Aortocaval Shunt Rats Research Articles

Preserved Norepinephrine Reuptake but Reduced Sympathetic Nerve Endings in Hypertrophic Volume-Overloaded Rat Hearts

Background In congestive heart failure (CHF), an activation of the cardiac sympathetic nervous system results in depleted cardiac norepinephrine (NE) stores. The underlying regulatory mechanisms are discussed controversially and were investigated in the present study in CHF resulting from volume overload. Methods and Results Aorto-caval shunt (AVS) was performed in rats. Plasma NE levels were determined by radioenzymatic assay, left ventricular NE by high-performance liquid chromatography, endothelin-1 by enzyme-linked immunosorbent assay. Tyrosine-hydroxylase (TH)– and nerve growth factor (NGF)–mRNA was determined by Northern blot analysis and ribonuclease-assay. Cardiac [ 3H]-NE uptake was measured in isolated perfused hearts. Glyoxylic acid–induced histofluorescence was used to quantify cardiac sympathetic nerves. Compared with sham-operated animals (SH), AVS rats were characterized by depleted cardiac NE stores and enhanced NE plasma levels. Neither TH-mRNA levels in stellate ganglia, nor cardiac [ 3H]-NE-uptake were reduced in AVS. The left ventricular density of sympathetic nerves was markedly decreased. Gene expression of myocardial NGF (a positive regulator of NE reuptake and cardiac sympathetic nerve density) and left ventricular endothelin-1 (a negative regulator of NE reuptake and positive regulator of cardiac NGF expression) were unchanged. Conclusion In volume-overloaded hypertrophic hearts, depletion of cardiac NE stores is caused by a reduction of the sympathetic nerve density, whereas cardiac NE reuptake is preserved.

Comparison of the Angiotensin II Type 1-Receptor Antagonist YM358 and the Angiotensin-Converting Enzyme Inhibitor Enalapril in Rats With Cardiac Volume Overload

We evaluated the effects of chronic oral administration of an angiotensin II type 1 (AT1)-receptor antagonist YM358 and an angiotensin converting enzyme inhibitor enalapril on hemodynamics and cardiac hypertrophy in rats with volume overload-induced heart failure. We assessed changes of cardiac hemodynamics and cardiac hypertrophy at 2 and 4 weeks after administration of YM358 (3, 30 mg/kg per day) or enalapril (30 mg/kg per day) in abdominal aortocaval shunt rats. YM358 (30 mg/kg) attenuated increases of left ventricle (LV)/body weight (BW), left atrium (LA)/BW, right ventricle (RV)/BW and heart/BW ratios, but did not affect cardiac hemodynamics in shunt rats. Enalapril also reduced the increased LV/BW and heart/BW ratios together with significant reductions of systolic blood pressure, left ventricular systolic pressure and the first derivative of left ventricular pressure. These data suggest that the effects on attenuation of the development of cardiac hypertrophy are not different for YM358 and enalapril, although the effects on cardiac hemodynamics are different for the same dosages. The attenuating action of YM358 on cardiac hypertrophy was independent of the action on hemodynamics and indicated the direct action of the AT1 receptor on the heart.

Myocardial beta-adrenergic reactivity in volume overload-induced cardiac hypertrophy in the rat.

The purpose of this study was to evaluate the changes in myocardial beta-adrenergic reactivity in animals undergoing a 4 week cardiac volume overload. Aortocaval shunt (ACS) or sham operation (sham) were performed in male Wistar rats, and 4 weeks later, isoproterenol dose-effects (chronotropic, inotropic and lusitropic properties) were studied after pithing. Noradrenaline (NA) and adrenaline (A) concentrations and NA turn-over index were evaluated in plasma and heart ventricles, while beta-adrenoceptor characteristics in ventricle homogenates and slices with [125I]iodocyanopindolol, and the beta(1)/beta(2)-adrenoceptor ratio were estimated. Four weeks of cardiac volume overload resulted in a 55% increase in ventricle weight/body weight ratio (from 2.5 +/- 0.1 to 3.9 +/- 0.1 mg/g in sham and ACS rats, respectively) and a 20% increase in protein contents (from 11.3 +/- 0.7 to 13.8 +/- 1.1 mg/100 mg ventricles in sham and ACS rats, respectively). Furthermore, NA and A concentrations and NA turn-over index were increased in ACS rats (14, 40 and 80% versus sham, respectively). A shift to the right of the responses in heart rate, left ventricular systolic pressure, +dP/dtmax and -dP/dtmax responses following increasing doses of isoproterenol was observed, without change in the dose inducing maximum effect. Total beta-adrenoceptor characteristics and beta(1)/beta(2) ratio were unchanged. However, beta(1)-adrenoceptor density increased in epicardium while decreasing in endocardium of left ventricle from ACS rats. Rightward shift at lower doses of isoproterenol-induced cardiac responses in volume-overloaded rats are not likely due to overall beta-adrenoceptor density changes.

Regulation of fibrillar collagen gene expression and protein accumulation in volume-overloaded cardiac hypertrophy.

Interstitial collagen accumulation has been extensively demonstrated to be increased at both mRNA and protein levels in pressure-overloaded cardiac hypertrophy. However, few data are available regarding the effects of volume overload on myocardial collagens. To determine whether the alterations of collagens may occur in volume-overloaded cardiac hypertrophy, we measured collagen types I and III mRNA levels and protein accumulation in left ventricular (LV) myocardium of rats at 3, 7, and 28 days after the creation of an aortocaval (AC) shunt. Eccentric LV hypertrophy was produced in rats with AC shunting. Northern blot analysis on RNA extracted from LV tissue indicated that the steady state mRNA levels for both type I and III collagen were persistently upregulated in AC shunt rats compared with sham-operated operated control rats. In contrast, the biochemical collagen protein concentration and morphometric collagen volume fraction were comparable between sham-operated control and AC shunt rats at any study time point. Furthermore, the immunohistochemical staining of types I and III collagen and Sirius red staining on myocardial tissue sections revealed no significant alterations in the distribution or density of fibrillar collagens in AC shunt rats. Tissue collagenase activity was not different between control and AC shunt rats after 28 days. Cardiac volume overload increases LV collagen mRNA as does pressure overload. However, in contrast to pressure-overloaded hypertrophy, the upregulation of collagen transcriptional activity does not result in subsequent myocardial fibrosis in volume-overloaded hypertrophy due to AC shunting. Therefore, the upregulation of collagen gene expression and protein accumulation might be different in pressure-overloaded and volume overloaded hypertrophy.

Early changes in the functions of cardiac sarcoplasmic reticulum in volume-overloaded cardiac hypertrophy in rats.

By sequestering activator calcium, the sarcoplasmic reticulum (SR) plays the central role in the excitation-contraction (E-C) cycle of cardiac muscle. Hence, functional changes in the SR in diseased myocardium might critically determine its mechanical characteristics. Previously, we demonstrated that both Ca2+ release and uptake were increased in SR isolated from hearts showing compensatory left ventricular (LV) hypertrophy taken from pressure-overloaded rats. However, it has not been elucidated whether such alterations also occur in the volume-overloaded myocardium. Rats in which volume-overloaded hypertrophy had been induced by aortocaval shunt 12 weeks prior to the investigation were compared to sham-operated controls in terms of SR Ca2+ uptake and release, and density of Ca2+ releasing channels (ryanodine receptors, RyR). Isometric tension and intracellular Ca2+ transients were also measured using the bioluminescent Ca2+ indicator, aequorin, in isolated LV papillary muscles. The extent of hypertrophy was verified by measuring the ratio of biventricular weight to body weight. In vivo, the aortocaval shunt rats showed normal LV contractility and slightly depressed LV relaxation, indicating a compensatory (adaptive) stage of LV function. In contrast, Ca2+ release, uptake, and maximal number of [3H]-ryanodine binding sites were all significantly lower in aortocaval shunt rats than in controls. Both the Ca2+ transients and isometric relaxation of the isolated myocardium were significantly prolonged in aortocaval shunt rats, though their amplitudes were similar in the two groups. Thus, the volume-overloaded cardiac hypertrophy, even at its hemodynamically compensatory (adaptive) stage, (i) was accompanied by abnormal Ca2+ handling, as indicated by prolonged intracellular Ca2+ transients and isometric tension traces, (ii) seems to involve subcellular mechanisms related to decreases in SR Ca2+ release and uptake functions, as well as to a decrease in the number of RyR. Therefore, changes in the intracellular processes underlying cardiac E-C coupling, including SR function, precede the development of this type of heart disease.

Cardiac renin-angiotensin system in the hypertrophied heart.

The cardiac renin-angiotensin system (RAS) has been suggested to play an important role in heart failure and cardiac hypertrophy. In the present study, we evaluated the expression of each component of the RAS in hypertrophied heart induced by aortocaval shunt. The expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin II type Ia and Ib receptor (AT1aR and AT1bR) mRNA were determined by the reverse transcription-polymerase chain reaction method owing to the relatively low expression levels of these mRNAs in the ventricle. The expression level of renin or angiotensinogen mRNA in the ventricle was very low, more than 1000-fold lower than that in the kidney or liver, respectively. The expression of ACE mRNA in the ventricle was relatively abundant and was increased in the hypertrophied ventricle in this model, whereas no significant increases in the expression levels of AT1aR and AT1bR mRNA were observed. Administration of lisinopril attenuated the development of left and right ventricular hypertrophy in this model and was accompanied by an attenuation of the upregulation of the ACE, collagen type I-alpha, and vimentin mRNAs. Because the activity of the circulating RAS in the aortocaval shunt rats was not higher than that in the sham-operated rats, the effects of lisinopril in attenuating the ventricular hypertrophy may be due to inhibition of the increased ACE in the ventricle. The present study supports the importance of ACE expressed in the ventricle in the development of hypertrophy induced by aortocaval shunt.

Captopril treatment does not restore either the renal or the ANF release response during volume expansion in moderate to severe high output heart failure

Atrial natriuretic factor (ANF) release and the renal response to moderate volume expansion have been shown to be conserved in rats with a mild to moderate degree of high output heart failure (aortocaval shunt). The aim of this study was to investigate whether these variables are also conserved in animals with moderate to severe heart failure induced by an aortocaval shunt. The effect of angiotensin converting enzyme (ACE) inhibition by captopril on these responses was also investigated. An aortocaval shunt was developed in Sprague-Dawley rats weighing 180-200 g; sham operated rats served as controls. Three weeks after surgery, three experimental groups were established: aortocaval shunt and sham operated controls, and aortocaval shunt rats treated with captopril during the last week before the experiments were started. Four weeks after surgery, haemodynamic variables, ANF release, diuresis, and natriuresis were evaluated following a moderate volume expansion. Mean arterial blood pressure was lower in shunt animals and still lower in the ACE inhibited group than in the sham operated controls. Central venous pressure and left ventricular end diastolic pressure (LVEDP) were significantly higher in untreated shunt rats than in their controls. ACE inhibition returned the raised central venous pressure, but not LVEDP, to control values. Shunt rats had lower baseline urinary sodium excretion (UNaV), urinary volume, and packed cell volume than their sham operated controls. ACE inhibition reversed baseline urinary volume to control values. Baseline COOH terminal and HN2 terminal ANF were greatly increased in both treated and untreated shunt rats. Volume expansion was performed three times in conscious animals at 15 min intervals with human plasma protein fraction. Its effect on LVEDP was similar in all three groups, but the increase in central venous pressure was much higher in untreated shunt animals. UNaV, urinary volume, and the release of COOH terminal and NH2 terminal ANF in response to volume expansion were blunted in both treated and untreated shunt rats when compared with their sham operated counterparts. Both absolute and relative heart weights were significantly lower in captopril treated shunt animals than in the untreated shunt group, the latter presenting very significant cardiac hypertrophy. Aortocaval shunt animals with moderate to severe heart failure show a blunted ANF release and renal response to volume expansion, which, despite significant haemodynamic improvement, are not restored by ACE inhibition.

Characterisation of plasma and tissue atrial natriuretic factor during development of moderate high output heart failure in the rat.

The aims were (1) to characterise plasma and tissue atrial natriuretic factor (ANF) levels, haemodynamic variables, and renal function at different stages of moderate chronic high output heart failure in the rat; and (2) to assess the contribution of the atria and ventricles to plasma ANF levels. Plasma and tissue ANF levels, haemodynamics, and renal function were evaluated at 1, 2, 4, 8, and 16 weeks after the development of aorto-caval shunts. Sham operated animals served as controls at identical time points. Male Sprague-Dawley rats weighing 225-275 g were used in all experiments. Mean arterial blood pressure was lower and right atrial pressure was higher in the aorto-caval shunt groups than in sham operated controls at all time periods. Left ventricular end diastolic pressure was increased significantly in aorto-caval shunt rats at 1, 2, and 4 weeks when compared with their control counterparts. Plasma COOH terminal and NH2 terminal ANF concentrations were increased significantly in aorto-caval shunt animals. Plasma ANF was positively correlated with right atrial pressure and left ventricular end diastolic pressure in aorto-caval shunt rats but not in sham operated controls. Aorto-caval shunt animals also developed marked cardiac hypertrophy with decreased atrial ANF concentration, but not content, and increased ventricular ANF concentration and content. Despite high plasma ANF concentrations, aorto-caval shunt rats had a lower packed cell volume at all observed periods and reduced urinary sodium excretion and urinary volume at 1 and 2 weeks, with trends to a reduction at 4, 8, and 16 weeks. Body weight was higher in aorto-caval shunt animals at 16 weeks than in sham operated controls. (1) Chronically increased cardiac filling pressure stimulated not only ANF release but also ANF synthesis in each cardiac chamber, which in turn contributed to raised plasma ANF concentrations in aorto-caval shunt rats; (2) an attenuated renal response to endogenous ANF and sodium and water retention were apparent in A-C shunt rats. Activation of neurohormonal vasoconstrictor systems and gradually decreased plasma ANF concentrations may contribute to sodium and water retention at different stages of this experimental model of heart failure.

Divergent regulation of atrial natriuretic factor receptors in high-output heart failure

We have investigated whether binding parameters and subtypes of glomerular, papillary, and vascular atrial natriuretic factor (ANF) receptors differ in rats with moderate high-output heart failure [aortocaval (AC) shunt] from their sham-operated controls. Body weight was lower and relative heart weight was higher in the AC shunt group than in the control group. Plasma renin activity (PRA) was also greater in AC shunt rats. Plasma COOH- and NH2-terminal ANF levels were higher in AC shunt animals than in their control counterparts. Total atrial ANF content was elevated in both the right and left atria of the AC shunt group. Glomerular and papillary ANF receptor density (Bmax) and ANF receptor affinity (Kd) were similar in both AC shunt and control rats. Vascular ANF receptor density and affinity were lower in AC shunt (Bmax = 65 +/- 13 fmol.mg protein; Kd = 467 +/- 52 pM) than in control rats (Bmax = 188 +/- 34 fmol.mg protein; Kd = 278 +/- 11 pM). Irreversible cross-linking of 125I-labeled ANF followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and radioautography demonstrated that both high- and low-molecular weight receptors were unchanged in glomerular membranes and downregulated in vascular membranes from AC shunt animals. However, guanosine 3',5'-cyclic monophosphate (cGMP) production by the isolated glomeruli of AC shunt rats was lower than that of controls. We conclude that in the presence of elevated plasma ANF levels, glomerular, papillary, and vascular ANF receptors may be regulated differently.

Chronic captopril and losartan (DuP 753) administration in rats with high-output heart failure.

We investigated the role of atrial natriuretic factor (ANF) and the renin-angiotensin system as well as the effects of losartan in rats with aortocaval (AC) shunts. Right atrial and left ventricular end-diastolic pressures (LVEDP) were higher and mean arterial blood pressure (MAP) was lower in AC shunt animals than in their controls. AC shunt rats presented marked cardiac hypertrophy, decreased right atrial ANF concentration, and increased ventricular ANF content and concentration. Plasma ANF levels were elevated, and hematocrit was lower in AC shunt animals than in controls. Captopril or losartan treatment decreased MAP and returned LVEDP to sham-operated control values. A clear regression of cardiac hypertrophy was evident in both treated AC shunt groups, with plasma ANF levels tending to follow those in sham-operated rats. Plasma COOH-terminal ANF levels were decreased and urinary volume and hematocrit were increased in losartan-treated AC shunt animals. We conclude that chronic angiotensin converting enzyme inhibition and angiotension II receptor antagonism improved hemodynamic conditions, diminished water retention, reversed cardiac hypertrophy, and restored plasma and tissue ANF to more "normal" levels in rats with moderate high-output heart failure.