INTRODUCTION

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₁-ADRENERGIC RECEPTORS (₁AR) belong to the superfamily of G protein-coupled receptors. Three distinct ₁AR cDNAs (_1AAR, _1BAR, and _1DAR) have been isolated and characterized by molecular techniques and have pharmacologically defined counterparts (12, 14). ₁AR agonists are effective activators of phospholipase C in cardiac tissue and induce the formation of the second messengers inositol trisphosphate and diacylglycerol, which in turn modulate intracellular Ca²⁺ concentrations and protein kinase C (PKC), respectively (12, 38). Experiments using cultured rat neonatal cardiomyocytes have shown that stimulation with ₁AR agonists can initiate a program of cardiac hypertrophy (36), and _1AAR subtype-mediated activation of inositide-specific phospholipase was implicated in this response (17). In addition, ₁AR have been shown to activate mitogen-activated protein kinase cascades in vitro, and these pathways have also been linked to cellular hypertrophy (40, 44).

₁AR have been identified in myocardial tissue of many mammalian species and play a role in the control of cardiovascular function under normal and disease conditions (39). Stimulation of ₁AR has been shown to protect the myocardium from ischemia-reperfusion injury and has been implicated in the generation of ischemia-induced cardiac arrhythmias (39). Several reports (6, 21, 30) have also shown that ₁AR and AR modulate each other's function, which may be important for the regulation of overall adrenergic activity in the heart. However, the specific contribution of the different ₁AR subtypes in cardiac homeostasis and the development of cardiomyopathies in vivo are unclear and have only recently begun to be addressed.

Insight into the role of cardiac ₁AR isoforms in vivo comes from mice with transgenic (TG) overexpression of a constitutively-active mutant of _1BAR, which demonstrated mild cardiac hypertrophy (23). In contrast, the characterization of TG mice overexpressing the wild-type form of _1BAR showed no overt signs of cardiac hypertrophy at a comparable age (3 mo) (1, 13). However, isolated perfused hearts from these mice revealed an impaired left ventricular (LV) function, suggesting a negative modulatory effect of _1BAR on contractility in vivo (13). The functional deficits were accompanied by alterations in AR signaling and differential regulation of PKC isoforms (1, 20).

In the present study, we report that overexpression of wild-type _1BAR, which serves as a model for chronic activation of the downstream signaling pathways, predisposes to the development of dilated cardiomyopathy (DCM). These TG mice showed progressive decrease of LV function, chamber dilation, and genetic reprogramming resembling features of idiopathic DCM in humans. Furthermore, TG animals died prematurely at middle age (~9 mo), presenting signs of heart failure (HF). These findings demonstrate an important role for ₁AR in cardiac homeostasis in vivo.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Transgenic mice. The generation and initial characterization of the _1BAR TG mouse lines have been described (1, 13). Two lines showed the dilated cardiac phenotype, whereas a third line with lower transgene expression did not develop the dilation within the 12 mo of observation. The line with the higher incidence of DCM was chosen for detailed analysis. Transgene-positive animals were identified by PCR on tissue biopsies using transgene-specific primers. Heterozygous TG mice of both sexes and their nontransgenic (NTG) littermates were analyzed. All animals were housed in accordance with institutional and federal guidelines.

Histology. After CO₂ asphyxiation, hearts were placed in relaxing buffer (5% dextrose amd 25 mmol/l KCl in PBS) and fixed in 10% buffered formalin. Paraffin-embedded serial sections of 6 µm thickness were stained with hematoxylin and eosin solution (Sigma).

Imaging procedure. Echocardiographic studies were performed as described (11) under light anesthesia (2.5% Avertin, 7 µl/g body wt) and spontaneous respiration using a Hewlett-Packard Sonos 5500 and S-12 transducer. We chose to use area measurements instead of M mode to minimize the possible effects of translation and rotation of the heart within the chest cavity that occur within the cardiac cycle (37). Therefore, to adequately assess the globular geometry of the LV typically encountered in DCM, short-axis two-dimensional (2D) images at the midpapillary level were acquired. Fractional area change (FAC) was calculated using the following formula: FAC = (LVEDA  LVESA)/LVEDA, where LVEDA is the papillary LV end-diastolic area (largest) and LVESA is the LV end-systolic area (smallest).

RNA isolation and blots. Total RNA preparation and RNA dot blots and hybridizations to specific radiolabeled oligonucleotides were performed as described (13). Briefly, RNA was isolated using TriReagent (Sigma) and quantitated using a spectrophotometer. Equal amounts of RNA were applied to nitrocellulose membranes using a Bio-Rad dot blot apparatus. Hybridizations to a probe specific for glyceraldehyde-3-phosphate dehydrogenase were carried out for the purpose of internal standardization. Results were quantitated using a Molecular Imager System (Bio-Rad). Northern blots were performed by separating 7.5 µg of total cellular RNA on a 0.7% agarose gel containing 2.2 M formaldehyde followed by capillary blotting. Specific transcripts were detected with an oligonucleotide probe directed against the 3'-untranslated region of -myosin heavy chain (MHC) RNA (32).

Protein detection and Ca²+-ATPase activities. Mouse ventricles were frozen quickly in liquid nitrogen. Samples were extracted by homogenization (Polytron, Brinkmann) in a buffer containing 1.5 ml of 10 mmol/l Tris · HCl (pH 7.4) and 250 mmol/l sucrose. Protein concentrations were determined by Bradford assay (Bio-Rad). Ventricular homogenates were resolved on 7.5-20% acrylamide-gradient SDS-PAGE gels, blotted onto nitrocellulose, and probed with antibodies specific for sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2), phospholamban (PLB), calsequestrin (26), and Na⁺/Ca²⁺ exchanger (Swant). Immunocomplexes were detected by enhanced chemiluminescence (ECL; DuPont-NEN), and quantitation was performed using a Molecular Imager (Bio-Rad). The activities of sarcolemmal Ca²⁺-ATPases were determined as previously described (28).

Statistics. Results are expressed as means ± SE. Unpaired Student's t-tests were performed for pairwise comparisons. A level of P < 0.05 was considered significant. Two-way ANOVA with Bonferroni's post hoc analysis was performed for comparisons between groups.

	RESULTS

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Cardiomyopathic phenotype. The cardiac-specific overexpression of the wild-type _1BAR has been previously shown to result in reduced contractility in isolated work-performing hearts of young animals (13). We now show that continued elevated expression leads to a late-onset DCM phenotype and death. During the course of this study, 24 TG animals (7 males and 17 females) presented with clinical signs of HF, including fatigue and dyspnea, followed by generalized edema and death. This phenomenon was never observed in NTG age-matched mice. The average age of premature death was 8.6 ± 0.7 mo. Gender-specific differences were observed in relation to the age of death. TG males were older than females and died at 12.4 ± 0.7 mo compared with 7.0 ± 0.7 mo for females.

View larger version (84K): [in this window] [in a new window]   Fig. 1.   Morphological and histological analyses. A: representative example of mouse hearts at 9.5 mo of age. TG, transgenic; NTG, nontransgenic littermate. Note the massive enlargement of the four cardiac chambers. Bar, 10 mm. B and C: paraffin-embedded thin sections of TG and age-matched NTG controls were stained with hematoxylin and eosin. B: enlargement of the four cardiac chambers is evident in addition to the distortion of both ventricles. Atrial thrombi are frequently observed. RV, right ventricle; LV, left ventricle. C: high magnification (×40 microscope objective) of the septum. Increased intercellular spacing and myocyte disarray are found in the TG ventricles.

Cardiac dimensions and contractile function. To evaluate the development of LV enlargement and systolic dysfunction, we analyzed TG and NTG cohorts at 3, 6, and 9 mo of age by echocardiography. Representative 2D images of NTG and TG hearts are shown in Fig. 2. When the different age groups were analyzed, small but statistically nonsignificant increases in LVESA and LVEDA with age were noted for NTG animals (Table 2). The calculation of FAC, an index of LV systolic function, also revealed no significant differences between the NTG age groups. These measurements highlight the normal age-related development in these animals. Among the TG hearts, however, both LVESA and LVEDA were consistently higher at 6 and 9 mo compared with 3-mo-old animals, suggesting a more progressive LV enlargement with age in the TG hearts (Table 2). In particular, LVESA increased over 80% from 3 to 9 mo (compared with 16% for NTG littermates). Also, FAC decreased between 3 and 9 mo by ~20%, indicating a loss in systolic function in the TG hearts. A comparison between NTG and TG hearts showed that LVESA and LVEDA were larger in TG hearts at all time points examined except for LVESA at 3 mo. Furthermore, the absolute increase in chamber dimension was more pronounced in TG hearts. This significant LV enlargement of the TG hearts in vivo corroborates the histological findings of chamber dilation (Fig. 1).

View larger version (109K): [in this window] [in a new window]   Fig. 2.   Representative echocardiographic two-dimensional images in 9-mo-old animals obtained from short-axis imaging at the midpapillary level. Note that the NTG heart shows smaller end-systolic and end-diastolic dimensions, whereas the TG heart has LV dilation and a decrease in systolic thickening. Arrowhead, endocardial border.

Gene expression program. Specific alterations in cardiac gene expression have been associated with cardiomyopathy. To determine whether _1BAR overexpression altered the genetic program of the mouse heart, we isolated RNA from mouse ventricles of 3, 6, and 9 mo of age and examined the expression of a selected panel of genes by RNA dot blot and Northern blot analysis. Samples from end-stage HF TG animals were also analyzed. Atrial natriuretic factor (ANF) and -MHC are classical hypertrophy and HF markers and are often upregulated, whereas -MHC may be downregulated. SERCA2 was chosen because of its prominent role in diastolic function. Titin, an important structural component of the sarcomere, has been previously found to be downregulated in DCM. As shown in Fig. 3A, the mRNA expression level of -MHC, ANF, and titin were altered with age in NTG hearts. ANF and -MHC mRNA content were increased with age, whereas titin mRNA levels were decreased. In the TG group, -MHC levels tended to decrease with age, as did those of SERCA and titin, whereas ANF was upregulated. A comparison between NTG and TG groups revealed that ANF mRNA levels were increased in the TG ventricles at all time points analyzed. At 9 mo, levels similar to those found in HF animals were reached. In contrast, mRNA abundance of -MHC and SERCA was decreased in the TG ventricles relative to controls, with levels at 9 mo comparable with those found in the HF group. A different pattern of expression was observed for titin mRNA levels. At 3 mo, levels were increased in TG samples compared with controls. However, mRNA content was decreased at 9 mo and was similar to that of the HF group. The relative difference of mRNA levels between the age groups is summarized in Table 3. Furthermore, Northern blot analysis using a -MHC-specific probe demonstrated an upregulation of -MHC mRNA levels only in the HF group (Fig. 3B).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Comparison of cardiac-specific mRNA levels in hearts of TG and NTG mice at different ages. A: ventricular RNA was analyzed by hybridization of RNA dot blots with specific radiolabeled oligonucleotide probes. Signals were digitized, quantitated, and normalized for loading to glyceraldehyde-3-phosphate dehydrogenase. Data are means ± SE and are expressed relative to NTG signals at 3 mo, which were set to 1. *P < 0.05, TG vs. NTG of 3-mo-old animals; P < 0.05, TG vs. NTG within the age-matched groups (unpaired Student's t-test). n = 4 mice/group. MHC, myosin heavy chain; ANF, atrial natriuretic factor; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase. B: Northern blot of ventricular RNA from 9-mo-old animals with (TG HF) or without (TG 9 mo) overt signs of heart failure (HF). The blot was hybridized to a -MHC-specific oligonucleotide probe.

Sarcoplasmic reticulum proteins. In patients and in animal models of DCM, alterations in sarcoplasmic reticulum (SR) protein expression have been linked to the disease phenotype (reviewed in Refs. 2 and 15). Prompted by our observation of a decrease in cardiac function, we compared the protein levels of SR proteins in TG and NTG samples at 3 and 9 mo using Western blotting with specific antibodies. A representative result is shown in Fig. 4A. Quantification of Western blot signals showed that levels of SERCA2 were dramatically decreased in the TG group (37-45%, 3 and 9 mo, respectively) compared with age-matched NTG hearts (Fig. 4B). This loss of protein content in TG ventricles was paralleled by a loss of SERCA2 function. Measurement of the thapsigargin-inhibitable Ca²⁺-ATPase activity in 3-mo-old ventricular heart samples showed a 34% decrease in TG preparations (NTG 8.58 ± 0.306 vs. TG 5.64 ± 0.225 µmol phosphate · mg¹ · h¹, n = 5, P < 0.0001). Calsequestrin, whose expression is usually unchanged in hypertrophy and DCM (3), was only modestly decreased, as were Na⁺/Ca²⁺ exchanger protein levels. Interestingly, _1BAR overexpression slightly increased the PLB protein level at 3 mo, but no further upregulation was detected at 9 mo. Taken together with the decrease in SERCA2 protein content, TG hearts exhibited increased PLB-to-SERCA2 ratios, which may contribute to the decrease of SERCA2 activity.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of 1B-adrenergic receptor overexpression on sarcoplasmic reticulum protein levels. Homogenates from TG and NTG mouse hearts at 3 and 9 mo were subjected to 7.5-20% gradient SDS-PAGE. After electrophoretic transfer, samples were probed with antibodies against SERCA2, calsequestrin (CSQ), Na+/Ca2+ exchanger (NCX), and phospholamban (PLB). A: representative immunoblots using ventricular homogenates of hearts from 3-mo-old animals. Positive control (C) is a canine purified sarcolemmal fraction (P0). The pentameric (P) and monomeric (M) forms of PLB are indicated. B: histogram showing average levels of the sarcoplasmic reticulum proteins in TG hearts. Signals from Western blots were digitized, quantitated, and normalized to age-matched NTG littermates, which were set to 1 (dotted line). Data are presented as means ± SE. *P < 0.05, TG vs. NTG within the age-matched groups (unpaired Student's t-test). n = 4 mice/group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Chronic receptor activation was achieved by overexpression of the _1BAR subtype in cardiomyocytes. The phenotype of the _1BAR TG animals showed features that are typical of cardiac dilation and HF and are found in experimental models as well as in failing human hearts. The morphological changes induced in the TG hearts included globular geometry of the LV and dilation of all four cardiac chambers. In addition, histological analyses revealed cardiomyocyte disarray. These findings are similar to those found in patients with idiopathic DCM. The echocardiographic analyses confirmed the LV enlargement and demonstrated a concomitant progressive loss of LV systolic function.

The overexpression of wild-type _1BAR resulted in the production of physiological levels of second messenger. An ~1.5-fold increase of intracellular diacylglycerol content was measured in TG hearts, indicating the production of second messenger in the absence of exogenous ligand (1). Responses of similar magnitude were elicited by the induction of ischemia, which led to a 1.5-fold increase in diacylglycerol content in perfused rat hearts (19). Similarly, stimulation of rat hearts with the ₁AR agonist phenylephrine resulted in a 1.8-fold increase in diacylglycerol production in rat hearts (29). Thus this level of chronic _1BAR activity in the TG hearts is physiologically relevant.

To explore the mechanism by which chronic ₁AR signaling contributes to the induction of HF, a number of cardiomyocyte mRNA transcripts and proteins were analyzed. We found changes in the expression of the sarcomeric components, MHCs, and titin (Fig. 3). These changes were most pronounced in older animals, suggesting an influence on the contractile behavior of the cardiomyocyte at a more advanced age. A shift in the -MHC-to--MHC ratio may directly affect cardiac contractility because -MHC has a threefold higher intrinsic ATPase activity (5). We observed a downregulation of -MHC and an upregulation of -MHC in failing hearts, suggesting that this isoform shift is correlated to the development of HF. Similar changes in the MHC expression profile also occur in the human heart, where they have been linked to myocardial failure (22, 27). Another affected sarcomeric protein is titin, which is decreased at 9 mo and in the failing hearts despite an initial upregulation. Abnormalities of titin expression may be important in the development of dilation because titin significantly influences elastic properties of the sarcomere (43) and is necessary as a template for the organization of newly synthesized sarcomeric filaments (16). Similar biphasic variations were found in a guinea pig model of decompensated hypertrophy (8). Decreased titin mRNA and protein levels were also documented in end-stage failing human hearts (16, 25).

An early molecular event that paralleled the decrease of Ca²⁺ transient amplitudes described earlier (13) was the reduction of SERCA2 protein abundance and activity. Similar to these findings in the intact animal, a recent study (33) in cultured adult rat cardiomyocytes also found a decrease in SERCA2 mRNA levels after 48 h of ₁AR stimulation with a concomitant reduction in the amplitude of Ca²⁺ transients. SERCA2 and its negative regulator, PLB, modulate cardiac relaxation via the regulation of Ca²⁺ uptake into the SR during diastole. AR-driven phosphorylation of PLB relieves the inhibition of SERCA2 and increases the rate of Ca²⁺ uptake. A decrease in Ca²⁺ reuptake is an important feature of HF in humans and in animal models. Decreases of the SERCA2-to-PLB ratio and diminished PLB phosphorylation correlate to the progression of HF (18, 34, 35). The importance of the SERCA2-PLB complex is also highlighted by an analysis of the muscle LIM protein knockout mouse, a model of DCM and HF with depressed SR Ca²⁺ uptake (4). The HF phenotype in these animals is rescued when PLB is ablated as well, suggesting that decreasing the inhibition of SERCA2 function by PLB is beneficial in averting cardiac decompensation (24). Chronic _1BAR signaling both in vitro and in vivo disrupts SR function by decreasing SERCA2 expression levels. Given the importance of the SERCA2-PLB complex in contractile homeostasis, this decrease must be considered a central determinant of the progression to HF in the _1BAR TG animals. Future work is necessary to determine the _1BAR-dependent intracellular signaling events that regulate SERCA2 expression. In addition to biochemical approaches in vitro, cross-breeding to other mouse lines with defined molecular alterations in the adrenergic signaling pathways may help to determine the molecular consequences of chronic _1BAR activity in vivo.

The adverse effects of chronic _1BAR signaling were already detectable in 3-mo-old animals, the earliest time point examined. Nevertheless, the decompensation process was protracted, with overt HF occuring only at ~9 mo. The initial progression of the DCM phenotype did not involve longstanding concentric cardiac hypertrophy as a primary compensatory mechanism. This was apparent from the largely unchanged ventricular wall thicknesses observed by histological and echocardiographical analyses (Fig. 1 and data not shown, respectively). Interestingly, TG mice with cardiac-specific overexpression of the _1AAR subtype also showed no hypertrophy (I. Lin, A. Owens, and R. Graham, personal communications), suggesting that the chronic activation of the ₁AR signaling pathway is not a primary stimulus for hypertrophic growth in the mouse in vivo. The absence of cardiac hypertrophy in vivo is in contrast to the development of hypertrophy in cultured rat neonatal cardiomyocytes on acute ₁AR stimulation (36). Differences in the ₁AR subtype prevalence or functional coupling in the neonatal cardiomyocytes may account for this apparent discrepancy. Furthermore, species differences in adrenergic responses have been described recently (31). However, mice overexpressing a constitutively active mutant (CAM) _1BAR (23) or signaling components downstream of the receptor, such as G_q (9) or activated PKC-II (41), demonstrate a hypertrophic response even in young animals. This suggests that regulation of receptor activity may be an important control point for late-onset eccentric hypertrophy and dilation. It is possible that this allows for compensatory mechanisms to be activated that prevent, or delay, the onset of hypertrophy and eventually HF. Thus the wild-type _1BAR may be more reflective of the interplay between receptor systems that plays a potentially important role in the progression to HF.

Despite the apparent lack of hypertrophic propensity, chronic _1BAR signaling in vivo seems to predispose the heart to decompensation and failure in synergy with additional stresses. This speculation draws support from a recent investigation of the CAM _1BAR mouse model. Without additional challenge, cardiomyocyte size was either moderately increased (23) or unchanged (42), and cardiac performance was normal. Transaortic constriction (TAC) induced cardiac hypertrophy to the same extent in both controls and CAM _1BAR TG hearts, as determined by the increase of myocyte cross-sectional area. However, CAM _1BAR hearts failed to produce a concomitant increase in LV contractility, suggesting cardiac decompensation and progression to failure. In addition, TAC TG animals showed a much higher incidence of atrial thrombus formation and pleural effusion, which also supports the notion of cardiac decompensation (42). A predisposition to decompensation and failure was also seen in the wild-type _1BAR TG animals. A high incidence of peripartum lethality combined with signs of HF in reproducing females suggests that the cardiovascular stress of pregnancy exacerbated the otherwise slower progression of the phenotype (unpublished data).

The reason for the relatively late onset of the HF phenotype in TG animals overexpressing the wild-type _1BAR is unclear. In light of the observation that an additional stress exacerbates the predisposition to HF by chronic _1BAR signaling, one might speculate that the normal aging process provides a sufficient stress to trigger decompensation. The aging heart undergoes functional and molecular changes similar in direction, albeit less severe, to those found in failing hearts. This includes altered function of the AR-G protein-adenylyl cyclase complex, such as decreases in AR expression levels and agonist affinities, and increases in the expression of the inhibitory G protein, G_i (for a review, see Ref. 7). While in itself not detrimental to cardiac function, an age-related functional decline might nevertheless exacerbate the DCM phenotype in the TG situation. Alternatively, it is possible that the late-occuring sarcomeric changes (MHC, titin) play a major role in triggering the HF phenotype. Imposing experimentally controlled stresses, such as TAC in the presence or absence of pharmacological interventions, on the wild-type _1BAR TG animals may serve to further explore the apparent predisposition to cardiac decompensation in vivo.

In conclusion, chronic ₁AR signaling has adverse effects on cardiac function. Increased activity in vivo was achieved by specific overexpression of the _1BAR subtype in cardiomyocytes of TG mice. Here, we show that chronic signaling through the wild-type _1BAR subtype pathway in TG mouse hearts predisposes these animals to develop DCM. Among the characteristic features of this cardiomyopathy is the relatively slow progression, the late presentation with HF, the dilation of all four chambers, and specific changes in the cardiac gene expression program. Many of these features mimic the prevalent idiopathic DCM phenotype in humans, such as sudden death prevalence and the late onset development of the disease (10). Given the high incidence of idiopathic DCM and its largely unknown etiology, particularly regarding its underlying molecular mechanisms, these TG animals may provide an interesting model to study the contribution of ₁AR to this disease.