Chapter 8
ON THE CARDIAC RENIN ANGIOTENSIN SYSTEM: THE HEART AS A SOURCE OF ANGIOTENSIN I1
Walmor C. De Mello, M.D., Ph.D.
Department of Pharmacology Medical Sciences Campus University of Puerto Rico San Juan, Puerto Rico
Evidence is available that angiotensin I1 (Ang 11) regulates heart contractility (Koch Weser, 1995), cell coupling and impulse propagation (De Mello, 1994; De Mello, 1996; De Mello et al, 1997; De Mello, 1999) and is responsible for remodeling and the induction of apoptosis (Horiuchi et al, 1999). Moreover, local renin angiotensin systems have been described in heart and other tissues (Dzau 1987).
The synthesis of some of the RAS components inside the heart cell is however, controversial. The levels of cardiac renin are extremely low in nephrectomized animals (Katz et a1.1997) suggesting that cardiac renin is dependent upon uptake from plasma at least in the normal heart (Danser et al, 1994; Campbell, Valentin 1994). Furthermore, the source of angiotensinogen in the heart, for instance, is not known. Although angiotensinogen is shown in the heart, no angiotensinogen is released from the perfused rat heart (de Lannoy et al, 1997) and no angiotensinogen is present in the supernatant of serum deprived neonatal cardiac cells (van Kesteren et al, 1999) what suggests that cardiac angiotensinogen is coming from the circulation. Similar observations have been published with respect to cardiac renin. Indeed, renin and renin mRNA levels in normal heart tissues are very low or even undetectable (de Lannoy et al, 1997; van Kestem et al, 1999) suggesting that, under normal condictions, cardiac renin is coming from plasma through diffusion in the interstitial space. Recently, renin receptors have been described in several tissues (Nguyen et a1 2004).
The contribution of plasma renin to cardiac renin was tested by
92 The Local Cardiac Renin Angiotensin-Aldosterone System
using transgenic mice expressing human renin in the liver (TTRhRen- A3) which were mated to mice expressing human angiotensinogen exclusively in the heart (MHChAgt-2). The results indicated low or undetectable angiotensin peptide in the heart of single transgenic animals while double transgenic mice showed a remarkable increase in cardiac levels of Ang I and Ang 11, indicating that plasma renin is able to act on its substract within the heart (Prescott et al, 2000).
However, it has been demonstrated that in some tissues a second renin gene transcription start site can be utilized leading to the synthesis of renin but lacking the secretory signal peptide. Initially found in the brain, the non-secreted transcript was found also in the myocardium, particularly during myocardium infarct (Clausmeyer et a1 2000), when the cardiac renin angiotensin system is activated. Indeed, the expression of exon 1A-renin mRNA in the left ventricle was found to be stimulated 4-fold during myocardial ischemia supporting the view of an intracellular function of renin (Clausmeyer et al, 2000).
Other studies indicated that overexpression of angiotensinogen gene in normal heart muscle cells of mice, for instance, caused an increase of angiotensin I1 concentration in the right and left ventricles and elicited hypertrophy of both ventricles without any change in arterial blood pressure (Mazzolai et al, 1998). These observations substantiate the notion of a cardiac renin angiotensin system at the level of cardiac myocytes. Upregulation of cardiac ACE mRNA as well as protein has been reported in salt-sensitive types of hypertension supporting the view that the cardiac RAS is in parte responsible for the detrimental action of salt-overload (Zhao et a1 2000).
Evidence of an intracrine renin angiotensin system has been previously described (De Mello 1994; De Mello, Danser 2000; De Mello, Re 2003; Cook et a1 2001) and is particularly important under pathological conditions when an increased expression of renin and angiotensinogen genes seems to occur in cardiac muscle. Evidence has been presented, for instance, that stretch of myocardial fibers enhanced the expression of theses genes (Malhotra et a1 1999; Tamura et a1 1998).
On the other hand, it is known that ACE and Ang 11, at cardiac sites, are increased after myocardial infarction and ventricular hypertrophy induced by pressure-overload (Yamada et al, 1991). Cardiac Ang I1 generation increases the expression of renin, AOGEN, AT1 and AT2 forming a positive feedback loop (Tamura et a1 1998). The left ventricular hypertrophy induced by aortic coarctation leads to an increase of renin and AOGEN mRNA in the left ventricle while the renin levels in plasma are only transiently elevated (Baker et al, 1990). The major question remains: which is the physiological or physiopathological
The Heart As A Source OfAngiotensin 11 93
meaning of a cardiac renin angiotensin system and particularly of an intracellular system?
It is known that intercellular communication is impaired in the failing heart (De Mello, 1999). Indeed, the values of gap junction conductance measured in cell pairs isolated from the failing heart of cardiomyopathic hamsters, showed areas in which the gap junction conductance was extremely low (0.8 -2.5 nS) and incompatible with impulse propagation (De Mello, 1996).
The activation of the plasma renin angiotensin system during the process of heart failure, is largely responsible for the impairment of heart function and the remodeling of the ventricle (Dzau, 1987; Lindpaintner et al, 1990). Furthermore, the activation of a local renin angiotensin in the failing heart (see for review De Mello, Danser 2000), might be implicated in cell abnormalities seen during this condition.
IS ANGIOTENSIN I CONVERTED AT HEART CELL MEMBRANE?
ACE is expressed in many cells especially in endothelial cells and consists of a cytosolic domain, a hydrophobic transmembrane domain and an extracellular domain subdividied in two homologous domains: a residue N and a domain called C (Soubrier et a1 1988).
Although molecular cloning has provided details about the function and structure of ACE, several questions remain about the functions of the enzyme in the heart.
Previous observations of de Lannoy et a1 (1994) showed that Ang I is converted to Ang I1 at the interstitium of cardiac tissue.
Recently, we found that Ang I is converted to Ang I1 at the level of cardiac cell membrane. Indeed, administration of Ang I to the extracellular fluid causes an appreciable increase of cardiac excitability of and generation of cardiac arrhythmias in the rat ventricle (De Mello 2004a).This effect of Ang I is related to its conversion to Ang I1 because in enalapril maleate suppressed this effect of Ang I. The possibility that Ang I can be converted to Ang I1 at the level of cell membrane was studied in myocytes isolated from the same preparation. The results indicated that Ang I reduced the inward calcium current-an effect abolished by enalaprilat (De Mello 2004a).
Previous studies showed that Ang 11, per se, reduced the inward calcium current in rat cardiomyocytes and increased it in hamsters cells (De Mello 1998). These findings support the view that Ang I1 can be formed at the level of cardiac cell membrane making possible a fast
94 The Local Cardiac Renin Angiotensin-Aldosterone System
interaction of the peptide with AT1 receptors. The generation of Ang I1 at the surface cell membrane provides a mechanism of paracrine and autocrine action of the peptide and indicates that the heart is a source of Ang 11.
RENIN ANGIOTENSIN SYSTEM AND INTERCELLULAR SIGNALING
Although contraction is the most important h c t i o n of the heart we cannot forget that the cardiac muscle is a complex electrochemical machine in which the generation of electrical propagated responses and its propagation fkom cell-to-cell is essential for the trigger of the contractile process. An increase in resistance at the level of the gap junctions, for instance, results in impairment of impulse propagation and cardiac arrhythmias (De Mello, 1999). Moreover, the suppression of cell communication reduces the heart contractions by sequestering a large number of cells which are not able to participate in the contraction process. Histologic studies indicated interstitial fibrosis, necrosis and calcifications in the failing heart (see De Mello 1999). The renin angiotensin aldosterone system is involved in the decline of cell communication and the generation of fibrosis. Both processes contribute to the sequestration of large number of heart cells @e Mello 2004b).
Several observations indicated that at an advanced stage of heart failure, Ang I1 added to the bath caused cell uncoupling abolishing intercellular communication and impulse propagation (De Mello 1996).This effect of the peptide is related to the activation of PKC and consequent phosphorylation of gap junction proteins.The possibility exists that tyrosine kinase is also involved in the Ang II- mediated signal transduction (see Hunter 1996).
ON THE INTRACRINE RENIN ANGIOTENSIN SYSTEM
The possible internalization of prorenin and the formation of renin inside the cardiac myocyte under pathological condictions might lead to two possible consequences: formation of Ang I1 inside the cell (Peters et a1 2002) with consequent intracellular action of the peptide or release of renin to the extracellular medium and formation of Ang I1 outside the cell with activation of AT1 receptors. Although intracellular Ang I1 can be the result of synthesis or internalization of the peptide, the presence of an intracrine RAS has been supported experimentally.
The Heart As A Source Of Angiotensin I1 95
Indeed, it was found that intracellular administration of Ang I abolished cell communication an effect suppressed by intracellular enalaprilat or losartan (De Mello 1996; De Mello Danser 2000).
Recently, it has been shown that intracellular dialysis of Ang I in cells of 2 month-old cardiomyopathic hamsters in which the ACE activity is not increased, caused a small decline of junctional conductance (33 %) while in 6 month-old animals in which the ACE is enhanced Ang I abolished cell coupling (De Mello 2003b). In these animals in which the ACE activity is enhanced the intracellular dialysis of Ang I1 also caused cell uncoupling, an effect not abolished by losartan added to the extracellular fluid.
The possibility that endogenous Ang I1 contributes to the regulation of cell communication was investigated dialyzing enalaprilat inside the cell. In 2 month-old animals, for instance, in which the ACE is not increased, enalaprilat caused no change of gj while in 6 month- animals the increase of gj was of 72% (De Mello 2003b). These observations suggest that there is ACE in the cytosol. The presence of a soluble form of ACE inside the cell might explain these results. Evidence exists that a post-translational proteolytic cleavage of membrane bound ACE by secretases, release the protein in a soluble form (see Hooper, Turner 2000). It is conceivable that the transport of the soluble form into the cell might represent an important source of intracellular ACE but further studies are needed to confirm or discard this hypothesis.
INWARD CALCIUM CURRENT IS MODULATED BY
EXTRACELLULAR AND INTRACELLULAR ANGIOTENSIN
Further support for the existence of an intracrine RAS system is the finding that intracellular Ang I1 injection increases Ica density in the failing heart (De Mello, Monterrubio 2004). Althouth similar results were found with extracellular Ang 11, only intracellular Ang I1 increased
E,
inactivation.This effect on Ic, inactivation might be related to the release of Ca by the SR through an activation of ryanodine receptor.Indeed, thapsigargin which depletes the SR of Ca ions, abolished the effect of intracellular Ang I1 on
L-,
inactivation. Moreover, intracellular Ang I also increased Ic, density and enhanced the rate of Ica inactivation (De Mello, Monterrubio 2004) an effect abolished by intracellular enalaprilat. The effects of intracellular Ang I1 are not the same found when Ang I1 is added to the extracellular fluid because only intracellular Ang I1 increased the rate of I,-, inactivation. Although intracellular Ang I1 can be the result of internalization it is unlikely that this be the case in96 The Local Cardiac Renin Angiotensin-Aldosterone System
our experimental conditions. It is important to emphasize that the overexpression of renin and angiotensinogen genes during the process of heart failure can provide the elements for the synthesis of intracellular Ang 11. An alternative hypothesis is that intracellular Ang I1 might be synthesized by internalization of both renin and angiotensinogen.
The role of a possible intracellular ACE on the intracellular conversion of Ang I remains to be determined. Internalization of the enzyme associated with a monoclonal antibody has been described in endothelial cells (Muzykantov et a1 1996) rising the possibility that similar phenomenon occur in cardiac myocytes.
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