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Am J Physiol Heart Circ Physiol 281: H2150-H2158, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 5, H2150-H2158, November 2001

Losartan prevents contractile dysfunction in rat myocardium after left ventricular myocardial infarction

Marcel C. G. Daniëls1, Rebecca S. Keller2, and Pieter P. de Tombe2

1 Department of Cardiology, Groot Ziekengasthuis, 5200 ME Den Bosch, The Netherlands; and 2 Program in Cardiovascular Sciences, Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the effects of chronic losartan (Los) treatment on contractile function of isolated right ventricular (RV) trabeculae from rat hearts 12 wk after left ventricular (LV) myocardial infarction (MI) had been induced by ligation of the left anterior descending artery at 4 wk of age. After recovery, one-half of the animals were started on Los treatment (MI+Los; 30 mg · kg-1 · day-1 per os); the remaining animals were not treated (MI group). Rats without infarction or Los treatment served as controls (Con group). MI resulted in increases in LV and RV weight and unstressed LV cavity diameter; these were partially prevented by Los treatment. The active peak twitch force-sarcomere length relation was depressed in MI compared with either Con or MI+Los. Likewise, maximum Ca2+ saturated twitch force was depressed in MI, whereas twitch relaxation and twitch duration were prolonged. Myofilament function, as measured in skinned trabeculae, was not significantly different among the Con, MI, and MI+Los groups. We conclude that Los prevents contractile dysfunction in rat RV trabeculae after LV MI. Our results suggest that the beneficiary effect of Los treatment results not from improved myofilament function but rather from improved myocyte Ca2+ homeostasis.

heart failure; ventricular hypertrophy; angiotensin II type I receptor blocker; skinned fiber; Ca2+ sensitivity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCARDIAL INFARCTION (MI) may result in remodeling and progressive contractile dysfunction of the left ventricle. Several large-scaled clinical trials have convincingly shown the potential of angiotensin-converting enzyme drugs (ACE inhibitors) to ameliorate this process (2, 29). Consequently, these drugs are used extensively, particularly after larger infarctions. The exact mechanisms accounting for this cardiac protective effect are not yet completely understood but probably relate to better loading conditions on the heart as well as a blunted hypertrophic response. ACE inhibitors also influence the bradykinin system, which is widely held responsible for side effects such as cough. Furthermore, alternative pathways of angiotensin II formation have been identified that do not rely on the ACE. Therefore, drugs that directly interact with the angiotensin receptor at the cellular level have been introduced to more specifically and completely inhibit the target site of angiotensin II. However, despite their clinical use, little is known about the effects of these drugs in the setting of experimental MI, especially on contractile parameters in multicellular preparations. In a previous study (11), we demonstrated the negative impact of left ventricular infarction on right ventricular contractile function. In the present study, we investigated the potential of potassium losartan, one of the angiotensin II receptor antagonists, to prevent contractile dysfunction in both intact and skinned right ventricular trabeculae that were isolated from rats 12 wk after sustaining a large left ventricular MI.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study model. We adopted a model described previously by us (11), in which right ventricular trabeculae were studied from rats with or without a left ventricular anterior wall MI. The thin right ventricular trabeculae allow for reliable sarcomere length assessment by laser diffraction techniques (overcoming the problem of nonlinear behavior of series elastic elements if muscle length itself were taken as a measure), optimal equilibration with the superfusate, and effective membrane permeabilization in the case of the skinning procedure. All procedures that were used in the current study were in accordance with institutional guidelines regarding the care and use of laboratory animals.

MI was induced in 4-wk-old female Sprague-Dawley rats by the supplier (Charles River) using the procedure described by Selye et al. (26). Briefly, the left anterior descending coronary artery was ligated with a silk suture after an incision in the fourth intercostal space under appropriate anesthesia. The wound was then closed with metal clips, and the rats were allowed to recover for several days before they were shipped to our animal resources center. The control group (Con) of animals consisted of Sprague-Dawley rats of matched age; these animals were not operated on.

Drug administration. As soon as the animals arrived at our institution they were assigned to one of two feeding programs. Con animals that had not suffered an MI received a normal diet with normal drinking water to which no drugs were administered. The rats that had their left anterior descending coronary artery ligated received a normal diet with either normal water ad libitum or water mixed with potassium losartan (provided by Merck). On the basis of daily measurements of water consumption and regular weighing of the animals, losartan was added to the drinking water to obtain an average daily drug intake of 30 mg/kg for the next 12 wk.

Isolation of muscle preparations. The procedures followed for the isolation of right ventricular trabeculae have been described in detail previously (7, 10, 11, 32). Briefly, at the age of 16 wk, the animal was anesthetized using halothane inhalation, the chest was opened subcostally, and the heart was rapidly removed, transferred to a dissection chamber, and retrogradely perfused with an oxygenated modified Krebs-Henseleit solution (see Solutions) after cannulation of the aorta. Under a binocular microscope (Olympus; ×10-40 magnification), one leg of a pair of scissors was positioned in the pulmonary artery; the right ventricle was opened by cutting along the interventricular septum toward the apex of the heart. Remaining right atrial tissue was removed along the atrioventricular ring. Thin and unbranched trabeculae, running between the right ventricular free wall and the atrioventricular ring, were dissected by cutting through the atrioventricular ring on one end and by removing a small part of the right ventricular wall with the site of attachment of the muscle on the other end. The left ventricle was then cut transversally, approximately at the base of the papillary muscles so as to measure its unstressed diameter; all remaining left and right ventricular tissue was blotted briefly and weighed.

After the heart was removed, the main parts of lungs and liver of the rats were removed for determination of the wet-to-dry weight ratio. Wet weight was measured immediately after removal of the organs and brief blotting. The material was then dried in an oven at 80°C for 4 h.

Isolation and mounting of muscle preparations. For measurements of contractile function in intact trabeculae, the dissected muscle was moved from the dissection dish into a 250-µl bath (milled from plastic with a microscope coverslip as bottom) that formed part of the microscope stage in the experimental setup (7, 10, 32). The muscle was mounted horizontally in this bath by positioning its muscular end in a platinum cradle attached to a tension transducer (Semiconductor Strain Gauge, model AE 801, Sensonor) and by attaching its valvular end to a hook. Both attaching devices were controlled by micromanipulators. Finally, the bath was covered with a glass slide. The muscle was observed with the use of an inverted microscope (Olympus) and a video system (Panasonic). The temperature of the superfusate in the muscle bath was controlled at 25°C within 0.1°C using a cryostat and glass heat exchanger at the inflow line to the bath; flow rate of the superfusate was kept constant at 3-4 ml/min.

Muscles that were to be used as skinned trabeculae were transferred from the dissection dish into a smaller dish containing cold standard relaxing solution, to which 1% (vol/vol) Triton X-100 was added to chemically permeabilize (skin) the preparation. The composition of this skinning solution was the following (in mmol/l): 7.3 Na2ATP, 10.6 MgCl2, 20 EGTA, 10 phosphocreatine, 100 N,N-bis[2 hydroxyethyl]-2-aminoethanesulfonic acid (BES), and 100 µM leupeptin; pH 7.0, adjusted with KOH, ionic strength 200 mmol/l (adjusted with potassium propionate). The preparations were left in this cold solution for at least 2 h to allow solubilization of virtually all of the membranous structures. A skinned trabecula was then attached to aluminum T clips (15) and mounted in the experimental setup.

Solutions. All of the chemicals used were purchased from Sigma and were of the highest purity available. The standard modified Krebs-Henseleit solution used for intact trabeculae was composed of (in mM) 142.5 Na+, 5.0 K+, 127.5 Cl-, 1.2 Mg2+, 2.0 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 21 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 10 D-glucose, and Ca2+ as indicated. During dissection of the trabeculae the solution contained a high (15 mM) K+ and low (0.2 mM) Ca2+ concentration ([Ca2+]) to prevent spontaneous beating of the heart and thus to facilitate further dissection. The solutions were in equilibrium with 95% O2-5% CO2 (pH 7.4 at 25°C). Three bathing solutions were used for the determination of stress development and ATP hydrolysis rate in skinned trabeculae (9, 35): a relaxing solution, a preactivating solution with low Ca2+ buffering capacity, and an activating solution. The relaxing solution was composed of (in mM) 7.76 MgCl2, 5.86 Na2ATP, 20 EGTA, 0 HDTA, 0 CaEGTA, and 38.1 potassium propionate (Kprop). The preactivating solution contained 7.35 MgCl2, 5.86 Na2ATP, 0.5 EGTA, 19.5 HDTA, 0 CaEGTA, and 38.9 Kprop. For the activating solution, these values were 7.24 MgCl2, 5.97 Na2ATP, 0 EGTA, 0 HDTA, 20 CaEGTA, and 38.6 Kprop. In addition, all solutions contained 0.9 mM NADH, 100 mM BES, 10 mM phosphoenolpyruvate, 1 mg/ml pyruvate kinase (500 U/mg), 0.05 mg/ml lactate dehydrogenase (870 U/mg), 20 µM P1,P5-di(adenosine-5') pentaphosphate, 10 µM leupeptin, 10 µM pepstatin, 10 µM phenylmethylsulfonyl fluoride, 5 mM sodium azide, and 10 µM oligomycin B. The ionic strength of the solutions was kept at 200 mM by adding the appropriate amount of Kprop. The pH was adjusted to 7.0 at 20°C with KOH. The compositions were calculated using the methods described by Fabiato and Fabiato (12). The free Mg and MgATP concentrations were calculated at 1 and 5 mM, respectively. To achieve a range of free [Ca2+], activating and relaxing solutions were appropriately mixed, assuming an apparent stability constant of the Ca-EGTA complex of 106.58.

Measurement of tension and sarcomere length. The generated tension was calculated as force divided by the cross-sectional area calculated from the muscle dimensions.

Sarcomere length was measured by laser diffraction techniques, as described in detail previously (10, 32). Briefly, the cross striations of cardiac muscle will act as an optical grating for an incident laser light beam and will diffract the beam into zero-order and higher-order bands. The angle between the zero-order band and the first-order diffraction band is proportional to the sarcomere length and the wavelength of the laser light. To measure this angle, one of the first-order diffraction bands was projected onto a 512-element photodiode array (Reticon) that was scanned every 0.5 ms. The median position of the first-order intensity distribution was determined by analog circuitry after correction for the zero-order diffraction band and scatter; sarcomere length was calculated on-line. The spatial resolution of the system was ~10 nm. This method was used for the intact trabeculae. In the skinned preparations, sarcomere length in the relaxed state was measured from a calibrated screen and set to 2.3 µm (9, 35).

Measurement of ATP consumption in skinned trabeculae. The method used to measure the rate of ATP hydrolysis as function of tension development, which has been described in detail previously (9, 35), is based on the principle that NADH (but not NAD) absorbs ultraviolet light at 340 nm. Ultraviolet light from a 75-W halogen lamp (World Precision Instruments) was projected through the bath below the muscle, through a beam splitter, and subsequently onto two photodiodes via 340- and 410-nm interference filters (9, 35). The logarithm of the ratio between the light intensity at 340 nm (being sensitive to the NADH concentration in the bath) and 410 nm (serving as a reference signal) was calculated on-line. Because 1 mole of NADH is converted to NAD for every mole of ATP converted to ADP, the slope of the decay of the absorbance signal during activation of the muscle represents the rate of ATP consumption. The signal was calibrated by repeated injections of a known small amount (250 pmol) of ADP into the solution (using a motor-controlled calibration pipette) after the muscle has been relaxed after an activation-relaxation cycle.

Experimental protocol in intact trabeculae. After the trabeculae were mounted in the experimental setup, the preparation was stimulated through two platinum electrodes running parallel to the muscle at a rate of 0.5 Hz. Stimulus intensity was 50% above threshold, and stimulus duration was 2-5 ms. Muscles were stretched to a resting sarcomere length of ~2.10 µm, a length at which passive tension was usually 5-10% of active tension, and left to equilibrate for at least 1 h in an extracellular [Ca2+] ([Ca2+]o) of 1.5 mM. After this equilibration period, the muscle was restretched to a resting sarcomere length of 2.10 µm. If peak twitch tension production had decreased to <70% of control at this time, the preparation was discarded.

The study protocol started with the assessment of the tension-sarcomere length relationship (32). The muscle was stimulated at a frequency of 0.5 Hz in 1.5 mM [Ca2+]o and a diastolic sarcomere length of 2.10 µm. Every seven beats, the length of the preparation was changed for five beats; at the fifth beat, systolic length was plotted against developed tension. After these five beats, the muscle was again set at the initial sarcomere length for seven beats, and the protocol was repeated until a dataset was obtained covering sarcomere lengths from virtual slack length to a length at which high passive tensions were developed. Care was taken to measure sarcomere length at a predetermined site of the preparation so as to avoid motion artifacts.

Once a tension-sarcomere length relation was obtained, [Ca2+]o was reduced to 0.1 mM and tension was measured at steady state and a systolic shortening to 2.0 µm, again at the predetermined site of the muscle. The [Ca2+]o level was then increased to 2.5 mM with additional tension measurements at 0.3, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 mM. At maximum developed tension, five twitches were recorded and averaged to allow for the assessment of tension, time between 50% tension and maximum tension, and time between maximum tension and decline to 50% of peak tension (a measure of relaxation velocity). Ca2+ was then reduced to 0.6 mM and 0.2 µM isoproterenol (final concentration) was applied; twitch characteristics were recorded both before and after isoproterenol application.

Experimental protocol in skinned trabeculae. After being mounted, the muscle was bathed in a series of solutions with different activating Ca2+ levels so as to compile a tension-Ca2+ relationship. During each series of measurements, the trabecula was incubated in the relaxing solution for 3 min, in the preactivating solution for 3 min, in the activating solution for ~2 min, and from there back into the relaxing solution. During activation of the muscle, the decline in the NADH concentration is stochiometrically coupled to the rate of ATP consumption. This rate was plotted as function of active tension development; the slope of this relationship represents the energetic cost of tension maintenance during isometric contraction.

Data analysis. All measurements were compared for statistical significance among the three groups [Con, infarction without losartan (MI), and infarction with losartan (MI+Los)] using analysis of variance, followed by Student-Newman-Keuls test for multiple comparisons. A P value of <0.05 was considered significant. Data are expressed as means ± SE.

The data from the tension-sarcomere length relationships were grouped in 0.05-µm-wide bins of sarcomere length. Within each bin, data obtained in the three types of muscles were compared for a significant difference. For display purposes, a second-order polynomial fit was calculated through the data points.

Sigmoidal tension-Ca2+ relationships of each individual muscle (both intact and skinned) were fit using a nonlinear fit procedure to a modified Hill equation
T<IT>=</IT>T<SUB>max</SUB><IT>·</IT>[Ca]<SUP><IT>H</IT></SUP><IT>/</IT>([Ca]<SUP><IT>H</IT></SUP><IT>+</IT>EC<SUP><IT>H</IT></SUP><SUB>50</SUB>) (1)
in which T is developed tension, Tmax is the maximum saturated value that T can attain, EC50 is the [Ca2+] at which T is 50% of Tmax, and H represents the slope of the relationship (the Hill coefficient).

In the experiments with skinned trabeculae, the relationship between tension and ATPase for each muscle were fit by linear regression and the slope of these lines were used to test for differences among the groups of animals.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

For further presentation and discussion, the study groups are marked as Con, MI, and MI+Los.

Clinical consequences of infarction and differences between groups. No clinical signs of heart failure were noted in any of the animals after 12 wk of MI. During their stay at our department, 4 of 30 MI+Los rats died versus 2 of 28 within the MI group. Table 1 shows the anatomic findings in the animals we studied. Body weight in the losartan-treated group was lower than in the other groups. The wet-to-dry weight ratios for the lung and liver were not different between the groups, reflecting the absence of clinical signs of heart failure. Table 1 also shows the significant larger right ventricular weight in the animals from the MI group versus Con and MI+Los animals; the same held true for left ventricular weight. Both intact and skinned trabeculae were thicker in infarcted rats, with a tendency to a reduced hypertrophic response in the MI+Los group. Although losartan did blunt the increases in left ventricular and right ventricular weight that occurred after infarction, it did not influence the remodeling process as assessed by the unstressed left ventricular diameter measurement. That is, unstressed left ventricular diameter was increased in the infarcted animals but no difference was noted between MI+Los and MI and Con animals.

                              
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  		Table 1.   Anatomic characteristics in the three study groups

Tension-sarcomere length relationships in intact trabeculae. In each group, eight trabeculae were studied (we used only one trabecula per animal). The most impressive difference in the tension-sarcomere length relationships was the downward shift of the active tension production in the muscles from infarcted hearts if not treated with losartan. Thus, for a given systolic sarcomere length, less tension was produced after MI, whereas this decrease was prevented with losartan treatment. Figure 1 shows the aggregate curves for each group. Figure 1A depicts the results obtained in the Con animals. Figure 1B shows the data from the MI group with reference to the averaged fitted relationship reproduced from Fig. 1A (dotted line). Figure 1C compares the relationships between the MI+Los group and the group data shown in A and B (dotted lines). Tension generation in the MI group (Fig. 1B) was significantly depressed compared with the Con and MI+Los groups. This was most apparent at sarcomere length >1.8 µm. Trabeculae from hearts with a MI not treated with losartan tended to have a steeper passive tension-sarcomere length relationship, being significant at the highest sarcomere length range studied.


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  		Fig. 1.   Average active and passive tension-sarcomere length relationships in intact right ventricular trabeculae from the control (Con) group (A), the myocardial infarction (MI) group (B), and the MI+losartan (Los) group (C), shown as black-triangle, , and , respectively. Dotted lines in B and C are reproduced from A and B, respectively, for ease of comparison. After MI, active tension was significantly depressed (B), a process that was partially reversed by chronic treatment with losartan (C). Passive force was significantly higher in the MI group (B) compared with both other groups for the bin composed of sarcomere lengths from 2.1 to 2.2 µm.

Tension-Ca2+ relationships in intact trabeculae. Figure 2 shows the pooled tension-[Ca2+]o relationships for the eight trabeculae within each group. The average parameters obtained from the fit of the data to the modified Hill equation in the individual muscles are shown in Table 2. Tmax (see Table 2) was 31% lower in the MI group and 8% lower in the MI+Los group than in the Con group. The sensitivity of twitch tension for the [Ca2+]o (EC50 parameter) was 29% higher in the MI group, albeit that this difference did not reach statistical significance. The Hill coefficient, a measure of the steepness of the tension-[Ca2+]o relationship, was not significantly different among the groups, however.


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  		Fig. 2.   Average twitch tension-extracellular Ca2+ concentration ([Ca2+]o) relationships in intact trabeculae for the Con group (A), the MI group (B), and the MI+Los group (C). Dotted lines in B and C are reproduced from A and B, respectively, for ease of comparison. After MI, losartan significantly blunted the depression of the tension-Ca2+ relationship.


                              
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  		Table 2.   Average fit parameters for the tension-Ca2+ relationships

With increasing extracellular Ca2+ in the bathing medium, spontaneous activity increased in all groups, sometimes resulting in the occurrence of propagating contractile waves and synchronous "triggered" twitches. These more advanced forms of spontaneous activity were observed in none of the muscles in the Con group and in three and two muscles in the infarcted animals with and without losartan treatment, respectively. No effort was made to further quantify possible differences in the occurrence of spontaneous activity.

Twitch characteristics in intact trabeculae. Twitch characteristics were measured at saturated [Ca2+]o and at 0.6 mM, the latter both before and after application of 0.2 µM isoproterenol to the bathing solution.

Figure 3A shows the average twitch characteristics of the groups at saturated extracellular Ca2+ in the bath. The time to peak twitch tension was not different among the groups, whereas relaxation time was 29% longer in the MI group and 7% longer in the MI+Los group compared with the Con group. Because of this, total twitch duration was significantly prolonged in the untreated infarcted animals versus the other groups.


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  		Fig. 3.   A: average time between half-maximum and maximum twitch tension (TTP), between maximum twitch tension and decline to half-maximum tension (Relax), and their sum, total twitch duration (TWD), at saturated Ca2+ levels in the Con group (open bars), the MI group (gray bars), and the MI+Los group (solid bars). Twitch duration was significantly prolonged after MI, mostly due to a prolonged relaxation. This response was blunted by losartan. *P < 0.05 vs. Con group. B: response on maximum twitch tension after the addition of isoproterenol in the three groups (0.2 µM at [Ca2+]o = 0.6 mM). At baseline (open bars), twitch tension was comparable between the groups. After application of isoproterenol (gray bars), it increased significantly in the Con and MI+Los groups but not in the MI group. *P < 0.05 vs. baseline conditions.

At [Ca2+]o = 0.6 mM, peak tension was comparable in all three groups, reflecting the fact that the EC50 for [Ca2+]o of the tension-[Ca2+]o relations was lower in the MI group (see Fig. 2). However, isoproterenol increased peak tension by 113% in the Con group, by 127% in the MI+Los group, but only by 27% in the MI group (see Fig. 3B).

Tension-Ca2+ and tension-ATPase relationships in skinned trabeculae. We studied 13 trabeculae in the Con group, 11 in the MI group, and 12 in the MI+Los group. Figure 4A shows the pooled relationships between free [Ca2+] and isometric steady-state tension development in skinned trabeculae in the three groups. The average parameters obtained from the fit of the data to the modified Hill equation in the individual muscles are shown in Table 2; no differences were detected in the maximum tension production or in the Hill coefficient. However, a small but statistically significant difference was observed in the EC50 parameter, which was 14% and 15% higher, respectively, in the MI and MI+Los groups.


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  		Fig. 4.   A: average tension-free [Ca2+] relationship in skinned trabeculae from the Con (black-triangle), MI (), and MI+Los () groups fitted to a modified Hill equation. No significant differences were observed in maximum tension and the slope of the relationships (Hill parameter). B: average tension-ATPase activity relationship in skinned trabeculae from the three groups. No differences in the energetic cost of tension maintenance were observed between the groups.

Figure 4B shows the pooled relationship between steady-state tension development and the rate of ATP consumption in the skinned trabeculae. The average slope of this relationship, obtained from the linear regression fit of the data in individual muscles in the three groups, is presented in Table 2. On average, this slope was not significantly different between the groups, indicating no change in the energetic cost of tension maintenance by the myofilaments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our study shows that left ventricular MI in rats has a negative impact on right ventricular contractile function, even in the absence of clinical signs of congestive heart failure, and that losartan partially prevents this dysfunction. Also, our findings provide some insight into the cellular mechanisms underlying the right ventricular dysfunction.

Right ventricular contractile dysfunction after left ventricular infarction. Several studies (5, 11, 17, 22, 36) have demonstrated that ligation of the left anterior descending artery (with subsequent MI) not only induces dilatation of the left ventricular cavity and an increase in left ventricular weight but also has an impact on the right ventricle: right ventricular weight increases and its contractile function or that of isolated trabeculae or myocytes becomes impaired. Our findings are in line with these observations, in that the diameter of the left ventricle and its weight increased after the infarction, the right ventricle showed a hypertrophic response as judged both by its weight and by the thickness of the isolated trabeculae, and the contractile function of the right ventricular trabeculae was impaired. The latter was demonstrated by a 31% decrease in peak tension at saturated extracellular Ca2+ levels in the MI group compared with the Con group (Fig. 2), a depressed active tension-sarcomere length curve with an increase in passive diastolic tension (Fig. 1), and a prolonged twitch relaxation (Fig. 3).

The reasons for the anatomic and functional changes in the right ventricle are not yet completely understood. It might be argued that pulmonary artery pressures increase after left ventricular infarction; indeed, the findings reported in earlier studies, in which rats showed signs of right-sided congestion, would fit this explanation (11). In our rats, however, no clinical signs of heart failure were present and the wet-to-dry weight ratios of both the lungs and liver were similar for the animals with and without MI. Thus, because pulmonary artery pressures were not measured directly, we cannot exclude other possible factors impacting on the right ventricle, e.g., humoral ones.

Whether initiated by an increased pulmonary artery pressure or other factors, our observations offer some insight into the cellular defects underlying the right ventricular contractile dysfunction after left ventricular infarction. Potential explanations include changes in the Ca2+ handling of the cell, a change in the myofilament sensitivity for Ca2+, and/or a change in the extracellular matrix. In our studies, we did not find a change in the tension-Ca2+ relation in the skinned preparations after a MI (Fig. 4). This virtually excludes decreased myofilament sensitivity to Ca2+ or a change in the isoform distribution of the myofilament contractile proteins as the major explanation for the decreased tension generated by the intact trabeculae 12 wk after MI. The observed hypertrophic response in both left and right ventricle after MI suggests a change in the extracellular matrix; in general, such changes have predominantly been related to diastolic dysfunction and may therefore explain the increase in passive tension in our study. An increase in extracellular matrix may also have (falsely) caused a decrease in active tension because this would have decreased the number of contractile units per square millimeter. If this were the case, however, one would have expected a decreased maximally activated tension in the skinned trabeculae as well. That is, a depression of the complete tension-Ca2+ relation in both intact and skinned trabeculae would have been anticipated. In fact, we did not observe such changes. The unchanged EC50 and Hill coefficient in the tension-Ca2+ relation, and the unchanged time to peak tension of the twitch in the intact trabeculae after a MI, indicates that the processes involved in the systolic release of Ca2+ from the sarcoplasmic reticulum have not slowed down. Thus the most important cellular defect underlying the contractile dysfunction that we observed in our postMI model probably involves an abnormal Ca2+ reuptake in the sarcoplasmic reticulum. Obviously, the increased passive tension and prolonged twitch relaxation would be consistent with such a depressed reuptake of Ca2+ into the sarcoplasmic reticulum. A less than optimal diastolic filling of the sarcoplasmic reticulum would explain a decrease in tension at saturated Ca2+ levels. Nevertheless, from our studies, we cannot exclude the possibility that an increased extrusion of Ca2+ from the cell by enhanced function of the Na-Ca2+ exchange mechanism (18, 30), a lower threshold for Ca2+ release from the sarcoplasmic reticulum, or a decoupling of the Ca2+-induced Ca2+ release process (16) contributed to some of the changes observed.

Once again, it should be noted that our animals showed no signs of heart failure. It seems logical to assume that the contractile dysfunction that we observed would over time have caused symptoms. Indeed, we (11) previously observed overt congestive heart failure in rats 24 wk after their infarction, at that moment having a 40% depressed peak tension, a value not much different from the one that we currently find in asymptomatic animals. Of interest is the fact that the skinned trabeculae from the infarcted animals in our previous study (11) showed a depressed maximally activated tension. This indicates that the cellular changes in our model are time dependent. Apparently, an asymptomatic phase with clear alterations in contractile function in twitching isolated myocardium due to abnormal Ca2+ handling precedes the symptomatic phase, in which changes in the myofilament sensitivity to Ca2+ occur. More detailed studies are required to address the time course of these cellular changes with the development of congestive heart failure.

In the present study, 12 wk following MI, right ventricular trabeculae displayed depressed function (cf Table 2, Figs. 1 and 2). We adopted this small rodent model because the right ventricle of rats can provide thin and homogeneous cardiac trabeculae (7, 10, 11, 32). This feature is essential to ensure both the metabolic stability of the preparation and to allow for the measurement and control of sarcomere length by laser diffraction techniques (7, 10, 32). Furthermore, by studying trabeculae from the right ventricle, we ensured that only noninfarcted tissue was studied. The relation between active force and sarcomere length in intact, electrically stimulated right ventricular trabeculae that were isolated from animals with MI was significantly depressed compared with the relation in trabeculae obtained from control noninfarcted operated animals (cf Fig. 1). It has been suggested that the active force-sarcomere length relation underlies the end-systolic pressure-volume relationship of the intact ventricle (32). Thus this observation suggests that the contractile state of the right ventricle was depressed in the infarcted group of animals. This may seem surprising because right ventricular dysfunction was induced by left ventricular infarction in our study. However, in this model, both the right and left ventricle are presumably exposed to increased mechanical load and neurohormonal stimuli after left ventricular MI, and it may be that some of these factors affect both ventricles in a similar manner. Consistent with this notion is the observation that similar changes in contractile proteins and Ca2+ handling proteins have been observed in the left and right ventricle during the development of heart failure in this model, albeit with a prolonged time course in the right ventricle (1, 3, 13). Further investigation is required to determine which of these factors is responsible for the decrease in myocardial function.

Effects of losartan on right ventricular contractile dysfunction. In the animals with MI, the addition of a daily dose of 30 mg/kg losartan clearly prevented the hypertrophic response in both left and right ventricle, the decrease in peak tension at saturated Ca2+, the depression in the active tension-sarcomere length relationship, the increase in passive tension, the prolongation in twitch relaxation, and the blunted response to isoproterenol.

Several mechanisms may have contributed to these beneficial effects. A vast amount of data demonstrates that losartan either has a preventive or regressive effect against hypertension-induced cardiac hypertrophy (6, 14). In our experiments, a losartan-mediated decrease in systemic blood pressure or pulmonary artery pressure may have reduced the loading conditions imposed on the heart with subsequent prevention of a hypertrophic response. In addition, stimulation of the angiotensin II receptor has been shown to induce a growth response (22, 24), a signal that may have been blocked by losartan. A recent study (8) in rats showed that losartan reduced the fibrotic response at and remote of the site of infarction 4 wk after coronary artery ligation. Reduction of cellular hypertrophy may have contributed to the observed decrease in passive tension after treatment with losartan.

In our experiments, we noted a blunted response to isoproterenol in the infarcted animals, an effect described previously in postinfarct rat left ventricular preparations (19). This may relate to a downregulation of beta -receptors or an uncoupling of the receptor from its second messenger system as a consequence of chronic activation of the neurohumoral system and consistently elevated levels of circulating catecholamines. As pointed out by Litwin and Morgan (19), a reversal of this blunted response (as seen with losartan) may be explained by a general improvement in hemodynamics, such that the animals no longer are dependent on a constant adrenergic drive. Alternatively, losartan may affect the processes that are involved in the action of catecholamines. Indeed, angiotensin II receptor activation decreased beta 1-adrenergic responsiveness in rats in a study by Schwartz and Naff (25), an effect that may have been prevented by losartan. From our studies, the possibility that losartan directly or indirectly influences the Ca2+ reuptake into the sarcoplasmic reticulum cannot be refuted. However, a direct effect has, to our knowledge, never been described. On the other hand, an effect of losartan on the Ca2+ handling of the cell is not that elusive because the angiotensin II receptor is coupled to Ca2+ regulation by activation of phospholipase C and its second messenger inositol trisphosphate, and thus activation of protein kinase C (31). Stimulation with angiotensin II in cardiomyocytes from spontaneously hypertensive rats (33), in cultured neonatal rat atrial and ventricular myocytes (34), and in rat myocytes after coronary artery constriction (17) has been shown to increase intracellular Ca2+ levels. Furthermore, cardiac dysfunction after MI in rats appears to be associated with an upregulation of angiotensin II type 1 receptors and by an enhanced phosphoinositol turnover in myocytes from both left and right ventricle (17, 20, 22). It is not known, however, to what extent such alternative Ca2+ regulating pathways play a role in heart failure.

Other studies of losartan after experimental MI. Although the effects in hypertension are well documented (14), only a few other studies have addressed the potential of losartan to beneficially influence the natural course after MI. In a study without the Con group, Milavetz et al. (20) showed that losartan had similar effects as the ACE inhibitor captopril on 1-yr survival after MI in a rat model without differences in parameters of left ventricular remodeling. Schieffer and co-workers (23) demonstrated, also in rats, that both losartan and the ACE inhibitor enalapril reduced the infarct-related increases in left and right ventricular weight. Smits et al. (28) also noted the prevention of left ventricular hypertrophy after MI by losartan, whereas in another study (8), losartan reduced the postinfarct fibrotic response. Losartan prevented the increase in left ventricular end-diastolic pressure (4, 21, 27), the increase in the relaxation constant tau  (4, 27), and the increase in right ventricular pressures (27) in invasive studies after coronary artery ligation in rats.

In summary, we have shown that left and right ventricular hypertrophy, right ventricular contractile dysfunction, prolonged twitch relaxation, and a blunted response to inotropic intervention exist in the asymptomatic, subacute phase after MI in a rat model, whereas the myofilament sensitivity to Ca2+ is unaltered. The deleterious effects are prevented by the daily administration of the angiotensin II receptor blocker losartan. Our results suggest that the beneficiary effect of losartan treatment results not from improved myofilament function but rather from improved myocyte Ca2+ homeostasis.


    ACKNOWLEDGEMENTS

This study was supported by the Stichting Stibeca (Den Bosch, The Netherlands) (to M. C. G. Daniels) by National Heart, Lung, and Blood Institute Grants RO1-HL-52322 and PO1-HL-62426, Project 4 (to P. de Tombe), and by National Institutes of Health Grant T32-07692 (to R. S. Keller). P. de Tombe was an Established Investigator of the American Heart Association during the time this study was performed.


    FOOTNOTES

Address for reprint requests and other correspondence: P. de Tombe, Dept. of Physiology and Biophysics, MC 902, Sarcomere Dynamics Laboratory, MBRB Rm. 1370, 900 S. Ashland Ave., Univ. of Illinois at Chicago, Chicago, IL 60607-7171 (E-mail: pdetombe{at}uic.edu).

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

Received 30 April 2001; accepted in final form 1 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(5):H2150-H2158
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