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Content archived on 2024-05-28

Functional role of the beta3-adrenoceptor in modulating the remodeling of the myocardium submitted to stress

Final Report Summary - FRB3AR (Functional role of the beta3-adrenoceptor in modulating the remodeling of the myocardium submitted to stress.)

Central hypothesis: The activation of the beta3-AR protects against hypertrophic remodeling of the heart through a eNOS and PKG -dependent mechanisms. In order to restrict the focus of our investigation, we proposed to pursue the following specific aims:
1) Characterization of the beta3-AR - induced protection in vivo in models of harsh pathological remodeling.
The host laboratory set out to study transgenic mice that had been previously generated in collaboration with the University of Toulouse – Rangueil (Prof D. Langin). These mice carry a transgene coding for the human beta3-AR (beta3-AR-tg mice) under the control of a cardiac specific promoter and are therefore an invaluable tool to study the impact of cardiac myocyte -restricted activation of the beta3-AR on cardiac pathophysiology. Having provided evidence that beta3-AR cardiac specific expression impedes hypertrophy induced by chronic (2 weeks) neurohormonal infusion (ISO and Ang II), the phenotype of these mice was examined under more severe stress that leads to advanced dysfunction and heart failure. Beta3-AR-tg mice were submitted to hemodynamic overload induced by transaortic constriction. Cardiac function and ventricular mass were assessed by echocardiography on anaesthetized animals using a Visualsonic® ultrasonic probe in collaboration with Pr Christophe Beauloye (CARD Unit/ Bruxelles). Hemodynamic parameters were studied on the same animals with a high-fidelity SPR-671 Millar pressure catheter (Millar Instruments, Houston, TX) advanced into the left ventricle. Analysis of morphometric changes (heart and left ventricle (LV) weight / tibial length), histology of paraformaldehyde-fixed ventricular sections including individual myocyte size (Wheat Germ Agglutinin staining), fibrosis (picroSirius Red staining) and capillary density (isolectin staining) were studied. Measurement of the expression of “fetal genes” including #-myosin heavy chain and atrial natriuretic peptide were performed by reverse transcription of total mRNA from left ventricle followed by real time PCR.
After sacrifice at 3 and 9 weeks, we observed that TAC induced a significant increase in left ventricular weight / tibial length ratio compared to the sham-operated mice (p<0.01). This increase was significantly attenuated in the Beta3-AR-tg mice 3 weeks after TAC (p<0.05).

2) Investigating the relevance of the coupling of beta3-AR with NOS in the regulation of pathological remodeling.
First, a preliminary set of data obtained using the in vivo model of beta3-AR-tg mice indicates that NO synthesis mediates beta3-AR -induced protection against hypertrophy. We observed that beta3-AR-tg mice under ISO stimulation when given the NOS inhibitor L-NAME in drinking water presented an increase of the myocytes size, unlike their untreated beta3-AR-tg littermates. We tested this hypothesis in a model of more severe stress that leads to advanced dysfunction and heart failure, by submitting beta3-AR-tg mice to Isoproterenol administration, 50mg/kg/d, i.p. 10d with/without L-NAME.
Contrary to the low-dose group, non-specific NOS inhibition did not restore the hypertrophic remodeling in response to the high dose Iso in TG mice. However, when LVNIO, a specific inhibitor of nNOS was used instead of L-NAME together with high dose Iso, hypertrophy was fully restored in TG mice. Notably, LNAME did reverse the anti-fibrotic effect of ß3-AR transgenic overexpression in TG under high-dose Iso, measured histologically.
This set of data is now being completed by investigating which NOS isoform is responsible for the production of the pool of NO that is promoted by hBeta3-AR in our models. As the eNOS isoform is likely to have such a role in the heart, we have first cross-bred beta3-AR-tg mice with eNOS -/- mice to ascertain the specific involvement of eNOS. Next, we cross-bred our beta-3 AR-tg mice with nNOS -/-; all these mice are viable and the phenotyping is ongoing as above.
In order to confirm these results in an alternative model, we used the well-described model of cultured neonatal rat ventricular myocytes (NRVMs). We have optimized the protocol for infection of these primary cultured NRVMs with adenoviral particles carrying the human beta3-AR gene (Ad-hBeta3-AR), allowing the cells to have a constitutive, moderate expression of the hBeta3-AR.
Ad-hBeta3-AR –infected cells were compared to NRVMs infected with a control adenoviral construct.

We showed that hBeta3-AR overexpression blunted the increase of cell size (standard readout for hypertrophy) that is observed in control NRVMs under stimulation with pro-hypertrophic agonists such as phenylephrine (PE). Moreover, co-incubation with L-NAME abolished the protection conferred by Ad-Beta3-AR under PE stimulation, indicating that, in this model, the hBeta3-AR antihypertrophic effect is coupled to NO production.
To account for hypertrophy, protein synthesis was measured 24 hours after stimulation with PE and compared to non –stimulated cells. We showed that hBeta3-AR overexpression blunted the increase of protein synthesis that is observed in control NRVMs under stimulation with PE. The coupling state of NOS (coupled, producing NO, versus uncoupled, producing O2.-) was also examined. As oxidative stress and uncoupled NOS were reported in hearts of Beta3-AR -/- mice, we investigated whether NOS is, conversely, maintained “coupled” in our models of Beta3-AR over expression. This was carried out using spin-trapping agents and electron paramagnetic resonance (EPR), a gold standard technique for measurement of free radicals and NO (expertise provided in our laboratory by I. Lobysheva and B. Gallez (Unit of Biomedical Magnetic Resonance, UCL). Control myocytes had measurable NO production (by EPR-spin-trapping), which was sensitive to L-NAME. This NO signal was increased upon stimulation with Nebivolol, which we showed to combine beta1 antagonistic with Beta3 agonistic properties. Consistent with B3AR coupling to NOS in these myocytes, hB3AR-overexpressing myocytes exhibited higher basal and stimulated NO signals as well. NOS involvement was then examined by treating cardiac myocytes with L-NAME together with PE. L-NAME alone had no effect on cell size in any group; in controls, co-treatment with L-NAME resulted in a slight increase in the PE-induced hypertrophic response. However, in Beta3-av infected cells, L-NAME fully restored the hypertrophic response to PE. Of note, when the nNOS specific inhibitor, LVNIO, was used instead of L-NAME in similar experiments, we observed a striking increase in cell mortality in PE-treated myocytes (regardless of ß3-AR expression) which precluded any size measurement. Such increased mortality was not observed in absence of PE stimulus.
In summary we now have robust evidence that hBeta3-AR activation can exert an antihypertrophic regulation under adrenergic and non adrenergic stimuli, both in vivo and in vitro. We also have compelling data showing that NOS is mediating a part of this effect.

3) Analysis of signalling pathways downstream to the Beta3-AR.
As the results suggest that NOS activity is critical for the Beta3-AR effects to occur, we explored the involvement of the downstream cGMP / PKG1 pathway, using both classic and novel pharmacological agents including the PKG-blocker polypeptide DT2. Among the downstream effectors, activation of the classic Ca2+ - sensitive calcineurin – NFAT hypertrophic pathway was analyzed, using a NFAT-Luc reporter assay. We also examined activation of the adenosine monophosphate activated protein kinase (AMPK) pathway, as it was described as an anti-hypertrophic effector and amenable to NO regulation in other cell types. This was monitored by studying the phosphorylation state of AMPK and its substrate, ACC and S6 both in tissue homogenates and NRVMs overexpressing the human Beta3-AR. In PE-treated control myocytes and after TAC in WT mice, respectively, we have shown a significant decrease of phosphorylated-AMPK and phosphorylated-ACC associated with an increase in phosphorylated-S6, all of which were prevented under Beta3-AR overexpression and in the Beta3-AR-tg mice. This was associated with a preservation of autophagic flux. Moreover, using silencing experiments to downregulate AMPKalpha1/2, we found that the anti-hypertrophic effect of the beta3-AR was lost, meaning that the protective effect of the beta3-AR is partially due to AMPK signaling pathway.

4) Colocalization studies of beta3-AR and its potential effectors.
One characteristic of eNOS is its tight and complex regulation by caveolin proteins, particularly caveolin type-3 (Cav-3) in cardiomyocytes. We observed a co-localization between beta3-AR together with eNOS but also with nNOS or AMPK in caveolae of cardiac myocytes, with and without neurohormonal stimulation. This was done by immunocytochemistry using confocal microscopy and by immunoblotting following either subcellular fractionation for analysis of caveolin-enriched membrane fractions or co-immunoprecipitation experiments with anti-Cav-3 antibodies. We completed this analysis with the proximity ligation assay (PLA)

5) Development and study of a cardiac-specific, inducible beta3-AR -/- mouse model in the context of pathologic cardiac remodeling.
Our data about the protection from hypertrophy in beta3-AR-tg mice suggest an intrinsic effect of the receptor in the cardiomyocyte. However, even if hBeta3-AR over-expression is moderate in this model, the use of a cardiac-specific, inducible, deletion of the beta3-AR will provide an even more convincing demonstration of the in vivo role of beta3-AR in impeding cardiac remodeling. The development of such a murine model using the Cre/lox strategy has been undertaken in collaboration with the GenOway Company (France). Breeding was made to obtain double transgenic mice carrying both the “floxed” Beta3-AR and the alpha MHC promoter - driven MerCreMer transgenes. This allows to “silence” the endogenous Beta3-AR gene before or after the development of cardiac dysfunction after stress (as described above). This model is an invaluable tool for studying the causal influence of cardiac Beta3-AR in the context of cardiac injury and remodeling, and for identifying a therapeutic window when the activation of this Beta3-AR can be either beneficial of deleterious.
Attention was recently drawn about the possible cardio-toxicity of the Tamoxifen / MerCreMer recombinase system. The protocol for choosing the adequate estrogen receptor ligand (tamoxifen) and administration protocol has been optimized in our hands in order to limit the occurrence of hypertrophy in the absence of any stress. Homozygote double transgenic mice have been treated with progressive doses of tamoxifen and the resulting loss of cardiac expression of the beta3-AR DNA was monitored by PCR. We have shown that 40 mg/kg/day is a dose sufficient to induce the deletion of beta3-AR DNA. This protocol, contrary to 60 and 80 mg/kg/day, limits the occurrence of hypertrophy in the absence of any stress (measured by LVW/TL ratio). Unfortunately, this dose induced a cardiac fibrosis. We performed subsequent tests with a lower dose such as 20 mg/kg/day for 3 days. This dose is sufficient to induce the deletion of beta3-AR DNA without any fibrosis. The Tamoxifen protocol is now optimized and mice have been included in TAC experiment for further investigation.