Altered Ventricular and Myocyte Response to Angiotensin II in Pacing-Induced Heart Failure
Abstract Alterations in the cardiac response to angiotensin II (Ang II) may contribute to the functional impairment in tachycardia-induced heart failure (congestive heart failure [CHF]). Accordingly, we studied the response to Ang II in eight conscious instrumented dogs before and after inducing CHF. Left ventricular (LV) performance was assessed by measuring LV pressure and LV volume. Isolated myocyte function was evaluated using computer-assessed videomicroscopy. In conscious animals before CHF, Ang II produced a load-dependent slowing of the time constant of LV relaxation (τ) and did not depress intact LV contractile function. After CHF, although Ang II produced a similar increase in LV systolic pressure, the increases in LV diastolic pressure and time constant τ were much greater, and contractile performance was depressed. These changes persisted when the elevation of end-systolic pressure was prevented by nitroprusside. Similar changes were also present after autonomic blockade. In isolated myocytes, before CHF, Ang II (10−6 mol/L) produced a slight positive inotropic effect. In contrast, after CHF, Ang II produced a negative inotropic effect and slowed the rate of relengthening. The effects in the intact LV and myocytes were reversed by an Ang II AT1 receptor blocker (losartan). We conclude that pacing-induced CHF alters the LV and myocyte response to Ang II, so that Ang II produces direct depressions in intact LV contraction, relaxation, and filling and exacerbates myocyte contractile dysfunction. These effects are mediated through the activation of AT1 receptors.
Congestive heart failure (CHF) is accompanied by LV dilation and activation of the renin-angiotensin system.1 2 3 4 Ang II stimulates myocyte and fibrous growth,5 6 7 8 and treatment with ACE inhibitors attenuates the progression of LV dilatation and improves survival in patients with CHF.9 10 11 ACE inhibitors delay or prevent the development of CHF in rats with ascending aortic stenosis8 and in dogs with rapid ventricular pacing.4
Whether Ang II plays a role in contractile behavior in CHF remains undetermined12 13 ; Ang II has been reported variably to have a positive inotropic effect,14 15 16 no effect,17 negative inotropic effect,18 19 or biphasic20 inotropic effects on normal LV contractile performance. These disparate observations may be due to the confounding effects of anesthesia, open-chest surgery, tissue preparations, loading conditions and dosage of Ang II, and species differences.21
The effects of Ang II on normal myocardial contraction may be altered in pathological states.16 19 20 22 The direct effect of Ang II to impair myocardial relaxation and diastolic function is greater in hypertropic than normal myocardium,23 24 perhaps resulting from the interaction of Ang II with the impaired calcium handling seen in hypertrophy. Since calcium handling is also altered in CHF, the failing myocardium may be more sensitive to Ang II. If this is correct, Ang II may exacerbate LV systolic and diastolic dysfunction and contribute to the functional impairment of CHF. In the present study, we evaluated the effect of Ang II on LV systolic and diastolic performance in conscious dogs and in cardiomyocytes obtained from the LV of these animals, both before and after pacing-induced CHF.
Materials and Methods
Eight healthy, adult, heart worm–negative mongrel dogs (weight, 25 to 34 kg) were instrumented under anesthesia, which was induced with xylazine (2 mg/kg IM) and sodium thiopental (6 mg/kg IV) and maintained with halothane (0.5% to 2%). They were intubated and ventilated with oxygen-enriched room air to maintain arterial oxygen tension at >100 mm Hg and pH between 7.38 and 7.42. A left lateral thoracotomy was performed aseptically. Micromanometer pressure transducers (Konigsberg Instruments) and polyvinyl catheters for transducer calibration (internal diameter, 1.1 mm) were inserted into the LV and LA. Three pairs of ultrasonic crystals (5 MHz) were implanted in the endocardium of the LV to measure the anterior-posterior, septal-lateral, and base-apex (long-axis) dimensions, using methods we have previously described.25 Hydraulic occluder cuffs were placed around the superior and inferior venae cavae. Two 54-cm sutureless myocardial leads (model 4312, Cardiac Pacemakers, Inc) were implanted within the myocardium of the right ventricle and right atrium, and the leads were attached to a unipolar multiprogrammable pacemaker (model 8329, Medtronics, Inc) positioned under the skin of the chest. Two ultrasonic transit-time flow probes (model 2R or 3R, Transonic Systems, Inc) were placed on the proximal left circumflex and left anterior descending coronary artery. The wires and tubing were tunneled subcutaneously and brought out through the skin of the neck.
Data Collection and Calculation
Studies were performed in unanesthetized dogs (after full recovery from instrumentation, ≈2 weeks after the original surgery) as they lay on their right sides in slings. The LV and LA catheters were connected to pressure transducers (Statham P23DB) and calibrated with a mercury manometer. The signal from the micromanometer was adjusted to match that of the catheters. The transit time of 5-MHz sound between the crystal pairs was determined and converted to distance, assuming a constant velocity of sound in blood of 1.55 m/ms. The analog signals were recorded on a 16-channel oscillograph (Astro-Med), which was digitized with an on-line analog-to-digital converter (Data Translation Devices) at 200 Hz. Each data acquisition lasted 15 to 20 seconds, spanning several respiratory cycles. The derivatives of LV pressure and volume were calculated using the five-point Lagrangian method.26
LV Function Evaluation
Studies Before CHF With Reflexes Intact
Data were initially recorded with the animals lying quietly on their sides without medication to obtain baseline values. Three sets of variably loaded pressure-volume loops were generated by sudden transient occlusion of the cavae. This caused a progressive fall in LV PES-VES over a 15-second recording period. Immediately after the recording period, the caval occlusion was released, and hemodynamic parameters were allowed to restabilize. After all parameters returned to their baseline levels, Ang II (300 to 500 ng/kg) was injected intravenously over 2 minutes and then infused at 100 ng·kg−1·min−1, which increased PES by ≈40 mm Hg. After 5 to 8 minutes, when the arterial pressure had reached a stable level, steady state and caval occlusion data were again collected.
To assess the direct effects of Ang II, independent of its effect on systolic load, during the Ang II infusion, NTP (0.7 to 1.0 mg/min) was administrated to restore the PES to the control level. After stabilization, the steady state and caval occlusion data were obtained.
Ten minutes after stopping NTP infusion, a second set of control and Ang II steady state data, as well as caval occlusion data, were collected, and then the Ang II AT1 receptor blocker LOS (1 mg/kg plus 50 ng·kg−1·min−1 IV) was substituted for NTP. We confirmed that this dosage of LOS produced adequate AT1 blockade by evaluating the response of arterial pressure to an infusion of Ang II. Consistent with previous studies in dogs, this dosage of LOS completely blocked the pressure response to Ang II.27 28
Since heart rates were higher in animals after the development of CHF than before CHF, we compared the response of Ang II before and during RA pacing in a subgroup of four conscious normal dogs. Data were collected at baseline, and then the heart rate was increased with RA pacing to the rate (130 to 140 bpm) that matched the heart rate with CHF. After the animals were stabilized for 10 to 15 minutes, both steady state and caval occlusion data were obtained, and then Ang II was infused. After 5 to 8 minutes, the data collections were repeated.
Studies Before CHF With Autonomic Blockade
To assess the interactions of Ang II with autonomic reflexes, the Ang II protocol was repeated after administering metoprolol (0.5 mg/kg IV) and atropine (0.1 mg/kg IV). The adequacy of the autonomic blockade was confirmed in each animal by the lack of an increase in heart rate after arterial pressure was reduced by 30 mm Hg by caval occlusion.
Studies During the Development of CHF
After completion of the baseline studies, the pacing rate was adjusted to 220 to 250 bpm using the external magnetic control unit. The pacemaker rate was adjusted below the spontaneous rate three times per week. The animal was allowed to equilibrate for 30 minutes, and then data were collected. The pacing rate was returned to 220 to 250 bpm. After pacing for 4 to 5 weeks, when the LV PED during the nonpaced period had increased by >15 mm Hg over the prepacing control level and the animals had begun to show clear evidence of CHF (anorexia, ascites, and pulmonary congestion), the pacing was disconnected, and CHF data were obtained.
Studies After the Onset of CHF
The pacemaker was turned off, and the animal was allowed to stabilize for at least 30 minutes. Data were recorded with the animals lying quietly on their sides. Then the above protocols were repeated both with and without autonomic blockade.
Data Processing and Analysis
To account for respiratory changes in intrathoracic pressure, steady state measurements were averaged over a 15- to 20-second recording period that spanned multiple respiratory cycles.25 The stored digitized data were analyzed by computer algorithm. End systole was defined as the upper left corner of the LV pressure-volume loop, identified using the iterative technique described by Kono et al.29 End diastole was defined as the relative minimum following the A wave of the high-fidelity LV pressure tracing. The time of mitral valve opening was defined to be when LV pressure fell below LA pressure. LV pressure and volume were measured at end diastole, end systole, and minimum LV pressure. LA pressure was measured at the time of mitral valve opening (peak V wave) and at the peak of the A wave. The mean LA pressure was also determined.
The LV volume was calculated as a modified general ellipsoid using the following equation: VLV=(π/6)DAPDSLDLA, where VLV is volume of LV, DAP is the anterior-posterior LV dimension, DSL is the septal-lateral LV dimension, and DLA is the long-axis LV dimension. This method of volume calculation gives a consistent measure of LV volume despite changes in LV loading conditions, chamber configuration, and inotropic state. It is similar to that used and validated by others,30 except that we determined endocardial dimensions directly, making the subtraction of LV wall thickness or volume unnecessary.
SW was calculated by point-by-point integration of the LV pressure-volume loop for each beat as described by Glower et al.31 Stroke volume was calculated as LV VED minus VES. The rate of LV relaxation was analyzed by determining the time constant of the isovolumic fall of LV pressure. LV pressure from the time of minimum dP/dt until mitral valve opening was fit to an exponential equation: P=PA·exp(−t/τ)+PB, where P is LV pressure, t is time, and PA, PB, and τ are constants determined by the data. Although the fall of isovolumic pressure is not exactly exponential,32 the time constant, derived from the exponential approximation, provides an index of the rate of LV relaxation.33 TSR was estimated as PES divided by cardiac output, as suggested by Sunagawa et al.34 Ea was calculated as LV PES divided by stroke volume. The coronary blood flow was calculated as the sum of the flow of the left circumflex and left anterior descending coronary arteries.
Only caval occlusions that produced a fall in LV PES of at least 30 mm Hg were analyzed. Premature beats and the subsequent beat were excluded from analysis. The LV PES-VES, dP/dtmax-VED, and SW-VED data during the fall of LV pressure, produced by each caval occlusion, were fit using the least-squares technique, as we have previously described.35
Cardiomyocyte Function Evaluation
Myocardial biopsies were obtained at the time of surgery and after 4 to 5 weeks of pacing before the animals were killed. Using biopsy forceps, tissue samples were obtained from the epicardium of the LV anterior wall and placed in cold (4°C) BDM (Sigma) protective solution (pH 7.3; osmolality, 285 mOsm/L) consisting of basal solution HBS-1, which contained (mmol/L) NaCl 130, KCl 5.4, NaHCO3 2.0, glucose 15.0, and HEPES 10.0; MgSO4·7H2O (1.2 mmol/l) plus BDM (30 mmol/L) and 10 μL insulin (0.01 U/mL) were added to the solution. The tissue specimens were maintained in this preoxygenated solution for 30 minutes and then minced into 1-mm3 cubes. After they were rinsed several times with basal solution HBS-1, the minced tissue specimens were transferred to a centrifuge tube that contained 3 mL prewarmed 0.2% trypsin solution made of 6 mL basal solution HBS-1 plus 12 mg trypsin type XI (diphenyl carbamyl chloride treated, 0.2% [wt/vol], Sigma) and were gently agitated in a water bath for 5 minutes at 35°C. The supernatant was removed, and enzyme solution (3 mL), basal solution HBS-1 plus 20 mg collagenase (142 U/mg, type I, Worthington No. F3K009), and 40 mg of BSA (fraction V, fatty acid free, Sigma) were added to the trypsinized tissue for 15 minutes. The supernatant was removed, and fresh enzyme solution (3 mL) was added and allowed to incubate for another 15 minutes. Then the cells were allowed to settle by gravity. The final pellet was resuspended in HBS-1 solution. After each settling, the HBS-1 solution was changed stepwise to increase calcium concentrations (ie, 250, 500, and 1000 μmol/L). Finally, the cells were suspended in HBS-1 with 1.8 mmol/L CaCl2 and stored at room temperature until ready for use. The number of viable cells was counted at ×100 magnification using a hemocytometer (R-J Cambridge Instruments, Inc).
After 2 hours of stabilization, the isolated myocytes were placed in superfused culture dishes. The myocytes were imaged with an inverted microscope using a ×40 phase-contrast objective. The image was entered into the image analysis system through a high-resolution monochrome video camera. Measurements of myocyte dimensions were made in 40 to 50 randomly selected rod-shaped cells from each experiment.
Determination of Contractile Responses of Myocytes to Ang II Before and After CHF
Isolated myocytes were placed in an open thermostatically controlled (22°C), flow-through, T-culture dish and continuously superfused with oxygenated HBS-1 solution. The myocytes were imaged using a ×40 long-working-distance Hoffman modulation contrast objective attached to an inverted microscope, and the myocyte contractions were elicited by field stimulation at a frequency of 1.0 Hz, at 1.2 times the contraction threshold and a duration of 5 milliseconds. Myocyte motion signals and contraction amplitude were measured using a video-dimension analyzer (model M303, Instrumentations for Physiology and Medicine, Inc). Stimulated myocytes were allowed a 5-minute stabilization period, after which steady state data were collected for 20 seconds. The myocytes were then exposed by Ang II (10−6 mol/L). Data were acquired after 3 to 5 minutes of drug exposure and 5 minutes after drug washout. In a subset of these cells, an Ang II AT1 receptor blocker, LOS (10−5 mol/L), was added to the superfusion in the continued presence of Ang II. Percent shortening was determined as the percent difference between maximum and minimum cell length of each contraction; the maximum rate of shortening (+dL/dt) and relengthening (−dL/dt) were obtained by differentiating the digitalized contraction profiles. The time to peak contraction was computed by calculating the time required for the velocity profile to reach zero velocity after contraction.
Data were summarized as mean±SD. Multiple comparisons were performed using ANOVA. When a significant overall effect was present, intergroup comparisons were performed using a Bonferroni correction for multiple comparisons. Myocyte functional data were analyzed using the mean measurements obtained for each dog before and after CHF. Significance was accepted at P<.05.
At the conclusion of the studies, the animals were killed by a lethal injection of sodium pentobarbital (100 mg/kg IV), and the hearts were examined to confirm that the instrumentation was properly positioned.
Effects of Pacing-Induced CHF
Steady state measurements before and after CHF are summarized in Tables 1⇓ and 2⇓. After 4 to 5 weeks of rapid pacing with reflexes intact, LV PED progressively increased from 9.0±2.2 to 19.6±1.6 mm Hg (P<.01). VES and VED (49.6±8.0 versus 39.7±4.0 mL, P<.01) also increased, whereas cardiac output decreased because of the marked reduction of stroke volume (10.0±1.2 versus 14.8±4.6 mL, P<.05). Although mean LA pressure was significantly increased (20.4±3.8 versus 5.2±1.4 mm Hg, P<.01), dV/dtmax decreased (173±47 versus 200±40 mL/s, P<.05) because of a marked increase in minimum LV pressure (13.9±3.5 versus 1.0±1.6 mm Hg, P<.05). The time constant τ (38.7±4.3 versus 26.3±2.8 milliseconds, P<.05), TSR, and Ea (10.4±1.8 versus 8.2±3.6 mm Hg/mL, P<.05) all increased. We have calculated the time constant τ using both zero (Weiss algorithm) and nonzero asymptote (by LV P dP/dt) methods, and the results were similar with both techniques, indicating that during Ang II infusion, PB was decreased.
LV pressure-volume analysis before and after CHF are summarized in Tables 3⇓ and 4⇓. After 4 to 5 weeks of rapid pacing with reflexes intact, the slopes of the LV PES-VES relation (5.1±0.5 versus 7.4±0.8 mm Hg/mL), the dP/dtmax-VED relation (59.3±12.3 versus 103.6±13.3 mm Hg/sec/mL), and the SW-VED relation (60.8±3.7 versus 73.3±3.2 mm Hg, P<.05) all significantly decreased, and all three relations were shifted toward the right, consistent with a depression of LV contractile performance. Similar changes were also present after autonomic blockade. These observations are consistent with our previous reports36 37 and other reports4 demonstrating that chronic rapid pacing produces cardiac dilation and impairment of LV systolic and diastolic performance. In addition, the animals had clinical evidence of pulmonary congestion and ascites.
Effect of Ang II, Independent of Systolic Load
The steady state hemodynamic data during control and Ang II infusion before and after CHF are summarized in Tables 1⇑ and 2⇑. Before CHF, with reflexes intact, Ang II (500 ng/kg plus 100 ng·kg−1·min−1 IV) increased PES (37±9 mm Hg), TSR, Ea, minimum LV pressure, and the time constant τ. The mean coronary flow and heart rate were relatively unchanged. These effects on LV relaxation, τ, and minimum LV pressure were reversed when PES was returned to control with NTP (Table 1⇑ and Fig 1A⇓). In contrast, after CHF, Ang II produced a similar increase in PES but larger increases in τ, minimum LV pressure, mean LA pressure, and PED (Table 1⇑) and produced an upward shift of the early diastolic position of the LV pressure-volume loop (Fig 1⇓). These changes persisted when elevations of PES were prevented with NTP (Fig 1B⇓) but were completely reversed by LOS, a selective AT1 receptor antagonist (Fig 1C⇓).
The pressure-volume data during Ang II infusion before and after CHF are summarized in Tables 3⇑ and 4⇑. Before CHF, with reflexes intact, as shown in Fig 2A⇓, although Ang II caused a parallel leftward shift of the PES-VES relation (Fig 5⇓), the slope was not altered. Furthermore, both the positions and slopes of the dP/dtmax-VED and SW-VED relations remained unchanged, indicating that in the normal intact LV, Ang II had no direct inotropic effect. As shown in Fig 2B⇓, similar findings were also observed after autonomic blockade. After CHF, the LV response to Ang II was altered. Ang II produced significant decreases in the slopes of the PES-VES relation (1.4±0.3 mm Hg/mL, P<.05), dP/dtmax-VED relation (9.8±0.9 mm Hg·s−1·mL−1, P<.05), and SW-VED relation (10.7±2.8 mm Hg, P<.05) and shifted these relations to the right. The decreases in slopes and rightward shifts of all three relations after Ang II are indicative of a direct depressant effect on LV contractility. Similar observations were also obtained after autonomic blockade. Examples of three LV pressure-volume relations derived from the pressure-volume loop generated during Ang II infusion after autonomic blockade are presented in Fig 3B⇓. Ang II produced rightward shifts of the PES-VES, dP/dtmax-VED, and SW -VED relations, with reduced slopes. Ang II–induced cardiac depressants were also present after autonomic blockade. The Ang II–induced depression of LV pressure-volume relations persisted when the elevation of PES was returned to control with NTP but were restored to their control position and slopes by LOS (Tables 3⇑ and 4⇑ and Fig 4⇓).
Ang II Levels
We have previously reported that the resting value of plasma Ang II was 34.8±10.9 pg/mL before CHF and increased to 138±56 pg/mL after CHF.38 In two animals, Ang II levels were measured and increased to >500 pg/mL both before and after CHF during Ang II infusions. By comparison, plasma Ang II increased to a similar level (482±129 pg/mL) during exercise after CHF.38
Effect of Heart Rate
As shown in Tables 5⇓ and 6⇓, before CHF, compared with the unpaced control condition, RA pacing produced an increase in heart rate similar to that occurring with CHF (109±9 to 136±51 bpm). As heart rate increased, the minimum LV pressure (1.0±1.7 versus −0.9±2.2 mm Hg, P<.05), PED, VED, VES, and τ all significantly decreased. Compared with the unpaced control condition, Ang II caused similar increases in PES, LV PED, minimum LV pressure, and mean LA pressure during RA pacing. Furthermore, Ang II induced a similar parallel leftward shift of the PES-VES relation (Fig 5⇓), and both the positions and slopes of dP/dtmax-VED and SW-VED relations remained unchanged with RA pacing, indicating that before CHF, the response to Ang II was not altered by the increase in heart rate.
Effect of Rapid Pacing on Cardiomyocyte Structure and Contractile Performance
The yield of viable myocytes from the LV biopsies was similar (70% to 80%) both before (Fig 6A⇓) and after (Fig 6B⇓) CHF. At room temperature (22°C), the isolated myocytes maintained a rod-shaped morphology for >18 hours. After isolation, cell viability at 13 to 18 hours was not significantly lower in the CHF heart (71±4.6% versus 78±3.5%, P=NS). An overall viability of 70% is usually indicative of a good-quality isolation39 and is consistent with previous studies.39 40 As shown in Fig 6⇓, with CHF, the length of myocyte was increased (58±9%, from 124±4 to 196±8 μm; P<.05), and the length-to-width ratio was greater (59±7%, P<.05) than in the normal cells.
Myocyte contractility was markedly depressed after CHF. There was a 41% (8.1±0.3% versus 14.5±0.5%, P<.05) decrease in the extent of shortening, a >38% reduction in peak velocity of shortening (96±8 versus 155±9 μm/s, P<.05), and a 26.4% reduction in peak velocity of relengthening (78±5 versus 106±7 μm/s, P<.05). The time to peak contraction was markedly prolonged (259.0±19.3 versus 219.0±15.7 ms) after CHF.
Effects of Ang II on Cardiomyocyte Contraction
As presented in Table 7⇓, before CHF, superfusion with Ang II (10−6 mol/L) slightly increased the amplitude of myocyte contraction with increased percent shortening (16.6±0.4% versus 14.5±0.5%, P<.05) and +dL/dtmax (198±10 versus 155±9 μm/s, P<.05) and relatively unchanged values for the peak velocity of relengthening (116±8 versus 106±7 μm/s, P=NS) and the time to peak contraction (233.0±13.2 versus 219.0±15 ms, P=NS). As shown in Fig 7⇓, myocyte response to Ang II was changed after CHF. Ang II caused a marked decrease in cell contraction with significantly reduced percent shortening (4.6±6.5% versus 8.1±0.3%, P<.01), +dL/dtmax (76±6 versus 96±8 μm/s, P<.05), and −dL/dtmax (57±7 versus 78±5 μm/s) after CHF. The time to peak contraction was also prolonged (298.0±13.8 versus 259.0±19.3 milliseconds). These effects were completely reversed after washout of the Ang II. Furthermore, addition of the Ang II AT1 receptor blocker LOS (10−5 mol/L) to the superfusion solution in the continued presence of Ang II completely reversed the effects.
The major findings of this investigation are as follows: (1) In conscious dogs, before CHF, Ang II has no direct depressant effect on LV contractility, relaxation, and LV filling. (2) After pacing-induced CHF, Ang II depresses LV contraction and relaxation. (3) The altered response of LV chamber performance to Ang II in CHF results from an enhanced sensitivity of the failing LV to increased arterial pressure and a direct depression of cardiomyocyte contractile performance and relaxation mediated through activation of myocardial AT1 receptors. This suggests that Ang II may contribute to the functional impairment of both systolic and diastolic performance in CHF. Thus, renin-angiotensin system activation with CHF may have adverse functional consequences that are due to an altered response to Ang II in CHF in addition to effects on cardiac structure.
Ang II is a potent vasoconstrictor. Consistent with previous observations, Ang II produced increases in PES and VED in our animals before and after CHF (Tables 1⇑ and 2⇑). The failing heart is more sensitive to increased afterload41 42 43 ; thus, some of the decreased LV systolic performance and slowed relaxation we observed with Ang II in CHF was due to an Ang II–induced increase in arterial pressure.
To avoid the potentially confounding effects of these changes in loading conditions on conventional measures of LV performance, we evaluated LV contractile performance in the pressure-volume plane. We found that before CHF, Ang II had no direct inotropic effects (Tables 3⇑ and 4⇑), although the PES-VES relation was shifted to the left (Fig 2⇑) because of Ang II–induced arterial constriction.35 44 45 In contrast, after CHF, Ang II produced a significant depression in LV contractile performance as indicated by the decreased slopes and rightward shift of the LV pressure-volume relations (Table 3⇑). The depression of LV contractile performance with Ang II after CHF was apparent with intact reflexes (Fig 3A⇑) as well as after autonomic blockade (Table 4⇑ and Fig 3B⇑) and persisted when the increase in PES was prevented. Furthermore, this response to Ang II was not present when the heart rate before CHF was increased to the faster resting rate present with CHF (Table 6⇑ and Fig 5⇑). Thus, the negative inotropic effects of Ang II after CHF were not dependent on changes in loading conditions, heart rate, or reflex activation. In addition, the depression of LV contractile performance induced by Ang II was completely reversed by the infusion of an Ang II AT1 receptor antagonist (Tables 1 through 4⇑⇑⇑⇑ and Fig 4⇑). This indicates that the direct negative inotropic effect of Ang II in CHF is mediated exclusively through activation of Ang II AT1 receptors.
Our findings of no direct inotropic effect in the normal LV in conscious dogs is consistent with previous observations in anesthetized dogs46 47 but differs from the work of Kobayashi et al15 and Ahmed et al.48 Ahmed et al, who studied myocardial contractile work in normal and diseased hearts of intact dogs and humans, found that Ang II had cardiodepressant effects, whereas Kobayashi et al, using isolated muscle preparations and isolated working heart preparations, observed a slight positive inotropic action. These inconsistent results may have resulted from the influence of Ang II–produced changes in loading conditions on conventional measures of LV performance and variable effects of anesthesia.
After CHF, the Ang II AT1 receptor blocker LOS not only prevented Ang II–induced depression of LV contractile performance but also augmented the intact LV contractile performance above the control level both before and after autonomic blockade. Two possible mechanisms may account for this finding. First, the higher circulating levels of Ang II in CHF may have been contributing to a depression of LV contractile performance at baseline that was reversed by LOS. A second possibility is that the AT2 receptor that is not blocked by LOS may produce a positive inotropic effect after CHF.49 This second effect may be enhanced by the increase in circulating Ang II produced in the intact circulation by LOS.49
We also studied the direct effects of Ang II on cardiac myocytes isolated from the LV before and after CHF. These studies removed the effects of extracardiac factors, which may influence contractility. Similar to the findings in the intact LV, the response of the cardiac myocytes to Ang II was altered after CHF. Before CHF, Ang II produced no depression of the rate or extent of contraction or of the speed of relengthening. After CHF, Ang II produced a clear depression of myocyte contraction and relaxation that was reversed with an AT1 receptor blocker (Table 7⇑ and Fig 7⇑). This confirms that the negative inotropic effects we observed in the intact LV of the animals with CHF were due to a direct receptor-mediated effect on the myocytes.
Before CHF, we found that Ang II produced no apparent inotropic effects in the intact LV but did increase the rate and extent of shortening of the myocytes isolated from the LV of these animals. The difference in the responses may be related to a reduced ability to detect a positive inotropic effect in the intact LV. Another possibility is the difference in loading conditions. The intact LV contracts against a varying afterload, whereas the isolated myocytes are unloaded. Li et al20 recently described a length-dependent modulation of the inotropic effects of Ang II, with a greater positive inotropic effect in normal myocardium at shorter diastolic lengths. This effect may have contributed to the greater positive inotropic effect of Ang II seen in the normal myocytes contracting from their slack length than in the intact LV, which is distended at end diastole. There may be other possible explanations for the difference in the inotropic response of the normal intact LV and isolated myocytes to Ang II.
In addition to depressed LV contractile performance in animals with CHF, Ang II also caused further impairment of LV relaxation and diastolic filling. Before CHF, Ang II slowed the rate of LV relaxation without a marked change in the peak LV filling rate. This effect was reversed when the increase in arterial pressure was prevented. After CHF, Ang II more markedly slowed LV relaxation and increased early diastolic LV pressure with an upward shift of the early diastolic portion of LV pressure-volume loop. This decreased the early diastolic pressure gradient across the mitral valve, thus reducing the rate of early LV filling.25 Part of this effect was due to an enhanced sensitivity of LV relaxation and LV filling to increases in systolic pressure after CHF. However, Ang II directly impaired the rate of LV relaxation, because the Ang II–induced slowing of LV relaxation in CHF was not completely reversed when PES was returned to control with NTP. Furthermore, these effects were completely reversed by an AT1 receptor antagonist. The direct effect of Ang II was also demonstrated in isolated CHF myocytes, where Ang II caused a marked reduction in the velocity of relengthening.
Our finding of an altered response to Ang II in CHF resulting in a direct depression of LV myocardial contraction and relaxation should be compared with findings in previous studies. Li et al20 studied rat papillary muscles removed from normal and CHF rats. When studied at the muscle length that resulted in maximum force development, Ang II had a threefold greater depression of peak contraction in failing than in normal LV myocardium. At shorter lengths, Ang II had a positive contractile effect in the normal myocardium. Similarly Capasso et al19 found a greater negative inotropic effect of Ang II in noninfarcted myocardium from rats 2 days after myocardial infarction than in myocardium from control rats. In addition, Moravec et al16 found that the positive inotropic effects of Ang II on normal human and hamster myocardium were reduced in CHF. Thus, these findings and the present study all demonstrate a similar directional alteration in the inotropic response to Ang II in CHF: either less augmentation or greater depression of contraction. Furthermore, our finding that Ang II directly slows the rate of LV relaxation and increases diastolic LV pressures after CHF is similar to previous findings involving the effects of Ang II in hypertrophic myocardium.23 24
What is the mechanism of Ang II’s depression of LV contraction and relaxation in pacing-induced CHF? Since these effects are present in the isolated myocytes, they are not due to extramyocardial factors such as increased arterial pressure or myocardial ischemia. Although β-adrenergic receptors are downregulated and uncoupled in CHF, Ang II receptors are not reduced in CHF and may be increased.50 The effects of Ang II on myocardial contraction may be partially mediated through the inositol pathway that changes the mobilization and reuptake of cytosolic Ca2+13 17 20 22 51 and alters myofibrillar Ca2+ sensitivity.52 53 In addition, Ang II promotes intracellular alkalinization by enhancing Na+-H+ exchange by activation of protein kinase C.22 54 55 These changes may result in the positive inotropic response seen in normal myocytes. In CHF, there is altered calcium handling.56 57 The Ang II–induced activation of inositol trisphosphate and protein kinase C may exacerbate the dysfunctional Ca2+ homeostasis, which might account for the further impairment of myocardial contraction and relaxation that we observed after CHF. The altered response to Ang II in CHF may also be related to the greater end-diastolic loading in CHF.20
There are several methodological issues that should be considered in interpreting our data. First is the experimental model of CHF. Although rapid pacing produces an animal model of CHF that closely mimics the clinical picture of a congestive cardiomyopathy,4 37 58 59 we cannot be certain our results apply to CHF that is due to other causes.
Second, the PES-VES relation may be curvilinear when evaluated over a wide range.60 61 62 Thus, the slope may depend on the portion of the PES-VES curve that is evaluated. In the present study, the relations were determined in overlapping ranges both before and after Ang II. Thus, possible curvilinearity should not have affected our results. Furthermore, the depression of LV performance with Ang II after CHF was apparent as a rightward shift of the PES-VES relation and similar responses of both the dP/dtmax-VED and SW-VED relations.
Third, we used enzymatic dissociation to isolate myocytes from biopsied canine heart tissue before and after CHF. Since not all cells recover after enzymatic dissociation, there is the potential sampling bias toward those cells that survived. This bias could be further complicated by the possibility that a different subpopulation of cells survived from the CHF heart tissue. However, previous studies have reported that the individual myocyte isolated by this technique retains the morphological and contractile properties similar to those observed in intact muscle. Consistent with previous observations,3 4 40 we have obtained a high yield of viable myocytes by this preparation from both normal and CHF heart tissues. Furthermore, evidence of CHF-induced changes was clearly demonstrated by the alterations in the morphology, contraction, and relengthening of myocytes isolated after CHF. Thus, our observation of altered response to Ang II in CHF myocytes is unlikely to be due to sampling bias or artifacts introduced by the enzymatic isolation process.
In conclusion, pacing-induced CHF in dogs is associated with an altered LV and myocyte response to Ang II. After CHF, Ang II produces a direct depression in LV contraction and relaxation and exacerbates the reduced myocyte contractile performance. These alterations are mediated through activation of AT1 receptors and may play an important role in the development of functional impairment in CHF.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|CHF||=||congestive heart failure|
|LA||=||left atrium, left atrial|
|LV||=||left ventricle, left ventricular|
|RA||=||right atrium, right atrial|
|TSR||=||total systemic resistance|
This study was supported in part by grants from the National Institutes of Health (HL-45258 and HL-42364) and the American Heart Association. We gratefully acknowledge the computer programming of Ping Tan, the technical assistance of Mack Williams, the review by Dr Gregory Freeman, and the secretarial assistance of Judy F. McClenny.
Previously presented as an abstract at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994 (Circulation. 1994;90[suppl I]:I-16).
- Received December 8, 1995.
- Accepted February 14, 1996.
- © 1996 American Heart Association, Inc.
Armstrong PW, Stopps TP, Ford SE, DeBold AJ. Rapid ventricular pacing in the dog: pathophysiologic studies of heart failure. Circulation. 1986;74:1075-1084.
Travill CM, Williams TDM, Pate P, Song G, Chalmers J, Lightman SL, Sutton R, Noble MIM. Hemodynamic and neurohormonal response in heart failure produced by rapid ventricular pacing. Cardiovasc Res. 1992;26:783-790.
Spinale FG, Fulbright BM, Mukherjee R, Tanaka R, Hu J, Crawford FA, Zile MR. Relation between ventricular and myocyte function with tachycardia-induced cardiomyopathy. Circ Res. 1992;71:174-187.
Spinale FG, Holzgrefe HH, Mukherjee R, Hird RB, Walker JD, Arnim-Barker A, Powell JR, Koster WH. Angiotensin-converting enzyme inhibition and the progression of congestive cardiomyopathy: effects on left ventricular and myocyte structure and function. Circulation. 1995;92:562-578.
Suzuki J, Matsubara H, Urakami M, Inada M. Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy. Circ Res. 1993;73:439-447.
Sadoshima J, Izumo S. Molecular characterization of angiotensin II–induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: critical role of the AT1 receptor subtype. Circ Res. 1993;73:413-423.
Weinberg EO, Schoen FJ, George D, Kagaya Y, Douglas PS, Litwin SE, Schunkert H, Benedict CR, Lorell BH. Angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis. Circulation. 1994;90:1410-1422.
Pitt B. Use of converting enzyme inhibitors in patients with asymptomatic left ventricular dysfunction. J Am Coll Cardiol. 1993;22:158A-161A.
Dzau VJ. Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation. 1988;77(suppl I):I-4-I-13.
Haber HL, Powers ER, Gimple LW, Wu CC, Subbiah K, Johnson WH, Feldman MD. Intracoronary angiotensin-converting enzyme inhibition improves diastolic function in patients with hypertensive left ventricular hypertrophy. Circulation. 1994;89:2616-2625.
Fowler NO, Holmes JC. Coronary and myocardial actions of angiotensin. Circ Res. 1964;14:191-201.
Kobayashi I, Furukawa Y, Chiba S. Positive chronotropic and inotropic effects of angiotension II in the dog heart. Eur J Clin Pharmacol. 1978;50:17-25.
Moravec CS, Schluchter MD, Paranandi L, Czerska B, Stewart RW, Rosenkranz E, Bond M. Inotropic effects of angiotensin II on human cardiac muscle in vitro. Circulation. 1990;82:1973-1984.
Baker KM, Singer HA. Identification and characterization of guinea pig angiotensin II ventricular and atrial receptors: coupling to inositol phosphate production. Circ Res. 1988;62:896-904.
Allen IS, Cohen NM, Dhallan RS, Gaa ST, Lederer WJ, Rogers TB. Angiotensin II increases spontaneous contractile frequency and stimulates calcium current in cultured neonatal rat heart myocytes: insight into the underlying biochemical mechanisms. Circ Res. 1988;62:524-534.
Capasso JM, Li P, Zhang X, Meggs LG, Anversa P. Alterations in ANG II responsiveness in left and right myocardium after infarction-induced heart failure in rats. Am J Physiol. 1993;264:H2056-H2067.
Li P, Sonnenblick EH, Anversa P, Capasso JM. Length-dependent modulation of ANG II inotropism in rat myocardium: effects of myocardial infarction. Am J Physiol. 1994;266:H779-H786.
Mochizuki T, Eberli FR, Apstein CS, Lorell BH. Exacerbation of ischemic dysfunction by angiotensin II in red cell-perfused rabbit hearts. J Clin Invest. 1992;89:490-498.
Neyses L, Vetter H. Impaired relaxation of the hypertrophied myocardium is potentiated by angiotensin II. J Hypertens. 1989;7(suppl 6):S104-S105.
Schunkert H, Dzau VJ, Tang SS, Hirsch AT, Apstein CS, Lorell BH. Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy: effects on coronary resistance, contractility, and relaxation. J Clin Invest. 1990;86:1913-1920.
Cheng C-P, Freeman GL, Santamore WP, Constantinescu MS, Little WC. Effect of loading conditions, contractile state, and heart rate on early diastolic left ventricular filling in conscious dogs. Circ Res. 1990;66:814-823.
Chan DP, Aarhus LL, Heublein DM, Burnett JC Jr. The role of angiotensin II in the regulation of basal renal and cardiovascular function. Circulation. 1991;84(suppl II):II-107. Abstract.
Kono A, Maughan WL, Sunagawa K, Kallman C, Sagawa K, Weisfeldt ML. The use of left ventricular end-ejection pressure and peak pressure to estimate the end-systolic pressure-volume relationship. Circulation. 1984;70:1057-1065.
Olsen CO, Tyson GS, Maier GW, Spratt JA, David JW, Rankin JA. Dynamic ventricular interaction in the conscious dog. Circ Res. 1983;52:85-104.
Glower DD, Spratt JA, Snow ND, Kabas JS, Davis JW, Olsen CO, Tyson GS, Sabiston DC, Rankin JS. Linearity of the Frank-Starling relationship in the intact heart: the concept of preload recruitable stroke work. Circulation. 1985;71:994-1009.
Yellin EL, Hori M, Yoran C, Sonnenblick EH, Gabbay S, Frater RWM. Left ventricular relaxation in the filling and nonfilling intact canine heart. Am J Physiol. 1986;250:H620-H629.
Gilbert JC, Glantz SA. Determinants of left ventricular filling and of the diastolic pressure-volume relation. Circ Res. 1989;64:827-852.
Sunagawa K, Maughan WL, Sagawa K. Optimal arterial resistance for the maximal stroke work studied in isolated canine left ventricle. Circ Res. 1985;56:586-595.
Little WC, Cheng CP, Mumma M, Igarashi Y, Vinten-Johansen J, Johnston WE. Comparison of measures of left ventricular contractile performance from pressure-volume loops in conscious dogs. Circulation. 1989;80:1378-1387.
Cheng C-P, Noda T, Nozawa T, Little WC. Effect of heart failure on the mechanism of exercise-induced augmentation of mitral valve flow. Circ Res. 1993;72:795-806.
Cheng CP, Pettersson K, Little WC. Effects of felodipine on left ventricular systolic and diastolic performance in congestive heart failure. J Pharmacol Exp Ther. 1994;271:1409-1417.
Cheng CP, Suzuki M, Ohte N, Little WC. Functional significance of RAS activation during exercise with heart failure. Circulation. 1995;62(suppl I):I-258. Abstract.
Stemmer P, Wisler PL, Watanabe AM. Isolated myocytes in experimental cardiology. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE, eds. The Heart and Cardiovascular System. 2nd ed. New York, NY: Raven Press Publishers; 1992:387-404.
Yu Z, Tibbits GF, McNeill JH. Cellular functions of diabetic cardiomyocytes: contractility, rapid-cooling contracture, and ryanodine binding. Am J Physiol. 1994;266:H2082-H2089.
Little WC. Enhanced load dependence of relaxation in heart failure: clinical implications. Circulation. 1992;85:2326-2328.
Cheng CP, Little WC. Enhanced load sensitivity of left ventricular relaxation and early diastolic filling on congestive heart failure. Circulation. 1993;88(suppl I):I-527. Abstract.
Eichhorn EJ, Willard JE, Alvarez L, Kim AS, Glamann DB, Risser RC, Grayburn PA. Are contraction and relaxation coupled in patients with and without congestive heart failure? Circulation. 1993;85:2132-2139.
Maughan WL, Sunagawa K, Burkhoff D, Sagawa K. Effect of arterial impedance changes on the end-systolic pressure-volume relation. Circ Res. 1984;54:595-602.
Freeman GL, Little WC, O’Rourke RA. Effect of vasoactive agents on the left ventricular end-systolic pressure-volume relation in closed-chest dogs. Circulation. 1986;74:1107-1113.
Kaneko Y, McCubbin JW, Page IH. Ability of vasoconstrictor drugs to cause adrenal medullary discharge after ‘sensitization’ by ganglion stimulating agents. Circ Res. 1961;9:1247-1254.
Freer RJ, Pappano AJ, Peach MJ, Bing KT, McLean MJ, Vogel S, Sperelakis N. Mechanism for the positive inotropic effect of angiotensin II on isolated cardiac muscle. Circ Res. 1976;39:178-183.
Gwathmey JK, Hajjar RJ. Effect of protein kinase C activation on sarcoplasmic reticulum function and apparent myofibrillar Ca2+ sensitivity in intact and skinned muscles from normal and diseased human myocardium. Circ Res. 1990;67:744-752.
Moorman JR, Kirsch GE, Lacerda AE, Brown AM. Angiotensin II modulates cardiac Na+ channels in neonatal rat. Circ Res. 1989;65:1804-1809.
Sen L, O’Neill M, Marsh JD, Smith TW. Myocyte structure, function, and calcium kinetics in the cardiomyopathic hamster heart. Am J Physiol. 1990;259:H1533-H1543.
Morgan JP, Erny RE, Allen PD, Grossman W, Gwathmey JD. Abnormal intracellular calcium handling, a major cause of systolic and diastolic dysfunction in ventricular myocardium from patients with heart failure. Circulation. 1990;81(suppl III):III-21-III-32.
Komamura K, Shannon RP, Pasipoularides A, Ihara T, Lader AS, Patrick TA, Bishop SP, Vatner SF. Alterations in left ventricular diastolic function in conscious dogs with pacing-induced heart failure. J Clin Invest. 1992;89:1825-1838.
Cheng CP, Igarashi Y, Klopfenstein HS, Applegate RJ, Shihabi Z, Little WC. Effect of vasopressin on left ventricular performance. Am J Physiol. 1993;264:H653-H660.
Kass DA, Maughan WL. From ‘Emax’ to pressure-volume relations: a broader view. Circulation. 1988;77:1203-1212.
Little WC, Cheng CP, Peterson T, Vinten-Johansen J. Response of the left ventricular end-systolic pressure-volume relation in conscious dogs to a wide range of contractile states. Circulation. 1988;78:736-745.
Van der Velde ET, Burkhoff D, Steendijk P, Karsdon J, Sagawa K, Baan J. Nonlinearity and load sensitivity of end-systolic pressure-volume relation of canine left ventricle in vivo. Circulation. 1991;83:315-327.