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Am J Physiol Heart Circ Physiol 280: H142-H150, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 1, H142-H150, January 2001

Myocardial remodeling and arrhythmogenesis in moderate cardiac hypertrophy in rats

Donatella Stilli1, Andrea Sgoifo1, Emilio Macchi1, Massimiliano Zaniboni1, Sergio De Iasio2, Elisabetta Cerbai3, Alessandro Mugelli3, Costanza Lagrasta4, Giorgio Olivetti4, and Ezio Musso1

1 Dipartimento di Biologia Evolutiva e Funzionale-Sezione Fisiologia, 2 Dipartimento di Biologia Evolutiva e Funzionale-Sezione Biologia Umana, and 4 Istituto di Anatomia Patologica, Università degli Studi di Parma, 43100 Parma; and 3 Dipartimento di Farmacologia Preclinica e Clinica, Università degli Studi di Firenze, 50134 Florence, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In 47 male adult Wistar rats with 4-wk aortic coarctation (AC) and 39 age-matched sham-operated rats (SO) chronically instrumented for telemetry electrocardiogram recording, we investigated the mechanisms of arrhythmogenesis in moderate cardiac hypertrophy, with an approach from "in vivo" toward the cellular level, analyzing 1) stress-induced cardiac arrhythmias in all rats and 2) myocardial fibrosis in 35 animals and action potential duration and density of hyperpolarization-activated current in 19 others at the ventricular level. Aortic banding increased arterial blood pressure, cardiac weight, and ventricular myocyte volume by 11, 25, and 14%, respectively (P < 0.001-0.05). Ventricular arrhythmias occurred at similar rates in AC and SO rats throughout the stress procedure. Action potential duration and hyperpolarization-activated current were about twice as great and myocardial fibrosis about four times greater in AC animals (P < 0.005-0.05). Electrocardiogram data also revealed more supraventricular arrhythmias in AC rats during the baseline period and after stress and fewer atrioventricular block episodes after stress (P < 0.05). Thus stress-induced supraventricular and atrioventricular nodal, but not ventricular, arrhythmias were affected in moderate cardiac hypertrophy when ventricular morphofunctional alterations were evident.

mechanisms of cardiac arrhythmias; myocardial fibrosis; membrane potential; hyperpolarization-activated current; social stress


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC HYPERTROPHY IS ASSOCIATED with abnormal electrical activity leading to a considerable propensity to arrhythmias (18, 30). The biological determinants of arrhythmogenicity are in all probability myocardial fibrosis and/or changes in protein membrane composition of hypertrophied myocytes resulting in altered cellular excitability (29). Although there is a large body of information concerning the time course of structural and electrophysiological changes in the heart during the development of hypertrophy, it remains to be established which of these changes, or combination of changes, actually triggers cardiac arrhythmias "in vivo" at the various stages of the disease. We recently evaluated the arrhythmogenic effect of an acute social challenge (social stress) in healthy, freely moving rats by means of telemetry electrocardiogram (ECG) recordings (25). This stress procedure has been reported to produce a greater neuroendocrine stimulation (23) than other stressors and provides a powerful tool to gain insight into the arrhythmogenic pathways that are activated in the real life of a social animal.

In the present investigation, we exposed rats with a mild form of cardiac hypertrophy induced by 1 mo of abdominal aortic banding to social stress; the rats were chronically instrumented with an ECG telemetry system. The aim of the study was to establish whether morphofunctional changes occurring in moderate cardiac hypertrophy at its initial stages facilitate stress-induced ventricular arrhythmias. To test this hypothesis, we adopted an experimental approach from the in vivo toward the tissue/cellular level by analyzing 1) ventricular arrhythmias occurring during exposure to a social stressor in all experimental animals and 2) the amount of myocardial fibrosis or action potential duration (APD) and density of hyperpolarization-activated inward current (If) in left ventricular tissue or isolated myocytes in selected subgroups. To our knowledge, this approach has never before been adopted to evaluate the mechanisms of arrhythmogenesis in cardiac hypertrophy. A second novelty was the use of stressors belonging to the environment of the animal to trigger arrhythmic events via naturally induced autonomic stimulation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The experimental protocol was approved by the Veterinary Animal Care and Use Committee of the University of Parma. All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (14a).

Animals and Housing

The study population consisted of 86 male 7-mo-old Wistar rats weighing 548 ± 9.0 g. The animals were kept in groups of five in clear Makrolon cages measuring 55 × 35 × 20 cm from weaning to the beginning of the study. The animals were housed in a temperature-controlled room at 20-24°C, with lights on between 8 AM and 8 PM. The bedding of the cages consisted of wood shavings, and food and water were freely available.

Experimental Protocol

The animals were randomly subdivided into two groups, one undergoing aortic coarctation (AC group, n = 47) and the other sham operation (SO group, n = 39). Seventy-five animals (36 AC and 39 SO) were chronically instrumented with a miniature transmitter for telemetry ECG recording [models TA11CTA-F40 (transmitter) and CTR85-SA (receiver), Data Sciences, St. Paul, MN]. During all surgical procedures, the animals were anesthetized with droperidol plus fentanyl citrate (Leptofen, Farmitalia-Carlo Erba, Milan, Italy; 0.6 ml/kg im). The ECG response to social stress was evaluated 1 mo after surgery. In 27 animals (11 AC and 16 SO), mean blood pressure was directly measured from the carotid artery during anesthesia (Leptofen, 0.6 ml/kg im) with a pressure transducer (Stoelting, Wood Dale, IL) connected to a chart recorder (UNIRECORD 7050, Basile, Varese, Italy). At death, 19 rats (9 AC and 10 SO) were used for electrophysiological studies by means of the patch-clamp technique and 35 for morphological measurements comprising myocardial weights, amount of myocardial fibrosis (18 AC and 17 SO), and mean myocyte cell volume (18 AC and 7 SO).

Chronic Instrumentation for Telemetry ECG Recordings and Aortic Coarctation

Transmitters were implanted according to a recently described procedure that enables the recording of high-quality signals (26). The body of the transmitter was placed in the abdominal cavity; one lead was fixed on the dorsal surface of the xiphoid process, and the other wire was subcutaneously tunneled toward the anterior mediastinum, so that the tip of the recording lead was close to the right atrium.

The aorta between the two renal arteries was dissected free, and a 1.3-mm-diameter blunted needle was laid alongside the exposed segment of the vessel. A silk ligature was passed under the aorta and the needle and tied securely between the two renal arteries (9). Then the needle was carefully withdrawn and the abdominal wall was sutured. The procedure led to an ~40% reduction in the lumen of the aorta. In SO rats, the ligature was not tied. Finally, the animals were given antibiotic therapy with amikacin for 4 days (Amikavet, PrM, Milan, Italy; 0.2 ml/kg im) and individually housed for 4 wk.

Social Stress and ECG Acquisition and Processing

Social stress was achieved by placing the instrumented animal in the territory of an aggressive male (resident-intruder test) (25, 26). The recording session consisted of three 15-min periods, during which the experimental animal (intruder) was first left alone and undisturbed in its cage (baseline period), then exposed to social stress (test period), and finally returned to its cage for recovery (posttest period). At the beginning of the stress period, the intruders actively explored the resident cage. After receiving a few biting attacks by the resident, they typically exhibited a passive/submissive coping strategy, mainly consisting of immobility and upright defensive posture at resident approach. The recovery period was characterized by autogrooming behavior reflecting the process of dearousal due to the termination of the stressful situation (26). The three periods were set for 15 min on the basis of the change in norepinephrine plasma levels with time during the resident-intruder test procedure. Specifically, norepinephrine levels, which reached values ~10 times greater than baseline levels during the test period, decreased to values 1.5 greater than baseline at the end of the 15-min recovery period and remained constant for the subsequent 30 min (22). The telemetry ECG receiver was placed under the experimental cage. ECGs, provided as analog signals at the output of the receiver, were monitored on an oscilloscope and simultaneously routed to a Macintosh Quadra 700 computer via an analog-to-digital conversion board (12 bits, 1,000-Hz sampling rate; National Instruments, Austin, TX). A real-time acquisition program, developed in our laboratory, allowed the digital signal to be continuously recorded and stored on hard disk. The data were subsequently transferred to a VAX Station 3100 (Digital Equipment, Maynard, MA) and permanently stored on tape for off-line processing. ECG data were interactively analyzed by using a custom-made software package for determining R-R interval and rhythm disturbances. Within 1 wk of the ECG recordings, the animals were killed, and the heart was used for cellular electrophysiological or morphological studies.

Cellular Electrophysiological Studies

Single left ventricular myocytes were isolated from AC and SO rats, as previously described (5), by perfusing the rat heart with a low-calcium solution (LCS) and then with LCS plus 1 mg/ml collagenase (type I, Sigma, Milan, Italy), 0.03 mg/ml dispase (Boehringer, Milan, Italy), and 1 mg/ml albumin (fatty acid-free fraction V, Serva, Milan, Italy). The left ventricle and the septum were removed and cut into chunks, and the pieces were stirred in the LCS. Cardiomyocytes that appeared in the supernatant were purified by gravity sedimentation, collected, and stored at room temperature in LCS supplemented with 1 mM CaCl2 and 4% penicillin/streptomycin (GIBCO BRL, Milan, Italy). For electrophysiological recordings, cells were placed in an experimental bath on the platform of an inverted microscope (Diaphot TMD, Nikon, Tokyo, Japan). The patch-clamp technique (whole cell configuration) was used to measure the electrophysiological properties of the isolated myocytes [action potential, membrane capacitance (Cm), and If]. Experiments were performed using a patch amplifier (Axopatch-1B, Axon Instruments, Foster City, CA) interfaced to a personal computer by means of a DAC/ADC interface (Labmaster Tekmar, Scientific Solutions, Mentor, OH). Data were viewed on-line on an analog 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). Cells were superfused with normal Tyrode solution or Tyrode solutions modified to measure If. Temperature was maintained at 36-37°C. Patch-clamp pipettes, prepared from glass capillary tubes (Garner Glass, Claremont, CA) by means of a two-stage vertical puller (Hans Otchoski, Homburg, Germany), had a resistance of 1.5-2.5 MOmega when filled with the internal solution. The patch-clamped cell was superfused by means of a custom-made temperature-controlled microsuperfuser, which allowed for rapid changes in the solution bathing the cell. Data analysis and fitting were performed using the Origin 4.1 program (MicroCal Software, Northampton, MA) running on a personal computer. Cm was measured by applying a ±10-mV pulse starting from a holding potential of -70 mV and calculated as described previously (4).

To evaluate the steady-state values of If, 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 Cm (current density, in pA/pF). As for the activation curve, specific conductance (gf) measured in individual cells from SO or AC rats was plotted as a function of membrane potential (5). Experimental data points were fitted to a Boltzmann distribution, which allowed for the automatic computation of the maximal gf (in pS/pF), the potential for half-maximal activation (in mV), and the slope of the activation curve (in mV).

Action potentials were elicited at a rate of 0.2 Hz and sampled at 1 kHz. The following parameters were measured: action potential amplitude, overshoot, resting potential, and APD at -20 mV (APD-20 mV) and -60 mV (APD-60 mV). In preliminary experiments, the action potential parameters were also measured at a more physiological pacing rate (5 Hz). Twenty-four isolated left ventricular myocytes from 6 AC rats and 16 from 5 SO animals were studied. Action potentials were elicited through brief (2-3 ms) current injections using an Axoclamp-2B amplifier (Axon Instruments) in current-clamp mode and sampled at 5 kHz. Other experimental conditions were comparable to those previously described, with the following exceptions: 1) aortic coarctation was greater than that adopted here, leading to a reduction of ~70% in the vessel lumen, and 2) no EGTA was present in the pipette solution.

Solutions. LCS was composed of (in mM) 120 NaCl, 10 KCl, 1.2 KH2PO4, 1.2 MgCl2, 10 D-(+)-glucose, 20 taurine, and 10 HEPES-NaOH (pH 7.0). Normal Tyrode solution was composed of (in mM) 140 NaCl, 5.4 KCl, 1.5 CaCl2, 1.2 MgCl2, 10 glucose, and 5 HEPES-NaOH (pH 7.35). Modified Tyrode solution was composed of (in mM) 140 NaCl, 25 KCl, 1.5 CaCl2, 1.2 MgCl2, 5 BaCl2, 2 MnCl2, 0.5 4-aminopyridine, 10 glucose, and 5 HEPES-NaOH (pH 7.35). This solution allowed for the amplification of If and the reduction in interference from other currents, i.e., L-type calcium current, inward rectifier-like current, and transient outward potassium current. Pipette solution was composed of (in mM) 130 potassium aspartate, 5 Na2-ATP, 2 MgCl2, 5 CaCl2, 11 EGTA, and 10 HEPES-KOH (pH 7.2, pCa 7.0).

Morphological Studies

Hearts were arrested in diastole with 1 ml of cadmium chloride solution (100 mM iv) and excised. Subsequently, the right ventricle and the left ventricle inclusive of the septum were separately weighed and fixed in 4% paraformaldehyde. Left ventricular volume was obtained by dividing ventricular weight by the specific gravity of the muscle tissue (1.06 g/ml). The left ventricle was transversely cut into 9-10 1-mm-thick slices, and the 2 intermediate rings were embedded in paraffin. Sections (5 µm thick) stained with hematoxylin and eosin were used for morphometric analysis.

The morphometric methods adopted have been described extensively elsewhere (2, 16) and are summarized briefly. The amount of fibrotic tissue present in the left ventricular myocardium was determined in 60 randomly selected consecutive fields from the endocardium, midmyocardium, and epicardium in each animal at a calibrated magnification of ×250 with a reticle containing 42 sampling points defining a tissue area of 0.160 mm2. The fraction of points underlying myocardial scarring was measured to calculate the volume fraction of replacement and perivascular fibrosis in the myocardium. In addition, the number of fibrotic foci per unit area of myocardium and the cross-sectional area of the lesion profiles were computed.

Mean myocyte cell volume per nucleus was obtained by counting the total number of myocyte nuclear profiles per unit area of myocytes in myocardial regions in which myofibers were transversely sectioned, as described above (15). The myocardium was examined at a magnification of ×1,000, viewing 15 consecutive fields in the endocardium and epicardium. A square tissue area equal to 0.008 mm2 was delineated in the microscopic field by the ocular reticle described above. The number of nuclei in the sampled area was counted following the criteria described by Gundersen (11) to estimate their numerical density. At the same magnification, the fraction of sampling points overlying myocyte profiles was also counted to determine the volume fraction of myocytes within the myocardium. Nuclear counts were calculated with respect to myocyte area to minimize variations in the interstitial space. Average myocyte nuclear length [D(n)] was determined by measuring 25 nuclear profiles in longitudinally oriented fibers at a magnification of ×1,000.

The number of myocyte nuclei per unit volume of myocytes was then obtained by dividing the number of nuclei per unit area of myocytes by their mean nuclear length
N(n)<SUB>V</SUB><IT>=N</IT>(n)<SUB><IT>A</IT></SUB><IT>/D</IT>(n) (1)
The total number of nuclei in the ventricle [N(n)T] was then calculated according to the equation
N(n)T<IT>=N</IT>(n)<SUB>V</SUB><IT>·</IT>V(myo)T (2)
where V(myo)T represents the total volume of myocytes in the ventricle obtained by the volume fraction of myocytes and myocardial volume. It follows that the aggregate volume of myocytes divided by the total number of myocyte nuclei yields the average myocyte cell volume per nucleus.

Statistical Analysis

The SPSS statistical package was used (SPSS, Chicago, IL). The normal distribution of variables was checked by means of the Kolmogorov-Smirnov test. Statistics of variables normally distributed (most variables except arrhythmias) included means ± SE, Student's t-test, ANOVA (post hoc analysis by Bonferroni's test), and linear regression analysis. Information on the incidence of arrhythmias in the various groups and experimental conditions was provided as a percentage of animals showing arrhythmic events and the number of events. Additional statistical treatment of arrhythmias comprised Poisson distribution probability, the Mann-Whitney U-test, and rank correlation coefficient (Kendall). Statistical significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vivo Measurements

Arterial blood pressure in anesthetized animals. One month after aortic ligature, mean arterial pressure, directly measured from the right carotid artery, was 11% higher in AC rats (110 ± 3 mmHg, n = 11) than in control animals (99 ± 3 mmHg, n = 16, P < 0.05).

Telemetry ECG data. The time course of the R-R interval during the three recording sessions was similar in the AC and SO groups, with a marked decrease during stress (P < 0.001) followed by a partial recovery in the posttest period (Fig. 1A). The mean R-R interval in baseline conditions was 189 ± 4 and 183 ± 4 ms in AC and SO rats, respectively, decreasing by approximately one-third during the test period (P < 0.001) and eventually reverting to ~80% of baseline values in the posttest period (P < 0.001; Fig. 1B).


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  		Fig. 1.   A: time course of the R-R interval every 3 min during baseline, test, and posttest periods in sham-operated (SO) and aortic-banded (AC) rats. Values are means ± SE. *All test and posttest values were significantly lower than baseline values in both groups. B: R-R interval during the entire baseline, test, and posttest periods (15 min each) in SO and AC groups. Values are means ± SE. *P < 0.001, ANOVA; post hoc analysis by Bonferroni's test.

In the AC and SO groups, most arrhythmias consisted of premature supraventricular and ventricular beats (PSVBs and PVBs, respectively) and first- and second-degree (mainly 2:1) atrioventricular block episodes (AVBs). However, analysis of AVBs was limited to second-degree episodes, since the use of one-lead ECG telemetry recording in freely moving animals did not allow for a reliable evaluation of first-degree episodes.

As a general rule, the time course of rhythm disturbances in AC and SO rats throughout the experimental trial was characterized by a rise caused by the exposure to stress and a subsequent progressive decrease to values similar to baseline at the end of the posttest period. In most animals with arrhythmias, the number of episodes in baseline, test, and posttest periods for each kind of arrhythmia ranged from one to five, with the exception of PSVBs during the test period and AVBs during the posttest period, when more numerous arrhythmic events (up to 20) were observed in several animals.

The percentages of rats with arrhythmic events in the three recording periods are reported in Table 1. A higher percentage of animals with PSVBs was found in the AC than in the SO group in the baseline condition (50 vs. 33%, P < 0.005), extending to ~90% of the animals in both groups during stress and significantly decreasing during recovery but to a lesser extent in AC rats (75 vs. 54%, P < 0.005). The number of animals with PVBs was extremely low in the baseline condition, significantly increasing to about two-thirds in the AC and SO groups during the test period (P < 0.01) and decreasing during the posttest period to ~20% in both groups (P < 0.01). AVBs were negligible in the baseline condition, although in the AC group a slightly higher percentage of rats with AVBs was found (P < 0.05). The percentage of SO rats with AVBs increased during stress and to an even greater extent in the recovery period (P < 0.001). In the AC rats, the changes were limited to the recovery period, when the percentage of animals with AVBs was significantly higher than in the baseline condition without, however, attaining the posttest values measured in the SO group (P < 0.001). Thus the incidence of PVBs was unaffected by aortic banding, while PSVBs and AVBs were significantly modified during the baseline (higher PSVBs) and posttest periods (higher PSVBs and lower AVBs).


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Body weights. The body weights in the 47 AC and 39 SO rats were 541 ± 11 and 556 ± 10 g, respectively, at surgery (aortic coarctation and/or telemetry implant) and 551 ± 6 and 557 ± 16 g, respectively, before death, with no significant difference between the groups.

Morphological Studies

The weights of the heart, left ventricle, and right ventricle and the corresponding ratios to body weight are reported in Table 2. The various weights were significantly higher in AC rats, with average increments of 23-30%. In AC rats, the left ventricular weight-to-body weight ratio was positively correlated to blood pressure (r = 0.62, P < 0.05) and R-R interval (r = 0.63, P < 0.005).

                              
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  		Table 2.   Cardiac weights and myocyte cell volumes

Left ventricular myocyte volume in the endocardium was 25% greater in AC than in SO rats (P < 0.001). In the epicardium, mean myocyte volume was also slightly higher than in the control group, although the difference was not significant. When the results from the endocardium and the epicardium were combined, myocyte cell volume increased by 14% in AC rats (P < 0.05; Table 2).

Small amounts of interstitial and replacement fibrosis were clearly visible in the left ventricular wall of normal and hypertrophied hearts, while signs of structural damage were less definitely detectable in the right ventricle. Therefore, detailed quantitative measurements of myocardial fibrosis were limited to the left ventricle. The volume fraction of diffuse fibrosis and the numerical density of fibrotic foci in the epicardial, middle, and endocardial layers were significantly higher in the AC group, with about a fourfold increase in each layer compared with the corresponding layer in SO rats (P < 0.05-0.01; Table 3).

                              
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  		Table 3.   Myocardial fibrosis in the three layers of the left ventricular myocardium in SO and AC groups

No significant correlation was found between ventricular arrhythmias and cardiac weights and morphometric measurements.

Cellular Electrophysiological Studies

Figure 2A shows representative action potentials recorded from left ventricular myocytes isolated from 9 AC or 10 SO rats. It is apparent that APD is prolonged in the AC myocytes. This was a consistent finding, as shown in Fig. 2B, which summarizes the results obtained in myocytes isolated from SO and AC rats. Cm measured in AC rats did not significantly increase compared with SO rats (226 ± 7.3 vs. 219 ± 6.2 pF). The resting potential was similar in both groups (-72.0 ± 0.8 and -71.4 ± 0.8 mV in AC and SO, respectively, not significant), while APD was markedly prolonged in AC vs. SO rats [69 ± 8.7 vs. 35 ± 4.6 ms at -20 mV (P < 0.05), 162 ± 10.9 vs. 130 ± 12.6 ms at -60 mV (P = 0.06)]. Left ventricular myocytes paced at 5 Hz also indicated a prolongation of APD-20 mV in banded rats compared with SO rats (47.4 ± 5.7 vs. 28.2 ± 5.2 ms, P < 0.02), while resting potential was similar in the two groups.


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  		Fig. 2.   Action potential characteristics in SO and AC rats. A: superimposed representative action potentials recorded from left ventricular myocytes from SO and AC rats. B: summary of membrane capacitance (Cm) and action potential duration (APD) measurements in SO (open bars) and AC (solid bars) left ventricular myocytes at -20 mV (APD-20 mV) and -60 mV (APD-60 mV). Each column represents mean ± SE of 111 SO and 95 AC cells (Cm) and 31 SO and 26 AC cells (APD). *P < 0.05, Student's t-test.

Finally, previous data showed that the expression of If in rat ventricular myocytes is influenced by factors such as hypertension and aging (5, 6). In myocytes isolated from SO (n = 10) and AC rats (n = 9), a hyperpolarizing step to -120 mV elicited a time-dependent, cesium-sensitive inward current (If) in >50% of cells (57 of 87 cells from SO and 54 of 96 cells from AC rats; Fig. 3A). However, If amplitude and density (i.e., amplitude normalized to Cm) were different in the two groups, being significantly higher in myocytes from AC rats (Fig. 3B). The representative recordings of Fig. 4A show that If density is clearly greater in the AC group at all voltages. This is even more evident from the mean conductance-voltage relationship: maximal gf was 21.7 ± 3.1 (n = 40) and 47.5 ± 7.4 pS/pF (n = 32) in the SO and AC groups, respectively (P < 0.05; Fig. 4B). However, the voltage dependence of If activation was not modified, potential for half-maximal activation being -83.1 ± 1.3 and -86.7 ± 1.7 mV in SO and AC rats, respectively (Fig. 4B).


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  		Fig. 3.   Summary of occurrence (A) and amplitude and density (B) of hyperpolarization-activated inward current (If). Occurrence of If was tested in 87 SO and 96 AC cells by applying a step to -120 mV from a holding potential of -40 mV. If density was obtained by normalizing amplitude with respect to Cm. Each column represents mean ± SE. *P < 0.005, Student's t-test.



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  		Fig. 4.   If properties in SO and AC rats. A: representative recordings obtained in SO (top) and AC (bottom) cells by hyperpolarization to increasing negative potentials (from -60 to -120 mV) from a holding potential of -40 mV. B: average activation curves obtained by plotting If specific conductance (gf) as a function of hyperpolarizing step potential (in mV) recorded in SO (open circle ) and AC () left ventricular myocytes. Each point represents mean ± SE of 30-40 cells. Curves represent fitting of data points with a Boltzmann function.

No attempt was made to correlate If density and APD with ventricular arrhythmias, since the experimental conditions employed to measure the cellular electrophysiological parameters were not comparable to those encountered in conscious animals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrated that 1 mo of aortic banding increased mean arterial pressure, cardiac weights, average myocyte cell volume, and the amount of reparative fibrosis in the left ventricular myocardium. Ventricular myocytes obtained from AC rats had longer APD and higher amplitude and density of If. The incidence of ventricular rhythm disturbances (PVBs) did not significantly increase in AC compared with SO rats during intense autonomic stimulation after exposure to social stress. Interestingly, telemetry ECG revealed that the incidence of atrial arrhythmias (PSVBs) during the baseline period and recovery from stress was higher in AC animals. In addition, the increase in PSVBs in the recovery period was associated with a reduced incidence in AVBs.

The coarctation of the abdominal aorta between the two renal arteries, as adopted here, has been reported to have effects similar to the two-kidney one-clip model of hypertension (9) and has the characteristic that mechanical and humoral factors participate in cardiac hypertrophy and hypertension (28). In our case, the slight increase in arterial pressure (+11%) was coupled with a hypertrophic response of the entire heart of ~25%. This result was due to a similar increase in left and right ventricular weights and can be considered a moderate form of cardiac hypertrophy (29). The occurrence of right ventricular hypertrophy in AC animals may be attributed more to the action of circulating growth factors than to a mechanical stimulus, although the specific signaling pathways have not been completely elucidated (19, 21).

The response of myocytes to the aortic banding resulted in a differential growth adaptation of the cells located in the different layers of the left ventricular wall. The increase in mean myocyte cell volume was greater in the endocardium than in the epicardium, averaging 14% (P < 0.001) in the entire wall. This transmural gradient of hypertrophy may reflect the different amount of stress induced by the increased left ventricular pressure on single myocytes, which, according to Laplace's Law, is higher in the layers adjacent to the ventricular chamber (15). On the other hand, the slight increase in Cm in AC compared with SO rats (+3%) was not statistically significant. We are aware that cardiac hypertrophy caused by several weeks of aortic coarctation produces a parallel increase in myocyte Cm and cell volume, leaving their ratio unaltered (10). The discrepancy between the two measurements of myocyte size found here could be explained by considering that 1) experimental studies have shown that the enlargement of myocyte cell volume exceeds that of the surface area of the external sarcolemma, in agreement with the geometrical properties of regular solids (1), and 2) to the best of our knowledge, the relative time course of cell volume and cell surface changes, complicated in rat cells by the presence of membrane invaginations and caveolae (33), in our model of cardiac hypertrophy remains to be established. The finding that, in this model, myocyte cell volumes in AC rats were greater at the endocardial level than in other myocardial layers implies that the use of myocytes from the entire left ventricular wall for Cm evaluations might lead to a considerable dispersion of the data, making it difficult to detect possible slight differences between AC and SO animals, should be considered as well. Finally, it should be pointed out that the higher dispersion of capacitance-to-volume ratio found by Satoh et al. (20) in adult rat myocytes than in the rabbit and ferret makes any speculation regarding mild hypertrophy-induced changes in Cm more complex.

The increase in heart rate during the test period in AC and SO rats and its progressive reduction during recovery were in agreement with previous studies in which the stressor induced significant increments of plasma levels of norepinephrine, which peaked during the initial phases of the test period and lasted, although progressively decreasing, until the end of the posttest period (22). The incomplete recovery of the R-R interval to baseline values was most probably due to the high level of somatomotor activity shown by the rats in the posttest period (mostly grooming behavior) (24). Reflex mechanisms in heart rate control mediated by the vagus nerve, which were greater the higher the pressure overload and cardiac hypertrophy, represent one possible explanation for the positive correlations in the baseline condition in the AC group between the R-R interval and left ventricular weight-to-body weight ratio and blood pressure.

In SO animals, rhythm disturbances were similar to those previously described in conscious rats during 24-h Holter monitoring (3) and/or during exposure to adverse social stimuli (22). Specifically, the presence of PSVBs during the baseline period was in agreement with the spontaneous supraventricular electrical instability usually found in the rat, augmenting with age (3). The increased PSVBs and PVBs during the test period were expected as a result of the stress-induced neuroendocrine response (22). During recovery from stress, the high level of PSVBs and AVBs could be attributed to the persisting effects of sympathetic stimulation and the accentuated vagal recruitment occurring after stress exposure (22). Aortic coarctation had no effect on the incidence of PVBs compared with the SO group throughout the experimental trial, while significant differences were observed in PSVBs and AVBs, the former increasing during the baseline and posttest periods and the latter decreasing during the posttest period.

PVB data suggested that in our model of mild hypertrophy the threshold to ventricular arrhythmias as modulated by sympathetic activation was unchanged, although ventricular structural and electrophysiological alterations were clearly evident. In fact, the hypertrophic remodeling of the myocardium in AC animals was associated with an increased percentage of myocardial fibrosis due to the appearance of a higher number of microscopic scarrings homogeneously distributed through the left ventricular wall. These findings are in agreement with the structural changes reported in different models of pressure-overload cardiac hypertrophy of long duration (2, 17) in which pharmacological treatments able to attenuate myocardial damage have decreased arrhythmia vulnerability (17). It is conceivable that the limited amount of damage found in the present study did not significantly affect the propensity of the heart to ventricular arrhythmias. The finding indicating that, in the initial stages of moderate cardiac hypertrophy, the degree of involvement of the ventricles is not in itself arrhythmogenic under basal conditions and does not facilitate arrhythmias under stress constitutes new information.

A prolonged APD is a well-recognized effect of hypertrophy in rat ventricular myocytes (4, 31). Recent data (27) have shown that, after catecholamine-induced left ventricular hypertrophy, consistent prolongation of APD at 25% of recovery occurs in subepicardial and midmyocardial myocytes, while changes in subendocardial myocytes are negligible. These results strengthen our findings of an overall prolongation of APD in AC rats, in that we randomly chose cells from the entire free wall of the left ventricle, and we therefore expected that, in our sample, subepicardial cells were less representative than midmyocardial and subendocardial myocytes, which statistically represent the majority of the enzymatically isolated cell yield (32). A pacing frequency as low as 0.2 Hz was adopted to prevent possible APD changes induced by hypertrophy from being masked by factors such as calcium current rundown, ATP depletion, and incomplete recovery of transient outward potassium current from inactivation, which can also affect APD but should play a minor role at low pacing rate. Our experimental protocol enabled us to conclude that the ionic mechanisms leading to APD prolongation in ventricular myocytes were indeed modified in mild ventricular hypertrophy, although the incidence of stress-induced PVBs in AC rats did not differ from that in SO animals. This conclusion is also supported by the data obtained in ventricular myocytes paced at 5 Hz, suggesting that, even at physiological pacing rate, APD was significantly prolonged in AC compared with SO rats. Thus our findings documented the presence of APD changes induced by mild hypertrophy and their lack of arrhythmogenic effects in the ventricles under social stress, at least at these stages of the disease.

As reported for other models of hypertrophy (5, 6) and heart failure (7, 12), the present data show that the density of the pacemaker current If is already enhanced in the initial stages of moderate hypertrophy. The high density of If in hypertrophied ventricular myocytes may represent a reexpression of a fetal program. In line with this, very recent data have demonstrated that the occurrence and density of If are maximal after birth and progressively decrease with age, suggesting that the gene encoding for If is turned off by some unknown mechanism during development (8). In our experimental model, the changes in If properties were less pronounced than those measured in myocytes obtained in other rat models with various degrees of hypertrophy (5, 6), providing a reasonable explanation for their inability to increase ventricular electrical instability. However, the demonstration of increased If density in our model of hypertrophy is important, since it could play an arrhythmogenic role in more advanced stages of hypertrophy.

The lack of any structural and electrophysiological measurements at the atria and of atrioventricular conduction system levels prevented us from providing a definite explanation for the increased incidence of PSVBs (baseline and posttest periods) and reduced AVBs (posttest period) observed in AC compared with SO rats. All these changes may be related to the increased adrenergic activity induced by hypertrophy, which can result in greater effects at the atrial and atrioventricular conduction system levels, provided that these tissues are affected less than the ventricles by aortic coarctation and, therefore, preserve a better beta -adrenergic response (29). As an alternative, PSVB and AVB changes could be due to a shift in the sympathovagal balance toward a sympathetic prevalence resulting from reduced efficiency in the parasympathetic input to the heart in pressure overload hypertrophy, as recently described (13, 14). Finally, it should be mentioned that, in a recent Holter monitoring study (3) performed in conscious rats with pressure overload hypertrophy induced by abdominal aortic stenosis, most spontaneous arrhythmias were of supraventricular origin, and this finding was considered to be mainly due to the very specific calcium metabolism of the rat ventricle, possibly resulting in a greater protection of the hypertrophied ventricle against acute changes in internal calcium. In that study, however, no conduction abnormality was found, and no information was available on stress-induced changes in cardiac excitability.

In conclusion, we provided experimental evidence that morphofunctional changes generally considered to be involved in the arrhythmogenesis of the hypertrophied heart, including myocardial fibrosis, prolongation of APD, and abnormal occurrence of If, were already present in a mild form of ventricular hypertrophy. At these initial stages of the disease, however, the hypertrophic damage was quite limited, and the changes did not succeed in significantly lowering the threshold to ventricular arrhythmias triggered by naturally induced autonomic stimulation. In the absence of increased PVBs when ventricular morphofunctional changes were clearly documented, aortic coarctation facilitated PSVBs during the baseline period and recovery from stress and reduced AVBs after stress.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (COFIN97: "Role of ionic carriers and channels in cellular physiology and pathology" and "The failing heart") and Italian National Research Council Grant 96.03447.CT04.


    FOOTNOTES

Address for reprint requests and other correspondence: D. Stilli, Dipartimento di Biologia Evolutiva e Funzionale-Sezione Fisiologia, Università degli Studi di Parma, Parco Area delle Scienze 11 A, 43100 Parma, Italy (E-mail: stilli{at}biol.unipr.it).

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 17 February 2000; accepted in final form 8 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(1):H142-H150
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