The Effect And Ion Channel Mechanism Of Hydrogen Sulfide On Rat Heart Function

Hydrogen sulfide (H₂S) is considered as an endogenous gasotransmitter and plays important roles in physiological and pathophysiological processes in the cardiovascular system. It is reported that H₂S is an inhibitor of L-type calcium channel and may reduce isolated rat heart cardiac function in a dose-dependent manner. However, perfusion experiments did not clamp down on the heart rate (HR), while HR change affected the cardiac function in rats. We found in our previous preliminary experiments that H₂S, at a level below the physiological concentration improved the diastolic function of the isolated heart .Therefore, it is interesting to study the protective effect and mechanism of H₂S at sub-physiological concentrations on cardiac function.ObjectiveThe present study aimed to study the effects of H₂S on heart function and ion channel currents including ICa,L, INaCa and I_KATP in rat ventricular myocytes in an attempt to further clarify the ion channel mechanism of protective effects of H₂S on heart function in rats.MethodsIsolated rat hearts were connected via the aorta to a Langendorff apparatus and perfused with Krebs-Henseleit solution in a retrograde manner at a constant pressure of 80 cm H2O, and clamped at a rate of 300bpm. After stabilization for 15 min, KH solution with four different concentrations of NaHS (6.25,12.5,25, 50, 100 and 200μmol/L) was perfused for 15 min, followed by washing for 20 min. Left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), the maximum rate of rise and decrease of Left Ventricular pressure (±dp/dt) and coronary flow (CF) were measured continuously.Isolated rat ventricular myocytes were acquired by perfusing the isolated rat heart with collagen solution using a Langendorff apparatus. ICa, L, INaCa and I_KATP in the isolated rat ventricular myocyte membrane were recorded and analyzed by whole cell patch clamp technique before and after treatment with NaHS at different concentrations. Factors of ICa, L such as normalized activation conductance curve, the current-voltage relationship(I-V) of ICa, L, steady-state inactivation, inactivation time were also analyzed.Results1. Effects of H₂S on isolated perfused rat heart functionThere were no significant differences in CF, LVDP, LVEDP and±dp/dt between the groups during the stabilization period. Treatment with 25μmol/L NaHS for 15min decreased LVEDP significantly (P<0.01, n=8), while treatment with NaHS at 50μmol/L did not affect LVEDP significantly, and NaHS at 100, 200μmol/L enhanced LVEDP. We also observed that NaHS at 25μmol/L, which was lower than the physiological concentration, increased the CF, while NaHS at higher concentrations decreased CF in a time-dependent manner. These results suggested that NaHS at sub-physiological concentrations could relax the coronary artery when HR was fixed.The suppression percentage of LVDP was 10±6%, 19±2%, 51±5% and 76±6% at the concentration of 25, 50, 100 and 200μmol/L 15 mini after NaHS treatment, respectively, all of which were significantly higher than that of the control (3±4%). The suppression percentage of +dp/dtmax was 11±8%, 42±12% and 50±7%, all of which were significantly higher than that of the control (7±6%). The suppression percentage of–dp/dtmax was also significantly higher than that of the control. These results suggested that NaHS had the effect of suppressing heart contraction in a dose dependent manner. 2 Effects of H₂S on ion channel current in isolated rat ventricular myocytes2.1 Effects of H₂S on ICa,L in isolated rat ventricular myocytes NaHS dose-dependently inhibited ICa,L in rat ventricular myocytes. ICa,L deceased by 4±2% and 8±3% after NaHS perfusion at 6.25μmol/L for 1 min and 5 min, and by 8±3% and 14±5% after NaHS perfusion at 12.5μmol/L for 1 min and 5 min respectively, but no such change was observed in the control group. ICa,L in rat ventricular myocytes decreased 13±2%, 19±2%, 23±4% and 27±5% respectively after NaHS treatment at 25, 50, 100 and 200μmol/L for 1 min, versus 3±1% in the control (P<0.05, n=8). ICa,L decreased by 22±4%, 25±3%, 29±7% and 52±9% respectively after NaHS perfusion at 50, 100 and 200μmol/L for 5 min, versus 6±3% in the control (P<0.01, n=8). Treatment with 200μmol/L NaHS for 1 min shifted the I-V curve upward, but did not alter the I-V relationship and reversal potential of ICa,L. The value at V1/2 of the normalized activation conductance curves was -19.52±0.54 mV with a slope factor (k) of 4.85±0.52mV for the control, and -19.03±0.59 mV with a k value of 5.43±0.55 mV mV for 200μmol/L NaHS. In the absence of NaHS, V1/2 of the steady-state inactivation was -23.61±0.15mV with a k value of 5.32±0.13 mV mV. In the presence of NaHS, V1/2 of the steady-state inactivation was -24.57±0.15 mV with a k value of 5.44±0.13. These results suggested that NaHS did not alter the activation and inactivation gating property of L-type calcium channel.2.2 Effects of H₂S on INaCa in isolated rat ventricular myocytes NaHS enhanced inward and outward INaCa in rat ventricular myocytes. A 5±2% decrease inward INacCa and a 3±3% decrease outward INaCa were observed in the control group without NaHS treatment, while a 15±6%,20±5%,33±8%,16±4%,25±3%,29±7% ( P<0.05, n=10) increase inward INaCa at 6.25,12.5,25,50,100,200μmol/L, and a 2±1% (P >0.05, n=10), 6±3%,10±2%,11±2%,21±4%,25±3% (P <0.05, n=10) increase outward INaCa were observed in the NaHS perfusion groups.2.3 Effects of H₂S on I_KATP in isolated rat ventricular myocytes A ramp pulse protocol of 125 ms form 50 to -100 mV was used to record the quasi-steady-state current (I_KATP). Pinacidil produced a marked increase in the current, which was completely inhibited by glibenclamide, indicating that the outward current was I_KATP. I_KATP was not detectable in myocytes perfused with 50, 100 and 200μmol/L NaHS for 15 min, suggesting that I_KATP channels were not opened. In 400, 800 and 1600μmol/L NaHS treated groups, the I_KATP current concentration was 17.25±8.45 pA/pF, 20.33±8.56 pA/pF and 22.87±6.23 pA/pF, respectively. There were no significant differences between NaHS treated groups, or NaHS treated groups versus the control group, suggesting that NaHS at a comparable high concentration could open I_KATP channels in isolated rat ventricular myocytes.ConclusionsThe present study demonstrated that H₂S significantly suppressed heart contraction in the isolated perfused rat hearts and inhibited ICa ,L in a dose-dependent manner, enhanced INaCa and opened the ATP sensitive potassium channels. The data show that H₂S may produce an effect on heart function by inhibiting ICa ,L and enhancing INaCa and opening the ATP sensitive potassium channels. H₂S significantly lowered LVEDP, suggesting that H₂S improved the diastolic function by partially enhancing inward INaCa.