| The renin-angiotensin system (RAS) plays a pivotal role in maintaining cardiovascular homeostasis but may contribute to cardiac arrhythmias in various cardiovascular diseases. Large scale clinical trials have provided evidence that inhibition of angiotensinⅡ(AngⅡ) synthesis by angiotensin- converting enzyme inhibitors or direct blockade of angiotensin receptor type 1 (AT1) with the antagonist losartan results in a significant decrease in the risk of arrhythmias associated with heart failure. However, little is known about the mechanism underlying the arrythmogenisis of RAS. It has been demonstrated that cardiac-specific over-express of AngⅡgene or angiotensinⅡtype 1 receptor (AT1R) in mouse heart tissue can prolong action potential and increase the incidence of spontaneous ventricular arrhythmias. Particularly, the young transgenic animals exist delayed repolarization and a high incidence of arrhythmias without cardiac hypertrophic remodeling. The results indicate that AngⅡmay exert a direct modulation on cardiac ionic channels. However, it is limited about the knowledge of electrophysiological responses to the stimulation on AT1 receptor. Thus, our long-term goal is to understand the direct modulations of AngⅡon cardiac ion channels. We have recently demonstrated that AngⅡinhibits rapidly activated delayed rectifier K+ currents (Ikr) in guinea-pig ventricular myocytes.The sinoatrial node (SAN) is responsible for initiating the heart rhythm and plays a pivotal role for cardiac physiological function. Evidence has shown that patients with chronic heart failure have significant sinus node remodeling characterized by a reduction in functional sinus node reserve. Although most sudden deaths in chronic heart failure can be attributed to ventricular arrhythmias, bradyarrhythmias are also an important cause and account for up to 42% of sudden deaths in hospital. Thus, it is of vital importance to understand the mechanism underlying the spontaneous heart beat. It is generally thought that cardiac pacemaker initiation result from interplay of various voltage-sensitive ionic membrane currents including a hyperpolariztion-activated pacemaker currents, If, which corresponds to the range where diastolic depolarization occurs. Because the open and close of membrane ionic channels dependent on the membrane potential, this process is referred to as"membrane clock". Recently, the spontaneous sarcoplasmic reticulum (SR) Ca2+ release has been suggested as an additional mechanism of action potential generation in SNA, also known as"Ca2+ clock". The Ca2+ clock is manifested by local Ca2+ release from SR that appear shortly before firing of next AP. These Ca2+ releases activate Na+-Ca2+ exchanger (NCX) and prompt the membrane clocks to generate rhythmic APs. Up to now, the knowledge about modulation of AngⅡon spontaneous firing rate of SNA is limited and controversial. In addition, human Kv1.5 gene encodes ultra-rapid delayed rectifier K+ channel, which carries the current IKur exclusively present in atrial cells. Little is known about the modulation of AngⅡon the channel.The present study was designed to examine the possible acute regulation of spontaneous firing rate in isolated guinea pig sinoatrial node cells and hKv1.5 channel in heterologous expression system by AngⅡusing the whole-cell patch-clamp technique.Part 1 AngⅡdecreases the spontaneous firing rate in guinea-pig sino- atrial node cellsAim: To assess the effect of AngⅡon spontaneous firing rate and ionic currents in guinea pig sinoatrial node cells.Methods: Single sinoatrial node cells were enzymatically dissociated from the heart of adult guinea pigs. For action potential recordings in single SA node cells, the patch pipettes were backfilled with amphotericin (240μg/ml). Pipette solution contained (in mM) KCl 140, MgCl2 5, K2ATP 1, and HEPES 3 (pH 7.2 with KOH). The external solution contained (in mM) NaCl 138, KCl 4, MgCl2 1, CaCl2 2, NaH2PO4 0.33, glucose 10, and HEPES 10 (pH 7.4 with NaOH).For If recordings, the external solution contained (in mM) NaCl 132, KCl 4, MgCl2 1.2, CaCl2 1.8, glucose 5, and HEPES, 10 (pH 7.4 with NaOH). The pipette solution contained (in mM) KCl 150, K2ATP 1, MgCl2 5, and HEPES 3 (pH 7.2 with KOH). Amphotericin B (Sigma-Aldrich) was used at 240μg/ ml. Single SA node cells were hyperpolarized from a holding potential of -40 mV to test potentials of -50 mV to -110 mV for 3000 ms to elicit currents, which were completely blocked by CsCl (2 mM), supporting a nature of If currentFor Iks recordings, the external solution contained (in mM) NaCl, 132; KCl, 4; CaCl2, 1.8; MgCl2, 1.2; glucose, 5 and HEPES 10 (pH 7.4 with NaOH). Nimodipine (1μM) was added to the external solution to block the L-type Ca2+ current. Ikr was blocked by the addition of 2μM N-[4-[[1-[2-(6- Methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide dihydrochloride (E-4031). The pipette solution contained (in mM) potassium asparate 70, KCl 50, KH2PO4 10, Na2ATP 3, LiGTP 0.1, EGTA, 5 and HEPES 5 (pH 7.2 with KOH). The Iks current was elicited from a holding potential of -50 mV to prepulse potentials of -40 mV to 50 mV for 2 s duration and was followed by a test pulse to the holding potential to evoke outward tail currents.For ICaL recordings, the external solution contained (in mM) NaCl 135, CsCl 10, MgCl2 1, CaCl2 1.8, glucose, 10, HEPES 5 and tetrodotoxin 0.003 (pH 7.4 with NaOH). The pipette solution contained (in mM) aspartic acid 90, NaCl 10, CsOH 100, CsCl 30, MgCl2 2, EGTA 5, CaCl2 2, HEPES 10, ATPNa2 2, and GTPNa2 0.1 (pH 7.2 with CsOH). The ICaL was recorded by depolarizing SA node cell from a holding potential of -50 mV to test potentials of -40 mV to 50 mV for 300 ms.Experiments were performed at 36±1℃using an Axopatch 200B amplifier (Axon Instrument, Foster City, CA, USA). The patchclamped cell was superfused by means of a temperature controlled micro-superfusor (TC-324B, Warner instrument, USA). The electrical signals were sampled at 2.5-10 kHz and filtered at 1 KHz using a low-pass filter, and digitized with an A/D converter. A pClamp software (Version 8.1) was used to generate voltage-pulse protocols, acquire and analyze data.Results: The inhibition on the spontaneous activity of AngⅡshowed a concentration-dependent manner in the range of 11000 nM, with IC50 of 8.34 nM. The spontaneous firing rate and DD slope were reduced from the control values of 171±5 beats /min and 150±5 mV/s to 113±8 beat/min(P<0.05)and 70±6 mV/s (P<0.01), respectively. AngⅡshowed a negtive effect on the spontaneous activity of SA node cells. The reduction of spontaneous beating rate occurred within 3 min and reached saturation about 10 min after washing AngⅡinto the bath. AngⅡproduced an inhibitory effect on the firing rate of SA node cells via AT1 receptor. AngⅡhad no significant effect on the current amplitude. AngⅡincreased both depolarizing currents at prepulse potentials and the tail current. The tail current density of Iks was increased from 7.0±0.5 pApF-1 to 9.0±0.5 pA/pF at prepulse potential of 50 mV (n=6, P<0.01). The ICaL was signifcantly inhibited on the exposure to AngⅡ. The peak current at 10 mV was decreased from 11.3±1.6 to 7.5±1.0 pA/pF (n=6, P<0.05).Conclusion: In summary, the present study demonstrated that AngⅡslowed the spontaneous firing rate of action potentials via increasing Iks and reducing ICaL in guinea-pig SA node cells.Part 2 Potential Ca2+ clock mechanism involving in the inhibition of AngⅡon spontaneous firing rateAim: To further examine whether the Ca2+ clock mechanism involving in the inhibitory action of AngⅡon spontaneous firing rate.Methods: The change of intracellular Ca2+ was monitored by confocal microscope. The sinoatrial node cells were loaded with 1μmol/L fluo-3 AM (Molecular Probes) for 30 min in the dark, and then re-suspended in fluofree Tyrode solution. Fluo-3 was excited at 488 nm. The action potential was recorded in guinea-pig sinoatrial node cells by using the same patch-clamp technique as it mentioned in the first part.Results: The positive control isoprenaline (ISO, 1μM) increased the intracellular Ca2+ for 51% (fluorescence intensity control 102.2±4.71 vs. ISO 154.5±4.13, P<0.01, n=5). AngⅡ(100 nM) significantly enhanced the SR Ca2+ release and increase the intracellular Ca2+ for 37% (fluorescence intensity in the control 102.2±4.71 vs. 154.5±4.13 after AngⅡ, P<0.01, n=5). The patch-membrane was ruptured by applying strong suction to the pipette containing 20 mM BAPTA (selective chelator of intracellular Ca2+ stores). As the cytosol was dialyzed with BAPTA, cell spontaneous activity disappeared within 60 s, reflecting chelation of bulk [Ca2+]i. 1 mM BAPTA showed a negative effect on the spontaneous activity, but didn't abolish it. The reduction of spontaneous beating rate reached saturation around 4 min. The spontaneous firing rate was reduced from the control values of 176±10 beat/min to 86.2±5 beat/min (P<0.01, n=6). AngⅡproduced a further inhibitory effect on pacemaker cells rhythm from 86.2±5 beat/min to 38.72±4 beat/min (P<0.01, n=6). SR Ca2+ release inhibitor, Ryanodine (2μM) significantly deceased the pacemaker beating rate from 182±12 beat/min to 91±6 beat/min and AngⅡdidn't show a further inhibition on spontaneous firing rate. However, at a lower concentration, ryanodine (100 nM) decreased the spontaneous firing rate from 122.4±7 beat/min to 100.8±6 beat/min and AngⅡfurther decreased spontaneous firing rate (P<0.01, n=6). In presence of nonspecific PLC inhibitor, U-73122, AngⅡproduced little effect on pacemaker cells rhythm.Conclusion: There is a interplay between membrane clock and Ca2+ clock mediating the inhibitory effect of AngⅡon pacemaker cells. AngⅡmay increase the beating rate through Ca2+ clock mechanism by enhancing the intracellular Ca2+ concentration and contrarily decrease the spontaneous rhythm through membrane clock. Obviously, the regulation on the membrane clock is predominant mechanism responsible for the inhibition of AngⅡon spontaneous rhythm, which results from the activation of PLC.Part 3 Effects of AngⅡon Kv1.5 current in heterologous expression systemAim: To further assess the effects of AngⅡon hKv1.5 channel in heterologous expression system using the whole-cell patch-clamp technique.Methods: Perforated patch technique was used to record in HEK293. For hKv1.5 recordings, the external solution contained(in mM) NaCl 140, KCl 5.4, MgCl2 1, CaCl2 2, glucose 10,HEPES 10 (pH 7.4 with NaOH). The pipette solution contained (in mM) KCl 140, Mg-ATP 4, MgCl2 1, EGTA 5,HEPES 10 (pH 7.2 with CsOH). The hKv1.5 was recorded by depolarizing SA node cell from a holding potential of -70 mV to test potentials of -60 mV to 40 mV for 100 ms to evoke outward tail currents . The cells were held at -100 mV, depolarized briefly at +40 mV, hyperpolarized for 2 s at -100 mV, then depolarized again for 5 s between -70 and +30 mV (10 mV steps) and finally the current was measured at +40 mV were the cell was held for 1 s. The data points were well fitted by a Boltzmann equation and constructed a inactivation curve.Results: The inhibition on hKv1.5 of AngⅡshowed a concentration -dependent manner in the range of 11000 nM, with IC50 of 10nM. The reduction of hKv1.5 occurred within 3 min and reached saturation about 20 min after washing AngⅡ(100 nM) into the bath. In the range of -4080 mV, AngⅡ(100 nM) showed a significant inhibition on Kv1.5 current. The tail current density of hKv1.5 was decreased from 18.22±0.4 pApF-1 to 3.06±0.5pA/pF at prepulse potential of +80 mV (n=6, P<0.01). Tail current amplitude, normalized to the maximum, was used to construct the activation curve. The activation curve showed that AngⅡdidn't influence the voltage-dependent activation of the channel. Both fast (τf) and slow (τs) components of deactivation were significantly shorten by AngⅡ(n=6, P<0.05). AngⅡdidn't influence the steady-state inactivation of the channel. In presence of nonspecific PKC activators, staurosporin(Stau), AngⅡproduced little further effect on pacemaker rhythm.Conclusion: AngⅡmay inhibit Kv1.5 current by speeding the deactivation of the channel through PKC pathway.Summary: The main findings of our study include:1 AngⅡslowed the spontaneous firing rate of action potentials via increasing Iks and reducing ICaL in guinea-pig SA node cells.2 Both membrane clock and Ca2+ clock are involve in the effect of AngⅡon pacemaker cells. The regulation on the membrane clock is predominant mechanism responsible for the inhibition of AngⅡon spontaneous rhythm, which results from the activation of PLC. 3 AngⅡmay inhibit Kv1.5 current by speeding the deactivation of the channel through PKC pathway.Our study demonstrates that AngⅡcan produce acute modulation on spontaneous firing rate in isolated guinea pig sinoatrial node cells and hKv1.5 channel. This is a potential mechanism by which elevated levels of AngⅡare involved in the occurrence of arrhythmias in pathological heart.
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