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Am J Physiol Heart Circ Physiol 281: H903-H914, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H903-H914, August 2001

Mechanisms underlying delayed afterdepolarizations in hypertrophied left ventricular myocytes of rats

János Mészáros1, Daniel Khananshvili2, and George Hart1

1 Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom; and 2 Department of Physiology and Pharmacology, Tel-Aviv University, Ramat-Aviv 69978, Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiac hypertrophy was induced in rats by daily injection of isoproterenol (5 mg/kg ip) for 7 days. Membrane voltage and currents were recorded using the whole cell patch-clamp technique in left ventricular myocytes from control and hypertrophied hearts. Ryanodine-sensitive delayed afterdepolarizations (DADs) and transient inward current (Iti) appeared in hypertrophied cells more often and were of larger amplitude than in control cells. DADs and Iti are carried principally by Na/Ca exchange with smaller contributions from a nonselective cation channel and from a Cl- channel. The latter is expressed only in hypertrophied myocytes. In hypertrophy, the density of caffeine-induced Na/Ca exchange current (INa/Ca) was increased by 26%, sarcoplasmic reticulum (SR) Ca2+ content as assessed from the integral of INa/Ca was increased by 30%, the density of Na-pump current (Ipump) was reduced by 40%, and the intracellular Na+ content, measured by Na+-selective microelectrodes was increased by 55%. The results indicate that DADs and Iti are generated by spontaneous Ca2+ release from an overloaded SR caused by a downregulated Na pump and an upregulated Na/Ca exchange. These findings may explain the propensity for arrhythmias seen in this model of hypertrophy.

transient inward current; sodium/calcium exchange current; chloride current; nonselective cation current; sodium-pump current; arrhythmia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VENTRICULAR ARRHYTHMIAS are common in cardiac hypertrophy and may contribute to sudden death in patients with this condition (28). Although cardiac hypertrophy is strongly associated with arrhythmias and mortality, cellular mechanisms for triggered arrhythmias have not been extensively investigated in hypertrophied hearts. Delayed afterdepolarizations (DADs) that are responsible for some forms of triggered arrhythmias have been demonstrated in hypertrophied myocardium from rats with renal hypertension (2), in diseased myocardium from rats with streptozotocin-induced diabetes (36), and in postmyocardial infarction remodeled hearts (41). Furthermore, augmented after-contractions have been observed in papillary muscles isolated from rats with cardiac hypertrophy (20). We have observed DADs in left ventricular trabeculae (34) in a model of cardiac hypertrophy induced in the rat by repeated administration of isoproterenol (34, 44). It has been known for a long time that the mechanism underlying DADs is the transient inward current (Iti) (23). Although it has also been well demonstrated that intracellular Ca2+ concentration ([Ca2+]i) overload must be present for the generation of Iti, its ionic mechanism is still a subject of debate and is undocumented in diseased cardiac myocytes. [Ca2+]i elevation can be induced by an increased intracellular Na+ level as a result of Na pump inhibition, which makes the Na/Ca exchange operate in reverse mode (2, 15, 23, 31). The elevated [Ca2+]i causes overloading of the sarcoplasmic reticulum (SR) with Ca2+ and induces spontaneous oscillatory release of Ca2+ from the SR after depolarization or an action potential (4, 23, 32). In nonhypertrophied tissue, Iti has been shown to depend in part on Na/Ca exchange (31). Na/Ca exchange current (INa/Ca) and protein expression are increased in hypertrophied myocytes (17, 19, 45, 51), but DADs have not previously been reported in hypertrophied cardiac myocytes. The aims of the present study were therefore to establish the mechanisms and properties of Iti and to characterize the ionic basis for this current in catecholamine-induced cardiac hypertrophy in the rat.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of cardiac hypertrophy. Cardiac hypertrophy was induced in male Wistar rats weighing 200-250 g by intraperitoneal injection of 5 mg/kg isoprenaline once daily for 7 days (34, 44). Age-matched control rats received the same volume of 0.9% NaCl solution. The animals were used for experiments 24 h after the last injection. The degree of hypertrophy was estimated by measuring blotted wet heart weight and the body weight and calculating heart weight-to-body weight ratio. These experiments were done on 10 pairs of animals. The investigation conforms with the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (Her Majesty's Stationery Office, United Kingdom).

Cell isolation. Single left ventricular myocytes used for these electrophysiological experiments were isolated from six pairs of animals. Cells were obtained from control and hypertrophied hearts according to the method described previously (45). Briefly, the animals received 500 units heparin intraperitoneally 20 min before death by cervical dislocation. The heart was removed and perfused retrogradely with a nominally Ca2+-free Tyrode solution at 35°C for 4 min and then perfused for 12 min with a Tyrode solution containing 1 mg/ml collagenase and 0.1 mg/ml protease. The heart was removed from the column, and the left ventricle and septum were dissected free, roughly chopped, suspended in the enzyme solution (but without protease), and stirred slowly on a heated plate. Fractions were removed at 5- to 10-min intervals and the cells were washed twice after centrifugation in a normal Tyrode solution containing 5 mg/ml bovine serum albumin. The cells were stored at room temperature in Dulbecco's modified Eagle's medium with 2 mg/ml Ultraser G (GIBCO-BRL, Paisley, UK). Cells were left for 1 h before use and were used within 8 h.

Electrophysiological protocols. Myocytes were layered on a glass coverslip that formed the base of a perfusion chamber. This was mounted on the stage of an inverted microscope situated on an isolation table. The flow rate of perfusion fluid was 2-3 ml/min. Temperature of the bath was maintained at 35 ± 1°C by a heating block surrounding the input line, which was controlled by a feedback circuit using a sensing thermocouple positioned in the chamber. The level of solution in the bath was controlled using a feedback circuit, the sensor for which was a piece of photographic film positioned on the meniscus and connected by a stainless steel rod to an Akers transducer (SensoNor; Horten, Norway). For experimentation, only those myocytes were used that showed rod shape, clear cross striations, no blebs on the surface, and no spontaneous contractions.

The whole cell voltage-clamp technique was used to record membrane currents using an Axopatch-200 amplifier (Axon Instruments; Burlingame, CA). Electrical compensation was made for whole cell capacitance and series resistance. Cell capacitance was measured directly from the Axopatch-200 after correction for series resistance. Patch pipettes were pulled from filamented borosilicate capillary glass (Clark Electromedical Instruments; Pangbourne, UK) on a microprocessor-based three-stage puller (Mecanex BB-CH-PC; Basel, Switzerland). The pipettes had resistances of 2-5 MOmega after filling with the appropriate internal solution. The resistance of the seals between the pipette tip and the myocytes was 5-10 GOmega .

To record DADs and Iti, micropipettes were filled with a solution containing (in mM) 140 KCl, 2 MgCl2, 5 Mg-ATP, 5 Na2-phosphocreatine, 10 HEPES (at pH 7.2, adjusted with KOH). Cells were superfused with an external solution containing (in mM) 134 NaCl, 5.4 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES (at pH 7.4, adjusted with NaOH). Action potentials were initiated by depolarizing current pulses (2-5 ms duration) at a frequency of 0.3 Hz under whole cell current clamp conditions. Iti was induced by applying depolarizing voltage pulses from a holding potential of -40 mV at a frequency of 0.3 Hz under whole cell voltage-clamp conditions.

The caffeine-dependent INa/Ca was induced at a holding potential of -80 mV by short application of 20 mM of caffeine onto the whole cell voltage-clamped myocyte after a 1-min rest period following a train of stimulation to ensure that the SR was consistently loaded with Ca2+ (54). Rapid solution changes were evoked through a system of solenoids. In these experiments, the external solution contained 1 mM CaCl2, to which 5 mM 4-aminopyridine and 0.1 mM BaCl2 were added to block potassium currents, and 10 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were added to block chloride current. All of the other components of the external and internal solutions were the same as described above. To assess SR Ca2+ content, INa/Ca was integrated and converted to total Ca2+ fluxes (53, 54). Considering that INa/Ca is generated mostly by Na/Ca exchange but is 1) contaminated by other [Ca2+]i-activated currents, and 2) caffeine-released Ca2+ can also be removed from the cell by mechanisms other than Na/Ca exchange, INa/Ca was first corrected for these mechanisms by multiplying using a factor of 1.5 (53, 54). Cell volume was calculated from the membrane surface area obtained from the membrane capacitance assuming a capacitance-to-volume ratio of 6.76 pF/pl for rats (47, 53, 54).

To study the Na-pump current (Ipump), micropipettes were filled with a solution containing (in mM) 86 CsCl, 2 MgCl2, 10 Mg-ATP, 5 Na2-phosphocreatine, 20 tetraethylammonium-chloride, 5 EGTA, and 5 HEPES (at pH 7.2, adjusted with CsOH). Cells were superfused with an external solution containing (in mM) 143 NaCl, 5.4 KCl, 1 MgCl2, 5.5 glucose, 2 BaCl2, 2 CoCl2, and 5 HEPES (at pH 7.4, adjusted with NaOH). Temperature was 35 ± 1°C. Ipump was activated on returning to 5.4 mM external K+ after 2 min in K+-free solution at a holding potential of -40 mV (10). Ipump was measured either as a constant holding current or during ramp pulse protocols. A quasi-steady-state current-voltage relationship was determined by applying slow voltage ramp (53 mV/s) between +40 and -120 mV from a holding potential of -40 mV (49). The negative ramp was used to prevent activating the voltage-gated sodium channel.

Intracellular Na+ concentration ([Na+]i) was measured in control and hypertrophied ventricular myocytes using voltage-sensitive and Na+-sensitive microelectrodes. The latter were made with the Na+ ionophore I (ETH-227, Fluka; Buchs, Switzerland) and filled with 150 mM NaCl as described by Rodrigo and Chapman (43). [Na+]i was obtained by interpolating on the Na+-electrode calibration curve the difference between potentials measured by the Na+-sensitive and voltage-sensitive electrodes.

The cyclic hexapeptide FRCRCFa (828 mol wt) was obtained as a powder, made up as a 1 mM stock solution in distilled water, and kept in the freezer. FRCRCFa was applied in the pipette solution to inhibit Na/Ca exchange from the cytoplasmic side.

Chemicals. dl-Isoprenaline hydrochloride (Saventrine Intravenous, Pharmax; Bexley, Kent, UK), collagenase type IV (Worthington Biochemical), Ultraser G (GIBCO-BRL), and FRCRCFa were synthesized by D. Khanashvili (25). All of the other chemicals were obtained from Sigma (St. Louis, MO).

Data analysis. Commercial software (pCLAMP 5.5.1 and 6.02, Axon Instruments) was used for generating voltage pulses, data acquisition, and analysis of digitized whole cell currents. The current signals were filtered with an eight-pole Bessel filter (5-kHz cutoff frequency) and sampled at 3-10 kHz. All values are presented as means ± SE. Unpaired Student's t-tests were used to evaluate the statistical significance of differences between means. Values of P < 0.05 were considered to indicate significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of hypertrophy. At day 7 after the start of isoproterenol injections, this model demonstrated compensated, moderate left ventricular hypertrophy without heart failure. At this stage, rats did not exhibit fatigue nor hyperpnea. When the body cavity was opened to remove the heart, pulmonary edema, pleural effusion, ascites, and hepatomegaly were never observed in any treated animal. The heart weight gradually increased throughout. The body weight of the animals increased similarly in control and treated animals. The heart weight-to-body weight ratio, as an indicator of the degree of hypertrophy, was increased from 3.21 ± 0.05 mg/g (control) to 4.16 ± 0.07 mg/g (hypertrophy) (n = 10, P < 0.05). The lung weight-to-body weight ratio was unchanged at 5.04 ± 0.15 mg/g (control) and 5.12 ± 0.11 (hypertrophy) (n = 10, not significant), as was the liver weight-to-body weight ratio at 41.8 ± 1.9 mg/g (control) and 42.2 ± 1.4 mg/g (hypertrophy) (n = 10, not significant). No serous cavity effusions were observed.

Changes in the action potential. The resting potential recorded from 29 hypertrophied left ventricular myocytes was depolarized compared with 23 control myocytes (-76.8 ± 1.1 mV, control; -71.2 ± 0.9 mV, hypertrophy, P < 0.05, n = 3 pairs of animals). The amplitude of the overshoot was unchanged in hypertrophy. Action potential duration was prolonged in the hypertrophied myocytes (Fig. 1). Action potential duration at 25% repolarization was 290% of the control value (8.1 ± 0.9 ms, control; 23.5 ± 2.7 ms, hypertrophy, P < 0.01), and action potential duration at 95% repolarization (APD95) was 155% of the control value (99.4 ± 3.6 ms, control; 152.7 ± 5.4 ms, hypertrophy, P < 0.01).


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Fig. 1.   Representative action potentials in a control (C) and a hypertrophied (H) left ventricular myocyte. Note that in hypertrophy the action potential is prolonged and a delayed afterdepolarization can be seen after repolarization.

Occurrence and ionic properties of DADs. Spontaneous DADs were frequently observed in the hypertrophied myocytes after completion of a prolonged action potential, but they were rarely seen in control myocytes (Fig. 1). The incidence of DADs was 9 of 32 in control and 23 of 31 in hypertrophied myocytes. The amplitude of DADs averaged 2.9 ± 0.4 mV in control, and 12.4 ± 1.3 mV in hypertrophied myocytes (P < 0.05). In five pairs of hypertrophied ventricular myocytes, we studied the pharmacological properties of DADs. In the group of myocytes in which DADs were recorded without FRCRCFa (a specific blocker of Na/Ca exchange) in the pipette, the amplitude of DADs was 13.2 ± 1.6 mV. After inclusion of 10 µM FRCRCFa in the pipette, the amplitude of DADs was 2.8 ± 0.3 mV (P < 0.05). Application of 10 µM DIDS into the superfusing solution reduced the DADs amplitude further to 1.7 ± 0.3 mV (P < 0.05).

These results indicate that in the hypertrophied ventricular cells, DADs are carried principally but not exclusively by Na/Ca exchange. Minor components are carried by a [Ca2+]i-activated nonselective cation current and by [Ca2+]i-activated Cl- current. The relative contributions of these components to the overall amplitude of DADs are ~80% Na/Ca exchange, 7% Cl- current, and 13% nonselective cation current.

Induction of Iti. To elucidate the membrane currents underlying the hypertrophy-induced arrhythmias, cells were voltage-clamped using the whole cell configuration. Figure 2A shows representative currents induced by depolarizing voltage pulses of different amplitudes (0, +30, and +60 mV for 2 s) from a holding potential of -40 mV in a control cell (middle) and in a hypertrophied cell (bottom). Iti of very small amplitude could be seen in 12 of 30 control cells. However, this oscillatory current was seen in 23 of 31 hypertrophied cells at the termination of the long depolarizing pulses (Fig. 2A, bottom) at each of the potentials shown. The pattern of Iti appears to be different, i.e., in some cells it is single, and in other cases it is oscillatory. Furthermore, in some cases, the Iti is superimposed on a slowly decaying inward current, which appears to be carried by Na/Ca exchange. Similar patterns have also been reported by others in control cells in which Iti was induced by reoxygenation (4) or oxidative stress (32). The amplitude of Iti increased after depolarizing to more positive potentials. Only the first oscillatory current component was used for evaluation, the amplitude of which was measured as the difference current between the maximum inward peak and a baseline level constructed from the tangent joining the two neighboring nadirs as described by Benndorf et al. (4). The mean amplitude of Iti induced by +60 mV depolarization was -32 ± 6 pA in 12 control cells and -225 ± 11 pA in 23 hypertrophied cells (P < 0.001).


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Fig. 2.   A: induction of transient inward current (Iti) in control and hypertrophied left ventricular myocytes. Depolarizing voltage pulses lasting 2 s were applied from a holding potential of -40 mV to different potentials as indicated (top) in a representative control and hypertrophied cell. Iti can be seen on repolarization in the hypertrophied (bottom) but not in the control cell (middle). Note that Iti becomes larger as the amplitude of the depolarizing pulse increases. B: mean Iti density as a function of voltage. Amplitude of Iti recorded after termination of 2-s voltage pulses of different amplitude has been corrected for cell capacitance and plotted against the magnitude of the depolarizing voltage step. Data are means of 12 control cells and 23 hypertrophied cells. C: dependence of the time to peak current on the depolarizing voltage. Note that peak current occurs earlier after more positive voltage pulses and in hypertrophied myocytes.

The dependence of Iti density on the magnitude of the preceding voltage step is shown in Fig. 2B. Increasing the amplitude of the depolarizing voltage pulse increases the amplitude of Iti induced on repolarization. Depolarization to potentials more positive than +20 mV is required to elicit measurable Iti in control cells, but the current is inducible after much smaller depolarizations in hypertrophied cells. The mean value of Iti density at +60 mV was -0.28 ± 0.05 pA/pF (n = 12) in control cells and -1.39 ± 0.12 pA/pF in hypertrophy (n = 23, P < 0.001). Figure 2C shows that increasing the amplitude of a depolarizing voltage pulse reduces the time to peak current.

It is generally accepted that oscillatory release of Ca2+ from the SR associated with intracellular Ca2+ overload is involved in the generation of Iti (5, 23). In five ventricular myocytes taken from three hypertrophied hearts, we have therefore studied the effect of ryanodine, which interferes with the SR Ca2+-release channel (33) and blocks the [Ca2+]i-activated Iti (6), consequently abolishing DADs (14, 31). Figure 3 shows an example of the results obtained after 2-min exposure to 10 µM ryanodine, which would be expected to abolish any SR contribution to current generation. Figure 3A illustrates Iti on repolarization after a 2-s pulse to +60 mV in a hypertrophied cell. This large oscillatory current component is completely abolished by ryanodine (Fig. 3B). In addition, ryanodine also inhibits current oscillations, which may be seen during the long depolarizing voltage pulse.


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Fig. 3.   Ryanodine blocks Iti. A: without ryanodine. B: 2 min after application of 10 µM ryanodine, a Ca2+-release channel blocker at the sarcoplasmic reticulum (SR). Currents were elicited by applying a depolarizing voltage pulse from a holding potential of -40 mV to +60 mV for 2 s, followed by repolarization to the holding potential. Note that after perfusion of the cell with ryanodine, the oscillatory currents are no longer observed either on repolarization or during the depolarizing step.

Ionic basis of Iti. Current-voltage relationships for Iti density were constructed by applying a double-pulse protocol (16, 32) illustrated in Fig. 4A, top. A prepulse to +60 mV of 2-s duration was applied from a holding potential of -40 mV. The membrane was then repolarized to different test potentials for 2 s before being returned to the holding potential. Figure 4A shows representative current traces from a hypertrophied cell. The amplitude of Iti varies markedly with repolarization voltage, the largest current being seen at -40 mV. The mean current density-voltage relationship obtained from nine hypertrophied myocytes is shown in Fig. 5A (curve a). Iti values recorded during the test pulses are plotted against the corresponding test voltage. Iti amplitude reached a peak of -1.49 ± 0.08 pA/pF at -40 mV and decreased on either side of this voltage. The current remained inward over the voltage range tested (-60 mV to +60 mV) with no apparent reversal potential.


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Fig. 4.   Dependence of Iti on repolarization voltage under different pharmacological conditions. Top inset, the voltage protocol. A: Iti recorded without channel blockers at different repolarization potentials after a constant voltage step of 2 s to +60 mV from a holding potential of -40 mV. Iti becomes larger at more negative repolarization potentials. Currents remain inward at all potentials tested. B: Iti recorded with 10 µM FRCRCFa in the pipette and using the same pulse protocol. Outward current oscillations can be seen at positive repolarization potentials and during the long depolarizing prepulse. The inward current component is markedly reduced. C: Iti recorded under the same pulse protocol with 10 µM FRCRCFa in the pipette and 10 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, in the superfusate. Note that DIDS abolished only the outward current oscillations at positive potentials and during the long depolarizing prepulse.



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Fig. 5.   Separation of the ionic components of Iti. A: current density-voltage relationships for Iti. Curve a, mean current density from 9 hypertrophied left ventricular myocytes plotted against repolarizing voltage. Curve b, current density-voltage relationship from 12 cells obtained with 10 µM FRCRCFa in the patch pipette. B: curve a, current density-voltage relationship for Iti obtained from 12 hypertrophied myocytes, using 10 µM FRCRCFa in the patch pipette. This is the same curve as curve b in A. Curve b, current density-voltage relationship for the same cells using 10 µM FRCRCFa in the pipette and 10 µM DIDS in the superfusate. Curve a-b, difference of curves a and b showing the DIDS-sensitive (Cl- current) portion of Iti. Standard error bars, mean data points.

The contribution of Na/Ca exchange to the Iti was investigated in hypertrophied myocytes by using the Na/Ca exchange blocker FRCRCFa (25). This compound has been developed as a cyclic hexapeptide analog of a molluscan cardioexcitatory peptide, and it is a potent and selective blocker of Na/Ca exchange (21). Because FRCRCFa acts from the cytoplasmic side, we have applied the peptide in the pipette solution. After whole cell configuration was established, the myocyte was repetitively stimulated with a 2-s depolarizing pulse from a holding potential of -40 to 0 mV for 1 min. We have proved that this allowed sufficient time for the peptide to dialyze into the cell (results not shown). The pulse protocol was then changed to the protocol used above. Figure 4B shows that under these conditions, Iti amplitude is markedly reduced. Inward current oscillations are present only at negative potentials. At positive potentials, outward current oscillations can be observed both during the depolarizing prepulse and on repolarization. At 0 mV no current oscillation can be seen. The mean current density-voltage relationship constructed from data obtained in 12 hypertrophied ventricular myocytes is shown in Fig. 5A (curve b). This curve represents the small proportion of Iti carried by mechanisms other than Na/Ca exchange. The current density-voltage relation reverses at +2.5 ± 2.0 mV.

To investigate whether a [Ca2+]i-activated outward Cl- current (57) contributes to the generation of Iti in hypertrophied rat myocytes, we added the specific Cl- channel blocker DIDS (16, 50) to the superfusate in a concentration of 10 µM, while at the same time including 10 µM FRCRCFa in the pipette solution. Figure 4C shows that under these conditions, outward current oscillations during the long depolarizing prepulse and on repolarization are markedly suppressed, but inward current oscillations at negative potentials are only slightly reduced compared with Fig. 4B. Figure 5B shows that the mean current density-voltage relationship (curve b) from 12 cells is depressed and the reversal potential (+15.0 ± 4.0 mV) is shifted toward more positive potentials, compared with the curve obtained after block of Na/Ca exchange in the absence of DIDS. The DIDS-sensitive component of the current (shown as a dashed line in Fig. 5B) is an outward rectifier current with a reversal potential of -4.0 ± 3.1 mV close to the calculated Cl- equilibrium potential of -1.8 mV. The current remaining after block of Na/Ca exchange and Cl- current shows marked inward rectification (Fig. 5B, curve b).

These results demonstrate that in hypertrophied ventricular myocytes, similarly to DADs, Iti is carried principally but not exclusively by Na/Ca exchange. Minor components are carried by a [Ca2+]i-activated nonselective cation current and by [Ca2+]i-activated Cl- current. The relative contributions of these components to the overall amplitude of Iti density at -40 mV are ~82% Na/Ca exchange, 5% Cl- current, and 13% nonselective cation current.

In five pairs of control myocytes, we studied the pharmacological properties of Iti to determine whether Iti has the same charge carriers in control cells that was observed in hypertrophied cells. In the group of myocytes in which Iti were recorded without FRCRCFa (a specific blocker of Na/Ca exchange) in the pipette, the density of Iti was -0.32 ± 0.06 pA/pF. After inclusion of 10 µM FRCRCFa in the pipette, the density of Iti was -0.05 ± 0.02 pA/pF at -40 mV (P < 0.05). Application of the Cl- channel blocker DIDS (10 µM) into the superfusate failed to exert any further effect on the density of Iti. The result suggests that in control ventricular myocytes, Iti is carried principally but not exclusively by Na/Ca exchange with a smaller contribution from a [Ca2+]i-activated nonselective cation current but with no contribution from a [Ca2+]i-activated Cl- current. This result emphasizes that Cl- current component of Iti is expressed in hypertrophy.

Changes in Na/Ca exchange current and SR Ca2+ content. In a series of experiments, we wanted to study whether there is any change in the SR Ca2+ content as a possible mechanism underlying the appearance of arrhythmogenic DADs and Iti in hypertrophied myocytes. Caffeine-dependent inward INa/Ca was induced at a holding potential of -80 mV by a short application of 20 mM caffeine onto the myocytes after a 1-min rest period following a train of stimulation to ensure that the SR was consistently loaded with Ca2+ (54). The amplitude of INa/Ca was increased in hypertrophied myocytes from -103.5 ± 5.2 pA in control myocytes (n = 40) to -179.9 ± 6.9 pA in hypertrophied myocytes (n = 41, P < 0.001). Examples of these changes are shown in Fig. 6. The density of INa/Ca was increased by 31% in hypertrophied myocytes (control, -1.04 ± 0.02 pA/pF; hypertrophy, -1.31 ± 0.02 pA/pF; P < 0.001).


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Fig. 6.   Effect of hypertrophy on Na/Ca exchange current (INa/Ca). Estimation of SR Ca2+ content in control and hypertrophied myocytes. Top, digitized recordings of membrane current taken at -80 mV holding potential to compare caffeine-dependent inward INa/Ca in a control (A) and a hypertrophied (B) myocyte. Caffeine (20 mM) was applied for the periods shown by the solid bars to release the SR Ca2+ content. Bottom, comparison of cumulative integrals of INa/Ca expressed as µmoles Ca2+ per liter total cell volume in a control (C) and a hypertrophied (D) myocyte.

The relationship between INa/Ca and cell capacitance for the control and hypertrophy group is illustrated in Fig. 7. Linear regression analysis was carried out using Eq. 1
y=a+bx (1)
where y is the current (in pA), a is the truncation, b is the slope, and x is the cell size (in pF). The data points were best fitted by two regression lines, the slopes of which were significantly different (control, 1.11 ± 0.06; hypertrophy, 1.33 ± 0.07; P < 0.001). Correlation coefficients were 0.943 ± 0.164 (P < 0.0001) in control and 0.956 ± 0.162 (P < 0.0001) in hypertrophy, suggesting a linear relationship between cell size and INa/Ca amplitude in both groups. Confidence interval analysis shows that the regression line in hypertrophy is shifted by 35 ± 4 pA to significantly higher INa/Ca values (P < 0.01). Thus two populations can be distinguished by this analysis, and we may conclude that the increase in INa/Ca is not simply explained by the increase in cell size in the hypertrophy group.


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Fig. 7.   Hypertrophy increases INa/Ca. Relationship between peak INa/Ca and cell membrane capacitance for control and hypertrophied cells. Two regression lines have been fitted to the data points. Dashed lines, ±95% confidence intervals. Regression line in the hypertrophy group is shifted to higher INa/Ca values.

To assess SR Ca2+ content, INa/Ca was integrated and converted to total Ca2+ fluxes (53, 54). Considering that INa/Ca is generated mostly by Na/Ca exchange, but 1) is contaminated by other [Ca2+]i-activated currents, and 2) caffeine-released Ca2+ can also be removed from the cell by mechanisms other than Na/Ca exchange, INa/Ca was first corrected for these mechanisms by multiplying with a factor of 1.5 (53, 54). Cell volume was calculated from the membrane surface area obtained from the membrane capacitance assuming a membrane capacitance-to-volume ratio of 6.76 pF/pl for the rat (47). SR Ca2+ content was increased by 30% in hypertrophied myocytes (control, 98.7 ± 1.5 µM, n = 40; hypertrophy, 128.3 ± 1.8 µM, n = 41; P < 0.001; Fig. 6).

Changes in Ipump current and intracellular Na+ content. In another series of experiments we investigated whether decreased Ipump could underlie the increase in SR Ca2+ content. Ipump was isolated using extracellular and intrapipette solutions that suppressed channel currents and INa/Ca (see MATERIALS AND METHODS). The Ipump was activated on returning to 5.4 mM extracellular K+ concentration ([K+]o) after 2 min in K+-free solution (10, 49) in both control and hypertrophied myocytes at -40 mV holding potential (Fig. 8, A and B). On reducing [K+]o to 0 mM, holding current shifted inward by about 20 pA in control and 15 pA in hypertrophied myocyte. Restoration of [K+]o to 5.4 mM after 2 min in K+-free solution transiently stimulated the Na pump to extrude Na+ that had accumulated intracellularly. The peak of the outwardly directed holding current reached about 130 pA in control and 100 pA in hypertrophied cells. This [K+]o-activated transient outward Ipump was completely prevented by adding 300 µM ouabain into the superfusate (Fig. 8, C and D) and was reactivated after ouabain has been washed out in both control and hypertrophied myocytes (Fig. 8, E and F). These results indicate that the [K+]o-activated Ipump is a ouabain-sensitive current and generated fully by the Na pump. Figure 9 illustrates current-voltage relationships for Ipump obtained by ramp pulses superimposed on the constant holding current as shown in Fig. 8. Current traces taken in 0 mM [K+]o show no change in the background current in hypertrophied myocytes compared with the control ones. In both control and hypertrophy, the current trace crosses the voltage axis at 0 mV. The current traces, taken in 5.4 mM [K+]o were shifted toward higher Ipump values. Averaged data show that Ipump, at 0 mV membrane potential, was 136.5 ± 6.6 pA in control cells (n = 36) and 117.3 ± 6.4 pA in hypertrophied cells (n = 33, P < 0.05) (Fig. 9, A and B). Figure 9C demonstrates the current density-voltage relationships of the difference currents obtained by subtracting current traces in 0 mM [K+]o from current traces in 5.4 mM [K+]o and normalized for cell capacitance in control and hypertrophied myocytes. These current density-voltage relationships for the [K+]o-activated Ipump increase gradually between -120 and -40 mV and saturate between -40 and +40 mV. Ipump density is substantially reduced in hypertrophied myocytes at 0 mV (control, 1.31 ± 0.05 pA/pF; hypertrophy, 0.79 ± 0.04 pA/pF, P < 0.001). The zero-current potential estimated by linear extrapolation averaged -158 ± 15 mV in control and -153 ± 18 mV in hypertrophied cells, in agreement with data reported by others (13).


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Fig. 8.   Effect of hypertrophy on Na pump current (Ipump). A and B: chart recordings of membrane current taken at -40 mV holding potential. Ipump was activated on returning to 5.4 mM extracellular K+ concentration ([K+]o) after 2 min in K+-free solution in a control and a hypertrophied myocyte. Top, solid bars show the time course of changes in [K+]o. Changing [K+]o from 5.4 to 0 mM inhibits resting Ipump, and restoring [K+]o to 5.4 mM stimulates Ipump transiently. Vertical excursions on the current traces indicate currents evoked by ramp pulses. C and D: application of ouabain (300 µM) in the external solution prevents activation of Ipump both in control and hypertrophied myocytes. E and F: 30 min after ouabain has been washed out, Ipump can be activated again both in control and hypertrophied myocytes.



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Fig. 9.   Effect of hypertrophy on current-voltage relationship for Ipump. Averaged current traces recorded with a ramp pulse at 0 mM [K+]o (Na pump inactive) and at 5.4 mM [K+]o (Na pump fully activated) in control myocytes (A, n = 36) and in hypertrophied myocytes (B, n = 33). Parameters of the slow negative ramp pulse (slope, 53 mV/s; magnitude, from +40 to -120 mV) applied at -40 mV holding potential on the holding current as shown in Fig. 8, A and B. C: density of the averaged difference currents ([K+]o-activated Ipump) obtained by subtracting current traces at 0 mM [K+]o from current traces at 5.4 mM [K+]o and normalized for cell capacitance in control and hypertrophy myocytes.

The relationship between Ipump and cell capacitance for the control and hypertrophy group is illustrated in Fig. 10. Linear regression analysis was carried out using Eq. 1. The data points were best fitted by two regression lines, the slope of which is significantly different (control, 1.05 ± 0.14; hypertrophy, 0.77 ± 0.12; P < 0.001). Correlation coefficients were 0.781 ± 0.174 (P < 0.0001) in control and 0.758 ± 0.183 (P < 0.0001) in hypertrophy, suggesting a linear relationship between cell size and Ipump amplitude in both groups. Confidence interval analysis shows that the regression line in hypertrophy is shifted by 52.7 ± 6.0 pA to significantly lower Ipump values (P < 0.01). Thus two populations can be clearly identified from this analysis, and the decrease in Ipump cannot be explained by the increase in cell size.


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Fig. 10.   Hypertrophy decreases Ipump. Relationship between peak Ipump and cell membrane capacitance for control and hypertrophied cells. Two regression lines have been fitted to data points. Dashed lines, ±95% confidence intervals. Regression line in hypertrophy group is shifted to lower Ipump values.

[Na+]i content measured by Na+-selective microelectrodes was significantly increased in hypertrophied left ventricular myocytes (control, 10.7 ± 0.6 mM, n = 6; hypertrophy, 16.8 ± 0.7 mM, n = 5; P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows for the first time that at the cellular level, both the incidence and magnitude of Iti is greatly increased in compensated left ventricular hypertrophy. The major charge carrier for this current is the electrogenic Na/Ca exchange with smaller contributions from a nonselective cation channel and from a Cl- channel. We suggest that the mechanism underlying Iti is spontaneous Ca2+ release from the SR, which is overloaded with Ca2+ as a consequence of Na-pump downregulation and Na/Ca exchange upregulation. Iti can account for triggered activity based on DADs, which leads to arrhythmias often observed in hypertrophied ventricular myocardium (2, 20, 34).

It could be postulated that effects seen in this model could be the direct response of the elevated level of circulating isoproterenol at the time of the experiments. However, it has been shown that catecholamine clearance kinetics in humans have two phases with half times of 2 and 34 min (11). In congestive heart failure, clearance is reduced to 67% of control (18), but it would still be expected that catecholamines would be completely cleared after ~4 h (5 half times). Catecholamine clearance levels in the rat are not available, but if it is assumed that the clearance rates are of the same order as those reported for humans, and because these animals were used for experimentation 24 h after the last injection of the catecholamine, any changes observed are unlikely to be due to a direct effect of the isoproterenol used to induce the hypertrophy, but rather to the effects of the hypertrophy itself.

Charge carriers for Iti. In different preparations cyclic release of Ca2+ from the SR can activate different charge-carrier mechanisms such as 1) [Ca2+]i-activated nonselective cation current (8, 16, 23), 2) [Ca2+]i-activated chloride current (16, 55), and/or 3) the electrogenic Na/Ca exchange (4, 12, 32), and thus induces Iti. Our data demonstrate that the major charge carrier for the Iti recorded in hypertrophy is Na/Ca exchange, and the current-voltage relation for this current shows no reversal potential because of the change in the reversal potential of the Na/Ca exchange (ENa/Ca) (4, 12, 16, 32). However, it has been reported that Iti may be comprised of more than one current mechanism in a given myocyte (16). Therefore, we wanted to investigate whether the Iti seen in this model of hypertrophy has more components than the Na/Ca exchange current. To exclude the exchanger we added a specific and selective exchange inhibitor, the cyclic hexapeptide FRCRCFa (21, 25), into the pipette solution, because the specific receptor sites for this peptide are on the intracellular side of the Na/Ca exchange molecule. This peptide gives us the possibility of inhibiting the exchanger in the presence of physiological concentrations of Na+ and Ca2+ on both sides of the sarcolemma. With FRCRCFa in the pipette, the current-voltage relationship for the remaining current component shows a reversal potential at +2.5 mV, thus indicating that Iti is carried not only by the Na/Ca exchange but also by one or more channels. Further proof for this view is provided by the fact that this remaining current component shows different voltage sensitivity from that which was recorded without FRCRCFa.

The current recorded with FRCRCFa in the pipette reverses close to the expected reversal potential of Cl- current (ECl) and consists mainly of an outwardly rectifying, Ca2+-activated Cl- current (57) and an inwardly rectifying Ca2+-activated nonselective cation current (8). The outward current oscillations, which may be visible during the depolarizing pulse and on repolarization to positive potentials, are markedly reduced after superfusion with 10 µM DIDS, a specific Cl- channel blocker (16, 50), thus proving that this outward component of the Iti depends on an outward Cl- current. The pure Cl- current component of the Iti was calculated by subtracting the DIDS-insensitive current from the total current. This difference current (Fig. 5B, dashed line) shows a reversal potential at -3 mV that is very close to the calculated ECl (-2 mV). However, the reversal potential of the small current that remains after block of both Na/Ca exchange and the Cl- current, is shifted to more positive potentials (+15 mV), suggesting a contribution for Na+ entry. It appears to be an inwardly rectifying Ca2+-activated nonselective cation current that can be carried by both Na+ and K+ (16).

Our present results indicate that in hypertrophied myocytes, Iti can be the underlying mechanism of DADs, because they are carried by the same charge carrier mechanisms both qualitatively and quantitatively. However, our results also suggest that Itis are not similar in control and hypertrophied cells because in control cells, Iti has no DIDS-sensitive component. These findings are in good agreement with those showing that control ventricular myocytes do not have Cl- current (27), but hypertrophied myocytes express it (3).

Mechanisms of induction of Iti. It is generally accepted that Iti is generated by an oscillatory release of Ca2+ from an overloaded SR that occurs when [Ca2+]i is elevated after a long depolarizing pulse or a prolonged action potential in myocytes having altered Ca2+ and/or Na+ handling capability (5, 23, 31). Our experimental data to support this idea are that increasing the amplitude or the duration of the voltage pulse, conditions which favor increased SR Ca2+ loading, cause an increase in the density of Iti and reduce the time to the peak of the first oscillatory component. The other supporting fact is that ryanodine, a Ca2+-release channel blocker at the SR (33), abolishes DADs and Iti, which is in common with previous reports (6, 14, 31, 34). Moreover, we could not elicit Iti after inclusion of EGTA in the patch pipette (data not shown). These experiments confirm that the Iti we have observed in hypertrophied myocytes depends on elevated [Ca2+]i causing oscillatory Ca2+ release from the SR, such as in normal tissue under conditions such as glycoside toxicity (23), reoxygenation (4), or oxidative stress (32).

In this study, we demonstrate an increased SR Ca2+ content in hypertrophied myocytes in which Iti density is elevated. This finding is consistent with the view that increased SR Ca2+ content can give rise to greater release of Ca2+ after a given rise in [Ca2+]i and, therefore, to a larger Iti in the hypertrophied myocyte. Upregulation of the SR functions, including increased uptake and release of Ca2+ associated with increased contractile force, have been reported in the early stage of both pressure overload-induced cardiac hypertrophy (29, 37) and catecholamine-induced hypertrophy (52). In addition, the SR Ca-pump activity has been reported to show no change in compensated hypertrophy (38). The SR Ca2+-release channel activity was found to be upregulated as reflected by an increased density of the ryanodine receptors in cardiomyopathic hamster hearts early in the development of the disease (46) and in pressure overloaded hypertrophy in the rat (42) in which the Ca2+-ATPase and ryanodine receptor mRNA levels are also increased (1).

What mechanisms can cause the SR to overload with Ca2+ in hypertrophied ventricular myocytes? Our data show that hypertrophy-induced reduction of Na-pump activity increases [Na+]i. In accordance with other reports (48), this increased [Na+]i reduces transmembrane [Na+] gradient, which then shifts the reversal potential of the ENa/Ca to more negative potentials closer to the resting membrane potential (Vm). This condition makes the Na/Ca exchange less able to extrude Ca2+ from the cell and eventually results in a net Ca2+ gain during diastole. If [Na+]i increases further, ENa/Ca can be shifted to potentials even more negative than Vm at which Na/Ca exchange works in reverse mode and now brings Ca2+ into the cell and causes a more elevated [Ca2+]i. A high [Ca2+]i can then overload the SR with Ca2+ (4, 23, 32). This Ca2+ loading can be increased further if the Na/Ca exchange is upregulated, i.e., its density is increased, as has been reported to occur in cardiac hypertrophy (24, 51). Under such circumstances, if a spontaneous Ca2+ release from the SR takes place, ENa/Ca is shifted to potentials less negative than Vm and Na/Ca exchange works in forward mode, takes the Ca2+ out of the cell, and generates a large Iti. Thus a downregulated Na pump could be one of the most likely candidates initially responsible for the appearance of Iti in hypertrophied myocytes.

What causes the Na pump to downregulate? Our findings of a reduced Ipump activity and an elevated [Na+]i in hypertrophied myocytes confirm that downregulation of the Na pump plays a crucial role in Iti generation. Although hypertrophy-induced Na+-K+-ATPase inhibition (26, 34, 35, 38) and [Na+]i elevation (22, 39) are not new, the present data show for the first time that hypertrophy decreases Ipump, a current generated by the Na-pump enzyme. Reduced Ipump activity can also be due to altered isoform expression of the alpha  subunit of the Na+-K+-ATPase, as it has been demonstrated by Kim et al. (26) in a similar catecholamine model in which hypertrophy was induced by chronic norepinephrine infusion. These authors found that in hypertrophy the myocardial ouabain-binding sites are reduced and the alpha 3-isoform protein of the Na+-K+-ATPase is decreased, but there is no significant change in the alpha 1-isoform protein. Similar results were reported by Charlemagne et al. (7) who observed an isoform shift in another hypertrophy model induced by aortic constriction in the rat, in which myocardial ouabain binding characteristics and Na+-K+-ATPase activity changed to those resembling neonatal form.

What causes the Na/Ca exchange to upregulate? We have found that INa/Ca is increased in this model of hypertrophy in line with several studies showing that Na/Ca exchange is upregulated in compensated cardiac hypertrophy on the membrane current level (19, 30, 40, 45), the mRNA expression level (24), and the protein expression level (51). Regression analysis of the Ipump and INa/Ca data plotted against cell membrane capacitance indicates a reciprocal regulation for these two currents, insofar as the regression line fitted to the Ipump data points is shifted to lower current values, whereas the regression line fitted to the INa/Ca data points is shifted to higher current values in hypertrophied myocytes (see Figs. 7 and 10). These observations lead us to postulate that first in the order of the events induced by hypertrophy is a reduced Na-pump activity and an increased [Na+]i, which then upregulate the Na/Ca exchange to take excess Na+ out of the cell at the expense of bringing Ca2+ in. This adaptational mechanism, however, may facilitate SR Ca2+ overload and the appearance of DADs and Iti. Actually, DADs or Iti can be regarded as a "protective" mechanism that removes Ca2+ from the diseased myocyte at the expense of generating arrhythmias (9).

Clinical implications. Cellular mechanisms for cardiac arrhythmias are receiving increasing prominence in the literature as the membrane current changes underlying early and DADs that give rise to triggered activity are being evaluated in a number of different arrhythmia substrates (17). Iti has been shown to underlie clinically important arrhythmias, based on DADs resulting from cardiac glycoside toxicity (23), reoxygenation (4), and conditions of oxidative stress (32). DADs are more easily induced in cardiac hypertrophy secondary to renal hypertension (2) after myocardial infarction (41), in diabetic cardiomyopathy (36), and in failing rabbit ventricular trabecula (56). Our finding of substantially increased Iti in hypertrophied myocytes provides a cellular basis for DADs and triggered arrhythmias in cardiac hypertrophy.

The results indicate for the first time at the cellular level that Ipump is decreased, INa/Ca is increased, SR Ca2+ content is increased, and, consequently, Iti is greatly increased in myocytes from a rat model of catecholamine-induced compensated cardiac hypertrophy. Our findings also show that Iti, which can be the underlying mechanism of DADs, depends principally on the electrogenic Na/Ca exchange with smaller contributions from a Ca2+-activated nonselective cation current and a Ca2+-activated Cl- current. The latter is only expressed in hypertrophied myocytes. The crucial factor for the generation of Iti is an increased [Na+]i, which then causes an intracellular Ca2+ overload through a reverse Na/Ca exchange and oscillatory release of Ca2+ from the overloaded SR. These findings, if confirmed in other models and humans, may explain the propensity of hypertrophied hearts to triggered arrhythmias based on DADs.


    ACKNOWLEDGEMENTS

The authors thank Jeremy J. Coutinho, Joanna K. Lawton, and Timothy Grocott for skilled and valued technical assistance.


    FOOTNOTES

This work was supported by the British Heart Foundation.

Address for reprint requests and other correspondence: J. Mészáros, Dept. of Medicine, University of Liverpool, Duncan Bldg., Daulby St., Liverpool L69 3GA, UK.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 November 2000; accepted in final form 24 April 2001.


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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 281(2):H903-H914
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society



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