Capsaicin Inhibits Multiple Voltage-Gated Ion Channels in Rabbit Ventricular Cardiomyocytes in TRPV1-Independent Manner

Capsaicin is a naturally occurring alkaloid derived from chili pepper which is responsible for its hot, pungent taste. It exerts multiple pharmacological actions, including pain-relieving, anti-cancer, anti-inflammatory, anti-obesity, and antioxidant effects. Previous studies have shown that capsaicin significantly affects the contractility and automaticity of the heart and alters cardiovascular functions. In this study, the effects of capsaicin were investigated on voltage-gated ion currents in rabbit ventricular myocytes. Capsaicin inhibited rapidly activated (IKr) and slowly activated (IKs) K+ currents and transient outward (Ito) K+ current with IC50 values of 3.4 µM,14.7 µM, and 9.6 µM, respectively. In addition, capsaicin, at higher concentrations, suppressed voltage-gated Na+ and Ca2+ currents and inward rectifier IK1 current with IC50 values of 42.7 µM, 34.9 µM, and 38.8 µM, respectively. Capsaicin inhibitions of INa, IL-Ca, IKr, IKs, Ito, and IK1 were not reversed in the presence of capsazepine (3 µM), a TRPV1 antagonist. The inhibitory effects of capsaicin on these currents developed gradually, reaching steady-state levels within 3 to 6 min, and the recoveries were usually incomplete during washout. In concentration-inhibition curves, apparent Hill coefficients higher than unity suggested multiple interaction sites of capsaicin on these channels. Collectively, these findings indicate that capsaicin affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when capsaicin is administered to carriers of cardiac channelopathies or to individuals with arrhythmia-prone conditions, such as ischemic heart diseases.


Introduction
Capsaicin (8-methyl-N-vanillyl-6-nonenamide), as an algogen and prototypical activator of transient receptor potential vanilloid type 1 (TRPV1) channels, has been used extensively in pain research. In addition, capsaicin has been shown to play functional roles in a wide range of pathophysiological conditions, ranging from cardiovascular to respiratory and urinary diseases [1,2]. The effects of capsaicin on the cardiovascular system have been known for several decades. Activation of TRPV1 channels on capsaicin-sensitive sensory nerves in the cardiovascular system has been shown to release various neuropeptides and play important roles in physiological regulation, as well as the pathophysiology of cardiovascular diseases such as heart failure, myocardial infarction, and hypertension [3,4]. The vascular effects of capsaicin and the role of TRPV1 channels in the pathophysiology of hypertension is a complex process involving the TRPV1-dependent and independent action of capsaicin on the vascular endothelium, smooth muscle myocytes, and the peripheral and central nervous system, which have been reviewed in earlier studies [3,[5][6][7].
In earlier studies, capsaicin has been shown to exert transient positive inotropic and chronotropic effects [8][9][10], which are reversed by TRPV1 antagonists such as ruthenium-

The Effects of Capsaicin on Na + Channels (I Na )
Inward I Na was elicited by step depolarizations from a holding potential of −100 mV to −20 mV at 30 s intervals. The effects of 6 min capsaicin (30 µM) application and 8 min recovery on superimposed traces of I Na are shown in Figure 1A. The bath application of capsaicin caused a gradually progressing suppression of I Na , which reached steadystate levels within 4-5 min ( Figure 1B). The recovery was partial during the experiments lasting up to 15 to 18 min. The current-voltage (I-V) relationship for I Na is presented in Figure 1C. No significant changes in the I-V relationship were observed; the threshold, peak, and reversal potentials for I Na remained unaltered (n = 5). The extent of the capsaicin suppression of I Na was not altered by changes in the test potentials ( Figure 1D). The concentration-dependency of capsaicin inhibition is presented in Figure 1E. The IC 50 value and apparent Hill coefficient were 42.7 µM and 1.4, respectively (n = 4-7). The bath application of capsazepine (3 µM) for 10 min did not alter the peak I Na (n = 4). The extent of capsaicin inhibition of I Na was not altered by the co-application of 3 µM capsazepine, an antagonist of the TRPV1 receptor, and 30 µM capsaicin (n = 6-7, p > 0.05; t-test).
2.2. The Effects of Capsaicin on L-Type Ca 2+ Channels (I L-Ca ) I L-Ca was studied in the presence of extracellular TEA + and intracellular Cs + to suppress K + currents. In addition, the contaminating Na + current was eliminated by applying test potentials from a relatively depolarized potential of −50 mV to induce the inactivation of I Na [20]. The superimposed traces of the currents elicited by the test potentials from −50 mV to +20 mV in the control, after a 5 min application of 30 µM capsaicin and 7 min recovery, are shown in Figure 2A. The bath application of capsaicin caused a steadily progressing inhibition of I L-Ca , which was detectable within 30 sec and reached a steady-state level within 3-5 min, and the recovery was partial ( Figure 2B). The I-V relationships in the control and in the presence of 30 µM capsaicin are presented in Figure 2C (n = 5). The relationship between the test potentials and the extent of capsaicin inhibition of I L-Ca was presented in Figure 2D (n = 5). Capsaicin inhibited I L-Ca in a concentration-dependent manner with an IC 50 value of 34.9 µM and an apparent Hill coefficient of 1.3 (n = 3-6; Figure 2E). The application of capsazepine (3 µM) for 10 min did not affect the peak I Na (n = 4). The extent of capsaicin inhibition of I Na was not altered by the co-application of 3 µM capsazepine, an antagonist of the TRPV1 receptor, and 30 µM capsaicin (n = 5-7, p > 0.05; t-test). IL-Ca was studied in the presence of extracellular TEA + and intracellular Cs + to suppress K + currents. In addition, the contaminating Na + current was eliminated by applying test potentials from a relatively depolarized potential of −50 mV to induce the inactivation  Figure 2D (n = 5). Capsaicin inhibited IL-Ca in a concentration-dependent manner with an IC50 value of 34.9 µM and an apparent Hill coefficient of 1.3 (n = 3-6; Figure  2E). The application of capsazepine (3 µM) for 10 min did not affect the peak INa (n = 4). The extent of capsaicin inhibition of INa was not altered by the co-application of 3 µM capsazepine, an antagonist of the TRPV1 receptor, and 30 µM capsaicin (n = 5-7, p > 0.05; t-test).

The Effects of Capsaicin on the Delayed Rectifier K + Channels (I K )
The delayed rectifier K + current in mammalian ventricular myocytes consists of a rapidly activating but inwardly rectifying I Kr and a slowly activating I Ks [21,22].
Rapidly activating delayed rectifier (I Kr ) was quantified by applying a test pulse from −40 mV to +40 mV and measuring the deactivating tail currents in response to repolarizing test pulse of −40 mV. The difference between the peak of the tail current and the current at the beginning of test potential was taken as an estimate of I Kr . The contribution of I Ks was suppressed by 3 µM of selective I Ks blocker HMR 1556 to increase the proportion of I Kr . Traces of tail currents elicited by test potentials from +40 mV to −40 mV are presented for control and after 6 min application of 10 µM capsaicin and 7 min recovery in Figure 3A. The bath application of 10 µM capsaicin caused the inhibition of I Kr , which was evident at 30 sec and reached a steady-state level within 5 min, and recovery was almost complete within the time course of the experiment ( Figure 3B). The effects of 3 µM capsaicin on the I-V relationship of I Kr are presented in Figure 3C (n = 5). The extent of the capsaicin inhibition at test potentials ranging from −20 to 50 mV is shown in Figure 3D (n = 5).  Slowly activating delayed rectifier (IKs) was measured as the time-dependent current induced by 500 ms pulses from −50 to +60 mV. The superimposed current traces are shown for the control after an 8 min application of 10 µM capsaicin and a 5 min recovery in Figure  4A. The application of capsaicin caused a gradually developing inhibition of IKs, which reached a steady-state level within 6 min and recovered partially during the experiments ( Figure 4B). Figure 4C shows the effect of 10 µM capsaicin on the I-V relationship of IKs (n = 5). The extent of capsaicin inhibition at test potentials ranging from −40 to 60 mV is shown in Figure 4D (n = 5). The concentration-dependent inhibition of IKs is presented in The concentration-dependent effects of capsaicin are shown in Figure 3E. The IC 50 value and apparent Hill coefficient were 3.4 µM and 1.4, respectively (n = 4-8). The bath application of 3 µM capsazepine did not affect the peak amplitudes of I Kr tails (n = 4). The extent of the capsaicin inhibition of I Kr was not altered by the co-application of 3 µM capsazepine and capsaicin (3 µM) ( Figure 3F; n = 5-6, p > 0.05).
Slowly activating delayed rectifier (I Ks ) was measured as the time-dependent current induced by 500 ms pulses from −50 to +60 mV. The superimposed current traces are shown for the control after an 8 min application of 10 µM capsaicin and a 5 min recovery in Figure 4A. The application of capsaicin caused a gradually developing inhibition of I Ks , which reached a steady-state level within 6 min and recovered partially during the experiments ( Figure 4B). Figure 4C shows the effect of 10 µM capsaicin on the I-V relationship of I Ks (n = 5). The extent of capsaicin inhibition at test potentials ranging from −40 to 60 mV is shown in Figure 4D (n = 5). The concentration-dependent inhibition of I Ks is presented in Figure 4E. The IC 50 value and apparent Hill coefficient were 14.7 µM and 1.3, respectively (n = 4-6). The bath application of 3 µM capsazepine did not affect the peak amplitudes of I Ks (n = 4). The extent of capsaicin inhibition of I Ks was not altered by the co-application of 3 µM capsazepine and 3 µM capsaicin ( Figure 4F; n = 5-6, p > 0.05).

The Effect of Capsaicin on Transient Outward K + Current (Ito)
Ito was activated by 400 ms test pulses from −80 mV to +60 applied at 30 s intervals and defined as the difference between the initial transient peak of the current and the maintained current at the end of the pulse. The superimposed current traces in the absence and 6 min presence of 10 µM capsaicin and during an 8 min recovery are presented in

The Effect of Capsaicin on Transient Outward K + Current (I to )
I to was activated by 400 ms test pulses from −80 mV to +60 applied at 30 s intervals and defined as the difference between the initial transient peak of the current and the maintained current at the end of the pulse. The superimposed current traces in the absence and 6 min presence of 10 µM capsaicin and during an 8 min recovery are presented in Figure 5A. The time course of the effect of bath application of capsaicin on the peak amplitudes of I to is shown in Figure 5B. The I-V relationships of I to in the absence and presence of 10 µM capsaicin are shown in Figure 5C (n = 7). The relationship between the test potentials and the extent of the capsaicin inhibition of I to was presented in Figure 5D (n = 7). Figure 5E shows the concentration-inhibition curve of capsaicin on I to (n = 4-7 cells). The IC 50 and apparent Hill coefficient were 9.6 µM and 1.4, respectively. Capsazepine (3 µM) alone did not affect the peak amplitudes of I to (n = 4). The extent of capsaicin inhibition of I to was not altered by the co-application of 3 µM capsazepine and 10 µM capsaicin ( Figure 5F; p > 0.05 t-test). IK1 was activated from the holding potential of −80 mV to the test potential of −120 mV. The amplitude of IK1 was determined by measuring the peak current relative to zero current. The traces of the currents in the control, in the presence of 30 µM capsaicin, and  (I K1 ) I K1 was activated from the holding potential of −80 mV to the test potential of −120 mV. The amplitude of I K1 was determined by measuring the peak current relative to zero current. The traces of the currents in the control, in the presence of 30 µM capsaicin, and during recovery are shown in Figure 6A. Figure 6B shows the time course of the capsaicin effect and washout on I K1 . The I-V relationships of I K1 in the absence and presence of 30 µM capsaicin are presented in Figure 6C (n = 5). The relationship between the test potentials and the extent of the capsaicin inhibition of I K1 is presented in Figure 6D (n = 5). Figure 6E demonstrates the concentration-inhibition curve of capsaicin on I K1 (n = 4-6 cells). The IC 50 and apparent Hill coefficient were 38.8 µM and 1.4, respectively. Capsazepine (3 µM) alone did not affect the peak amplitudes of I K1 . The extent of capsaicin inhibition of I K1 was not altered by the co-application of 3 µM capsazepine and 10 µM capsaicin ( Figure 6F; p > 0.05; t-test).

Discussion
The results have demonstrated that capsaicin at concentrations higher than those required for the activation of TRPV1 channels directly inhibits the functions of multiple ion channels in rabbit ventricular myocytes. Capsaicin appears to effectively inhibit delayed rectifier K + currents (IKr and IKs) and Ito, with IC50 values of 3.4 µM,14.7 µM, and 9.6 µM,

Discussion
The results have demonstrated that capsaicin at concentrations higher than those required for the activation of TRPV1 channels directly inhibits the functions of multiple ion channels in rabbit ventricular myocytes. Capsaicin appears to effectively inhibit delayed rectifier K + currents (I Kr and I Ks ) and I to , with IC 50 values of 3.4 µM,14.7 µM, and 9.6 µM, respectively. On the other hand, I Na , I L-Ca , and I K1 seem to be significantly less sensitive to capsaicin, with respective IC 50 values of 42.7 µM, 34.9 µM, and 38.8 µM.
Although this is the first report on the capsaicin inhibition of I Kr and I Ks , in earlier studies, capsaicin was reported to inhibit I to , and the I K1 , with IC 50 values of 6.4 µM and 46.9 µM, respectively, in rat ventricular myocytes [23]. Similarly, the inhibition of I to by capsaicin (IC 50 = 5 µM) was reported in rat atrial myocytes [24]. In addition, capsaicin suppresses I HERG , which corresponds to a rapid component of the delayed rectifier K + channel, expressed in Xenopus oocytes with an IC 50 of 17.5 µM [25]. These results are in agreement with our findings on K + currents in rabbit ventricular myocytes.
Capsaicin, at significantly higher concentrations, suppressed I Na , I L-Ca , I K1 with IC 50 values of 42.7 µM, 34.9 µM, and 38.8 µM, respectively. In an earlier study, capsaicin, at lower concentrations (0.4-4 µM), was reported to significantly suppress I Na in rat atrial myocytes [26]. Similarly, capsaicin, at a significantly lower concentration of 0.33 µM, inhibited Vmax during phase 0 of action potential in guinea-pig papillary muscle [10]. However, other studies reported that capsaicin suppresses Vmax of action potentials in the concentration range of 30-120 µM in guinea-pig papillary muscle [27]. In a recent study, in agreement with our findings (IC 50 = 42.7 µM), capsaicin inhibited cardiac Na + channels (Na V 1.5) with an IC 50 of 60.2 µM in HEK-293 cells [28]. Although the present study is the first to report the inhibition (IC 50 = 34.9 µM) of I L-Ca by capsaicin in cardiomyocytes, in agreement with our findings, capsaicin, at similar concentrations (10-30 µM) suppressed Vmax of Ca 2+ -dependent action potentials in rabbit atrioventricular node [29] and sinoatrial cells [30].
The simulation with LabHEART, a computer model of rabbit ventricular action potentials [31], revealed that the suppression of I Ks , I Kr , and I to at a concentration of 10 µM, capsaicin, as expected, causes a marked prolongation (39%) of action potential duration at 50% repolarization (APD 50 ), whereas the addition of the inhibition of I Na , I L-Ca , and I K1 at higher capsaicin concentration (30 µM) results in a 27% shortening of APD 50 ( Figure 7A). In earlier studies, capsaicin at concentrations higher than 1 µM has been reported to prolong APD in rat ventricular [23,24] and guinea-pig atrial [19] myocytes. On the other hand, capsaicin, at concentrations of 10 µM and higher, has been shown to decrease APD in guinea-pig papillary muscles [10,27,32] and canine cardiomyocytes for zucapsaicin [33]. Both the increase and decrease in APD can potentially have arrhythmogenic effects depending mainly on the underlying pathophysiological mechanisms, such as ischemic heart diseases. Our LabHEART simulation of rabbit ventricular myocytes also indicates that the high concentration of capsaicin (30 µM) can markedly depress intracellular Ca 2+ transients ( Figure 7B) and tension development ( Figure 7C). TRPV1 channels are not expressed in adult rat [34] and mouse [35,36] cardiomyocytes but may play roles at earlier developmental stages of myocytes [36,37]. In line with these findings, the effects of capsaicin on ion channels tested in this study (I Kr , I Ks , I to ) were not reversed by capsazepine, a TRPV1 antagonist. In addition, the bath application of capsaicin (1-30 µM), even at concentrations 10-100 times higher than those required to activate TRPVI channels, did not change the holding currents under patch-clamp conditions to record I Na and I L-Ca (n = 14, data not shown), suggesting that TRPV1 is not involved in the observed effects of capsaicin. Cannabinoid receptors (CB1 and CB2) have been shown to be expressed in rat hearts [38], and capsaicin at concentrations higher than 10 µM was reported to bind to CB1 and CB2 receptors [39]. However, the co-application of specific CB1 antagonist SR141716A (2 µM) or specific CB2 antagonist SR 144528 (2 µM) and capsaicin did not alter the extent of the capsaicin inhibition of I Na and I L-Ca (Paired t-test; p > 0.05; n = 4-5), suggesting that CB1 and CB2 receptors are not involved in the capsaicin inhibition of these ion channels. The effect of capsaicin on the ion channels tested in this study reached a maximal level within several minutes (3-6 min) of application. Similarly, the actions of several lipophilic modulators, including capsaicin [42,45,46], endocannabinoids [47][48][49], and general anesthetics [50,51] and steroids [52] on various ion channels require 5-15 min to reach their maxima with, in many cases, Hill coefficients of higher than unity in their concentration-effect curves, suggesting that the multiple binding sites for these allosteric modifiers are located inside the lipid membrane and require a relatively slow (in minutes) time course to reach equilibrium to modulate the functions of these channels. Thus, the accumulation of capsaicin in the lipid membrane appears to be a function of both the exposure It is likely that capsaicin, a highly lipophilic agent with a LogP (octanol-water partition coefficient) value of 3.8, permeates the lipid membrane and then diffuses into the lipid membrane, and then alters the functional properties of the ion channels. Molecular dynamics simulations suggested that capsaicin is localized to the bilayer/solution interface [40]. Capsaicin was reported to regulate the functions of voltage-gated Na + and K + channels, as well as antibiotic-induced ion channels [41], by altering the biophysical properties of the lipid membranes, such as the bilayer elasticity [40,42], dipole potential [43], and lipid disordering [44].
The effect of capsaicin on the ion channels tested in this study reached a maximal level within several minutes (3-6 min) of application. Similarly, the actions of several lipophilic modulators, including capsaicin [42,45,46], endocannabinoids [47][48][49], and general anesthetics [50,51] and steroids [52] on various ion channels require 5-15 min to reach their maxima with, in many cases, Hill coefficients of higher than unity in their concentrationeffect curves, suggesting that the multiple binding sites for these allosteric modifiers are located inside the lipid membrane and require a relatively slow (in minutes) time course to reach equilibrium to modulate the functions of these channels. Thus, the accumulation of capsaicin in the lipid membrane appears to be a function of both the exposure time and concentration of capsaicin. Thus, while low capsaicin concentrations require longer exposure times, higher concentrations reach a threshold concentration at faster time scales to affect channel functions. In line with this assumption, the application of low concentrations (0.1-0.3 µM) for relatively long exposures (10-20 min) causes gradually developing suppression of contractility in guinea-pig papillary [10] and ventricular [19] muscle and whole heart [11] preparations. In another in vitro study, the 30 min administration of capsaicin, at concentrations as low as 1 nM, significantly inhibited the contractions of rat ventricular papillary muscle in a reversible manner [17]. In agreement with these findings, we have found that time to reach steady-state inhibition by capsaicin was inversely correlated with capsaicin concentrations applied to the ion currents investigated in this study (Figure 8). In addition to partitioning into the lipid bilayer and subsequently altering the biophysical characteristics of the plasma membrane, capsaicin can bind directly to ion channel domains embedded in cell membranes and affect the energy requirements for the gating-related conformational changes in ion channels [49].
LabHEART simulations. (A) At 10 µM capsaicin concentrations, the inhibition of IKr, IKs, Ito was incorporated. Whereas, at 30 µM capsaicin concentrations inhibitions of IKr, IKs, Ito, IK1, INa, and ICa-L were incorporated into the model. At 10 µM concentration, capsaicin induced marked prolongation (39%) of APD50, while at higher concentration (30 µM) significantly shortened (27%) the APD50. (B) Capsaicin, at the concentration of 10 µM, caused a slight delay in decay phase of Ca 2+ transient. At higher concentration of 30 µM, capsaicin induced a marked suppression of Ca 2+ transient. (C) Capsaicin, at 10 µM concentrations, caused a slight delay in decay phase of force development, but at higher concentrations of 30 µM, capsaicin induced a marked inhibition of force development during a simulated twitch response of a rabbit ventricular cardiomyocyte.
The effect of capsaicin on the ion channels tested in this study reached a maximal level within several minutes (3-6 min) of application. Similarly, the actions of several lipophilic modulators, including capsaicin [42,45,46], endocannabinoids [47][48][49], and general anesthetics [50,51] and steroids [52] on various ion channels require 5-15 min to reach their maxima with, in many cases, Hill coefficients of higher than unity in their concentration-effect curves, suggesting that the multiple binding sites for these allosteric modifiers are located inside the lipid membrane and require a relatively slow (in minutes) time course to reach equilibrium to modulate the functions of these channels. Thus, the accumulation of capsaicin in the lipid membrane appears to be a function of both the exposure time and concentration of capsaicin. Thus, while low capsaicin concentrations require longer exposure times, higher concentrations reach a threshold concentration at faster time scales to affect channel functions. In line with this assumption, the application of low concentrations (0.1-0.3 µM) for relatively long exposures (10-20 min) causes gradually developing suppression of contractility in guinea-pig papillary [10] and ventricular [19] muscle and whole heart [11] preparations. In another in vitro study, the 30 min administration of capsaicin, at concentrations as low as 1 nM, significantly inhibited the contractions of rat ventricular papillary muscle in a reversible manner [17]. In agreement with these findings, we have found that time to reach steady-state inhibition by capsaicin was inversely correlated with capsaicin concentrations applied to the ion currents investigated in this study (Figure 8). In addition to partitioning into the lipid bilayer and subsequently altering the biophysical characteristics of the plasma membrane, capsaicin can bind directly to ion channel domains embedded in cell membranes and affect the energy requirements for the gating-related conformational changes in ion channels [49]. In recent years, capsaicin has been administered as a dietary supplement for weight management and appetite suppression. Pharmacokinetic studies indicate that about 80% of ingested capsaicin is absorbed through the gastrointestinal system, leading to peak concentrations at sub-µM levels with a half-time of 30-60 min [1,53]. Thus, the regular intake of low-dosage capsaicin supplements (1-30 mg/day) would not reach blood levels that would cause the significant inhibition of K + channels investigated in this study. However, as mentioned earlier, capsaicin is a highly lipophilic compound (LogP = 3.8), and its concentration in the cell membrane is expected to be considerably higher than blood levels. In animal studies, after intravenous or subcutaneous administrations, the capsaicin concentrations in the brain and spinal cord were found to be five-fold higher than that in the blood [54,55]. Thus, it is possible that chronic and frequent intake of high-dosage ( >100 mg/day) capsaicin may lead to significant capsaicin partitioning into lipid membranes and trigger cardiac arrhythmias, especially in patients with comorbid ischemic heart diseases.

Materials and Methods
Male New Zealand white rabbits (ca. 2-2.5 kg) were anesthetized by sodium pentobarbitone (40 mg/kg) injection into a marginal ear vein, and following anesthesia, the animal was killed by the removal of the heart. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Care Committee of Bogomoletz Institute of Physiology of the National Academy of Science of Ukraine (approval code: 345/5.7WK). Ventricular myocytes were isolated according to minor modifications of the method described earlier [56,57]. Briefly, the hearts were retrogradely mounted and perfused according to the Langendorff method at a constant flow of 10 mL g heart −1 min −1 and at 37 • C with a cell isolation solution containing (mM): 130 NaCl, 5.4 KCl, 1.4 MgCl 2 , 0.75 CaCl 2 , 0.4 NaH 2 PO 4 , 5 HEPES, 10 glucose, 20 taurine, and 10 creatine set to pH 7.3 with NaOH. After the stabilization of the heart contractions, perfusion was continued for 4 min with Ca 2+ -free isolation solution containing 0.1 mM EGTA, and then for 6 min with cell isolation solution containing 0.05 mM Ca 2+ -, 0.8 mg/mL collagenase (type 1; Worthington Biochemical Corp, USA) and 0.075 mg/mL protease (type X1 V; Sigma, Taufkirchen, Germany). Following enzyme treatment, the heart was removed from the Langendorff perfusion system, and the ventricles were excised, minced, and gently shaken in a collagenase-containing isolation solution supplemented with 1 % BSA. The cells were filtered from this solution at 4 min intervals and resuspended in an isolation solution containing 0.75 mM Ca 2+ . The shaking and filtration process was repeated up to five times.

Whole Cell Patch-Clamp Technique
Myocytes were dispersed and allowed to settle for at least one hour at room temperature (22-24 • C) prior to their use. The measurements were performed only in quiescent myocytes displaying normal morphology and clear striated appearance. The whole-cell patch-clamp technique was used to evaluate individual ionic currents using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) linked to an A/D interface (Digidata 1322; Molecular Devices). The analog signal was filtered using a four-pole Bessel filter with a bandwidth of 5 kHz and digitized at a sampling rate of 10 kHz under software control (PClamp 10.6.2.2, Molecular Devices, Sunnyvale, CA, USA). Heat-polished borosilicate glass pipettes (World Precision Instruments, Sarasota, FL, USA) with a tip resistance of 1 to 2 MΩ were used to establish GΩ seals and continuity with the intracellular medium. The cell capacitance (C m ) was calculated by integrating the area under an uncompensated capacity transient elicited by a 10-mV depolarizing pulse from a holding potential of −80 mV. The total series resistance (R s ) between the pipette interior and the cell membrane in the whole-cell configuration was calculated from the estimates of C m (172 ± 14 pF, n = 247 from 36 rabbits) and the time constant (τ c ) of the capacitative current decay from the equation τ c = R s × C m . The mean R s for the pathway between the pipette and the cell membrane after the rupture of the membrane seal were calculated to be 3.87 ± 0.32 MΩ (n = 247). After the establishment of the whole-cell configuration and the measurement of C m , the R s were compensated to > 60%. The junction potentials under our conditions were approximately −3 mV and were not corrected.
The experiments were performed at room temperature (22-24 • C). The changes of the external solutions and the application of drugs were performed using a multi-line perfusion system with a common outflow connected to the recording chamber. A perfusion rate of 2 mL/min was used routinely in a recording chamber with a volume of 200 µL.
Capsaicin was from Sigma (St. Louis, MO, USA). It was dissolved in 100 % DMSO, and final concentrations were diluted from stock solutions. The stocks were kept at −20 • C until their use. The highest final concentration of DMSO in the extracellular solutions (0.03% v/v) did not affect the amplitudes of the membrane currents investigated in this study. Capsazepine (dissolved in DMSO), other reagents, and chemicals used in our experiments were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Statistical Analysis
All of the cumulative results are expressed as mean ± SE or SEM as indicated. Statistical significance among groups was determined using t-test, ANOVA, or pairwise comparisons (Mann-Whitney U-Test) followed by Bonferroni Post-hoc analysis. Statistical analysis of the data was performed using Origin 7.0 software (OriginLab Corp., Northampton, MA, USA). p < 0.05 was considered statistically significant. The concentrationresponse parameters were obtained by fitting the data to the logistic equation in Origin 7.0 software, y = E max /(1 + [x/EC 50 ] n ), where x and y are the concentration and response, respectively, E max is the maximal response, EC 50 is the half-maximal concentration, and n is the slope factor (apparent Hill coefficient).