation and attenuation of recovery from inactivation. In contrast to reported presence of sodium current in ventricular fibroblasts (61%) [15], we found relatively few cells (1%) with detectable sodium current. This is consistent with previous reported presence of fast sodium current only in atrial myofibroblasts, but not in undifferentiated fibroblasts [28] like those used in our study.
BK Ca channels (encoded by KCa1.1) have been demonstrated to regulate proliferation of human cardiac ventricular fibroblasts [18] and endothelial cells [29]. Furthermore, inhibition of IK ir current suppressed proliferation of endothelial cells [30]. Similarly, inhibition of BK Ca (by paxilline), I to (by 4-AP) and IK ir (by Ba 2+ ) currents resulted in a significant reduction in fibroblast proliferation in our study. Consistently, suppression of the K + currents by NaHS inhibited atrial fibroblast proliferation in a dose-dependent manner. Furthermore, suppression of proliferation by NaHS in the presence of naringenin (channel opener of BK Ca ) or NS5806 (channel opener of I to ) suggested an additive inhibitory effect of H 2 S on BK Ca and I to channels in proliferation of atrial fibroblasts. Consistent with K ATP channel-activating effect of H 2 S [11,31], addition of NaHS recovered K ATP channel activity from glibenclamide inhibition. Nevertheless, suppression of cellular proliferation by NaHS in the presence of glibenclamide (specific inhibitor of K ATP ) or pinacidil (specific enhancer of K ATP ) indicated that K ATP channel was unlikely to be involved in proliferation of atrial fibroblasts. Consistently, H 2 S inhibition of lung fibroblast proliferation has been reported to be independent of K ATP channel [32].
Consistent with electrophysiological findings on the presence of BK Ca , I to and IK ir potassium currents, RT-PCR confirmed expression of KCa1 ated enhanced expression of Kv4.3 as well as KCa1.1 and Kir2.1. Effect of NaHS (exogenous donor of H 2 S) on expression of cystathionine clyase (CSE) that produces endogenous H 2 S is controversial, with reports of no effect in human airway smooth muscle cells [33] to inhibitory effect in mouse aortic smooth muscle cells [34]. However, in concordance with other reports [27,35], our results showed that NaHS enhanced CSE expression and further sustained its expression in the presence of DL-PPG [27] that strongly inhibited expression of CSE.
Myofibroblasts characterized by increased a-SMA expression are abundant in cardiac fibrosis [36] that has been associated with TGF-b-mediated [20] and Ang II-mediated [21] atrial fibrillation. Preventing myofibroblast differentiation from proliferating fibroblasts has been an attractive target in limiting cardiac fibrosis. Inhibition of TGF-b1 function by anti-TGF-b1 antibodies reduced myofibroblasts and lessened fibrosis [37]. Hydrogen sulphide was found to inhibit TGF-b-induced transformation of MRC5 lung fibroblasts to myofibroblasts [32]. Consistently, our results showed that NaHS effectively reduced proliferation of atrial fibroblasts in response to TGF-b1, Ang II or FBS. Furthermore, NaHS ameliorated transformation towards myofibroblasts whereby a-SMA expression and their stress fibres were significantly suppressed, although causal role of potassium channels in such transformation remained to be ascertained.
In summary, our study provides evidence of major K + channels in human atrial fibroblasts that share similar heterogenous expression as in human ventricular fibroblasts [15]. Hydrogen sulphide inhibits fibroblast proliferation probably through a combined modulation of BK Ca , I to , IK ir , but not K ATP , channels. Although roles of MAPK and ERK pathways in our atrial fibroblasts remain to be determined, they were implicated in H 2 S-mediated suppression of proliferation of vascular smooth muscle cells [38] and lung fibroblasts [32]. Both kinase pathways were linked to cell cycle progression in lung fibroblasts [39], which, in turn were reportedly regulated by Bk Ca in human ventricular fibroblasts [18]. However, K ATP was found to play no significant role in ERK-inhibiting effect of H 2 S [32], which may explain our observation in this study. Consistent with the observed beneficial effects of H 2 S on cardiac fibrosis in vivo [12,13], our results suggested that such effects may be partly mediated via selective inhibition of K + channels in atrial fibroblasts and suppression of their transformation to myofibroblasts. Such regulating role of H 2 S in atrial fibroblasts may have clinical value in targeting atrial fibrillation, which invariably linked to atrial fibrosis.

Conflicts of interest
The authors confirm that there are no conflicts of interest.

Supporting information
Additional Supporting Information may be found in the online version of this article: