Yakhin Shimoni

An obligatory role for nitric oxide in autonomic control of mammalian heart rate.

Cholinergic modulation of heart rate in isolated spontaneously beating single cells from the rabbit sino‐atrial node was investigated by measuring transmembrane ionic currents using the nystatin‐perforated patch whole‐cell voltage‐clamp technique. Carbamylcholine (CCh), a stable analogue of acetylcholine (ACh), significantly inhibited L‐type calcium currents (Ica(L) which had been augmented by beta‐adrenergic stimulation. In addition, CCh activated a potassium outward current (IK(ACh)). Both effects were blocked by atropine. The possible involvement of nitric oxide (NO) in these responses was evaluated by inhibiting NO synthesis. In the presence of NG‐monomethyl‐L‐arginine (L‐NMMA, 100 microM) or nitro‐L‐arginine methyl ester (L‐NAME, 1 mM), two specific inhibitors of nitric oxide synthase (NOS), CCh no longer inhibited ICa(L). IK(ACh) could still be activated. Co‐incubation of cells in L‐NAME or in L‐NMMA with arginine (the endogenous substrate of NOS) restored the CCh‐induced attenuation of ICa(L), indicating that L‐NAME or L‐NMMA did not interfere directly with the muscarinic action of CCh on ICa(L). Effects of the NO‐releasing agent molsidomine (SIN‐1) on CCh‐induced changes in ICa(L) were also investigated. After ICa(L) had been augmented by beta‐adrenergic stimulation, SIN‐1 (0.1 mM) inhibited ICa(L); however, SIN‐1 had no further inhibitory effect after a maximal CCh concentration had been applied. These findings suggest that NO generation is an obligatory process in cholinergic inhibition of ICa(L) in mammalian cardiac pacemaker tissue.

Shal‐type channels contribute to the Ca2+‐independent transient outward K+ current in rat ventricle.

1. The hypothesis that Kv4.2 and Kv4.3 are two of the essential K+ channel isoforms underlying the Ca2+‐independent transient outward K+ current (It) in rat ventricle has been tested using a combination of electrophysiological measurements and antisense technology in both native myocytes and a stably transfected mammalian cell line, mouse Ltk‐ cells (L‐cells). 2. The transient outward currents generated by Kv4.2 channels in L‐cells exhibit rapid activation and inactivation properties similar to those produced by It in rat ventricular cells. The current‐voltage relationships and the voltage dependence of steady‐state inactivation are also very similar in these two preparations. However, the recovery from inactivation of Kv4.2 is much slower (time constant, 378 ms) than that of It in rat ventricular cells (58 ms). 3. The K+ current due to Kv4.2 can be blocked by millimolar concentrations of 4‐aminopyridine in L‐cells; a similar pharmacological response has been observed in rat ventricular myocytes. 4. Quinidine inhibits Kv4.2 in L‐cells and It in rat ventricular cells in a similar fashion. In L‐cells quinidine reduced the amplitude of Kv4.2 and accelerated its time course of inactivation, suggesting that quinidine may act as an open channel blocker of Kv4.2, as has been described for It in rat ventricle. 5. To provide further independent evidence that Kv4.2 and Kv4.3 channel isoforms contribute to It in rat ventricular cells, the effects of 20‐mer antisense phosphorothioate oligodeoxynucleotides directed against Kv4.2 and Kv4.3 mRNAs were examined in ventricular myocytes isolated from 14‐ and 20‐day‐old rats, and in L‐cells. In both preparations, Kv4.2 antisense pretreatment significantly reduced the transient outward K+ current (by approximately 55‐60%). Similar reduction of It was produced by the Kv4.3 antisense oligonucleotide on the 14‐day‐old rat myocytes. 6. In 14‐day rat ventricular cells, combination of Kv4.2 and Kv4.3 antisense oligonucleotides did not produce a significantly larger reduction of It than that observed after pretreatment with either antisense oligonucleotide alone. 7. L‐cells stably transfected with Kv4.2 were treated with Kv4.3 antisense oligonucleotide to evaluate the possibility of cross‐reactivity between Kv4.3 antisense and Kv4.2 mRNA. This antisense treatment produced no change in It, verifying the lack of cross‐reactivity. 8. These biophysical and pharmacological results together with the antisense data show that Kv4.2 and Kv4.3 are essential components of the Ca2+‐independent transient outward K+ current, It, in rat ventricular myocytes.

Role of an inwardly rectifying potassium current in rabbit ventricular action potential.

1. Whole‐cell voltage‐clamp measurements were made of the time‐ and voltage‐dependent properties of the inwardly rectifying background potassium current IK1, in single myocytes from rabbit ventricle. The main goal of these experiments was to define the role of IK1 in the plateau and repolarization phases of the action potential (AP). 2. Action potentials from single ventricular myocytes were used as the command signals for voltage‐clamp measurements. In these ‘action potential voltage‐clamp’ experiments, IK1 was isolated from other membrane currents by taking the difference between control currents and currents in K(+)‐free bathing solution. The results show that IK1 is small during the plateau, but then rapidly increases during repolarization and declines in early diastole. 3. Evidence of an important functional role for IK1 in AP repolarization was obtained by comparing the magnitude of IK1 and the rate of change of membrane potential (dVm/dt) in the same cell during the AP. The time courses of IK1 and dVm/dt during the AP were closely correlated, indicating that IK1 was the principal current responsible for final repolarization. 4. Rectangular voltage‐clamp steps were used to study time‐ and voltage‐dependent changes in IK1 at membrane potentials corresponding to the repolarization phase of the AP. ‘Slow’ relaxations or tail currents, lasting 100‐300 ms, were consistently recorded when the cell was repolarized to potentials in the range ‐30 to ‐70 mV, following depolarizations between +10 and ‐10 mV. 5. The close correlation between the magnitude of the steady‐state IK1 (in an external K+ concentration of 5.4 mM), which was outward for membrane potentials in the range ‐30 to ‐70 mV, and the magnitude of the tail currents, suggests that they resulted from a slow increase, or reactivation, of IK1. 6. The component of the slow tails due to reactivation of IK1 can be separated from a previously described component due to Na(+)‐Ca2+ exchange since the IK1 component: (i) does not depend on the presence of the calcium current, ICa; (ii) can be recorded when internal EGTA (5 mM) suppresses large changes in [Ca2+]i; (iii) does not depend on the Na+ electrochemical gradient; (iv) is abolished in K(+)‐free external solution; and (v) is not present in rabbit atrial myocytes, in which IK1 is very small. 7. The time‐ and voltage‐dependent properties of IK1 revealed by these tail current experiments suggest that the measured magnitude of IK1 will be dependent on the voltage‐clamp protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-term diabetes alters K+ currents in rat ventricular myocytes.

The electrophysiological properties of single ventricular myocytes from control rats and from rats made diabetic by streptozotocin (STZ) injection (100 mg/kg body weight) have been investigated using whole-cell voltage-clamp measurements. Our major goal was to define the effects of diabetes on rate-dependent changes in action potential duration and the underlying outward K+ currents. As early as 4 to 6 days after STZ treatment, significant elevation of plasma glucose levels occurs, and the action potential duration increases. In both control and diabetic rats, when the stimulation rate is increased, the action potential is prolonged, but this lengthening is considerably more pronounced in myocytes from diabetic rats. In ventricular myocytes from diabetic rats, the Ca(2+)-independent transient outward K+ current (I(t)) is reduced in amplitude, and its reactivation kinetics are slowed. These changes result in a smaller I(t) at physiological heart rates. The steady-state outward K+ current (IK) also exhibits rate-dependent attenuation, and this phenomenon is more pronounced in cells from diabetic rats. These STZ-induced changes in I(t) and IK also develop when a lower dose (55 mg/kg) of STZ is used and measurements are made after 7 weeks of treatment. These electrophysiological effects are not related to the hypothyroid conditions that accompany the diabetic state, since they cannot be reversed by replacement of the hormone L-triiodothyronine to physiological levels. Direct effects of STZ could be ruled out, since preceding the STZ injection with a bolus injection of 3-O-methylglucose, which prevents development of hyperglycemia, prevents the electrophysiological changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon β-adrenergic stimulation in normal and failing hearts

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

THYROID HORMONE REGULATES POSTNATAL EXPRESSION OF TRANSIENT K+ CHANNEL ISOFORMS IN RAT VENTRICLE

1. The ability of thyroid hormone to regulate the postnatal changes of the Ca2+‐independent transient outward K+ current (It) was studied in rat ventricular myocytes. 2. In rat ventricle, It is very small at birth and then increases markedly between postnatal days 8 and 20. The time course of this increase in current density is similar to that of a significant rise in plasma thyroid hormone (T3) levels. 3. During early development, the density of expression of It can be altered by changes in thyroid hormone levels. Eight days after birth the density of It measured at +50 mV in control animals is 2.2 +/‐ 0.4 pA pF(‐1). This value is about 3‐fold larger (6.5 +/‐ 0.8 pA pF(‐1)) in myocytes from age‐matched hyperthyroid animals. When the plasma T3 level in newborn rats is not allowed to increase, or is decreased by making animals hypothyroid, this age‐dependent increase in It fails to occur. 4. Using RNase protection assays, Kv4.2 and Kv4.3 mRNA levels were measured in ventricular tissues obtained from age‐matched 8‐day‐old control and hyperthyroid rats. In hyperthyroid animals, where an approximately 3‐fold increase in It was identified, increases in the mRNA levels for Kv4.2 and Kv4.3 were 1.6‐fold and 2.6‐fold, respectively. 5. These results show that thyroid hormone can regulate the development of It in rat ventricle. Direct measurements of It density and mRNA levels as a function of development and thyroid hormone levels also strongly suggest that the Kv4.2 and Kv4.3 channels are essential components of It in rat ventricular cells.

Thyroid status and diabetes modulate regional differences in potassium currents in rat ventricle.

1. The rate dependence and recovery kinetics of the Ca(2+)‐independent transient (I(t)) and steady‐state or ‘pedestal’ (Iss) outward potassium (K+) currents were studied in single myocytes isolated from epicardial and endocardial regions of rat left ventricles. The whole‐cell, suction microelectrode method was used to measure baseline (fully reactivated) I(t), as well as its rate‐dependent attenuation. Results from a group of control animals were compared with data from three other groups having an experimentally altered hormonal status. 2. I(t) was significantly smaller in endocardial cells than in epicardial cells, in part due to a very large difference in the recovery kinetics of this current in endocardial cells. This was reflected in a pronounced rate‐dependent prolongation of endocardial action potentials. In contrast, the non‐inactivating ‘pedestal’ current, Iss, was very similar in magnitude and showed comparable rate dependence in cells from both epicardium and endocardium. 3. Changing the thyroid status had selective, differential actions on the amplitude and rate dependence of It in epicardial and endocardial cells. Under hypothyroid conditions there was a more pronounced reduction of baseline I(t) in epicardial than in endocardial cells. Moreover, a slowing of the recovery kinetics in epicardial cells resulted in an enhanced attenuation of this current at high rates. Changing thyroid status had no effect on the magnitude or rate dependence of Iss in cells from either region of the left ventricle. 4. Following establishment of hyperthyroid conditions, there was no significant change in I(t) magnitude at baseline. However, when compared with control data, the recovery of I(t) was considerably faster in endocardial cells, and marginally faster in epicardial cells. 5. Streptozotocin‐induced diabetic conditions resulted in a much greater attenuation of I(t) in epicardial cells than in endocardial cells. Epicardial action potentials in these conditions showed prominent rate‐dependent prolongation. Iss was reduced to a similar extent in cells from these two regions. 6. Our findings demonstrate that altered hormonal status can selectively change the amplitude and kinetics of It in the epi‐ and endocardium of rat left ventricle. These changes can reduce the epicardial‐endocardial gradients in the magnitude and recovery kinetics of It and hence diminish the intrinsic differences in both action potential duration and refractoriness.

Alpha-adrenergic modulation of the transient outward current in rabbit atrial myocytes.

1. A whole‐cell voltage‐clamp technique has been used to study the alpha‐effects of the adrenergic agonists noradrenaline, methoxamine and phenylephrine on the action potentials and membrane currents of rabbit atrial myocytes. Experiments were carried out at 22‐23 degrees C. 2. In the presence of 10(‐6) M‐propranolol, all three agents prolonged action potential duration. This change could be ascribed principally to changes in membrane current early during the plateau phase of the action potential. In the presence of 10(‐3) M‐4‐aminopyridine, no changes in calcium current (ICa) were observed on exposure to alpha‐agonists. No significant shift in the voltage dependence or change in the amplitude of the calcium current‐voltage relation was observed. 3. Exposure to 3 x 10(‐4) M‐CdCl2 to block ICa reduced the action potential prolongation caused by alpha‐adrenergic agonists. Measurement of unloaded cell shortening revealed that action potential prolongation caused by alpha‐agonists, especially at low stimulus rates, could contribute significantly to the positive inotropic effect of alpha‐adrenoceptor stimulation. 4. The voltage‐activated transient outward current (It) was markedly reduced during exposure to alpha‐adrenergic agonists in a dose‐dependent manner in the presence of CdCl2 (3 x 10(‐4) M) and propranolol in sufficient concentration to prevent beta‐adrenoceptor activation. Noradrenaline exhibited a higher potency for this effect than either methoxamine or phenylephrine. The noradrenaline concentration required to give 50% of the maximal effect was 6 x 10(‐6) M compared with 2.3 x 10(‐4) M for methoxamine. Noradrenaline reduced It by only about 60% of the maximum reduction produced by methoxamine suggesting that it could be classified as a partial agonist for this effect. 5. The reduction of It during exposure to alpha‐adrenergic agonists was rate dependent in that larger current reductions were observed at very low rates of stimulation (less than 0.1 Hz). 6. The magnitudes of current‐voltage relations for It were reduced over the entire voltage range studied during exposure to alpha‐adrenergic agonists and reductions were dose dependent. No shift of these relations along the voltage axis was observed. 7. The steady‐state inactivation relations for It were studied using two voltage clamp protocols. A two‐step method resulted in a relatively steep sigmoid ‘quasi‐steady‐state’ relation. The half‐inactivation potential of ‐27 mV was unaffected by alpha‐adrenergic agonists.(ABSTRACT TRUNCATED AT 400 WORDS)

K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292-296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

Inhibition of the formation or action of angiotensin II reverses attenuated K+ currents in type 1 and type 2 diabetes

1 Transient and sustained calcium‐independent outward K+ currents (It and ISS) as well as action potentials were recorded in cardiac ventricular myocytes isolated from two models of diabetes mellitus. 2 Rats injected (i.v.) with streptozotocin (STZ, 100 mg kg−1) 6–10 days before cell isolation developed insulin‐dependent (type 1) diabetes. It and ISS were attenuated and the action potential prolonged. Incubation of myocytes (6‐9 h) with the angiotensin II (ATII) receptor blockers saralasin or valsartan (1 μm) significantly augmented these currents. Inclusion of valsartan (1 g l−1) in the drinking water for 5–10 days prior to and following STZ injection partially prevented current attenuation. 3 Incubation of myocytes from STZ‐treated rats (6‐9 h) with 1 μm quinapril, an angiotensin‐converting enzyme (ACE) inhibitor, significantly augmented It and ISS and shortened the ventricular action potential. It augmentation was not due to changes in steady‐state inactivation or in recovery from inactivation. No acute effects of quinapril were observed. 4 The effects of quinapril and valsartan were abolished by 2 μm cycloheximide. 5 Myocytes were isolated from the db/db mouse, a leptin receptor mutant that develops symptoms of non‐insulin‐dependent (type 2) diabetes. K+ currents in these cells were also attenuated, and the action potentials prolonged. Incubation of these cells (> 6 h) with valsartan (1 μm) significantly enhanced the transient and sustained outward currents. 6 These results confirm recent suggestions that cardiac myocytes contain a renin‐angiotensin system, which is activated in diabetes. It is proposed that chronic release of ATII leads to changes in ionic currents and action potentials, which can be reversed by blocking the formation or action of ATII. This may underlie the proven benefits of ATII receptor blockade or ACE inhibition in diabetes, by providing protection against cardiac arrhythmias.