(Circulation. 1996;94:1674-1681.)
© 1996 American Heart Association, Inc.
Articles |
Occurrence and Properties of the Hyperpolarization-Activated Current If in Ventricular Myocytes From Normotensive and Hypertensive Rats During Aging
the Institute of Pharmacology, University of Ferrara, Italy, and the Department of Pharmacology (A.M.), University of Firenze, Italy.
Correspondence to Alessandro Mugelli, MD, Department of Preclinical and Clinical Pharmacology, University of Firenze, Viale G.B. Morgagni 65, 50134 Firenze, Italy. E-mail mugelli@stat.ds.unifi.it.
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Abstract |
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Background Cellular electrophysiological alterations may contribute to arrhythmogenesis in cardiac hypertrophy. An If-like current occurs in left ventricular myocytes (LVMs) isolated from the hypertrophied heart of old spontaneously hypertensive rats (SHR). Factors that influence If occurrence during development of cardiac hypertrophy were studied by determining its presence, amplitude, characteristics, and ß-adrenoceptor modulation.
Methods and Results Patch-clamped LVMs from young (2 to 3 months old) or old (18 to 24 months old) normotensive Wistar-Kyoto rats (WKY) and SHR were used. A diastolic depolarization phase was present in old SHR. An If-like current occurred in >90% of LVMs from old SHR and WKY and in 
15% of LVMs from young rats (P<.05). Activation curves of If were similar in old rats, with the midpoint at -92.9±2.9 mV in WKY (n=42) and -88.1±1.5 mV in SHR (n=25); maximal specific conductance was 54.4±1.7 in SHR and 20.1±0.5 picosiemens/picofarad in WKY (P<.05). In WKY, If amplitude was linearly related to membrane capacitance, an index of cell size (r=.53, P<.001). This relation was absent in SHR, in which a significant positive correlation was found between the heart weight to body weight ratio and If density. In both old WKY and old SHR, 0.1 µmol/L (-)-isoproterenol increased If amplitude by shifting its activation curve toward more positive potentials.
Conclusions In LVMs from both WKY and SHR, the occurrence of If increases with aging. Density appears linearly related to the severity of cardiac hypertrophy and increases with ß-adrenoceptor stimulation, which suggests that If may contribute to an increased propensity of the hypertrophied heart for arrhythmias.
Key Words: arrhythmia • electrophysiology • hypertrophy
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Patients with cardiac hypertrophy and heart failure often experience ventricular arrhythmias.1 2 Of the patients with heart failure who die, 35% to 50% of their deaths are sudden and unexpected.3
Many changes that occur in the hypertrophied heart are likely to predispose to arrhythmias, eg, fibrosis, loss of viable myocytes, and increased dispersion of refractoriness.4 Alterations in cellular electrophysiological properties have also been described in hypertrophied myocytes.5 Action potential prolongation has been consistently observed in association with cardiac hypertrophy in different animal models5 as well as in failing human hearts.6 We7 have recently defined the ionic basis of action potential prolongation in hypertrophied myocytes isolated from the hearts of SHR, which are widely used as a model for cardiac hypertrophy caused by pressure overload.5 The phenomenon appears to be due to a selective reduction of the repolarizing transient outward current, Ito.7 The decrease in Ito density has been described in almost all animal models that develop myocardial hypertrophy,7 8 9 10 11 12 and interestingly, a similar modification has been reported in human ventricular myocytes isolated from failing hearts.13 14
Hypertrophied myocytes isolated from SHR present another electrophysiological abnormality: they manifest a diastolic depolarization phase,15 also observed in multicellular preparations excised from the left ventricle of old SHR.16 More than 90% of myocytes isolated from the left ventricle of old SHR (ie, severely hypertrophied hearts) exhibit a time-dependent inward current, activated by hyperpolarization and which has the properties of the pacemaker current If,15 which is thought to contribute to pacemaker activity in both primary (sinus node) and secondary (Purkinje fibers) pacemakers.17 18 19 In contrast, an If-like current was recorded only in a minority of cells isolated from young SHR, in which the degree of myocardial hypertrophy was mild; in these cells, however, the prolongation of action potential duration was already statistically significant compared with age-matched normotensive rats (WKY).15 Thus, the two alterations (ie, reduction of Ito, leading to the prolongation of the action potential, and appearance of If, possibly related to the diastolic depolarization phase) seem to become apparent at different points in the development of cardiac hypertrophy.
If is generally thought to be present only in primary or secondary pacemakers.20 Its role in cardiac automaticity is still debated.21 22 It has been reported recently that in guinea pig and canine ventricular myocytes, an If-like current can be evoked consistently if a sufficiently negative hyperpolarizing step (ie, to -120 mV) is applied.23 24 These studies23 24 suggest that the shift in If activation to very negative potentials is a mechanism to avoid pacemaking in normal ventricular cells. Our results in rat hypertrophied myocytes15 are instead suggestive of a different regulatory mechanism for If occurrence in ventricular cells. We hypothesized15 that the gene that codifies for the If channel is turned off in "normal" ventricular myocytes (ventricular myocytes from young SHR that show a modest degree of hypertrophy) and can be switched on by the progression of hypertrophy. When expressed, the current activates at voltages near the physiological diastolic potential of the ventricular myocytes and consequently may play a functional role. We speculated15 that it might contribute to arrhythmogenesis in the hypertrophied myocardium. To obtain insight into the factors that influence If occurrence during development of cardiac hypertrophy due to pressure overload, we studied patch-clamped left ventricular myocytes isolated from the hearts of SHR and WKY of different ages (from 2 to 24 months), which therefore displayed different degrees of myocardial and cellular hypertrophy. In these cells, we determined If occurrence, amplitude, and characteristics and studied the influence of ß-adrenoceptor stimulation.
Portions of this work have been published previously in abstract form.25 26 27
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Methods |
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This investigation conforms to the rules for the care and use of laboratory animals of the European Community (Law No. 609 [1986] of the European Community). Male SHR and WKY were obtained from Charles River (Italy). They were maintained in our animal facility until they were killed. Systolic blood pressure, measured with a tail-cuff system (BP Recorder, Basile), was (WKY versus SHR) 121±6 versus 183±3 mm Hg at 2 months (n=7, P<.05) and 136±5 versus 208±14 mm Hg at 18 months (n=6, P<.05). Because SHR begin to have heart failure at the age of 18 months, old WKY and old SHR both were observed carefully on a daily basis for signs of cardiac decompensation, such as tachypnea and labored respiration. All animals used were free from these signs. The oldest animal used was 24 months old. Before sacrifice, the animals were injected with 500 U heparin IP (Liquemin, Roche) and anesthetized with ether. The hearts were rapidly excised, rinsed in cool LCS, and weighed.
Cell Isolation
Single left ventricular myocytes were isolated from young (2 to 3 months old) or old rats (18 to 24 months old) by use of a protocol based on previously described procedures.15 28 The time of perfusion with the enzymatic solution ranged from 5 minutes for 3-month-old rats to 20 minutes for 22-month-old rats, depending on the size of the heart. Perfusion was terminated when the heart became soft. After digestion of the heart, the left ventricle, including the septum, was cut off, chopped into small pieces, and gently stirred in LCS containing (in mmol/L) NaCl 120, KCl 10, KH2PO4 1.2, MgCl2 1.2, glucose 10, taurine 20, and pyruvate 5; the pH was adjusted to 7.2 with HEPES/NaOH. On occasion, cells were also obtained from the right ventricle. Cardiomyocytes that appeared in the supernatant were purified by gravity sedimentation, collected, and stored in LCS supplemented with albumin (Boehringer Mannheim, 1 mg/mL), penicillin (Gibco Laboratories, 50 U/mL), streptomycin (Gibco Laboratories, 50 µg/mL), and CaCl2 1 mmol/L. The yield of rod-shaped cells ranged from 75% at 2 months to 30% at 22 months, with no difference between WKY and SHR. Cells were stored at room temperature and used within 10 hours of their isolation.
Solutions
Cells used for the study were transferred to a small (0.2-mL) tissue bath and superfused by means of a peristaltic pump (Masterflex model 7524/05, Cole-Parmer Instrument Co) at a flow rate of 1.8 mL/min; a three-line system controlled by electronic valves allowed solutions to be changed rapidly. The recording chamber was mounted on the stage of an inverted microscope (TMS, Nikon). The control solution was a modified Tyrode's solution containing (in mmol/L) NaCl 137, KCl 5.4, CaCl2 1.5, MgCl2 1.2, HEPES 5, and glucose 10. The pH was adjusted to 7.35 with NaOH; the temperature was kept at 36±0.5°C. The internal pipette solution contained (in mmol/L) K-aspartate 130, MgCl2 2, Na2-ATP 5, CaCl2 5, EGTA 11, and HEPES 10 and was titrated with KOH to a pH of 7.2. To study the hyperpolarization-activated current, the extracellular control solution was modified to reduce the interference of components other than If15 by addition of (in mmol/L) BaCl2 8, MnCl2 2, CdCl2 0.2, and 4-aminopyridine 0.5, and the concentration of KCl was increased to 25 mmol/L (unless otherwise indicated). The liquid junction potential between the electrode tip and the extracellular solution was not corrected.15 When necessary, CsCl from a 1 mol/L stock solution was added to the Tyrode's solution to reach the final concentration of 4 mmol/L. A stock solution of (-)-isoproterenol (1 mmol/L) containing 1 gram of ascorbic acid per liter, was prepared on the day of the experiment and diluted in Tyrode's solution to reach a final concentration of 0.1 to 1 µmol/L. (-)-Isoproterenol hydrochloride and 4-aminopyridine were obtained from Sigma Chemical Co. All reagents were of analytical grade.
Experimental Protocols
The experimental setup was similar to that described by Cerbai et al.15 28 The whole-cell configuration of the patch-clamp technique was used to record action potentials and membrane currents. The electric signal was recorded by a patch amplifier (Axopatch 1D, Axon Instrument Inc), digitized (Labmaster TL-1 DMA, Scientific Solutions), and displayed on the monitor of a 386 personal computer and a digital oscilloscope (Nicolet 310, Nicolet Instrumentation Co). The cut-off frequency was 20 kHz. Current and voltage protocol generation, data acquisition, and analysis were performed with use of pClamp software (version 5.5.1, Axon Instrument Inc). MicroCal Origin (version 3.5, MicroCal Software Inc) was used for additional analysis.
After intracellular access was gained, whole-cell Cm was measured by application of ±10-mV voltage steps from a holding potential of -70 mV and calculated as described previously15 ; no capacitance correction was used.
Recording was started after 5 minutes of dialysis of the cell. Action potentials were elicited at a rate of 0.2 Hz and sampled at 2 kHz. Voltage-clamp experiments were performed during superfusion with the modified Tyrode's solution (see above); the presence of If (ie, of a hyperpolarization-activated, time-dependent, increasing inward current blocked by addition of 4 mmol/L CsCl) was ascertained by application of a hyperpolarizing step to -120 mV from a holding potential of -50 mV. Steps were applied at low frequency (maximum rate, 0.1 Hz) and currents sampled at 0.5 to 1 kHz. Amplitude of If was measured as the difference between the instantaneous current at the beginning of the hyperpolarizing step and the steady-state current recorded at the end of the step and was expressed as current density, ie, normalized to Cm. In most of the cells in which If occurred, an analysis of its voltage dependence was performed as previously reported.15 Current-voltage relations were generated by use of clamping from a holding potential of -40 to -50 mV to more negative voltages (-60 to -160 mV) in 10-mV increments; for each cell, specific conductance of If was determined as a function of membrane potential according to the equation g=I/(Vm-Vrev), where g is the conductance calculated at membrane potential Vm, I the current amplitude, and Vrev the reversal potential of the current calculated from the analysis of tail currents. The maximal specific conductance (gmax) was obtained by use of a computer-calculated Boltzmann fit according to the equation g=gmax/{1+exp[(VH-Vm)/k]}, where g is the conductance calculated at membrane potential Vm and k is the slope factor describing the steepness of the activation curve. The Vrev of If at 5.4 or 25 mmol/L extracellular potassium concentration was evaluated by measurement of the amplitude of tail currents elicited by steps in the range of -50 to +20 mV and after a hyperpolarizing step to -120 mV, which maximally activated If. Values, normalized to Cm, were fitted by a linear relation that intersected the x axis at Vrev.15
Data Analysis
The occurrence of If, expressed as the ratio between the number of cells that showed cesium-sensitive, barium-insensitive, time-dependent increasing inward current elicited by hyperpolarizing steps and the total number of cells studied, was compared in different groups of animals by means of the 
2 test. All other data are presented as mean±SEM. Intergroup comparisons were performed by means of Student's t test or by one-way ANOVA followed by Tukey test, as appropriate. Correlation between different variables was calculated by linear regression analysis. A value of P<.05 was considered significant.
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If in Young and Old WKY and SHR Myocytes
Both young and old SHR showed a prolonged action potential compared with WKY; a diastolic depolarization phase, however, was present only in old SHR (Fig 1
). We determined If occurrence and amplitude in the different groups, as described in "Methods." If occurred in >90% of myocytes isolated from old SHR and old WKY but only in a minority (15%) of cells from young animals (P<.05; Fig 2a). The traces in Fig 2b and 2c demonstrate that in the few myocytes from young WKY (5 of 29) and SHR (4 of 37) in which the current was found, its amplitude was small (mean density at -120 mV was 1.2±0.3 pA/pF in WKY and 2.1±0.7 pA/pF in SHR), and although it was larger in SHR than in WKY, no statistical comparison was attempted because of the very low frequency of observations. As demonstrated in the recordings shown in Fig 2d and 2e, despite a similar occurrence, If amplitude was markedly greater in myocytes from old SHR than in age-matched WKY (5.7±0.5 pA/pF, n=25 versus 2.1±0.3 pA/pF, n=48; P<.05).
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) or presence (
) of 4 mmol/L CsCl.
The voltage dependence of If activation in the two groups of old rats is reported in Fig 3. In cells from old WKY (Fig 3a) and old SHR (Fig 3b), current amplitude, here reported as current density, increased as a function of voltage. The subsequent step to +20 mV elicited tail outward currents that reflected If deactivation. Interestingly, tail currents were larger in WKY (Fig 3a) than in SHR (Fig 3b) because of the different densities of the transient outward current (Ito),7 which likely was activated by the depolarizing step. Mean current specific conductance, normalized to Cm, was plotted as a function of the hyperpolarizing step potential. For both WKY and SHR, the activation curve was fitted by a Boltzmann function and showed a similar threshold for current activation; the VH and the slope (k) were also comparable (VH=-92.9±2.9 mV in WKY, n=42, and -88.1±1.5 mV in SHR, n=25; k=-7.9±2.0 mV in WKY and -7.6±1.5 mV in SHR). Specific conductance, however, was higher in SHR for any level of potential (Fig 3c); the current was fully activated at -120 mV in both groups, but maximal specific conductance was 2.5 times larger in SHR (54.4±1.7 pS/pF) than in WKY (20.1±0.5 pS/pF; P<.05). Voltage dependence of If was also studied in a few myocytes from young WKY (2 cells) and SHR (4 cells) that showed If activation on hyperpolarization to -120 mV. Mean values of VH were -91 mV in young WKY and -87 mV in young SHR, a figure that was not different from that observed in old rats. Moreover, when a similar protocol was applied to 6 myocytes from young WKY and to 13 cells from young SHR that did not show the presence of If according to the previously stated criteria, it consistently failed to evoke If even at the most negative potentials.
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, n=42) and old SHR (
) of If activation in myocytes from old WKY (
Current recordings displayed in Fig 3a and 3b
show that the inward current was activated on hyperpolarization with similar kinetics in both WKY and SHR. Time constants calculated from the single exponential fitting of individual current traces were reported as a function of the step voltage (Fig 3d). It clearly appears that the two curves are fully superimposable. In myocytes from old WKY, Vrev was shifted by 10 mV toward more negative potentials as a result of a decrease in [K+]o from -22.9±3.0 mV at 25 mmol/L [K+]o (n=9) to -34.4±2.6 mV at 5.4 mmol/L [K+]o (n=3). The corresponding values in 18- to 20-month-old SHR were -17±3.8 mV and -25±2.7 mV, respectively.15 The permeability (P) ratio (PNa/PK) calculated from Vrev values at the two [K+]o levels was 0.25.
If and Degree of Cellular and Cardiac Hypertrophy
We tried to assess the relative importance of aging and of the mechanical overload on If occurrence and amplitude. If occurs in a minority of cells from young rats and in 90% of cells from old (18 to 20 months old) rats (see Fig 2a); If was present in 100% of the myocytes from older (23 to 24 months old) WKY (n=10) and SHR (n=39). An increase in cell size occurs physiologically during aging29 and pathologically in hypertension as a consequence of pressure overload.30 We looked at the relationship between cell dimensions and If amplitude by plotting individual current values obtained in myocytes from WKY or SHR of different ages (from 2 to 24 months) against the respective values of Cm (an index of cell size7 15 28 ). From the plot of Fig 4a, it appears that If amplitude in normotensive animals somehow is linearly related to Cm (r=.53, P<.001) in that the increase in cell size (ie, Cm) is, as expected, associated with a larger current. However, in WKY, If density, a measure that excludes the influence of cellular hypertrophy, did not change significantly with aging; in fact, if we group the cells according to the age of the normotensive animals (2 to 3, 18 to 20, and 23 to 24 months), the mean±SEM value of If density in each subgroup was 1.2±0.3 pA/pF (n=5), 2.1±0.3 pA/pF (n=48), and 1.9±0.5 pA/pF (n=10), respectively. Thus, the main effect of aging per se seemed to be an increase in the percent of cells in which If occurred, with no effect on its density.
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As shown in Fig 4b, in SHR myocytes, If amplitude was larger than in WKY and the Cm also was generally larger (as expected for more hypertrophied cells), but no apparent relationship between cell dimensions and If amplitude was present (r=.002, P=.98), which suggests that other factors may be implicated.
In SHR but not in WKY (Fig 5), If density was related to the HW/BW, which is a measure of cardiac hypertrophy; this suggests that the severity of cardiac hypertrophy is an important determinant of the size of If. Because the degree of hypertrophy in SHR depends at least in part on the duration of the hypertension, it is not surprising that If density in SHR increased with aging: 2.1±0.7 pA/pF (n=4), 5.7±0.5 pA/pF (n=25), and 8.2±2.0 (n=39) in 2- to 3-, 18- to 20-, and 23- to 24-month-old rats, respectively. The hypothesis that mechanical overload is an important determinant of the size of If is further supported by the observation that in myocytes isolated from the right ventricle of 18- to 20-month-old SHR, If density was smaller than that recorded in myocytes from the left ventricle and similar to that recorded in myocytes from the left ventricle of age-matched WKY. In fact, in 5 of 8 myocytes in which an activation curve was recorded, If density was 3.6±0.5 pA/pF (Cm=283.9±39.9 pF) and VH was -84.4±2.0 mV.
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Effect of (-)-Isoproterenol on If
The effect of (-)-isoproterenol (0.01 to 1 µmol/L) on If was studied. Preliminary experiments demonstrated that the increase in If amplitude, measured during a hyperpolarizing step to -80/-90 mV (ie, in the range of the VH) in myocytes from both old WKY and old SHR, was 40% with 0.01 µmol/L (-)-isoproterenol (n=3); 0.1 µmol/L caused an effect similar to that obtained with 1 µmol/L (n=8) and which was already maximal. Fig 6 shows a representative example of the effect of (-)-isoproterenol (0.1 µmol/L) on If amplitude in WKY (Fig 6a) and SHR (Fig 6b). When a second step to -140 mV was applied (at which the current is maximally activated), the amplitude of the current (ie, the difference between peak and steady-state current) was reduced in the presence of ß-adrenergic stimulation. This result suggests that in ventricular myocytes, as in pacemaker cells,20 the effect of (-)-isoproterenol on If is not due to an increase in conductance but to a shift of its activation curve toward more positive potentials. When If was not present, ie, in the majority of cells isolated from young WKY or SHR, the addition of (-)-isoproterenol never caused it to appear, even at very negative potentials. This is clearly demonstrated in Fig 6c, which shows the currents elicited by a step to -130 mV in a myocyte from 2-month-old WKY in the absence and presence of 1 µmol/L (-)-isoproterenol. Similar results were obtained consistently in all the cells in which this procedure was applied (n=3 and n=4 for young WKY and SHR, respectively). The shift of the activation curve of If caused by (-)-isoproterenol in a myocyte from an old SHR is better illustrated in Fig 7. In control conditions (Fig 7a), If is clearly activated only by hyperpolarizing steps to -80 mV or more negative; in the presence of (-)-isoproterenol (Fig 7b), the current is increased at all the potentials, and at -70 mV, the current is as large as it was at -80 mV in control conditions. The activation curves in the absence and presence of (-)-isoproterenol (Fig 7c) clearly document a parallel shift of 10 mV toward more positive values. (-)-Isoproterenol (0.1 µmol/L) increased If density in both old WKY (from 0.46±0.19 to 0.84±0.30 pA/pF, n=4; P<.05) and old SHR (from 1.4±0.5 to 2.2±0.5 pA/pF, n=3; P<.05). The percent increase in density caused by (-)-isoproterenol was consistently lower in SHR (79% with 0.1 µmol/L and 92% with 1 µmol/L) than in WKY (116% and 106%, respectively).
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Discussion |
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The present results demonstrate for the first time that the pacemaker current If in rat ventricular myocytes is regulated differently in normotensive rats and SHR. In both WKY and SHR, the percent of myocytes with If increases with aging. However, If amplitude appears to be under the control of factors other than aging. First, it appears to be larger in old SHR than in old WKY. Second, If density increases with aging only in SHR, in which it appears linearly related to the severity of myocardial hypertrophy. This suggests that it may play a more relevant role as the disease progresses. The finding that a diastolic depolarization phase can be recorded only in old SHR is in keeping with this view. The current is modulated by ß-adrenoceptor stimulation, which suggests that the sympathetic tone may influence its functional relevance.
We15 have previously demonstrated in myocytes from old SHR the presence of a current that has the electrophysiological characteristics of If in pacemaker tissues. It typically is elicited on hyperpolarization, is fully activated at -120 mV, is cesium sensitive and barium insensitive, and has a reversal potential consistent with selectivity for Na+ and K+. In the present study, we confirm and extend those results and show that the occurrence of If in left ventricular myocytes increases as a function of the age of the animal in both SHR and WKY; in fact, although it is present in a minority of myocytes isolated from young rats, it is present in all myocytes isolated from rats aged 23 to 24 months. However, the density of the current is greater in SHR than in WKY and reaches its maximal value in old SHR. If one assumes that the functional consequence of If is the presence of a diastolic depolarization phase, it may be hypothesized that this phase is evident only in those cells that have a certain "threshold" value of If density. In effect, a diastolic depolarization could be recorded only in myocytes from old SHR (Reference 15, present results), but an If of smaller amplitude appears to be present in old WKY also. This finding is largely in agreement with our previous observations in papillary muscles,16 in which a diastolic depolarization phase was present in 18-month-old SHR but not in young SHR and WKY. In papillary muscles from old WKY, a diastolic depolarization was also recorded, even if its amplitude and sensitivity to isoproterenol were significantly lower than in age-matched SHR.16
Regardless of the rate of occurrence of If or its density, the basic electrophysiological properties (eg, VH) are similar to those previously described.15 We also found that when If is not elicited on hyperpolarization to -120 mV, it cannot be activated at more negative voltages, nor can it be induced by isoproterenol. Thus, in the rat, the situation seems to be quite different from that reported by Yu et al23 24 in guinea pig and canine myocytes. In their experiments (performed in the presence of cAMP in the pipette or in the presence of external isoproterenol), an If-like current apparently is present in all the cells when a sufficiently negative hyperpolarizing step is applied. They suggested23 24 that the shifting of If activation to a very negative and unphysiological potential could be the mechanism for avoiding pacemaking in ventricular cells. Even if the factors that control If expression are largely unknown, we have hypothesized previously15 that a less wasteful approach is operative in the normal rat: the If gene could be turned off, and the If channel is not synthesized. This would explain why If is practically absent in young WKY and SHR. In the few cells of young SHR and WKY in which an If-like current was detected in the present study, it was activated at a similar voltage, but its density was lower than in old SHR and possibly not sufficient to play any role. It could be suggested that aging and/or the hypertrophic process somehow turns on the If gene. In old SHR, the If channel is oversynthesized and If density increases. This overactivation could explain the lack of correlation between cell size and If amplitude in SHR. Because the current is activated at voltages near the physiological diastolic potential in hypertrophied rat myocytes, we speculated15 that it might contribute to the increased propensity of the hypertrophied heart to experience arrhythmias, as observed experimentally16 31 32 and clinically.33 The present demonstration that If activation is shifted to more positive values by ß-adrenergic stimulation strengthens this hypothesis. The effect of ß-adrenergic stimulation appears to be less in SHR than in WKY; this aspect requires further experimentation but is not unexpected, because reduction in activation of adenylyl cyclase has been reported in old SHR,16 and a downregulation of ß-adrenoceptors may occur with aging.28
The SHR is a widely used model for human hypertension.34 SHR develop left ventricular hypertrophy in response to pressure overload through a process that evolves continually during the life of the animal.35 36 The morphometric changes that occur in the myocardium of SHR have been documented clearly.30 36 37 At 2 to 3 months of age, SHR present a mild degree of myocardial hypertrophy,36 37 with the myocyte dimension only slightly increased compared with normotensive rats.36 37 At 18 months of age, SHR develop severe left ventricular hypertrophy, and the myocytes manifest the highest value of cross-sectional area.30 Between the ages of 18 and 24 months, 57% of male SHR have evidence of cardiac decompensation, and only 13% survive to 24 months without evidence of heart failure.38 Of similarly followed WKY, none develop heart failure and 92% survive until 24 months of age.38
We have previously observed that papillary muscles16 and myocytes7 isolated from old (18 to 20 months) SHR without signs of heart failure have prolonged action potentials due to a selective reduction of the Ito density.7 A similar reduction of Ito density has been reported in other rat models of pressure overload.9 10 11 12 On the other hand, the L-type calcium current density is not modified in hypertrophied myocytes.7 39 40 These findings suggest that during hypertrophy, protein synthesis of channels may be regulated differentially: the increase in cell size and in synthesis of L-type calcium channels appears to be concomitant, whereas a dissociation exists for Ito and If channels. In the case of a hypertrophied cell, a decrease in density is likely the consequence of nonactivation of the genetic expression (eg, Ito channels in SHR7 or during aging41 ), no change in density is a consequence of an activation (eg, L-type calcium channels in SHR and/or during aging7 39 40 41 ; If channels in WKY during aging, present results), and an increase in density is a consequence of an overactivation (If channels in old SHR, present results). Furthermore, many studies42 43 support the idea that an increase in hemodynamic loading causes a general reexpression of genes that encode fetal proteins. For example, it is known that T-type calcium channels predominate over L-type calcium channels in embryonic ventricles44 ; these channels are absent in adult ventricular myocytes but are reexpressed in hypertrophied myocytes.45 Neonatal rat ventricular myocytes beat spontaneously,46 and If seems to be involved in the pacemaker activity of chick embryonic ventricular myocytes.47 Consequently, the expression of If channels in hypertrophied left ventricular myocytes could be due to their reentry into an active growth phase, stimulated by pressure overload.
The explanation for the increased occurrence of If in normotensive rats during aging is not obvious; the main factor that influences myocardial changes during aging is vascular senescence, and aortic impedance is the true load of the heart. Many of the cardiovascular structural and functional changes that occur during aging48 are similar to those associated with hypertension in the younger subject.49 For example, papillary muscles isolated from the heart of old or senescent rats have a prolonged action potential.50 A similar but more marked age-dependent prolongation of action potential duration is observed in papillary muscles isolated from hypertensive rats.16 Myocytes isolated from the heart of normotensive or hypertensive rats at different ages retain the electrophysiological properties of the multicellular preparations and have prolonged action potentials.7 28 41 The prolongation of action potential duration associated with senescence or hypertension appears to be due to the same change in ionic currents, ie, a selective reduction of the repolarizing potassium current Ito.7 41 All these data support the concept that hypertension may be seen as accelerated aging49 even from the electrophysiological point of view. This may in part explain our present results that show that If can also be present in old normotensive rats. Because we do not have a group of rats with an intermediate rate of occurrence of If, it is impossible to say whether the phenomenon, ie, the increase in percentage of cells in which If is expressed, occurs earlier in SHR than in WKY myocytes, in keeping with the previously described concept of hypertension as accelerated aging. The observation that If is also present in myocytes isolated from the right ventricles of old SHR, which should not be exposed to mechanical stress (but certainly are exposed to neurohumoral activation), should suggest that aging may be a sufficient stimulus for If expression. Further experimentation in this area, however, is required.
Our data indicate that the degree of cardiac hypertrophy, as expressed by the HW/BW, seems to be important in modulating the density of If, but they fail to clarify the role of hypertrophy as such. How the process of development of cardiac hypertrophy is able to influence If amplitude can be clarified only by studies that use different induction models of cardiac hypertrophy.
In conclusion, our results suggest that in rat left ventricular myocytes, the presence and amplitude of If are influenced by several factors that are difficult to dissect. They clearly demonstrate that (1) the activation voltage for If is shifted toward more positive values by ß-adrenergic stimulation, and (2) the density of the current is larger in myocytes isolated from severely hypertrophied hearts. On the whole, these results indicate that If may represent an important arrhythmogenic mechanism in the hypertrophied heart.
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Acknowledgments |
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This work was supported by grants from Telethon (project Nos. 526 and 709) and CNR (Target Project on Aging).
Received January 17, 1996; revision received April 14, 1996; accepted April 19, 1996.
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References |
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