Angiotensin II in the Evolution of Experimental Heart Failure
Although angiotensin II (Ang II) has been implicated in the pathophysiology of congestive heart failure, its temporal and regional changes during the development and progression of the disease are poorly defined. Our objective was to assess circulating, renal, cardiac, and vascular Ang II in a canine model of rapid ventricular pacing–induced heart failure that evolves from early left ventricular dysfunction to overt congestive heart failure. Ang II was measured by radioimmunoassay with low cross-reactivity to other angiotensins. Control, early left ventricular dysfunction, and overt congestive heart failure dogs were studied. Early left ventricular dysfunction was characterized by impaired cardiac function, cardiac enlargement, preserved renal perfusion pressure, maintained urinary sodium excretion, and normal plasma renin activity. Overt congestive heart failure was characterized by further impaired cardiac function and cardiac enlargement, reduced renal perfusion pressure, urinary sodium retention, and increased plasma renin activity and plasma Ang II. In early left ventricular dysfunction dogs, renal cortical, renal medullary, ventricular, and aortic Ang II were unchanged, and atrial Ang II was decreased. In overt congestive heart failure dogs, Ang II was increased in the kidney and heart compared with normal dogs and in all tissues compared with early left ventricular dysfunction dogs. The greatest increase in tissue Ang II occurred in the renal medulla. We conclude that early increases in local renal, myocardial, and vascular Ang II do not occur in this model of early left ventricular dysfunction and may even be suppressed. In contrast, increased myocardial and particularly renal Ang II in association with increased circulating Ang II are hallmarks of overt experimental congestive heart failure. These studies provide new insights into the temporal and regional alterations in Ang II during the progression of experimental congestive heart failure.
Angiotensin II (Ang II) plays an important role in the control of renal, myocardial, and vascular function. Under physiological conditions, Ang II modulates renal sodium excretion, myocardial contraction and relaxation, and peripheral vascular tone.1 2 3 In overt CHF, investigations support a key role for Ang II as a mediator of renal, myocardial, and vascular adaptations that characterize this syndrome. Specifically, locally bound and/or generated Ang II may participate in the enhanced tubular reabsorption of sodium,4 cardiac hypertrophy and fibrosis,5 6 and systemic and regional vasoconstriction that are hallmarks of overt CHF. Underscoring the importance of Ang II are investigations in animals and humans which document that pharmacological interventions that retard increases in Ang II or prevent Ang II receptor binding may delay the progression of CHF.7 8 9 These beneficial pharmacological effects have been linked to the inhibition of avid sodium reabsorption, ventricular enlargement, and systemic and regional vasoconstriction.
Despite the important role of Ang II in the pathophysiology of overt CHF, little information exists regarding Ang II concentrations in local tissues during the evolution of the disease. Specifically, there is a lack of information regarding renal, myocardial, and vascular Ang II in the evolution of CHF from early LV dysfunction, characterized by impaired cardiac function without sodium retention or edema, to overt CHF with avid renal sodium retention and edema.
Our objective in the current study was therefore to define the temporal and regional concentrations of Ang II during the development of experimental heart failure from early LV dysfunction to overt CHF. We used rapid ventricular pacing in dogs to induce experimental heart failure. We induced early LV dysfunction using a recently described pacing protocol characterized by significant LV systolic dysfunction and cardiac dilatation but maintained renal perfusion pressure and renal sodium excretion.10 11 Tachypacing-induced early LV dysfunction shares many similarities with human early LV dysfunction, including an activation of the natriuretic peptide system without increases in PRA. Dogs with overt CHF were subjected to a more prolonged pacing protocol that evolves from early LV dysfunction to overt CHF through incremental ventricular pacing.12 Experimental overt CHF is characterized by more severe cardiac dysfunction, increased PRA, renal sodium retention, and clinical symptoms of congestion. In the current investigation, we hypothesized that renal, cardiac, and aortic Ang II remain unchanged in experimental early LV dysfunction despite LV dysfunction and dilatation and that overt CHF is characterized by a generalized increase in renal, cardiac, and aortic Ang II. To address this hypothesis, we determined Ang II in renal cortex and medulla, atrial and ventricular myocardium, and aortic tissue in normal dogs and dogs with early LV dysfunction and overt CHF.
Fifteen male mongrel dogs were used for the study. Ten dogs underwent implantation of a programmable cardiac pacemaker (Medtronic) as described previously.10 11 With dogs under pentobarbital sodium anesthesia (30 mg/kg IV) and artificial ventilation (Harvard respirator), the heart was exposed via a small left lateral thoracotomy and pericardiotomy, and a screw-in epicardial pacemaker lead was implanted into the right ventricle. The pacemaker was implanted subcutaneously into the left chest wall and connected to the pacemaker lead. Five dogs additionally underwent implantation of a chronic femoral artery catheter (model GPV Vascular-Access Port, Access Technologies) implanted via the left femoral artery and subcutaneously connected to a port above the left upper hindlimb. All dogs were allowed to recover for at least 10 days after surgery and were weighed before the pacemaker was started for the induction of heart failure. All studies were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and conducted in accordance with the Animal Welfare Act.
Five dogs (overt CHF group) underwent pacing with a stepwise increase of stimulation frequencies over 38 days. During the first 10 days, dogs were paced at 180 beats per minute (bpm). As previously described,10 11 this protocol results in early LV dysfunction as defined by significant systolic dysfunction with decreased cardiac output, cardiac enlargement, and increased filling pressures but maintained systemic perfusion pressure and renal sodium excretion and no clinical signs of heart failure. The pacing rate was then increased weekly to 200, 210, 220, and 240 bpm, and early LV dysfunction evolved to overt CHF. Overt CHF was defined as pacing-induced systolic dysfunction with avid sodium retention and clinical signs of congestion.12 At baseline (control), after dogs had been paced at 180 bpm for 10 days (early LV dysfunction) and at the end of the protocol (overt CHF), urine was collected over 24 hours for measurement of sodium excretion; conscious mean arterial pressure was measured via the port catheter; a 2D guided M-mode echocardiogram was obtained; and arterial blood was drawn. In addition, cardiac filling pressures and cardiac output by thermodilution (model 9510-A, American Edwards Laboratories) were measured in conscious dogs at baseline (control) and at the end of the pacing protocol (overt CHF). Arterial blood was collected in EDTA tubes for measurement of ANP, BNP, cGMP, PRA, aldosterone, and Ang II and was immediately placed on ice. The tubes for plasma Ang II measurement contained final concentrations of 5 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L sodium tetrathionate for inhibition of proteases.13 Dogs were then killed (intravenous Sleepaway euthanasia solution, Fort Dodge Laboratories Inc) for rapid tissue harvesting. Hearts were rapidly trimmed and left ventricles weighed for subsequent calculation of the index of LV weight (grams) to body weight (kilograms). Kidneys were dissected in cortex and medulla. All tissue was snap-frozen in liquid nitrogen after harvesting and stored at −80°C until further processing. Blood was centrifuged at 2500 rpm and 4°C, and the plasma was stored at −20°C until analysis as described below. All pacemakers were checked at the time of programming and then weekly and at the day of death for proper pacing.
A second group of five dogs was paced at 180 bpm for 10 days only and served as tissue donors for the early LV dysfunction group; a third group of five normal dogs served as tissue donors for the control group. Again, invasive hemodynamic measurements were obtained for documentation of cardiac function; arterial blood was drawn; and dogs were killed and tissue rapidly harvested.
Urinary sodium was measured with ion-selective electrodes (Beckman Instruments). ANP, BNP, and cGMP,14 PRA,15 and aldosterone16 were determined by standard radioimmunoassay techniques. Because of species variability in BNP, we used a polyclonal antibody specific for canine BNP.17
Ang II was measured by radioimmunoassay. Plasma samples were extracted in phenyl-encapped cartridges, washed with 1 mL distilled water, eluted with 0.5 mL methanol, lyophilized, and reconstituted. For extraction of tissue Ang II, samples were pulverized frozen, boiled for 5 minutes in 10 vol of acetic acid (1 mol/L)/HCl (20 mmol/L),18 and homogenized at high speed (PT 1200, Polytron). The homogenate was then ultracentrifuged at 27 000g at 4°C, and the supernatant was stored at −20°C until radioimmunoassay. Before centrifugation, a sample of the homogenate was taken for measurement of tissue protein content according to the Folin phenol method of Lowry et al.19 For the radioimmunoassay, antigen in standards or samples was preincubated in coated glass tubes with a commercially available polyclonal rabbit antibody (Peninsula Ang II No. 7002) at 4°C for 24 hours. 125I-labeled Ang II (7500 cpm) was added to each tube and incubated for another 24 hours at 4°C. Bound antigen-antibody was separated with goat anti-rabbit γ-globulin, and the precipitate was counted with a gamma counter. Immunoreactive Ang II in plasma was expressed in picograms per milliliter. Immunoreactive Ang II in tissue was measured as picograms per milliliter homogenate, normalized for protein content, and expressed as picograms immunoreactive Ang II per milligram tissue protein.
In the evaluation of the Ang II assay, sensitivity was 7.5 pg per tube, and the standard curve ranged from 7.5 to 500 pg per tube. The mean recovery for Ang II was 93%. For determination of the cross-reactivity of the Ang II antibody, binding curves for Ang I and Ang III were established (Fig 1⇓). On the basis of the molar concentration that bound 50% Ang II antibody (IC50), cross-reactivities of 1.4% and 31% were determined for Ang I and Ang III, respectively. When plasma was spiked with 50 pg/mL synthetic Ang III, recovery was only 44%, again indicating incomplete cross-reactivity of the Ang II antibody with Ang III.
The echocardiogram (Toshiba) was obtained by an expert echocardiographer from the right parasternal window. LV end-diastolic (LVEDd) and end-systolic (LVESd) dimensions and diastolic LV posterior wall thickness were determined from three repeated 2D guided M-mode tracings. From these measurements, ejection fraction (EF) was calculated as EF=(LVEDd2−LVESd2)/LVEDd2.
Results of the quantitative studies are expressed as mean±SE. Comparison between the control, early LV dysfunction, and overt CHF groups were performed by ANOVA followed by Fisher's least significant difference test. Statistical significance was defined at a value of P<.05.
Hemodynamics, LV Function and Geometry, and Clinical Parameters
In experimental early LV dysfunction, mean arterial pressure did not differ from control (Table 1⇓). However, echocardiographic analysis revealed significant eccentric LV dilatation (LV end-diastolic diameter, +8%, P<.02; diastolic posterior wall thickness, −10%, P<.02) and LV dysfunction (ejection fraction, −39%, P<.01). In overt CHF, systemic perfusion pressure decreased (mean arterial pressure, 108.6±3.4 to 96.8±6.5 mm Hg, P<.02 versus control), right atrial pressure (5.6±1.2 to 14.5±1.2 mm Hg, P<.03) and pulmonary capillary wedge pressure (10.1±1.0 to 23.5±1.8 mm Hg, P<.01) increased, and cardiac output showed a tendency to decrease (5.2±0.5 to 2.4±0.4 L/min, P=.057). In addition, ventricles were further dilated (+15% versus early LV dysfunction, P<.01), and LV wall thickness and ejection fraction further decreased (−11% and −34% versus early LV dysfunction, both P<.04). Postmortem, the LV mass over body mass index in dogs with overt CHF showed a tendency to increase from 4.15±0.1 (control) to 4.71±0.3 g/kg (overt CHF, P=.06). All overt CHF dogs showed pulmonary edema and pleural effusion, and all but one had ascites. In contrast, control dogs and dogs with early LV dysfunction were free of symptoms of congestion, the latter despite ventricular dysfunction and dilatation.
Circulating Hormones and Sodium Excretion
As depicted in Table 2⇓, early LV dysfunction is characterized by an activation of the natriuretic peptides and their second messenger cGMP. Mean circulating ANP was increased by 268% (P=NS), BNP by 507% (P=.05), and cGMP by 164% (P<.01). These elevations occurred in the absence of activated plasma renin, aldosterone, or Ang II. Urinary sodium excretion was maintained. In overt CHF, dogs showed a further increase in circulating ANP and cGMP (+144%, P<.04, and +71%, P<.01, respectively) and BNP (+72%, P=.09 [nonsignificant]). Plasma renin (+759%, P<.01), aldosterone (+1055%, P<.03), and Ang II (+1021%, P<.03) were activated, and urinary sodium excretion was decreased (−78% versus normal, P<.01).
Renal, Cardiac, and Aortic Ang II
Fig 2⇓ (renal cortex and medulla) and Fig 3⇓ (atrial and ventricular myocardium and aorta) illustrate immunoreactive Ang II (picograms per milligram of protein) in tissues from control, early LV dysfunction, and overt CHF dogs. In early LV dysfunction, renal cortical and medullary Ang II (Fig 2⇓) were unchanged compared with control (7.1±0.4 versus 6.7±0.5, P=NS, and 10.7±0.8 versus 8.7±1.2, P=NS). Ang II was decreased in atrial tissue (Fig 3⇓) compared with control (5.4±0.4 versus 6.4±0.3, P<.05). In left ventricle and aorta (Fig 3⇓), Ang II showed a tendency to decrease compared with control (4.5±0.4 versus 5.2±0.3, P=.18 [nonsignificant], and 6.0±0.5 versus 7.7±0.7, P=.09 [nonsignificant]). In overt CHF dogs, Ang II concentrations increased in renal cortex (8.8±0.4 versus 7.1±0.4, P<.03, Fig 2⇓) and medulla (14.6±1.5 versus 10.7±0.8, P<.05, Fig 2⇓), left atrium (7.0±0.3 versus 5.4±0.4, P<.01, Fig 3⇓), left ventricle (6.4±0.4 versus 4.5±0.4, P<.01, Fig 3⇓), and aorta (9.5±0.8 versus 6.0±0.5, P<.01, Fig 3⇓) compared with early LV dysfunction dogs. Compared with control dogs, Ang II concentrations increased in renal cortex (+31%, P<.01), renal medulla (+67%, P<.01), and left ventricle (+23%, P<.03).
We designed the current study to define circulating, renal, myocardial, and vascular Ang II during the evolution of rapid ventricular pacing–induced heart failure in the dog. Ang II was measured in plasma, renal cortex and medulla, atrial and ventricular myocardium, and aortic tissue in experimental early LV dysfunction and overt CHF dogs. Experimental early LV dysfunction was characterized by eccentric LV dilatation, impaired LV systolic function, preserved renal perfusion pressure, maintained renal sodium excretion, the absence of edema, and an activation of the natriuretic peptide and cGMP system. PRA, Ang II, and aldosterone did not differ from normal. Although renal, ventricular, and aortic Ang II were unchanged in early LV dysfunction, we found left atrial Ang II to be decreased. When early LV dysfunction progressed to overt CHF, a further impairment in LV systolic function and LV dilatation occurred in association with a reduction in renal perfusion pressure, onset of renal sodium retention, and formation of edema. Despite a further increase in circulating ANP and cGMP, PRA, Ang II, and aldosterone were activated. All tissues demonstrated significant increases in Ang II compared with early LV dysfunction. Renal cortex, renal medulla, and ventricular myocardium showed significant increases in Ang II compared with normal, with the greatest increase in renal medulla. Thus, these studies demonstrate first that the canine tachypacing model of progressive heart failure is characterized by a lack of increase in circulating and tissue Ang II in early LV dysfunction despite ventricular dysfunction and dilatation. Indeed, atrial Ang II is decreased. Second, these studies demonstrate a generalized increase of circulating and tissue Ang II in overt CHF, with the greatest increase in the kidney.
In recent investigations,10 11 we characterized cardiorenal function and neurohormonal activation in tachypacing-induced early LV dysfunction. We demonstrated that experimental early LV dysfunction is characterized by systolic LV dysfunction, increased cardiac filling pressures, systemic vasoconstriction, and maintained renal sodium excretion.10 The neurohormonal response to experimental early LV dysfunction is characterized by an activation of the natriuretic peptides without changes in PRA or aldosterone and thus mimics human early LV dysfunction as described in the Studies of Left Ventricular Dysfunction (SOLVD).20 In a more recent study, we also demonstrated that suppression of the natriuretic peptides in early LV dysfunction by surgical biatrial appendectomy or natriuretic peptide receptor blockade leads to an increase in PRA and aldosterone in association with renal sodium retention and a blunted response to intravascular volume expansion.11 These previous studies underscore the potency of the natriuretic peptide system in suppressing the circulating RAS in vivo and compensate for impaired myocardial function in early LV dysfunction.
Although a lack of activation of the circulating RAS has previously been shown to be a hallmark of experimental early LV dysfunction, the current finding of unchanged renal, ventricular, and aortic Ang II in experimental early LV dysfunction is novel and complements recent elegant studies which have demonstrated that the angiotensinogen, renin, and ACE genes are transcribed in renal and cardiovascular tissues.13 21 22 23 24 25 26 In these previous studies, it has further been demonstrated that activation of the local renal13 21 and cardiac21 22 23 24 25 RAS in experimental and human disease26 can be induced rapidly and in part independently of the circulating RAS. In addition, a pathway of Ang II generation independent of ACE has been characterized.27 If we would have found tissue Ang II to be increased in experimental early LV dysfunction, this observation would have been consistent with an isolated activation of the local RAS in response to significant ventricular dysfunction. With our current finding of a lack of increase in tissue Ang II in early LV dysfunction and a decrease in atrial Ang II, we conclude that a functional activation of the tissue RAS as defined by increased tissue Ang II does not occur in this model of experimental early LV dysfunction despite established ventricular dilatation and reduced systolic function. It is tempting to speculate that such a potential activation is suppressed by the activation of the natriuretic peptide system and cGMP. Consistent with the observation of partial inhibition of the RAS in heart failure are recent studies by Huang et al28 that report inhibition of pulmonary ACE in ventricular dysfunction produced by myocardial infarction in the rat and studies by Kawaguchi et al29 which report that ANP inhibits the conversion of Ang I to Ang II in vitro.
The finding of increased tissue Ang II in overt CHF occurred in the presence of increased PRA and aldosterone. Increased LV Ang II was associated with a tendency for increased LV mass. Although significant increases in LV mass have previously been demonstrated in the current model,12 the contribution of Ang II remains controversial because the traditional tachypacing model of severe CHF is associated with increased LV Ang II but without increased LV mass.30 Therefore, other factors, such as prolonged pacing or the progressive pacing mode, may be necessary as additional permissive factors for the development of LV hypertrophy. Increased renal Ang II has been previously reported by Schunkert et al13 in the rat infarction model of severe CHF in conjunction with an activation of the circulating RAS. Increased local Ang II in the presence of an activated circulating RAS is consistent with increased uptake of circulating Ang II as well as increased local generation or both. An obvious question is why circulating and tissue Ang II levels increase despite continued increases in natriuretic peptides and cGMP. Although the mechanisms may be multifactorial, the marked decrease in renal perfusion pressure and decreased sodium excretion suggest as a stimulus a decreased delivery of sodium to the macula densa, with subsequent renin release by the kidney.
In the measurement of immunoreactive Ang II by radioimmunoassay, the cross-reactivity of the used antibody with other angiotensins is important. The binding curves established for the current antibody to Ang II showed cross-reactivities of only 1.4% and 31% with Ang I and Ang III, respectively (Fig 1⇑). This indicates a low cross-reactivity against the C-terminal end of Ang I and a high specificity for the N-terminal end of Ang II. It has recently been described that renal tissue concentrations of Ang I and II are in a very similar range.31 With 1.4% cross-reactivity, Ang I should therefore constitute only an insignificant source for anti–Ang II immunoreactivity. The moderate cross-reactivity of Ang III with anti–Ang II is of less concern because Ang III is an (active) Ang II metabolite. Particularly the finding of unchanged or decreased (atrial) Ang II cannot be explained by cross-reactivity, which has to be discussed in findings of increased immunoreactivity.
Although the current study focused on early LV dysfunction and overt CHF, it remains to be determined exactly when a reversal of decreased tissue Ang II and a subsequent increase occur during the evolution of CHF and may be of particular significance in our understanding of the progression of the disease. Also, although the current experimental studies cannot be entirely transferred to human CHF, they raise some interesting issues. As interruption of Ang II generation by ACE inhibition has been shown to reduce morbidity and mortality in overt CHF,8 9 it has also been associated with beneficial actions in early LV dysfunction.32 Yet the actual concentration of the effector peptide Ang II in human renal, cardiac, and vascular tissues has not been investigated. The current studies underscore a priority for such studies for furthering our understanding of Ang II in human CHF.
In summary, the current study provides important new insight into the temporal and regional regulation of Ang II in the evolution of experimental CHF. Ang II is present in normal renal, myocardial, and aortic tissues. Ang II is increased in renal cortex, renal medulla, and ventricular myocardium in overt experimental CHF and may mediate pathophysiological alterations that characterize this syndrome. Unchanged renal, ventricular, and aortic Ang II concentrationss in experimental early LV dysfunction indicate that the current model is not characterized by an early increase in tissue Ang II. Indeed, decreased atrial Ang II in early LV dysfunction may support an in vivo role for the natriuretic peptides in the suppression of Ang II.
Selected Abbreviations and Acronyms
|Ang I, II, III||=||angiotensin I, II, III|
|ANP||=||atrial natriuretic peptide|
|BNP||=||brain natriuretic peptide|
|CHF||=||congestive heart failure|
|PRA||=||plasma renin activity|
This work was supported by grants from the National Institutes of Health (HL-36634 and HL-07111), the National Kidney Foundation of the Upper Midwest, the Hearst Foundation, and the Mayo Foundation. Andreas Luchner is a recipient of a grant by the Deutsche Forschungsgemeinschaft (Lu 562/1-1). The authors thank Lawrence L. Aarhus and the Department of Veterinary Medicine for their professional assistance.
This work was presented as an abstract at the annual meeting of the American Heart Association, Anaheim, Calif, November 13-16, 1995. (Circulation. 1995;92:I-602).
- Received February 5, 1996.
- Revision received February 28, 1996.
- Revision received May 6, 1996.
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