Cardiovascular Research

Cardiovascular Research 2000 45(2):388-396; doi:10.1016/S0008-6363(99)00344-2
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Long-term treatment of spontaneously hypertensive rats with losartan and electrophysiological remodeling of cardiac myocytes

Elisabetta Cerbai^a, Antonio Crucitti^a, Laura Sartiani^a, Petra De Paoli^a, Roberto Pino^a, Maria Luisa Rodriguez^c, GianFranco Gensini^b and Alessandro Mugelli^a,*

^aDepartment of Preclinical and Clinical Pharmacology, University of Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
^bInstitute of Internal Medicine and Cardiology, University of Firenze, Firenze, Italy
^cInstitute of Pharmacology and Toxicology, Universitad Complutense de Madrid, Madrid, Spain

^* Corresponding author. Tel.: +39-55-427-1264; fax: +39-55-427-1285 mugelli{at}pharm.unifi.it

Received 15 June 1999; accepted 22 September 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 
Objective: Cardiac hypertrophy due to pressure overload is associated with several cellular electrophysiological alterations such as prolongation of action potential duration (APD), decrease in transient outward current (I_to) and occurrence of the pacemaker current I_f. These alterations may play a role in sudden arrhythmic death, which is a major risk factor in myocardial hypertrophy and failure. Since angiotensin II is a key signal for myocyte hypertrophy, we tested if an 8-week treatment of old spontaneously hypertensive rats (SHR) with the antagonist of type-1 angiotensin II receptor (AT₁), losartan (10 mg/kg/day), was able to influence the cellular electrophysiologic remodeling associated with cardiac hypertrophy. Methods: Left ventricular myocytes were isolated from control (CTR) or losartan-treated (LOS) 18-month old SHR. Patch-clamped LVM were superfused with a normal Tyrode's solution (to measure action potential) or appropriately modified Tyrode's solution (to measure I_to and I_f). Results: Heart weight to body weight ratio (HW/BW) was significantly smaller in LOS (5.69±0.25 mg/g) than in CTR rats (6.67±0.37 mg/g; P<0.05). Membrane capacitance, an index of cell size, was significantly reduced in LOS (342±12, n=92) vs. CTR (422±14 pF, n=96, P<0.001). APD was significantly shorter in LOS than in CTR (at –60 mV: 197±23 vs. 277±19 ms, n=28, P<0.001); this effect was paralleled by a larger maximum I_to density in the LOS group (LOS: 15.1±1.4 pA/pF, CTR: 10.0±0.8 pA/pF) (n=27, P<0.02). I_f, elicited by hyperpolarizing steps (range: –60 to –130 mV), was consistently recorded in SHR cells; however, its maximal specific conductance was significantly lower in LOS than in CTR rats (28.6±3.6 vs. 54.2±8.0 pS/pF, n=55, P<0.001). Voltage of half-maximal activation (V_1/2) of both I_to and I_f was unchanged by the treatment. Conclusions: AT₁ receptor blockade with losartan prevents the development of myocyte hypertrophy and associated electrophysiological alterations in old SHR.

KEYWORDS APD, action potential duration; AT₁, type-1 angiotensin II receptor; C_m, membrane capacitance; HW/BW, heart weight to body weight ratio; I_f, pacemaker current; I_to; transient outward current; RAS, renin–angiotensin system; SHR, spontaneously hypertensive rats; V_1/2, voltage of half maximal activation

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 
Sudden death due to ventricular arrhythmias is a major risk factor in patients with myocardial hypertrophy and failure [1–3]. Despite optimal medical treatment, mortality in heart failure patients remains high. Recently, the ELITE study reported that treatment of elderly heart failure patients with the type-1 angiotensin II receptor (AT₁) antagonist losartan was unexpectedly able to reduce mortality by lowering the incidence of sudden deaths [4]. In order to gain insight into the mechanism by which losartan exerts its favorable effect, we planned to study the effect of chronic treatment with losartan on the electrophysiological alterations occurring in SHR with severe cardiac hypertrophy.

Spontaneously hypertensive rats (SHR), a widely used genetic model of chronic hypertension, progressively develop myocardial hypertrophy, whose severity increases with aging and finally evolves toward failure [5]. In middle-aged SHR the incidence of ventricular arrhythmias (VAs) is higher than in age-matched normotensive rats, and can be significantly reduced by long-term treatment with angiotensin-converting enzyme inhibitors (ACE-I) [6,7]. The reduction in VAs seems to be correlated with the regression of both myocardial hypertrophy and fibrosis [6,7]. Indeed, recent evidence suggests an important role of angiotensin II produced within the myocardium in regulating fibroblast collagen turnover [8] and in contributing to cardiac myocyte hypertrophy [9–11].

However, structural changes occurring in the hypertrophied myocardium are not the only factors involved in the increased propensity of the hypertrophied heart to arrhythmias. Myocyte hypertrophy is a process always accompanied by cellular phenotypic alterations [12]. Development of cellular hypertrophy is associated with changes in the cellular electrophysiological properties of cardiac myocytes. These alterations have been widely studied in ventricular myocytes isolated from the hypertrophied heart of SHR and other animal models of hypertrophy and failure as well as in myocytes isolated from human failing hearts [13]. The most common electrophysiological alteration observed in the hypertrophied or failing myocyte is the prolongation of action potential duration. This phenomenon appears to be due to a selective reduction in the transient outward current I_to, and has been clearly documented both in myocytes isolated from 18-month old SHR [13] and in myocytes isolated from the failing human heart [14–16]. Another electrophysiological alteration recently described both in human and in rat hypertrophied myocytes (the latter isolated from the left ventricle of the 18-month old SHR) is the overexpression of an inward current having the characteristics of the pacemaker current I_f [17–20]. These cellular electrophysiological alterations are likely to contribute to the increased propensity of the hypertrophied or failing human heart to life-threatening arrhythmias [16,19].

Thus, the SHR represents a suitable and predictive model to study the cellular electrophysiological alterations occurring during hypertrophy: in fact the prolongation of the action potential duration and the underlying decrease in I_to as well as the increase in I_f density are phenomena which accompany the development of hypertrophy, or in other words the age of the animals. Furthermore, SHR rats are widely used to assess the effect of long term treatment with drugs. It has been demonstrated that treatment with ACE inhibitors is able to reduce the development of hypertrophy and fibrosis as well as to reduce the incidence of spontaneous or induced arrhythmias [6,7]. Recently it has been reported that treatment of SHR with captopril is able to influence the cellular electrophysiological properties of isolated cells, in that the prolongation of action potential duration is prevented [21].

ACE inhibitors affect both the angiotensin and bradykinin pathways. The availability of AT₁ antagonists will disclose the importance of angiotensin II in the progression of these alterations. For all these reasons, we thought it would be interesting to assess the effect of long-term treatment with losartan, a selective AT₁ antagonist, on the electrophysiological alterations occurring in SHR during the development of severe cardiac hypertrophy. To this aim, we studied cell capacitance (an index of cell size), action potential, and membrane currents (I_to and I_f) from left ventricular myocytes isolated from the heart of 18-month old SHR after 8-week of treatment with saline or losartan. Preliminary data have been published in abstract form [22].

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 
2.1 Experimental design
Twenty-four spontaneously hypertensive (SHR) male rats of 16 months (Charles River, Italy) were kept in our animal facility; 12 of them were given 10 mg/kg/day losartan (losartan-treated group) dissolved in tap water for 8 weeks; a control group of 12 rats received only tap water during the same period of time, that is, until the age of 18 months.

The water consumption by each animal was measured twice a week, and the amount of drug dissolved in drinking water adjusted, in order to keep the daily dosage at 10 mg/kg/day. At the end of treatment, systolic blood pressure was measured using a tail-cuff system (Basile, Italy). Despite the fact that systolic blood pressure was measured in only five animals for each group, and not routinely, due to the high risk that old SHR may die when submitted to the stress of manipulation, previous studies have clearly documented that losartan 10 mg/mg/day has a modest antihypertensive effect in SHR [23,24]; this is the case in our study as well.

2.2 Cell isolation
This investigation conforms to the rules for the care and use of laboratory animals of the European Community (86/609/CEE) and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Single left ventricular myocytes were isolated from control or losartan treated SHR using a protocol based on previously described procedures [14,17]. Rats were injected with 500 I.U heparin i.p., anesthetized with ether and decapitated. After thoracotomy, the heart was rapidly excised, weighed, mounted in a Langendorff apparatus and perfused for 20 min with a low-calcium solution (LCS, see Solutions) prewarmed to 37°C and equilibrated with 100% O₂. The solution was then quickly changed to LCS plus 1 mg/ml collagenase (Type I, Sigma, Italy), 0.03 mg/ml dispase (Boehringer, Italy), 1 mg/ml albumin (Fatty Acid Free Fraction V, Serva, Italy) for 25–30 min. The left ventricle and the septum were removed with fine scissors, minced and the pieces were stirred in the LCS. Cardiomyocytes that appeared in the supernatant were purified by gravity sedimentation, collected and stored in LCS at room temperature. The isolated cells were kept at room temperature in LCS, supplemented with 1 mM CaCl₂ and 4% penicillin/streptomycin (Gibco BRL, Italy) and used within the day.

2.3 Electrophysiological recordings
Cells were placed in an experimental bath on the platform of an inverted microscope (Nikon Diaphot TMD, Japan). The patch-clamp technique (whole cell recording) was used to measure electrophysiological properties of the isolated myocytes. Experiments were performed using a patch amplifier (Axopatch-1B, Axon Instruments, CA, USA) interfaced to a 486 personal computer by means of a DAC/ADC interface (Labmaster Tekmar, Scientific Solutions, USA). Data were viewed on-line on an analogic oscilloscope and on a computer screen. Experimental control, data acquisition and preliminary analysis were performed by means of the integrated software package pClamp (Axon Instruments, CA). Cells were superfused with normal Tyrode's solution or with Tyrode's solutions properly modified to measure the transient outward current (I_to) and the hyperpolarization-activated inward current (I_f) (see Solutions). Temperature was maintained in the range of 36–37°C. Patch-clamp pipettes, prepared from glass capillary tubes (Garner Glass Co., CA, USA) by means of a two-stage vertical puller (Hans Otchoski, Homburg, Germany), had a resistance of 1.5–2.5 M when filled with the internal solution (see Solutions). The patch-clamped cell was superfused by means of a temperature-controlled micro-superfusor, which allowed rapid changes in the solution bathing the cell.

2.4 Solutions
The composition of the solutions employed was as follows (in mM): Low Calcium Solution (LCS): NaCl 120, KCl 10, KH₂PO₄1.2, MgCl₂ 1.2, D(+)-glucose 10, taurine 20, HEPES–NaOH, 10 (pH 7.0). Normal Tyrode's solution: NaCl 140, KCl 5.4, CaCl₂ 1.5, MgCl₂ 1.2, glucose 10, HEPES–NaOH 5 (pH 7.35); Tyrode's solution for transient outward current: Normal solution plus 0.5 mM CdCl₂ used to block calcium current. Tyrode's solution for hyperpolarization-activated inward current measurements: NaCl 140, KCl 25, CaCl₂ 1.5, MgCl₂ 1.2, BaCl₂ 5, MnCl₂ 2, 4-aminopyridine 0.5, glucose 10, HEPES–NaOH 5 (pH 7.35); this solution allowed for the reduction of interference from other currents, i.e. L-type calcium current, inward rectifier-like current and transient outward potassium current. Pipette solution: K-aspartate 130; Na₂-ATP 5, MgCl₂ 2, CaCl₂ 5, EGTA 11, HEPES–KOH 10 (pH 7.2; pCa 7.0).

2.5 Data analysis and statistics
Data analysis and fitting were performed by using the program Origin 4.1 (MicroCal Software Inc., MA, USA) running on a Pentium personal computer. For fitting functions, non-linear models of convergence to solutions were used.

Action potentials were elicited at a rate of 0.2 Hz and sampled at 2 kHz. Depolarizing steps (700 ms) to positive potentials activated a 4-aminopyridine-sensitive outward current, which was composed of transient and steady-state component, as already described in rat ventricular myocytes [14]. Outward currents were evoked by steps in the range of –40 to +70 mV from a HP of –70 mV (sampling rate: 5 kHz); a pre-step to –40 mV was used to inactivate sodium current. Series resistance and membrane capacitance were compensated in order to minimize the capacitive transient. I_to was measured as peak outward current at the beginning of the depolarizing step, and normalized with respect to the membrane capacitance value (see below) [14]. Steady-state currents were measured at the end of the depolarizing steps.

I_f was defined as a time-dependent, hyperpolarization-activated inward current completely and reversibly blocked by adding 4 mM CsCl to the extracellular solution [17]. I_f was evoked by hyperpolarizing steps in the range of –60 to –140 mV from a HP of –40 mV (sampling rate: 0.5 kHz); the duration of the step was decreased (from 3200 to 1600 ms) as the activation became progressively faster. To evaluate steady-state values of the hyperpolarization-activated current, data were fitted to an exponential decay. Current amplitudes were measured as the difference between the value at steady state and that at the beginning of the test pulse, and normalized with respect to the membrane capacitance value (see below). Specific conductance was determined as a function of membrane potential according to the following equation:

where g_f is the conductance (in pS/pF) calculated at the membrane potential V_m, I the current density (in pA/pF) and V_rev the reversal potential of the fully activated current [17].

For both I_to and I_f, the Boltzmann function:

was used to fit the activation data, where V (mV) is the test membrane potential, V_1/2 (mV) is the fitted potential for half-maximal activation and k (mV) is related to the slope of the activation curve.

Cell membrane capacitance (C_m) was measured by applying a ±10-mV pulse starting from a holding potential of –70 mV. The current transient following this clamp protocol was fitted with a mono-exponential model to compute series resistance (R_s) and then C_m using the two equations given below:

and

where I_peak is the maximum level of current (relative to the holding current) following the depolarization and is the time constant of the exponential current decay. The membrane capacitance values obtained have been used to compute ionic current densities (I_to and I_f density in pA/pF).

All data are expressed as mean±standard error of the mean (S.E.M.). Statistical analysis was performed by means of the Graph Pad Instat program, using the Mann–Whitney test (non-parametric test) for comparing membrane capacitance values, and Student's t-test for grouped data (two-sided P value) for all other values. A probability value of less than 0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 
The dose of losartan (10 mg/kg/day) was chosen on the basis of literature data, showing that this dose causes a modest reduction in blood pressure, but significantly reduces cardiac hypertrophy [23,24]. The data have been confirmed in our study: in five rats for each group we measured blood pressure, which was only slightly reduced by losartan treatment (225±30 vs. 234±24 mmHg in control; not significant). Blood pressure measurements were not performed routinely, because old SHR may die suddenly when manipulated by the operator.

Despite the lack of a statistically significant reduction in blood pressure, 8 weeks of treatment with losartan was able to significantly reduce the degree of cardiac hypertrophy as measured by the HW/BW ratio. HW/BW was 6.67±0.37 mg/g in control rats and 5.69±0.25 mg/g in losartan-treated rats (P<0.05). At least part of the reduction in cardiac hypertrophy appears to be due to a decreased cell size. This is shown in Fig. 1, where the distribution of membrane capacitance in the control and treated groups is reported. Cell membrane capacitance is a good index of cell dimension; it can be appreciated that the two cell populations (96 cells from control and 92 from losartan-treated) have a different distribution. The cells isolated from the heart of rats treated with losartan show a distribution toward the smaller dimensions, the mean value being 342±11.6 vs. 422±13.9 pF in the control group. These data are consistent with our previous results in SHR and normotensive WKY rats, in which the cell capacitance measurement nicely reflected the degree of cell hypertrophy.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Frequency histogram of cell membrane capacitance (in picoFarad) as measured in the whole-cell configuration in single myocytes isolated from the left ventricle of control (a) or losartan-treated SHR (b). Lines represent the result of fitting with a Gaussian distribution; the difference between mean±S.E.M. values (a: 422.3±13.9 pF, n=96; b: 342.3±11.6 pF, n=92) was statistically significant (P<0.0001).

 
Fig. 2 shows representative action potentials recorded from ventricular myocytes isolated from losartan-treated or saline-treated SHR. It is apparent that action potential duration is shorter in the losartan-treated myocyte. This was a consistent finding, as shown in Fig. 3, which summarizes the results obtained in 28 myocytes. It is apparent that the only change found was on the action potential duration, which was significantly shortened either at –20 mV (92±15 vs. 141±14 ms) or at –60 mV (197±23 vs. 277±19 ms). The other action potential characteristics (measured in losartan vs. control) were completely unaffected: amplitude (104.9±2.1 vs. 102.4±1.7 mV) maximum diastolic potential (–73.5±1.0 vs. –74.2±1.0 mV) and overshoot (31.4±1.5 vs. 28.2±1.1 mV). The repolarizing current I_to largely controls action potential duration in the rat. A decrease in I_to is responsible for the prolongation of action potential duration in hypertrophied myocytes in SHR [14]. In order to assess the ionic basis of the effect of losartan on action potential duration, we studied the characteristics of I_to.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Typical action potentials recorded from single myocytes of control (C) and losartan-treated (L) SHR.

 

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Summary of action potential parameters measured in single left ventricular myocytes of control (open columns) and losartan-treated (black columns) SHR. MDP, maximum diastolic potential; OS, overshoot; AP, action potential amplitude; APD–20 and APD–60, action potential duration at –20 and –60 mV, respectively. * P<0.05; * P<0.01.

 
As shown in Fig. 4, the density of I_to was larger in myocytes from losartan-treated animals. The peak outward currents were larger following the protocol (see inset) used to elicit I_to. This finding was confirmed by the current–voltage relationship obtained in 27 cells from both control or losartan-treated rats: maximum current density was 15.1±1.4 pA/pF in losartan-treated rats and 10.0±0.8 pA/pF in control rats (P<0.02); the midpoint and slope of the activation curve were similar in control (32.8±1.7 and 16.1±1.6 mV) and in losartan-treated rats (34.7±1.2 and 16.4±0.6 mV). The steady-state currents, which likely represent I_K [14], were slightly but significantly increased in losartan-treated rats (at +60 mV: 6.7±0.6 vs. 5.2±0.4 pA/pF, P<0.05), and might contribute to APD shortening.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of losartan treatment on transient outward current density. Left panels: typical recordings of Ito evoked during depolarization from –40 to 70 mV, in cells from control (a) and losartan-treated SHR (b). Panel c: Average I–V relationships of Ito density (in pA/pF) as a function of step potential (in mV), obtained in control (open circles) and losartan-treated (closed circles) SHR. Lines are fitted using a Boltzmann function. The voltage clamp protocol is shown in inset.

 
Fig. 5 also shows that the pacemaker current I_f was affected by losartan treatment. However, the effect was in the opposite direction, in that I_f density was clearly reduced in myocytes from the losartan-treated group g_max, i.e., the maximal current specific conductance calculated by fitting the activation curve with a Boltzmann function, was 54.2±8.0 pS/pF in control and 28.6±3.6 pS/pF in losartan-treated rats (P<0.02). The representative recordings show that the current is clearly reduced to all voltages; this is even more evident from the mean current–voltage relationship. The activation of the current was not modified by the treatment, V_1/2 and k being, respectively, –84.7±1.1 and 8.3±2.8 mV in control and –87.8±1.3 and 7.5±2.4 mV in losartan-treated rats. In fact the relationship between the time constant of activation reciprocal (1/) and voltage is not different in the control and losartan groups.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of losartan treatment on If. Left panels: typical recordings of If evoked during hyperpolarization from –60 to –120 mV, in cells from control (a) and losartan-treated SHR (b). Panel c: Average activation curves obtained by plotting If specific conductance (in pS/pF) as a function of step potential (in mV) in control (open circles) and losartan-treated (closed circles) SHR. Lines are fitted with a Boltzmann function. Panel d: Plot of reciprocal of time constant for If activation (1/) versus step potential, in control (open circles) and losartan-treated (closed circles) SHR. The voltage clamp protocol is shown in inset.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 
In the present study we evaluated the effect of chronic treatment with losartan on the electrophysiological alterations occurring in old SHR, i.e. during a phase of their life during which the development of severe cardiac hypertrophy occurs. SHR is a widely used model for left ventricular hypertrophy; the old SHR may better mimic the clinical course of untreated or poorly controlled essential hypertension in humans [17,25]. The main and novel finding of this study is that blockade of AT₁ not only affects development of cardiac and cellular hypertrophy, but also affects the electrophysiological alterations, which characteristically occur in the hypertrophied myocyte. To our knowledge, this is the first demonstration that chronic blockade of AT₁ receptors affects cardiac ionic currents. It is already known that chronic treatment with ACE-inhibitors or an AT₁ antagonist is able to prevent action potential prolongation [21,26]; at least for captopril the effect is associated with restoration of the repolarizing current I_to [21], which in the rat is the main current controlling the repolarization phase. Those experiments have been, however, performed in quite young animals, i.e. 18-weeks old at the end of treatment, in which the development of hypertrophy is at its early stages and evaluating only the effect on APD and (for captopril) on I_to. Our results have been obtained in old SHR, that is in animals in which cardiac hypertrophy is already severe and the electrophysiological alterations (such as the action potential prolongation) already well-established [14]. We studied the effect of AT₁ blockade on APD, I_to and on the pacemaker current I_f which is expressed in old but not in young SHR [16,17]. Furthermore, the alterations so far found in myocytes isolated from the old SHR have been found also in myocytes isolated from the failing human heart [19,20]. Taken together, our data suggest that an 8-week treatment with losartan is able to counteract all these electrophysiological alterations when they are already established, and that this could occur also in human with obvious and important therapeutic implications. The present data, however, do not permit to definitely conclude that losartan causes a true regression, i.e. at the level of age-matched normotensive rats, of the electrophysiological remodeling associated with hypertrophy, rather than blocking the progression of the phenomenon. In fact, we lack experimental data in SHR at 16 month of age and historical data show a large variability in many parameters. This is especially true for cell capacitance, APD and consequently I_to, which vary continuously both in normal and in hypertrophied myocytes. I_f expression does not depend on cell size [18,27]. It is not present in normal myocytes and its density is directly related to the severity of hypertrophy [18]. 18 month-old SHR show a mean value of I_f density of 48 pS/pF (range: 35.3–54.4 pS/pF [17,18,28]), being 54.2 pS/pF in the present study. Losartan-treated 18-month old SHR have an I_f density of 28.6 pS/pF, a value which is lower than that found in all SHR of the same age we have used, and similar to that found in historical age-matched WKY (20–21 pS/pF [18,28]). I_f current, being expressed only in hypertrophy, can be considered a better index than cell capacitance, APD and I_to to assess the effect of losartan on electrophysiological remodeling. In so doing, a regression of electrophysiological alterations toward age-matched control by chronic treatment with losartan can be hypothesized. Studies in progress will clarify this point.

Interestingly, the effect of losartan was observed for a daily dosage, which does not significantly affect systolic blood pressure. This is not surprising since regression of cardiac hypertrophy in the absence of significant lowering of blood pressure has been reported for both ACE-inhibitors and AT₁ antagonists [23,26,29,30]. However, many other studies failed to show a regression in cardiac hypertrophy in the absence of lowered blood pressure [31–34]. The reasons for such discrepancies are not obvious, but are possibly the consequences of the different models of cardiac hypertrophy used; in fact cardiac hypertrophy appears to encompass more than one phenotype, even at the cellular level, and it is likely that multiple switches exist in the process of hypertrophy [35]. Recent evidence suggests that the AT₁-mediated angiotensin II signaling is not essential for the development of pressure overload-induced cardiac hypertrophy [36]. In fact, knockout mice for the A subtype of the AT₁ receptor, exhibit the same degree of cardiac hypertrophy of the wild type in response to aortic constriction. Thus the myocardium has alternative signaling mechanisms for the development of hypertrophy; in this line, recent evidence suggests that load, independent of the renin–angiotensin system (RAS) is capable of stimulating cardiac growth [37] and that component of the cardiac RAS are independently and differentially regulated by mechanical stretch and angiotensin II in neonatal cardiac myocytes [38]. On the whole, the activation of the RAS is certainly playing an important role, which, however, requires better understanding [39].

The present results add novel information on the effect of pharmacological treatment on electrophysiological remodeling, showing for the first time that AT₁ blockade with losartan is able to influence the electrophysiological alterations associated with cardiac and cellular hypertrophy; they also suggests an important role of angiotensin II and of the stimulation of AT₁ receptors in their development. Recent data demonstrating that long-term exposure of canine isolated myocytes to angiotensin II causes changes in the properties of I_to [40] which are responsible for altered in action potential configuration, support such a view. Our results indeed clearly demonstrate that action potential duration is shorter in losartan-treated myocytes, and that this effect is due to a greater I_to and possibly I_K. We [14] have previously demonstrated that the main ionic alteration responsible for action potential prolongation caused by chronic hypertension is a specific decrease in I_to, the other currents influencing repolarization (I_Ca and I_K) being unmodified. The present results are consistent with data obtained in SHR treated with captopril [21] and strongly support the idea that changes in I_to are major determinants in influencing action potential duration in cardiac hypertrophy [13]. Thus, it may be speculated that chronic exposure of ventricular myocytes to angiotensin II, as probably occurs in hypertension, may cause a decrease in I_to channel expression, and that blockade of the RAS system by ACE-inhibitors or blockers of AT₁ receptors may antagonize this phenomenon. The electrophysiological changes in action potential duration, I_to and I_K observed in the present study are likely independent of direct electrophysiological effects of losartan or its active metabolite (E3174), taking into account the recently reported actions of these compounds on potassium currents [41].

Losartan-treated myocytes also show an I_f current of smaller amplitude than saline-treated rats. The pacemaker current I_f is physiologically present in pacemaker cells [42]; in rat ventricular myocytes it is expressed during the early neonatal phase [27], is absent in adult ventricular myocytes [17], and is re-expressed during hypertrophy [18]. Its density is directly related to the severity of cardiac hypertrophy [18] and we have hypothesized that it may play a role in the genesis of ventricular arrhythmias in the hypertrophied heart. I_f is also present in ventricular myocytes isolated from the failing human heart [19,20] where its expression seems to depend on the etiology of the underlying disease [43]. Whether its re-expression in ventricular myocytes during cardiac hypertrophy and failure is due to the continuous stimulation of the AT₁ receptors is an appealing possibility, which requires further experimentation.

On the whole the overexpression of I_f and the decrease in I_to in ventricular myocytes in disease states associated with an increased propensity for ventricular arrhythmias strongly suggest their role in arrhythmogenesis. A decrease in I_to is the ionic mechanism likely responsible for the prolongation of action potential duration: being dishomogeneous, it may cause a dispersion of repolarization, which is per se an arrhythmogenic mechanism [16]. The overexpression of the depolarizing current I_f may increase the likelihood that a subthreshold delayed after depolarization (a frequent event in hypertrophied or failing myocyte, [44,45]) may reach threshold and trigger an arrhythmia in a heart where the substrate for reentry is certainly present.

The observation that a sub-antihypertensive dose of losartan is able to influence the cellular electrophysiological remodeling associated with cardiac and cellular hypertrophy, suggests that this phenomenon may be the electrophysiological basis for the reduced incidence of sudden deaths described in the ELITE study [4].

	5 Limitations

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 
One limitation of the study is that patch-clamp experiments were performed at a low stimulation rate (0.2 Hz); under these conditions the contribution of I_to to action potential duration is much higher due to its frequency-dependent inactivation. Furthermore, the role of I_to in controlling repolarization in large mammals is disputed. Despite the reported reduction in I_to in human failing hearts [46], simulation studies of human ventricular action potential showed a small contribution of this current to APD [47]. Both the fast and slow component of the delayed rectifier I_K resulted of utmost importance [47]. However, electrophysiological and molecular evidence for the presence of these currents in human ventricle and of their alterations in heart failure are still preliminary. The presence of I_f in both human and rat myocytes is clearly documented, but direct proof of its role in ventricular arrhythmogenesis is still lacking. Finally, many other cellular electrophysiological changes occurring in the hypertrophied and failing heart may contribute to the phenomenon of electrophysiological remodeling [48].

Time for primary review 23 days.

	Acknowledgements

 
This study was supported by a grant from MURST (prot. 9706217225_006) and Telethon (Project no. 1092); MLR was supported by a grant of the European Community (BIOMED Concerted Action Contract no. BMH4-CT96-0287). We wish to thank Merck Sharp & Dohme for providing losartan.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Limitations References

 

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