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Cardiovascular Research 1998 39(1):242-256; doi:10.1016/S0008-6363(98)00081-9
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Copyright © 1998, European Society of Cardiology

Alteration of intracellular Ca2+-handling and receptor regulation in hypertensive cardiac hypertrophy: insights from Ren2-transgenic rats

Oliver Zolk, Markus Flesch, Georg Nickenig, Petra Schnabel and Michael Böhm*

Klinik III für Innere Medizin der Universität zu Köln, Joseph-Stelzmann-Straße 9, 50924 Cologne, Germany

* Corresponding author. Tel.: +49-221-4786207; Fax: +49-221-4786550.

Received 20 October 1997; accepted 10 February 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Neuroendocrine mechanisms
 3 Conclusions and perspectives
 References
 
Abnormal intracellular Ca2+-handling appears to be a major cause of systolic and diastolic dysfunction in animals and humans with cardiac hypertrophy due to pressure overload and heart failure. However, the precise mechanisms which cause alteration of Ca2+-handling remain unclear. Several lines of evidence suggest that activation of neurohormonal systems may play a central role. In particular, widespread awareness of the importance of the renin–angiotensin system (RAS) has occurred since experimental and clinical studies have detailed the efficacy of angiotensin-converting enzyme inhibitors in reducing morbidity and mortality in patients with left ventricular dysfunction. To evaluate in vivo the role of activated RAS in the regulation of (a) cardiac receptor expression and signal transduction mechanisms and (b) Ca2+ homeostasis, transgenic TG(mREN2)27 rats harbouring the murine renin Ren2d gene were chosen. These animals develop fulminant hypertension and cardiac hypertrophy at an early age despite low levels of renin in the plasma. High expression of the transgene in the vasculature and the heart is associated with increased local formation of angiotensin II. In the Ren2-transgenic model alterations of β-adrenergic neuroeffector mechanisms, Ca2+-handling and {alpha}-adrenergic signal transduction are observed which are very similar to those observed in the myocardium of patients with end-stage heart failure. Moreover, treatment with specific inhibitors of the RAS, such as angiotensin-converting enzyme inhibitors or angiotensin II-receptor antagonists, largely reversed these defects. Studies on TG(mREN2)27 rats may provide new insights into the pathogenesis of hypertensive heart disease and mechanisms which promote disease progression to end-stage heart failure and also may have important implications with regard to therapeutics of heart failure in man.

KEYWORDS Ca2+-handling proteins; TG(mREN2)27 rat; Renin–angiotensin system; Cardiac hypertrophy; Heart failure; β-adrenergic signal transduction


   1 Introduction
Top
 Abstract
 1 Introduction
2 Neuroendocrine mechanisms
 3 Conclusions and perspectives
 References
 
Myocardial hypertrophy in response to hemodynamic overload is an established risk factor for cardiovascular morbidity and mortality. Data from the Framingham study demonstrate that cardiac hypertrophy due to long-standing arterial hypertension is associated with an increased incidence of heart failure [1]. Although the specific mechanisms which are involved in the pathological process that leads from hypertension to heart failure are still unknown, a large body of experimental and clinical evidence supports a central role for the cardiac renin–angiotensin system (RAS) [2]and the sympathetic nervous system [3, 4]: Cardiac expression of angiotensin-converting enzyme (ACE) and angiotensinogen is enhanced in experimental myocardial overload and in human end-stage congestive heart failure [5, 6]. Cardiac stores of norepinephrine and neuropeptide Y are depleted in animal models of arterial hypertension and in patients with end stage heart failure [7, 8]. In addition, an increased cardiac norepinephrine spillover to plasma is observed in essential hypertensive patients indicating increased cardiac sympathetic outflow [9]. Increased concentrations of norepinephrine and angiotensin II exert trophic effects on cardiac fibroblasts and myocytes [10–12]and induce alterations in cardiac gene expression resulting in activation or deactivation of contractile protein isogenes [13, 14]. Furthermore, chronic exposure to the sympathetic neurotransmitter norepinephrine causes a heterologous desensitization of the β-adrenergic signalling pathway. This desensitization process is associated with a subsensitivity of the heart to β-adrenoceptor-induced increase in inotropy [15]. In addition, several studies on experimental animal models with cardiac hypertrophy or failure as well as on myocardium from patients with hypertrophy or end-stage heart failure have shown abnormal intracellular Ca2+-handling under these conditions [16–18]. The common key observation was a prolonged diastolic Ca2+ transient with a diminished capacity to restore low resting Ca2+ levels, which has been attributed to a decreased expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase.

Due to the fact that the access to vital myocardium from hypertensive individuals is rather limited, experiments on alterations of receptor regulation and Ca2+-handling have first been performed in animal models for hypertensive cardiac hypertrophy. The widely used model is the spontaneous hypertensive rat (SHR) with a polygenic determination of hypertension has been a useful tool for the study of pathophysiological alterations in hypertensive heart disease [19]. The value of this strain, however, is reduced by the fact that the genetic basis of its hypertension is undefined. Recently, a monogenic rat model of arterial hypertension has been generated. In an effort to elucidate the contribution of the renin gene to the pathogenesis of hypertension, an additional renin gene, the murine Ren2d gene, has been introduced into the germ line of normotensive Sprague-Dawley rats by microinjection techniques. These animals develop severe hypertension and cardiac hypertrophy. Since the transgene is expressed at high levels in multiple extrarenal tissues including the vasculature and the myocardium, local tissue RAS might play a predominant role in the etiology of hypertension in this animal model. This notion is supported by the fact that plasma renin activity as well as plasma angiotensin II is unchanged or even lowered in these animals [20]. Moreover, specific inhibitors of the RAS, such as angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists, are capable of inducing regression of myocardial hypertrophy even at doses that only cause a slight reduction in blood pressure [21], whereas the direct vasodilator hydralazine failed to exhibit similar cardioprotective effects despite efficient blood pressure reduction [22]. Thus, local activation of the RAS contributes to the cardiovascular pathology in the TG(mREN2)27 model independent from hemodynamic mechanisms. This review will focus on the molecular mechanisms underlying the alterations of contraction and relaxation that occur during hypertrophy in TG(mREN2)27 rats. Furthermore, the question is addressed how increased RAS-activity influences signalling pathways involved in regulating intracellular Ca2+-handling and in turn myocardial force of contraction in this animal model.


   2 Neuroendocrine mechanisms
Top
 Abstract
 1 Introduction
 2 Neuroendocrine mechanisms
3 Conclusions and perspectives
 References
 
2.1 Cardiac renin-angiotensin system
In contrast to the polygenic cause of hypertension in SHR, transgenic rats expressing the murine Ren2 renin gene (TG(mREN2)27) are a well-established monogenic model of severe arterial hypertension and cardiac hypertrophy [20]. The originally described transgenic rats were heterozygous. In these animals, hypertension is fully established at the age of 8 weeks [23]resulting in systolic and diastolic blood pressure values about 230 mmHg and 180 mmHg respectively [22, 24]. In homozygous TG(mREN2)27 rats, doubling of the gene dose leads to a further increase of systolic blood pressure values reaching 300 mmHg. These malignant blood pressure values are accompanied by a higher mortality rate due to hemorrhagic stroke in homozygous rats [25]. Hypertensive blood pressures are effectively reduced by angiotensin-converting enzyme inhibition or by angiotensin II subtype 1 receptor antagonism, indicating that angiotensin II is decisively involved in the development of hypertension in this transgenic strain [23]. The finding that (a) the transgene is expressed at high levels in extrarenal tissues like the adrenal, thymus, small intestine, testis, ovary, kidney, brain, lung, blood vessels, pituitary, thyroid and myocardium [20, 26]and (b), serum levels of renin, angiotensin I and angiotensin II are normal to low despite elevated plasma prorenin levels [26]support the hypothesis that the tissue rather than circulating RAS may play an important role in the pathophysiology of hypertension in the transgenic TG(mREN2)27 rats. Accordingly, experiments using isolated perfused hindquarter preparations suggest an increased local angiotensin II production in the vascular tissue of transgenic rats [27]. There is also evidence for an activation of the tissue RAS in the myocardium. Semiquantitative detection of renin mRNA expression in left ventricular myocardium using RT-PCR techniques revealed a significant increase in renin mRNA levels in TG(mREN2)27 rats compared to non-transgenic controls (Fig. 1A), which is most likely due to the expression of the transgene. Although cardiac angiotensin-converting enzyme activity did not significantly differ between TG(mREN2)27 rats and Sprague-Dawley control animals [28], tissue angiotensin II concentrations have been found to be significantly increased in the left ventricular myocardium from TG(mREN2)27 rats (Fig. 1B) confirming an activation of the RAS in the myocardium of transgenic animals.


Figure 1
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  		Fig. 1 A. Semiquantitative renin RT-PCR. Total RNA isolated from left ventricles of TG(mREN2)27-rats and Sprague-Dawley control rats (SD) was reversely transcribed by M-MLV reverse transcriptase and amplified using primers specific for renin. Shown is a representative ethidium bromide stained agarose gel. B. Left ventricular myocardial angiotensin II concentrations of transgenic (TG(mREN2)27) and Sprague-Dawley control rats (SD) as determined by radioimmunoassay. (Modified from [24, 107]).

 
2.1.1 Angiotensin receptors
Based on their pharmacological properties (affinity for nonpeptide receptor antagonists, sensitivity to dithiothreitol), the two major isoforms of the angiotensin receptor, the type 1 (AT1) and type 2 (AT2) receptors, have been identified [29]. Most known effects of angiotensin II, such as increasing blood pressure, stimulating myocyte hypertrophy and increasing collagen synthesis are attributed to the AT1-receptor. In contrast, the exact function and the signal transduction pathways of the AT2-receptor are still not known. Recent observations support the notion that the AT2-receptor antagonizes mitogenic effects of the AT1-receptor [30, 31]. Moreover, AT2-receptors appear to counteract classical pressor responses to angiotensin II since basal blood pressure was increased and the pressor response to angiotensin II was enhanced in transgenic AT2-receptor-deficient mice [32]. Finally, the AT2-receptor has been suggested to mediate programmed cell death [33].

In accordance with findings from animal models and patients with cardiac hypertrophy [34–36], the AT1-receptor expression has been found to be markedly decreased in atrial and ventricular tissue of TG(mREN2)27 rats, as shown by radioligand binding assays (Fig. 2B) and quantitative RT-PCR experiments (Fig. 2A). Angiotensin II generally elicits a positive inotropic effect in the cardiac muscle of certain mammalian species including man [37]. These AT1-receptor mediated effects exhibit a wide species-dependence [38]. In addition, differences in the expression of the inotropic effect between atrial and ventricular myocardium have been observed [39–41]. Several studies suggest that angiotensin II does not have any positive inotropic effect on human ventricular trabecular muscle whereas it has significant positive inotropic effect on atrial myocardium [39, 41]. In parallel, contraction studies on isolated cardiac muscle strips reveal that angiotensin II exerts no positive inotropic effects on the left ventricular myocardium of both, TG(mREN2)27 and Sprague-Dawley control rats. In contrast, angiotensin II leads to a significant increase of force of contraction in the left atrium of control rats. This increase in contractility is not detectable in left atrial preparations of transgenic animals which may be a consequence of AT1-receptor downregulation in TG(mREN2)27 [42].


Figure 2
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  		Fig. 2 A. Relative AT1-receptor steady state mRNA levels in left ventricle (LV), left atrium (LA) and aorta from TG(mREN2)27 rats (TGR) and Sprague-Dawley control rats, assessed by quantitative RT-PCR. AT1-receptor mRNA levels are normalised to the signal from the deletion mutated AT1-receptor mRNA (mut AT1-R), which was used as an internal standard. The upper panel shows a representative ethidium bromide stained agarose gel. B. AT1-receptor density in the atrium and the aorta from TG(mREN2)27 rats (TG) and Sprague-Dawley (SP) control rats as assessed by [125I]-angiotensin II saturation binding assays. (Modified from [42]).

 
The precise mechanisms, which are involved in the AT1-receptor signal transduction process influencing intracellular Ca2+-handling and enhancing cardiac contractility seems to vary among the mammalian species [40, 47]and are the subject of current investigations. Conflicting observations may reflect important differences in coupling of the AT1-receptor to phosphoinositide hydrolysis via a G-protein mediated stimulation of phospholipase C and the relative contributions of subcellular phosphoinositide signalling pathways to the regulation of intracellular Ca2+ homeostasis in different species and different cardiac muscle preparations. Activation of PLC leads to the formation of the intracellular messenger inositol 1,4,5-trisphosphate (IP3) which has been shown to promote Ca2+ release from the sarcoplasmic reticulum in skinned cardiac muscle preparations [43]. Phosphoinositide hydrolysis may also result in production of diacylglycerol (DAG), which activates protein kinase C (PKC). PKC in turn has the potential for complex and potentially inhibitory interaction with the Ca2+-mobilizing effects of IP3 [44]. PKC has been shown to influence transsarcolemmal Ca2+ uptake which results in diminished sarcoplasmic Ca2+ loading and subsequent release [45, 46]. Furthermore, phosphorylation of thin filament regulatory proteins by PKC promotes a decrease in myofibrillar Ca2+-sensitivity as demonstrated in skinned and intact muscle fibres from human myocardium [46]. These negative inotropic actions of PKC may be cancelled by activation of sarcolemmal Na+–H+-antiporter causing intracellular alkalosis and enhanced Ca2+ sensitivity of the contractile proteins [47]. The cellular mechanism underlying the activation of the antiport are not yet clear. The observation that the cardiac Na+–H+-antiporter lacks any protein kinase phosphorylation site and is not phosphorylated by PKC in vitro confirm the suggestion that indirect mechanisms besides PKC such as an activation of a cascade of other kinases may be involved [48].

2.2 Cardiac sympathetic activity
From animal studies, multiple mechanisms of angiotensin II-dependent augmentation of cardiac sympathetic activity have been established [49]. Angiotensin II stimulates neural transmission across sympathetic ganglia [50, 51]. Furthermore, angiotensin II facilitates the exocytotical release of norepinephrine from the sympathetic nerve terminals due to stimulation of presynaptic angiotensin AT1-receptors [52, 53]. Finally, increased local sympathetic tone can be produced through a central mechanism via angiotensin II-mediated stimulation of the area postrema [54, 55]. From these reports, one would expect an increased sympathetic outflow in TG(mREN2)27 rats. Indeed, increased levels of circulating norepinephrine have been observed in these animals. However, circulating catacholamine levels may not reflect the pattern of sympathetic discharge in different organs. Myocardial concentrations of norepinephrine and neuropeptide Y, which are exocytotically coreleased from cardiac postganglionic nerve fibres [56], are significantly reduced in TG(mREN2)27 [75], reflecting an increase of local sympathetic activation. In addition, alterations of norepinephrine clearance have been observed in the left ventricular myocardium of TG(mREN2)27 rats. Under physiological conditions, norepinephrine is eliminated from the cardiac synaptic cleft primarily via neuronal uptake1 carrier. In TG(mREN2)27 rats, the density of uptake1 carrier sites is significantly reduced by about 50% (Fig. 3). This may lead to a delayed elimination and a prolonged action of norepinephrine on postsynaptic adrenergic receptors thereby enhancing the sympathetic stimulation on the heart. Since treatment with the ACE-inhibitor captopril and the AT1-receptor antagonist BAY 10-6734 normalizes myocardial NPY concentrations [71], it is likely that the strong activation of the cardiac sympathetic nervous system in TG(mREN2)27 is caused by angiotensin II actions.


Figure 3
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  		Fig. 3 Binding of [3H]-nisoxetine to left ventricular membranes of TG(mREN2)27 rats and Sprague-Dawley (SP) control rats. Inset: Radioligand bound was plotted as the function of the ratio of bound to free radioligand. Intercepts with the abscissa represents the maximal number of uptake1 carrier sites. (M. Böhm and O. Zolk, unpublished data).

 
It is well accepted that cardiac sympathetic nerve activity is increased in patients with severe heart failure [57, 58]. Accumulating evidence suggests that adrenergic mechanisms have important prognostic significance in chronic heart failure [59, 60]. However, data on the cardiac sympathetic profile in the early stages of chronic heart failure are limited. Although plasma catecholamine levels are found to be increased in patients with essential hypertension and asymptomatic left ventricular hypertrophy [61, 62], the overall sympathetic activity may not reflect the local sympathetic outflow in the heart. However, a recent study on patients with mild to moderate heart failure suggest that increased cardiac adrenergic drive precedes the generalized sympathetic activation [63]. Thus, increased cardiac sympathetic nervous outflow seems to be an early alteration in the pathophysiological process that leads from compensated cardiac hypertrophy to failure.

2.2.1 β-adrenoceptor signalling
The sympathetic nervous system plays an important role in maintenance of cardiac output in response to increasing demand of the organism both by increasing heart rate and contractility [64]. Fig. 4 is a schematic representation of the signalling system that mediates the effects of β-adrenoceptor stimulation on intracellular Ca2+ homeostasis and contractility of the cardiac myocyte. Norepinephrine released into the synaptic cleft from sympathetic nerve terminals binds to β-adrenergic (predominantly β1) receptors on the cardiac sarcolemma and activates the stimulatory guanine nucleotide binding protein Gs{alpha} by promoting the exchange of GDP for GTP. This reaction catalyzes the dissociation of the GTP bound Gs{alpha} subunit from the β{gamma}-dimer. The GTP-bound Gs{alpha} is a powerful stimulus for the activation of the effector enzyme adenylyl cyclase, which generates the second messenger cyclic AMP from ATP. cAMP accumulates intracellularly and in turn activates cAMP-dependent protein kinase A, which induces the phosphorylation of proteins in the sarcolemma, sarcoplasmic reticulum and contractile filaments. Phosphorylation of the L-type Ca2+ channel, for example, enhances Ca2+ entry into the cardiomyocytes which triggers the release of Ca2+ stored in the sarcoplasmic reticulum through the Ca2+ release channel (ryanodine receptor) and contributes to the positive inotropic effects of β-agonists. Following phosphorylation of phospholamban, the inhibition exerted by the nonphosphorylated form of phospholamban on the sarcoplasmic reticulum Ca2+ pump (SR Ca2+-ATPase, SERCA 2a) is relieved and its rate of Ca2+ uptake is increased. This leads to a more rapid decrease of the cytosolic Ca2+ concentration and in turn to an accelerated relaxation. The rate of dissociation of the troponin C-Ca2+ complex is enhanced when troponin I is phosphorylated, which also supports an accelerated relaxation. These latter events support the positive lusitropic effect observed following β-adrenoceptor stimulation. Besides the activation of the cardiac L-type Ca2+ channels via a cAMP dependent phosphorylation mechanism, Gs{alpha} activates the Ca2+ -channel directly. This dual regulation of the Ca2+ channel may be the molecular basis of a biphasic fast and slow response of Ca2+ currents to β-adrenoceptor stimulation [65].


Figure 4
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  		Fig. 4 Schematic representation of the signal transduction pathway for myocardial β-adrenoceptors. Norepinephrine is released from sympathetic nerve terminals into the synaptic cleft. The catecholamine binds to cardiac β-adrenoceptors and activates the stimulatory guanine nucleotide binding protein Gs by promoting the exchange of GDP for GTP. This reaction catalyzes the dissociation of the GTP-bound Gs{alpha} subunit from the β{gamma}-dimer. Gs{alpha} in tern activates the effector enzyme adenylyl cyclase, which generates the second messenger cyclic AMP from ATP. cAMP accumulates intracellularly and activates cAMP-dependent protein kinase A, which induces the phosphorylation of proteins in the sarcolemma, sarcoplasmic reticulum and contractile filaments. On phosphorylation of phospholamban, the inhibition exerted by the non-phosphorylated form of phospholamban on the sarcoplasmic reticulum Ca2+ pump (SERCA) is removed, and the rate of Ca2+ uptake is increased. The action of norepinephrine is predominantly terminated by reuptake into presynaptic stores. m-Cholinoceptors (m-CH) and type 1 adenosine receptors are coupled to inhibitory G-proteins mediating adenylyl cyclase inhibition and antiadrenergic effects. RYN: calcium release channel, ryanodine receptor; NPY: neuropeptide Y; NA uptake: neuronal norepinephrine uptake carrier.

 
In human and rat myocardium, as in many other species, both β1 and β2 adrenoceptors coexist and are coupled to adenylyl cyclase [66–69]. However, the stimulation of these receptor subtypes elicits qualitatively different cellular responses. Studies on rat ventricular myocytes showed that the effects of β1-adrenoceptor stimulation on Ca2+ transient and contraction amplitude and their kinetics closely paralleled the increase in cAMP [67]. In contrast, the effects of β2-adrenoceptor stimulation appeared to be uncoupled from the cAMP production and cAMP-dependent protein phosphorylation [67]. In human left ventricular myocardium, stimulation of β2-adrenoceptors by catecholamines activates adenylyl cyclase more effectively than that of β1 adrenoceptors, whereas the positive inotropic effect is mediated predominantly through β1-adrenoceptors [68].These findings indicate that the contributions of cAMP to influence intracellular Ca2+ handling are not equivalent when arising as a consequence of β1 or β2 adrenoceptor stimulation. Findings from transgenic β1-adrenoceptor null mutant mice even suggest that the β1-adrenoceptor subtype is a central mediator of the positive inotropic responses following catecholamine stimulation [70].

Chronic exposure of the heart to high levels of catecholamines leads to a desensitization of the β-adrenoceptor-G-protein-adenylyl cyclase complex. As shown in Fig. 5, isoprenaline and guanine nucleotide-stimulated adenylyl cyclase activity is significantly depressed in left ventricular myocardium of TG(mREN2)27 rats. Activity of the catalytic subunit of adenylyl cyclase was determined using the diterpen-derivate forskolin which stimulate the catalyst directly. Because forskolin-stimulated enzyme activity is modulated by G-proteins, experiments were performed in the presence of manganese ions which are known to uncouple the catalyst from the influence of G-proteins. These data show no significant difference in catalyst activity between TG(mREN2)27 and control rats (Fig. 5). Moreover, reconstitution experiments of Gs{alpha} into S49cyc cell membranes (Fig. 5) provide evidence for unchanged bioactivity of the stimulatory G-protein. Desensitization of adenylyl cyclase is accompanied by a downregulation of the total β-adrenoceptor number (Fig. 6A) and an increase of Gi{alpha} on the functional (pertussis toxin ADP-ribosylation) (Fig. 6B), protein (Western blots with Gi{alpha}1/Gi{alpha}2 specific antiserum) and mRNA levels [71, 107]. Increased Gi{alpha}2 mRNA levels after prolonged in vivo β-adrenoceptor stimulation are caused by enhanced transcription of Gi{alpha}2-mRNA in the ventricular myocardium of Wistar–Kyoto rats, as shown by Müller et al. in nuclear run-on assays [72]. Interestingly, the promoter region of the Gi{alpha}2 gene, which represents the predominant Gi{alpha} subtype in the mammalian heart, [73]possesses a binding domain for activator protein-2 (AP-2), which is a cAMP-dependent transcription promoting factor [74].


Figure 5
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  		Fig. 5 Basal, isoprenaline-, Gpp(NH)p-, forskolin-, and forskolin plus Mn2+-stimulated adenylyl cyclase activities in left ventricular membranes from Sprague-Dawley control rats (SD) and transgenic rats (TG(mREN2)27) treated with captopril, BAY 10-6734 or kept under control conditions. (Modified from [71]).

 

Figure 6
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  		Fig. 6 A. Total β-adrenoceptor density in left ventricular membranes from Sprague-Dawley control rats (SD) and transgenic rats (TG(mREN2)27) treated with the ACE inhibitor captopril, the AT1-receptor antagonist BAY 10-6734 or kept under control conditions. Receptor densities (Bmax) were obtained from saturation binding experiments using [125I]-cyanopindolol.B. Gi{alpha}-related pertussis toxin substrates (40 kDa) in left ventricular membranes from Sprague-Dawley control rats (SD) and transgenic rats (TG(mREN2)27) treated with captopril, BAY 10-6734 or kept under control conditions. Bar graph show mean values for incorporated [32P]ADP-ribose as determined by detecting the radioactivity from the 40 kDa-bands. (Modified from [71]).

 
The alteration of the sympathetic neuroeffector mechanism is associated with an attenuated inotropic response of the myocardium to β-agonist stimulation [75]. Furthermore, experiments on isolated electrically driven papillary muscle strips under basal conditions show a prolongation of time to peak as well as total duration of the contraction (Fig. 7), which indicates alterations of contraction and relaxation in the transgenic myocardium possibly due to alterations of Ca2+-handling (see below).


Figure 7
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  		Fig. 7 Time course of isometric contraction in isolated electrically driven papillary muscle preparations from transgenic (TG(mREN2)27) rats and Sprague-Dawley (SD) control rats. (Modified from [24]).

 
Compared with other animal models of hypertensive heart disease like renal hypertensive rats [76], deoxycorticosteroid-treated rats, Dahl salt hypertensive rats [77]or spontaneously hypertensive rats [19], the alterations of the β-adrenergic neuroeffector mechanisms seen in the Ren2-transgenic model are most similar to those observed in the myocardium of patients with end-stage heart failure where a downregulation of β1-adrenoceptors and an increase of Gi{alpha} is related to a desensitization of adenylyl cyclase [78]. In accordance with findings from animal studies, increased myocardial Gi{alpha} protein levels are already observed in patients with hypertensive cardiac hypertrophy [79].

Treatment studies have shown a strong correlation between cardiac sympathetic nervous activity and the degree of β-adrenergic desensitization in TG(mREN2)27 rats. The vasodilator hydralazine, which has no effect on cardiac sympathetic nervous activity although it reduces blood pressure significantly, fails to reverse β-adrenergic signal transduction defects [22]. By contrast, ACE-inhibitors and AT1-receptor antagonists normalize sympathetic outflow and resensitize the β-adrenoceptor-G-protein-adenylyl cyclase system [22, 71]. Thus, restoration of β-adrenergic balance might be a relevant mechanism in the beneficial effects of pharmacological RAS inhibition.

2.2.2 {alpha}-adrenoceptor signalling
{alpha}1-Adrenoceptors elicit also positive inotropic effects in the myocardium [80]. Comparatively, the response is less than that of β-adrenoceptors [81]. This difference includes a smaller Ca2+ transient, a slower onset of action, marked frequency dependence, and not being antagonized by muscarinic agonists [82]. The signal transduction pathway of {alpha}1-adrenoceptors is quite different from that of β-adrenoceptors and involves phosphoinositide hydrolysis and generation of the second messengers inositol 1,4,5-trisphosphate (1,4,5-IP3), inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4) and diacylglycerol (DAG) [83]. The main mechanisms which have been proposed to participate in the positive inotropic effects include (a) {alpha}1-adrenergic-induced prolongation of action potential duration [84, 85]and an indirect increase in Ca2+ inward current [85], (b) sensitization of myofibrils to Ca2+ subsequent to phosphorylation of contractile proteins and cytosolic alkalinization [86], (c) inhibition of cAMP breakdown [87]and (d) stimulation of inositol phosphate turnover and IP3 induced Ca2+-mobilization [88]. The latter mechanism has been a subject of controversy, as to whether or not IP3 is effective in releasing Ca2+ from stores in the sarcoplasmic reticulum. Studies in endoplasmic reticulum enriched membranes, permeabilized cells, skinned muscle fibres or intact neonatal cardiomyocytes have reported conflicting results of either no IP3 mediated Ca2+ mobilization or an IP3 induced release of Ca2+ sufficient to induce a contraction [88, 89]. These data in general have been interpreted that IP3 induced Ca2+ release is unlikely of major importance in vivo under physiological conditions [90].

In left ventricular myocardium, isolated from hearts of patients undergoing heart transplantation because of end stage heart failure due to dilated or ischemic cardiomyopathy, an increased {alpha}1-adrenoceptor density has been reported [91]. For this reason, it has been assumed that the {alpha}-adrenergic effects may compensate in part the reduced β-adrenergic response on contractility in heart failure. However, the {alpha}1-adrenoceptor mediated positive inotropic effects are unchanged or reduced in the failing myocardium compared with non-failing control preparations [92–94]and do not correlate with the increased {alpha}1-adrenoceptor density. Similar to findings from human failing hearts, {alpha}1-adrenoceptor density is found to be increased by 80%, whereas the number of β-adrenoceptors is found to be decreased in left ventricles of TG(mREN2)27 rats compared with Sprague-Dawley controls (Fig. 6A, 8A). Despite an increased {alpha}-adrenoceptor density, the positive inotropic response to {alpha}-adrenoceptor agonist stimulation was found to be decreased in isolated, electrically driven left ventricular papillary muscle strips of transgenic rats [95]. These findings suggest alterations of the signalling cascade distal to the receptor involving the guanine nucleotide binding proteins of the Gq-family or the effector enzyme, phospholipase C. Maximal PLC activity is significantly reduced in transgenic rats (Fig. 8B) whereas Gq-protein expression on mRNA and protein level is not altered [95]. These findings are consistent with a desensitization of the {alpha}1-adrenergic signal transduction pathway at the level of the effector, which may be responsible for an impaired inotropic response to {alpha}1-adrenoceptor stimulation in TG(mREN2)27 rats.


Figure 8
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  		Fig. 8 A. Alpha-adrenoceptor densities in left ventricles from TG(mREN2)27 rats and Sprague-Dawley control rats (SD). Receptor densities (Bmax) were obtained from saturation binding experiments using [3H]-prazosin. B. Phospholipase C activity measured by inositol phosphate formation in left ventricular myocardial membrane preparations from TG(mREN2)27 rats and Sprague-Dawley control rats. (Modified from [95]).

 
2.3 Intracellular Ca2+-handling
2.3.1 Ca2+-handling proteins of the sarcoplasmic reticulum
In myocardial cells, relaxation is primarily governed by the SR Ca2+-ATPase (SERCA) transporting enzyme, which regulates Ca2+ sequestration against a concentration gradient into the sarcoplasmic reticulum. The predominant isoform expressed in the heart is SERCA 2a. Phospholamban inhibits SERCA 2a, this inhibition being attenuated by phospholamban-phosphorylation. The role of phospholamban in the regulation of basal myocardial contractility has been recently elucidated through the generation of transgenic mice with different phospholamban protein expression levels ranging from 0% to 200% of the phospholamban levels present in wild-type mice [96–98]. Phospholamban knockout mice display increased systolic function and increased rates of left ventricular relaxation [96], whereas expression of additional phospholamban molecules results in contractile dysfunction due to an inhibition of the sarcoplasmic Ca2+-transport [97]. Since cardiac levels of the Ca2+-ATPase are not affected in these genetically altered mice, alterations in phospholamban levels displacing the relative stoichiometry of phospholamban to SERCA, are obviously associated with parallel alterations in cardiac contractile parameters. These observations suggest that the relative ratio of phospholamban to SR Ca2+-ATPase is the main determinant of the rate of Ca2+ uptake into the sarcoplasmic reticulum.

As shown in Fig. 9, a significant reduction in SR Ca2+-ATPase and phospholamban mRNA and protein levels are observed in the hypertrophied left ventricular myocardium of TG(mREN2)27 rats. The decrease in SERCA protein level was more pronounced than the decrease in phospholamban protein levels (Fig. 10), leading to an inversed SR Ca2+-ATPase/phospholamban ratio. Thus, the reduction of SERCA molecules could account for the reduction of diastolic Ca2+ loading into the sarcoplasmic reticulum and in consequence for the observed diastolic dysfunction. Decreased amounts of SERCA 2a proteins and desensitization of the β-adrenergic signal transduction pathway, which results in reduced cAMP dependent phospholamban phosphorylation, might act synergistically to attenuate Ca2+ uptake into the sarcoplasmic reticulum.


Figure 9
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  		Fig. 9 Northern blot analysis of SERCA2a, phospholamban and atrial natriuretic factor mRNA levels in left ventricular myocardium from transgenic rats (TG) and control rats (SD). The upper panel shows representative hybridization signals. The hybridization signal for GAPDH was used as a control for the amount of total RNA loaded. Bar graph shows mean values related to GAPDH mRNA levels (*p<0.05 vs. SD). (Modified from [24]).

 

Figure 10
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  		Fig. 10 Immunochemical detection of SERCA2a and phospholamban in left ventricular myocardium from transgenic rats (TG) and Sprague-Dawley control rats (SD). In order to obtain the monomeric form of phospholamban ({approx} 6kDa), membranes had been denatured by heating for 30 min. Upper panel shows representative Western blots (p<0.001 vs. SD). (Modified from [24]).

 
In animal models of left ventricular pressure overload by aortic banding, which produces a wide range of alterations from compensated cardiac hypertrophy to decompensated end-stage heart failure depending on the duration of the overload and the type of surgical intervention, a decrease in Ca2+ reuptake, in Ca2+-stimulated ATPase activity and in expression of SERCA2a mRNA and protein has been observed [99, 100](Table 1). Findings from human failing hearts are not so uniform. The majority of functional studies have demonstrated a decreased SERCA-activity and reduced Ca2+ reuptake [101, 102]. Moreover, a decrease in SERCA 2a protein quantity and steady state mRNA expression has been shown in large series of failing human hearts [103–105]. However, there are conflicting results from Movsesian et al., who observed neither a reduced sarcoplasmic reticulum Ca2+ uptake nor an alteration in SERCA 2a protein [106].


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  		Table 1 Alterations in cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase and phospholamban (PLB) expression and Ca2+-ATPase-activity in human heart failure and in animal models of compensated left ventricular hypertrophy and heart failure

 
2.3.2 Treatment effects on Ca2+-handling
Both treatment with the ACE-inhibitor captopril and the AT1-receptor antagonist BAY 10-6734 partly prevents the decrease in SERCA 2a expression in TG(mREN2)27 rats [107]. In ascending aortic-banded rats with left ventricular hypertrophy, long term fosinopril-treatment has been shown to improve myocyte shortening and responsiveness to Ca2+ [108]. Furthermore, in a rat model of hypertension due to suprarenal coarctation of the abdominal aorta, angiotensin-converting enzyme treatment normalized the depressed expression of SERCA 2a and the attenuated Ca2+-accumulation into sarcoplasmic vesicles [109]. Finally, stimulation of adult cardiomyocytes with angiotensin II has been shown to induce a downregulation of SERCA mRNA levels [110]. These findings indicate that the renin angiotensin system is involved in the regulation of SERCA2a expression and intervenes in the intracellular Ca2+ homeostasis in cardiac hypertrophy.

2.4 Alterations of gene expression and myocardial hypertrophy
Cardiac adaptation to chronic pressure overload is aided by hypertrophy of the cardiac myocytes resulting in more contractile elements. Cardiac hypertrophy is produced by a combination of increased myocyte stretch, increased neurotransmitter release, and a variety of autocrine and paracrine hormonal activities which together enhance cardiac myocyte growth. There is strong evidence that these growth-promoting mechanisms include {alpha}-, β-adrenoceptor and angiotensin II (AT1) receptor pathways [12, 111, 112]. Interestingly, in neonatal cardiomyocytes, induction of protein synthesis by β-adrenoceptor stimulation required cocultivation with non-cardiomyocytes or the addition of defined growth-regulating factors [113]. The process of hypertrophy after {alpha}1-adrenoceptor stimulation may reflect a reversion of the myocyte transcriptional activity to the fetal form with reactivation of a series of genes including atrial natriuretic factor, (ANF), β-myosin heavy chain (MHC) and skeletal {alpha}-actin [114–116].

In TG(mREN2)27 rats compared with normal Sprague Dawley-rats, absolute and relative heart weights are increased about 25 to 30% [22, 75]. Myocardial ANF expression, as revealed by Northern blot analysis, is increased about forty-fold in left ventricles of TG(mREN2)27 [24]. Moreover, extensive fibrosis and scarring has been observed in right and left ventricles [28]. Thus, a hypertrophic phenotype is occurring in the heart of transgenic rats. Consistent with the hypertrophic phenotype is a change in the β-MHC/{alpha}-MHC ratio with an increase in the relative amount of β-MHC [24, 117]. The differential expression of the myosin heavy chain isoforms, namely an induction of the β-MHC and the deinduction of the {alpha}-MHC, results in a slower rate of ATP cycling by myosin leading to a slower and energetically more efficient contraction. Thus, the MHC isoform shift may be responsible for the systolic dysfunction observed in TG(mREN2)27 rats. However, the potential for an increase in β-MHC depends upon the initial phenotype: it is high in rat ventricles and in human atria that normally contain about 90% {alpha}-MHC, and it is small in human ventricles, which contain mainly β-MHC.

The activated local RAS and increased tissue angiotensin II may be a critical factor mediating cardiac hypertrophy in TG(mREN2)rats. Several lines of evidence support this notion. Cell culture studies have shown that angiotensin II is a direct mediator of hypertrophy in rat myocytes [118, 119, 31]and of proliferation and collagen production in fibroblasts [120]. Moreover, angiotensin II facilitates the exocytotical release of norepinephrine from the sympathetic nerve terminals due to stimulation of presynaptic angiotensin receptors [52, 53]. Increased norepinephrine in the long term is a potent stimulus of cellular growth via {alpha}- and β-adrenergic mechanisms. Furthermore, recent studies on neonatal rat cardiac myocytes provide evidence that trophic effects of angiotensin II are brought about via local endothelin-1 generation and secretion upon AT1-receptor stimulation of neonatal rat ventricular non-myocyte cells in a paracrine fashion [121].

However, also the increased blood pressure in TG(mREN2)27 rats may be the causing factor of cardiac hypertrophy. It is well known that mechanical forces have growth promoting effects on cardiomyocytes. Stretching adult or neonatal cardiomyocytes in vitro by 10 to 20% above resting length causes an increse in protein synthesis, transcriptional activation of immediate-early genes, an induction of fetal genes (ANF, skeletal {alpha}-actin, β-MHC) and an activation of autocrine/paracrine growth factors such as the cardiac RAS [121–123]. In order to assess the relative contribution of pressure overload versus activated RAS in TG(mREN2)27 rats, treatment studies have been performed with the AT1-receptor antagonist Telmisartan in non-hypertensive doses [124]. This treatment regimen decreased heart hypertrophy markedly without influencing blood pressure indicating that angiotensin II is the major mediator of heart hypertrophy in this transgenic animal model [124].

Findings from animal studies and from patients provide evidence that in conditions of left ventricular hypertrophy and heart failure AT1-receptor densities and mRNA levels are decreased [34–36]. Downregulation of the AT1-receptor is accompanied by a relative increase in AT2-receptor density and an increase in available angiotensin II levels [34–36]. Thus, effects mediated by the AT2-receptor may become exaggerated. Although the functional role of the AT2-receptor remains unclear, there is increasing evidence that in some tissues the AT2-receptor mediates antimitogenic effects [30, 31]. These findings have interesting implications for treatment strategies which aim at prevention and regression of cardiac hypertrophy. AT1-receptor inhibition, which allows angiotensin II to act unopposed on the AT2-receptor, may be superior to nonselective RAS-inhibition. In TG(mREN2)27 rats, treatment with the AT1-receptor antagonist losartan and the ACE-inhibitor quinapril equally reversed cardiac hypertrophy [22]. Findings from other animal studies are inconsistent [125, 126]and depend on the animal model investigated. These disparate observations may be due to the confounding physiological responses beside RAS-inhibition. Schunkert et al. [127]provide data that demonstrate a negative feed back regulation of ACE-activity and ACE-mRNA-levels by angiotensin II. It is intriguing to speculate that chronic administration of ACE-inhibitors may induce ACE-synthesis thereby attenuating the tissue drug effect. Some of the beneficial effects of ACE-inhibitors have been attributed to the accumulation of the vasoactive peptide bradykinin and increased production of prostaglandins following kininase II-inhibition [128, 129]. However, alternative formation of angiotensin II by cardiac enzymes like chymase which are not blocked by ACE-inhibitors [130]may compromise the full effects of these drugs. The observation that local angiotensin II formation was completely blocked by ACE-inhibitors in isolated perfused rat hearts [131]suggest that ACE is the major angiotensin II forming enzyme in rat myocardium. Nevertheless, this mechanism could be relevant in humans [130].


   3 Conclusions and perspectives
Top
 Abstract
 1 Introduction
 2 Neuroendocrine mechanisms
 3 Conclusions and perspectives
References
 
The findings from the TG(mREN2)27 model of hypertensive cardiomyopathy show that in pathological situations, in which tissue RAS is activated, accompanying changes of the sympathetic nervous system occur and emphasize the close interrelation between these two systems in cardiovascular disorders. Increased levels of myocardial angiotensin II and plasma norepinephrine are accompanied by downregulation of myocardial β-adrenergic and angiotensin II AT1-receptors. Additionally, an upregulation in the functional activity and amount of the inhibitory G-protein and an isoform-shift of myosin heavy chain due to the hypertrophic phenotype is observed. Finally, alterations in Ca2+-handling occur, which are based on a downregulation of SERCA2a and phospholamban protein.

The molecular and biochemical alterations in the hypertrophied heart of TG(mREN2)27 rats result in a decrease in stimulated contractile function and abnormalities in the intrinsic contractile apparatus. Obviously, these defects occur already in the early stage of compensated cardiac hypertrophy. Therefore, one might speculate that these alterations, which are very similar to those observed in human end-stage heart failure, directly accelerate the transition from cardiac hypertrophy to failure. In addition, there is accumulating evidence from animal studies that alterations in cytosolic Ca2+ homeostasis plays an crucial role in the induction of programmed cell death [132]. A recent study demonstrates that ligand binding of AT1-receptors initiates apoptosis via an elevation of cytosolic Ca2+ in neonatal ventricular myocytes [133]. Thus, alterations in Ca2+-handling may induce further events on cellular level which may contribute to progressive deterioration of myocardial function.

Treatment studies have shown that direct inhibitors of the RAS are capable of reversing myocardial hypertrophy, neuroendocrine activation, sympathetic neuroeffector defects and alterations of intracellular Ca2+-handling in TG(mREN2)27 rats. Thus, the TG(mREN2)27 strain may be a suitable model to study the effects of drugs that interfere with the renin–angiotensin system, providing a basis for further studies in animal models and humans.

Time for primary review 23 days.


   Acknowledgements
 
Experimental work was supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (01 KS 9502). MB is a recipient of the Gerhard Hess Programm.


   References
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 2 Neuroendocrine mechanisms
 3 Conclusions and perspectives
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