Synergistic Exacerbation of Diastolic Stiffness From Short-term Tachycardia–Induced Cardiodepression and Angiotensin II

From the Division of Cardiology (H.S., P.H.P., D.A.K.), Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md; the Laboratory of Cardiovascular Science (Y.A.G., M.T.C.), Gerontology Research Center, National Institute on Aging, Baltimore, Md; and the Department of Physiology and Pharmacology (J.S.J.), Auburn University, Auburn, Ala.

Correspondence to David A. Kass, MD, Halsted 500, Division of Cardiology, Johns Hopkins Medical Institutions, 600 N Wolfe St, Baltimore, MD 21287. E-mail dkass{at}eureka.wbme.jhu.edu

	Abstract

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Abstract—Synergistic interaction between angiotensin II (Ang II) and evolving cardiodepression may play an important role in worsening chamber function, particularly in diastole. To test this hypothesis, Ang II was infused at 10 or 17 ng · kg^-1 · min^-1 in 18 conscious dogs 4 days before and during induction of subacute cardiodepression by 48-hour tachypacing. The lower dose yielded negligible systemic pressure changes. Twelve additional animals served as paced-only controls. Pressure-dimension relations were recorded, and serial endocardial biopsies were obtained to assess histological and metalloproteinase (MMP) changes. Forty-eight–hour pacing alone depressed systolic function but had little effect on diastolic stiffness. Ang II alone only modestly raised diastolic stiffness at both doses and enhanced contractility at the higher dose. These changes recovered toward baseline after a 7-day infusion. However, Ang II (at either dose) combined with 48-hour pacing markedly increased ventricular stiffness (110±26% over baseline) and end-diastolic pressure (22±1.7 mm Hg). In contrast, pacing-induced inotropic and relaxation abnormalities were not exacerbated by Ang II. Zymography revealed MMP activation (72- and 92-kD gelatinases and 52-kDa caseinase) after a 4-day Ang II infusion (at both doses), which persisted during pacing. Tachypacing initiated 24 hours after cessation of a 7-day Ang II infusion also resulted in diastolic stiffening and corresponded with MMP reactivation. Ang II also induced myocyte necrosis, inflammation, and subsequent interstitial fibrosis, but these changes correlated less with chamber mechanics. Thus, Ang II amplifies and accelerates diastolic dysfunction when combined with evolving cardiodepression. This phenomenon may also underlie Ang II influences in late-stage cardiomyopathy, when chamber distensibility declines.

Key Words: heart failure • diastole • metalloproteinase • myocardium • ventricle

	Introduction

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Toward the later stages of evolving heart failure, cardiac dysfunction is accompanied by activation of the RAS, which may hasten the progression of disease.^{1 2} Adverse interactions between the RAS and the failing heart are supported by data showing enhanced survival of patients receiving ACE inhibitors.^{3 4} Such hearts respond unfavorably to acute increases in Ang II stimulation,^{5 6} whereas sustained Ang II exposure itself induces myocardial remodeling and cell necrosis,^{7 8 9} hypertrophy,^{10 11} and alterations in the extracellular matrix.^{8 12} It is less clear, however, whether pathophysiological levels of Ang II modify developing cardiodepression or whether there is synergy between these factors contributing to dysfunction.

The short-term canine tachycardia-pacing model of dilated cardiomyopathy provides a useful approach to studying this question. Rapid pacing induces early-onset systolic depression, whereas diastolic abnormalities develop more slowly, becoming quite abnormal only after several weeks of pacing.^{13 14 15 16} Systolic dysfunction is associated with abnormal ß-adrenergic signal transduction^{17 18} and excitation-contraction coupling,^{19 20} as observed in human heart failure; however, the short-term pacing stimulus is too brief to activate the RAS.^{11 13 21}

The present study tested the hypothesis that the effects of chronic Ang II on cardiac function are synergistically augmented when combined with evolving cardiac depression, thereby accelerating diastolic dysfunction. To clarify this interaction, we modified the pacing model as follows. Animals were first exposed to 4 days of continuous Ang II infused at doses that either did or did not alter systemic pressures. Ang II–induced changes in cardiac mechanics, cellular histology, and metalloproteinases were assessed. Then 48 hours of tachycardic pacing was used to induce systolic depression while Ang II infusion continued. Results from these animals were contrasted with a separate control group that did not receive Ang II and with a nonpaced group that received Ang II for 1 week.

	Materials and Methods

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Chronic Preparation
Thirty mongrel dogs of either sex (45 to 65 lb) were anesthetized with 1% to 2% halothane after induction with sodium thiamylal. The chest was opened via lateral thoracotomy, an indwelling catheter was secured in the right atrium, and a flexible tube was placed in the LV apex to serve as a conduit for micromanometer placement (SPC 350, Millar Instruments). Endocardial sonomicrometers were inserted to measure anterior-posterior short-axis dimension, and a pneumatic occluder around the IVC facilitated load reduction to assess LV pressure-dimension relations.²² An epicardial pacing lead was attached to the right ventricular free wall and connected to a programmable stimulator (Spectrax, Medtronics) within a subcutaneous pocket. All catheters and leads were externalized to the mid scapulae and protected by an external jacket. Ang II was infused intravenously by osmotic pump (Alzet 2ML1) via the external jugular vein in 15 of the animals. Baseline data were measured after 10 to 14 postoperative days.

Protocol
Four animal groups were studied. Group 1 (n=12) dogs were subjected solely to 48-hour tachypacing (250 bpm). Data were measured before and after the pacing period. Group 2A and 2B animals (n=12) were first exposed to 4 days of continuous Ang II infusion (dissolved in 0.01N acetic acid), followed by 48-hour tachypacing while Ang II administration continued. In group 2A, the Ang II dose was titrated to raise systolic pressure by 30 mm Hg above baseline (mean dose, 17.3±6.3 ng · kg^-1 · min^-1; n=6), whereas group 2B received a lower dose to minimally alter systemic pressures (mean dose, 10.0+2.5 ng · kg^-1 · min^-1; n=6; P<.05 versus group 2A). Data were measured at baseline, after 4 days of Ang II infusion, and after 48 hours of pacing plus Ang II. As a further control, 6 animals were studied with Ang II infusion only (higher doses) for the full 1-week period (group 3) with no pacing stimulus. Additional data were obtained in these animals by removing the osmotic pump after the 1-week Ang II infusion, providing 24 hours to ensure a decline in Ang II, and then performing 48 hours of tachypacing.

Blood samples were collected to determine Ang II and Ang I plasma levels. Arterial blood (5 mL) was withdrawn into a tube containing 18 mg EDTA, 30 mg 1,10-phenanthroline, and 2.5 mg enalaprilat. Samples were immediately centrifuged at 2000g for 10 minutes, and plasma was extracted and frozen at -80°C until assayed.

LV pressure-dimension data were recorded in conscious animals in the absence of autonomic blockade, with rapid pacing suspended for at least 30 minutes. Data were measured at rest and during transient IVC occlusion. Hemodynamic data were very stable and reproducible for well over a 1-hour period. Animals were then sedated (2.2 mg/kg IM xylaxine and 4 mg butorphanol tartrate), and four to six LV endomyocardial biopsies (3.3 mm³ ) were obtained from the distal chamber, taking care to avoid the regions where sonomicrometers were inserted as well as the distal apex.¹⁴ Samples were placed in 10% buffered formalin for histological study or rapidly frozen in liquid nitrogen and stored at -80°C for metalloproteinase analysis.

Hemodynamic Analysis
Pressure-dimension signals were digitized at 250 Hz. Signal-averaged data from 5 to 10 consecutive beats were used to derive steady-state parameters, and data measured during transient IVC occlusion were used to assess pressure-dimension relations. Heart rate is generally little changed during rapid caval occlusion, but to further eliminate rate effects, only beats for which the heart rate varied <5% of baseline were used for analysis. Systolic function was indexed by fractional diameter shortening, peak rate of pressure rise at matched end-diastolic dimension (dP/dt_max), ESPDR,²² and the slope of the relation between dimension work (pressure-dimension loop area) and EDD (M_sw).¹⁴ The latter index has units of mm Hg. Diastolic function was indexed by EDP, the time constant of relaxation derived by linear regression of LV pressure versus dP/dt, and diastolic ventricular stiffness (b). Stiffness was derived by fitting EDPDR to a monoexponential relation: EDP=P_o+a(e^{b · EDD}-1), where P_o is pressure at EDD=0, a is the scaling coefficient, and b is the stiffness coefficient. Data were fit by nonlinear regression. The mean correlation coefficient derived from 16 randomly selected EDPDRs from control, 48-hour pacing, 4-day Ang II infusion, and Ang II plus 48-hour pacing data (four EDPDRs from each condition) was 0.987±0.002.

Plasma Angiotensin Assay
Plasma Ang I and Ang II peptide concentrations were determined by HPLC and RIA.^{23 24} Separation was performed by reverse-phase HPLC on a phenyl silica gel column with an eluent consisting of 20% acetonitrile on 0.1 mol/L ammonium phosphate buffer (pH 4.9). Aliquots (100 µL) of each relevant fraction of column effluent were subjected to RIA immediately on collection. Elution of standard angiotensin peptides under isocratic conditions revealed clear resolution of both Ang I and Ang II. The cross-reactivity of anti–Ang I antiserum with Ang II and of anti–Ang II antiserum with Ang I was <0.5%. Sensitivity of the RIA for Ang I and Ang II was 4 and 2 pg/mL, respectively.

Histological Examination
Formalin-fixed endocardial biopsies were embedded in paraffin, and 5-mm serial sections were stained with hematoxylin-eosin and the collagen-specific stain picrosirius red F3BA (PSR). Histological examination was performed by a single investigator (J.S.J.) blinded with respect to the experimental condition. Sections were qualitatively graded as none, mild, moderate, or severe for the presence of inflammation, myocyte necrosis, and interstitial fibrosis.

Extracellular MMP Activity
Assessment of metalloproteinase content and activity in the myocardium was performed by zymography.^{25 26} Frozen biopsy tissues were extracted by three freeze/thaw cycles on dry ice in 50 or 100 µL 2x tris-Glycine SDS sample buffer (Novex) nonreducing sample buffer (125 mmol/L Tris-HCl, pH 6.8, containing 20% glycerol, 4% SDS, and 0.005% bromophenol blue [wt/vol]) and proteinase inhibitors (5 mmol/L EGTA, 5 mmol/L EDTA, 21 µmol/L leupeptin, 1.5 µmol/L aprotinin, and 14.6 µmol/L pepstatin A). Extracts containing 40 µg protein were diluted to 1x sample buffer and run on 10% SDS-polyacrylamide gels (Novex Chemical) containing 0.1% (wt/vol) gelatin or casein. Gels were then washed twice with 2.5% Triton X-100 for 30 minutes at room temperature, incubated at 37°C for 18 hours in 50 mmol/L HEPES, pH 7.5, 0.2 mol/L NaCl, 5 mmol/L CaCl₂, and 20 µmol/L ZnCl₂, and subsequently stained with Coomassie blue R-250. Activated metalloproteinase produced clear areas of gel lysis. Conditioned medium from phorbol ester–treated HT1080 cells was used for MMP standards, as was recombinant human MMP-2 (generously provided by Dr Rafi Friedman, Wayne State University) converted to its active form as previously described.²⁷ Bands were graded on a semiquantitative scale from 0 (no activity) to 8 (maximal activity) on the basis of the intensity of gel lysis. Grading was performed by two individuals independently, both blinded as to biopsy source.

Statistical Analysis
Data are presented as mean±SEM. Within-group comparisons were made by repeated-measures ANOVA, with post hoc testing with paired or unpaired t tests and a Bonferoni correction. Between-group comparisons were analyzed by unpaired t test. Histological and metalloproteinase data were analyzed by nonparametric tests.

	Results

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Influence of Ang II Infusion Alone
Table 1 provides control hemodynamic data for the four animal groups. There were no significant differences between groups for any of the parameters at baseline. Subsequent absolute changes after 4-day (groups 2A, 2B, and 3) or 7-day (group 3 only) Ang II infusion are provided in Table 2. In group 2A and group 3 dogs, the higher dose of Ang II increased systolic pressure by 37±7 and 29±3 mm Hg, respectively (both P<.01 versus group 2B). Isovolumic relaxation was prolonged, and ventricular contractility was enhanced at this Ang II dose. These changes were not observed with the lower Ang II dose (group 2B). In contrast, ventricular stiffness and EDP rose modestly and similarly at both doses. There was negligible change in end-diastolic cavity dimension or fractional shortening.

End-systolic pressure remained elevated after the 7-day Ang II infusion (P<.01 versus baseline); however, there was no further progression of diastolic changes observed at the earlier time point. If anything, EDP declined slightly, and ventricular stiffness was no longer different from baseline (P=.38) with the more sustained exposure. Example pressure-dimension loops and relations from a representative group 3 animal are shown in Fig 1 and demonstrate these 4-day and 7-day mechanical responses.

View larger version (17K): [in this window] [in a new window]   Figure 1. Changes in cardiac mechanics from an example dog exposed to sustained Ang II infusion for up to 7 days in the absence of tachycardic pacing. Pressure-dimension loops measured over a preload range are shown at baseline and after 4 and 7 days of Ang II infusion. Ang II was administered at the higher dose, which increased vascular load. After 4 days, there were modest changes in diastolic stiffness and EDP and a leftward parallel shift of the ESPDR (upper left boundary) consistent with improved contractile function. After 7 days of Ang II, EDP and stiffness returned to baseline, and there was a decline in systolic changes as well.

Influence of 48-Hour Tachypacing Alone
Tachycardic pacing for 48 hours resulted in a significant decline in contractile function (dP/dt_max, -36.4±3.9%; M_sw, -30.7±4.3%; both P<.001) without a change in chamber dimensions, diastolic stiffness, or EDP (Fig 2, open bars). Isovolumic relaxation was prolonged by 7±1 milliseconds. These results, demonstrating substantial systolic depression, no chamber dilation, and relatively little alteration in diastolic pressures or stiffness, are consistent with previously reported data.^{13 15 16 19}

View larger version (38K): [in this window] [in a new window]   Figure 2. Mean percent changes for systolic (panel A) and diastolic (panel B) hemodynamic parameters after 48-hour tachypacing in group 1, 2A, and 2B animals. ESP indicates end-systolic pressure; FS, fractional diameter shortening; Ees, slope of end-systolic pressure-dimension relation; EDD, end-diastolic diameter; b, coefficient of diastolic stiffness; tau, time constant of isovolumic relaxation; A2, Ang II; and 48hP, 48-hour pacing. Systolic changes for all three groups were significantly different from baseline (P<.05). *P<.05 vs baseline; #P<.05 vs group 1.

Synergistic Influence of Ang II with 48-Hour Tachypacing
When 48 hours of tachypacing was superimposed on the Ang II infusion baseline, there was a marked exacerbation of passive diastolic failure without worsening of systolic depression or relaxation delay. Fig 3 displays representative pressure-dimension loops and relations from group 1, 2A, and 2B animals before and after 48-hour tachypacing. Corresponding prepacing baseline pressure-volume relations are delineated by dashed lines. Systolic relations shifted rightward with a reduced slope from 48 hours of pacing, and the magnitude of change was similar among groups. However, there was a striking difference in diastolic stiffness. Exposure to both Ang II and 48-hour pacing led to a steep EDPDR with elevated EDPs in both group 2A and 2B dogs. As previously noted, there was a minimal change in EDPDR or EDP in group 1 dogs. The exacerbated diastolic stiffening is highlighted in Fig 3D, which depicts an expanded pressure scale.

View larger version (47K): [in this window] [in a new window]   Figure 3. Representative pressure-dimension loops and relations from group 1 (panel A), group 2A (panel B), and group 2B (panel C) animals before and after 48-hour tachypacing. Respective prepacing ESPDRs and EDPDRs are shown by dashed lines. There was a similar decline in systolic function (rightward and downward shift of ESPDRs). However, there was a striking difference in passive diastolic stiffness, which rose markedly (as indicated by the steeper EDPDR) in the Ang II–pretreated animals (groups 2A and 2B). Panel D shows EDPDRs from the same set of data at baseline (dotted line), after 4 days of Ang II (dashed line), and after Ang II plus 48 hours of pacing (solid line) replot on an expanded pressure scale.

Mean data for systolic and diastolic responses to pacing in group 2A and 2B dogs are also shown in Fig 2. Neither systolic cardiodepression nor delayed relaxation observed with pacing alone were further amplified by exposure to Ang II. In contrast, the combination of Ang II and 48-hour tachypacing yielded much stiffer ventricles with higher EDPs. The diastolic stiffness coefficient rose by 59.6±18.3% above that after 4-day Ang II infusion, yielding a net gain of 109.9±26% from the original baseline, whereas EDP rose to 22.0±1.7 mm Hg. Both diastolic changes were virtually identical in the two Ang II dosage groups, supporting hormonal rather than load-mediated interactions. Results of the 7-day Ang II infusion studies had shown that diastolic deterioration in these animals was not due to progressive influences of Ang II alone but implied a synergistic interaction between stimuli.

To further explore the mechanism of pacing-induced cardiodepression and Ang II exposure, additional studies were performed in 4 of the group 3 animals that had already received Ang II alone for 7 days. The Ang II infusion was stopped by removal of the osmotic pump, and Ang II levels were allowed to return to baseline over 24 hours. Then, in the absence of continued Ang II stimulation, we tested whether myocardial effects from the prior exposure could still act synergistically with 48-hour pacing to alter chamber function. Summary data are provided in Table 3. Systolic indexes all declined similarly to what had been observed in animals with 48-hour pacing alone and in group 2A and 2B animals. However, both EDP (28±12%, P<.05 versus prepacing) and diastolic stiffness (38±13%, P<.05 versus prepacing) increased. Although the magnitude of change was less than that observed when pacing was superimposed on continuing Ang II infusion, it was significantly greater than that observed with pacing alone.

Plasma Ang I and II Levels
Plasma Ang II rose from 66.4±15.5 pg/mL (combined group 2) to 193±46.4 (P=.019) after 4-day Ang II infusion, levels similar to those reported in human²⁸ and experimental^{9 29} heart failure. Interestingly, there was no difference in plasma Ang II in the two subgroups (group 2A, 197±75; group 2B, 188±88 pg/mL) despite different infusion rates and systemic pressure responses. Ang II levels declined after the 48-hour pacing period to 128.7±15.3 pg/mL, but this remained elevated over baseline (P=.018). Plasma Ang I displayed reciprocal changes, declining from 882±265 to 318±79 pg/mL after 4-day Ang II infusion (P<.02 by Wilcoxon test) and rising to 607±292 pg/mL after Ang II plus 48 hours of pacing. This is consistent with a negative-feedback effect of Ang II on renin synthesis and release.^{30 31}

In group 3 animals in which Ang II infusion was provided without pacing, Ang II levels declined toward baseline by the 7-day time point (37±3.9–baseline; 135±40.4–4-day Ang II; 61.8±11–7-day Ang II, pg/mL, P<.05 for baseline vs 4-day Ang II only). This suggests some interaction between the effects of 48-hour tachypacing and Ang II infusion in maintaining somewhat higher plasma levels when both stimuli were combined (ie, in groups 2A and 2B). Data obtained after removal of the pump (subset of group 3) confirmed full return to baseline (43±5 pg/mL).

Histological and MMP Changes
Ang II infusion alone induced consistent marked changes in cellular histology and metalloproteinase activity. Fig 4A displays a representative photomicrograph after 4-day Ang II exposure, demonstrating extensive myocyte damage with mononuclear cell infiltrates and some reparative fibrosis. Similar changes were observed in 78% and 80% of group 2A and 2B biopsies, respectively, and they persisted in 62% and 80% of biopsies after the addition of 48-hour pacing. Only 13% of baseline and 24% of 48-hour pacing-only biopsies revealed any histological abnormalities at all (P<.001). There was no histological evidence of myocyte hypertrophy with Ang II infusion. After a 7-day infusion of Ang II alone, acute cellular damage diminished, and there was increased deposition of interstitial collagen (Fig 4B). Interestingly in those group 3 dogs in which the pump was subsequently removed and 48-hour pacing was applied, follow-up biopsies showed marked regression of this collagen (Fig 4C).

View larger version (83K): [in this window] [in a new window]   Figure 4. A, Representative photomicrograph (hematoxylin-eosin staining) of LV endomyocardial biopsy after 4-day Ang II infusion. There are diffuse inflammatory infiltrates, with myocytolysis and necrosis. B, Increased interstitial collagen delineated by PSR staining observed after 7-day Ang II infusion in a nonpaced animal (group 3). Myocytes can be seen surrounded by collagen fibers. C, Same animal 3 days later, after removal of the Ang II infusion pump and subsequent exposure to 48-hour tachypacing. There is marked regression of the interstitial collagen observed in the earlier specimen.

Fig 5A displays representative gelatin (upper) and casein (lower) zymography from group 1 and 2 dogs. There was minimal MMP activity at baseline or with 48-hour pacing alone. Ang II infusion for 4 days itself markedly increased MMP activity, including a 52-kD caseinase and 72- and 92-kD gelatinases. Based on relevant control bands, these likely corresponded to MMP-1, MMP-2, and MMP-9, respectively (eg, the activated MMP-2 band shown in the gelatin zymogram). These changes were not associated with cross-sectional dilation (Table 1) and also persisted after 48 hours of pacing was added to Ang II. Summary data are provided in Fig 5C. Increased levels of MMP-1 and MMP-2 were observed only with Ang II and Ang II plus 48-hour pacing at both doses of Ang II. MMP-9 levels trended above baseline with 48-hour pacing alone, but this did not reach significance.

View larger version (50K): [in this window] [in a new window]   Figure 5. A, Gelatin (upper) and casein (lower) zymogram showing metalloproteinase activation induced by Ang II infusion after 4 days (4dA2), with persistent changes with Ang II plus pacing (A2+48hP). M indicates the marker lane, with activated MMP-2 used as a control (in gelatinase zymogram). Activated MMPs appear as light bands, indicating lysis of the underlying gel substrate. Baseline (base) and pacing only (48hP) showed little MMP activity, whereas both 4dA2 and A2+48hP showed marked activity in a 52-kD caseinase (MMP-1) and 72- and 92-kD gelatinases (MMP-2 and MMP-9). Bands on the zymogram migrated at somewhat lower molecular weight than on a standard Western blot. B, Gelatin zymogram from group 3 animals. Similar metalloproteinase activation after 4dA2 infusion was observed as with the other groups, but this regressed with more sustained Ang II infusion for 7 days (7dA2). With cessation of Ang II infusion and subsequent pacing (**48hP), there was marked reactivation particularly of MMP-9. +Con indicates positive control band MMP-2 and MMP-9; +Con-A, activated MMP-9 plus control. C, Summary data for MMP activity.

Fig 5B shows gelatin zymograms from group 3 dogs. These studies reconfirmed the minimal MMP activity at baseline and the increased activity by 4 days of Ang II infusion alone. However, with 7-day Ang II infusion, there was regression of MMP activity, consistent with the evolution of interstitial fibrosis (ie, Fig 4B) and decline in plasma levels. Mean results (Fig 5C) were consistent with this example (P<.05 for 7-day versus 4-day Ang II infusion for all MMPs). Interestingly, when the Ang II infusion was subsequently stopped and 48 hours of pacing was applied, MMP activity again rose (46% in MMP2 and 32% in MMP9; see Fig 5B). This too was consistent with the regression of interstitial fibrosis observed in the biopsies (Fig 4C).

	Discussion

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The present study presents the first direct evidence of an adverse synergy between Ang II and cardiac depression on ventricular diastolic dysfunction. Despite modest diastolic changes and even slightly enhanced systolic function from Ang II alone, hearts with prior Ang II exposure were predisposed to develop severe diastolic failure after only 48 hours of rapid pacing. Pacing itself produced systolic depression and mild relaxation delay but relatively little diastolic stiffening. The synergistic effect of Ang II with short-term cardiodepression was selective, in that neither contractility nor relaxation abnormalities from tachypacing were worsened by Ang II. Ang II alone induced diffuse myocyte necrosis and inflamation, eventual fibrosis, and activation of MMPs. In particular, diastolic stiffening was most consistently observed when MMP activation occurred, suggesting that the synergistic influence may depend on increased turnover within the extracellular matrix. These observations may help explain a clinical and experimental association of RAS and MMP activation with rises in chamber diastolic pressure and stiffness, commonly observed in later stages of evolving heart failure.

Study Design and Limitations
The present study and model were designed to simplify the analysis of Ang II–heart failure interaction by artificially reversing the order in which the heart is exposed to these stimuli. Our goal was not to mimic normal heart failure nor to duplicate the standard 3-week pacing model but to test the extent to which Ang II and cardiodepression acts synergistically to worsen function. Indeed, the histological abnormalities observed in our model are not typical of those reported after more sustained pacing in this model.¹⁵ Furthermore, Komamura et al¹⁵ reported that the marked diastolic abnormalities observed after 3 weeks of tachypacing were due to loading conditions rather than changes in intrinsic chamber stiffness. In the present model, however, apparent diastolic stiffening was unlikely due to altered ventricular loading since end-diastolic and -systolic dimensions were unchanged and since in group 2B animals, there was no rise in systolic pressure. The pacing model itself telescopes heart failure into a brief period, and a variety of responses, such as hypertrophy or fibrosis, are not observed. Thus, it is likely that the diastolic stiffening observed in the present study reflected the specific synergy from the addition of Ang II at an earlier phase in the progression of LV dysfunction. Whereas prior studies have examined synergistic effects of Ang II and evolving hypertrophic disease,⁵ the present study is the first to evaluate this interaction in a systolic failure model. Last, rapid pacing even for only 48 hours induces molecular and biochemical changes similar to those identified in later-stage human heart failure,^{17 18 19 20} supporting its use in studying interactions with Ang II.⁶

Although Ang II infusion rates were different in group 2A and group 2B animals, measured plasma Ang II levels were similarly increased at the various time points. Since the infusion dose was titrated to systolic pressure and not plasma level, this suggests that the difference in pressure response was not related to the dose per se but more to individual animal differences in systemic vascular sensitivity to this dose. In contrast, cardiac diastolic responses, histological and MMP changes, and synergistic worsening of diastolic failure with 48-hour pacing were identical in both dose groups. This dissociation of cardiac from vascular effects cautions the use of arterial pressure as the sole or primary index for monitoring Ang II effects in experimental studies. Furthermore, activation of myocardial RAS may play an important role in cardiomyopathy,^{5 32} but the extent to which systemic and myocardial Ang II levels correlate in failure remains unclear. Recent studies have suggested compartmentalization of cardiac interstitial from intravascular Ang II in normal dogs given acute systemic Ang I.³³ Whether such compartmentalization is maintained with more chronic exposure or in failing hearts remains unknown.

We used a single cross-sectional cardiac dimension for this study rather than estimate chamber volume from three orthogonal axes. This simplified the surgical instrumentation, and prior studies have shown excellent correlations between dimension-derived parameters (eg, EDPDR) and chamber indexes.²² This is further supported by the M_sw values. This parameter has units of mm Hg and thus yields similar values from pressure-dimension or pressure-volume loops as long as there is a consistent linear relation between them. The values we obtained were very similar to those reported by using volume¹⁶ in control and short-term failure states. Last, systolic and diastolic changes based on dimension analysis were consistent with those dependent purely on pressure (ie, dP/dt_max and EDP), further supporting this correlation. Nonetheless, extrapolation of these data to chamber stiffness should be done with caution, since only one dimension was measured and ventricular remodeling can appear in the setting of cardiodepression.

Conscious resting heart rates ranged between 120 to 130 bpm, which is faster than rates measured by some other investigators using this model.^{6 15 17} This may be related to placing the animals upright in a sling to facilitate introduction of a sterile micromanometer catheter to the LV rather than lying the animals down on their sides. Nonetheless, it is unlikely to have contributed to the present findings, in that heart rate was similar in all groups and not significantly altered by the interventions.

Cardiac Effects of Sustained Ang II Exposure
Although many prior studies have reported on the myocardial cellular,^{9 10 11 34 35} morphological,^{7 8} and molecular³⁶ changes after sustained Ang II exposure, this is the first to assess its net influence on ventricular mechanical properties in a conscious animal. It seems surprising that given the extent of histological damage and altered activity of extracellular matrix enzymes, ventricular diastolic and systolic function were not more prominently influenced by Ang II alone. This result suggests considerable reserve in normal hearts and highlights an often weak correlation between histology and function. Nonetheless, the nature and diffuse distribution of histological damage in the present experiment is consistent with prior data obtained in rats,^{9 34 35} which has been attributed in part to Ang II–AT₁ receptor–mediated postsynaptic catecholamine release.³⁵ This may play a role in the benefits of ß-blocker therapy in failing hearts.

The present study is also the first to show marked increases in cardiac MMPs with early sustained infusion of Ang II alone at pathophysiological serum levels. ACE inhibitor therapy lowers MMP activity in the kidney³⁷ and increases the collagen weave during development of tachycardia-induced cardiomyopathy,³⁸ supporting a linkage between upregulated MMPs and Ang II. Furthermore, MMP activity plays a critical role in collagen regulation.³⁹ The kilodalton values for MMPs upregulated in the present study were consistent with MMP-1, which can reflect interstitial as well as neutrophil-derived collagenase, and MMP-2 and MMP-9 gelatinases, the latter being cytokine inducible and expressed by both fibroblasts and neutrophils. The subsequent inhibition of MMP activity with longer Ang II exposure, particularly MMP-1, was likely important for the observed increase in interstitial collagen and is consistent with the negative feedback of Ang II on MMP-1 previously reported.⁴⁰

Synergy Between Ang II and 48-Hour Tachypacing
The adverse synergy between Ang II and 48-hour tachypacing was specific for accelerating passive diastolic abnormalities (stiffness and EDP), whereas 48-hour pacing–induced systolic cardiodepression and relaxation delay were not altered much further by Ang II pretreatment. The lack of exacerbation of systolic depression by Ang II contrasts with recent data reported by Cheng et al,⁶ who administered Ang II acutely to intact hearts and isolated myocytes from control dogs and dogs with pacing-induced heart failure. As with the present study, these investigators observed disproportionate increases in EDP in failing hearts after Ang II infusion, but they also noted greater systolic depression at both whole-heart and myocyte levels. However, the Ang II administered was at a much higher dose, with plasma levels of >500 pg/mL. This is similar to levels measured during dynamic exercise^{6 29} but considerably higher than those measured at rest with evolving failure.^{10 28 29} The greater Ang II systolic effect could also relate to severe underlying cardiac failure induced by longer term pacing.

Several lines of evidence support the hypothesis that Ang II potentiation of diastolic stiffening with 48-hour pacing was in part related to MMP activation. First, MMPs were minimally changed by pacing alone but increased with Ang II plus pacing (groups 2A and 2B) and with pacing after the removal of Ang II (group 3). In each of these conditions, there was enhanced diastolic stiffening. Furthermore, MMP activation declined after 7-day Ang II exposure alone, when corresponding diastolic stiffness decreased. In this sense, diastolic stiffness correlated better with MMP activity than with histological evidence of interstitial fibrosis. The latter was greatest after 7-day Ang II infusion, whereas chamber diastolic properties were similar to baseline at that time. This suggests that although MMP activation alone is insufficient to induce diastolic stiffening, the synergy between Ang II and 48-hour tachypacing may depend on the presence of increased matrix protein turnover. One possibility is that MMP activity facilitated formation of interstitial edema, which can increase myocardial stiffness.⁴¹

Elevation of MMPs has been reported in several heart failure conditions, including a rise in a 92-kD protein (MMP-9) in late-stage failure induced after several weeks of tachypacing in the dog.⁴² MMP-1, MMP-2, and MMP-9 activation has been reported in a rat infarction model.⁴³ Although these studies have suggested a contribution of MMPs to chamber remodeling, the precise role of MMPs and their mechanofunctional consequences in evolving heart failure have remained unclear. The present results suggest important mechanical implications of MMP activity in the setting of cardiac failure, particularly with respect to diastolic chamber stiffening.

Several other mechanisms may also underlie Ang II–mediated potentiation of cardiac diastolic dysfunction. Hearts may become sensitized to Ang II–mediated mechanical dysfunction, as observed with late-stage failure.⁶ Such sensitization is supported by isolated myocyte data from volume-overload hypertrophied hearts showing enhanced Ca²⁺ responses to Ang II⁴⁴ and increased expression and posttranslational regulation of Ang II receptors with mechanical stretch.⁴⁵ Recent studies have shown selective downregulation of the AT₁ receptor in human heart failure,⁴⁶ although there remains a potential for altered downstream signaling.

Short-term tachypacing could also compromise reserve mechanisms triggered to minimize ventricular abnormalities with sustained Ang II exposure despite histological damage and matrix changes. Tachypacing downregulates ß₁-adrenergic receptors,^{17 47} reduces adenylate cyclase activity,^{17 18 47} and alters calcium handling,^{19 20 48} and many of these changes are observed after only 24 to 48 hours of pacing.^{17 19} Loss of these mechanisms could expose underlying Ang II–mediated myocardial dysfunction. Last, vasculotoxic⁴⁹ or vasoconstrictive⁵⁰ effects of Ang II may impair coronary flow reserve and vasomotor reactivity, limiting oxygen delivery during rapid pacing. However, one would expect more synergistic effects on systolic impairment, which were not observed.

Summary
In summary, our results demonstrate a strong synergy between Ang II and cardiac depression resulting in exacerbated and accelerated diastolic stiffening that is not observed with either stimulus alone. Augmented interstitial metabolism by Ang II likely contributes to this synergy. Ongoing studies targeting ß-adrenergic pathway signaling, ACE and AT₁ receptor antagonism, bradykinin, and anti-inflammatory agents should help clarify the pathways involved in this interaction and focus therapeutic approaches for improving diastolic failure.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme Ang I, Ang II = angiotensin I and II EDD = end-diastolic dimension EDP = end-diastolic pressure EDPDR = end-diastolic pressure-dimension relation ESPDR = end-systolic pressure-dimension relation IVC = inferior vena cava LV = left ventricle (ventricular) MMP = matrix metalloproteinase Msw = slope of stroke work–EDD relation RAS = renin-angiotensin system RIA = radioimmunoassay

	Acknowledgments

 
This study was supported by National Institutes of Heath Grants HL-47511 and HL P50 52307 and an Established Investigator Award from the American Heart Association (Dr Kass). We are very grateful to Drs Suzanne Oparil, Louis dell'Italia, Qing C. Meng, and Eduardo Balcells from the Division of Cardiology, Birmingham Veteran Affairs Medical Center, and the Department of Physiology and Biophysics, University of Alabama at Birmingham, for performing the Ang I and II assays. We also thank Medtronic Inc for providing pulse generators and pacing leads and Cordis Inc for providing endomyocardial bioptomes. Last, we thank Richard Tunin for his expert surgical and laboratory assistance.

Received October 7, 1997; accepted December 4, 1997.

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