Fabrice Jaffré

Overexpression of the Serotonin 5-HT2B Receptor in Heart Leads to Abnormal Mitochondrial Function and Cardiac Hypertrophy

Background—Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. We previously reported that 5-HT2B receptor ablation in mice leads to dilated cardiomyopathy. In this study, we investigated the pathological consequence of overexpressing 5-HT2B receptors in heart in vivo. Methods and Results—We have generated transgenic mice overexpressing the Gq-coupled 5-HT2B receptor specifically in heart. We found that overexpression of 5-HT2B receptor in heart leads to ventricular hypertrophy as the result of increased cell number and size. Increased atrial natriuretic peptide and myosin heavy chain expression demonstrated activation of the molecular program for cardiac hypertrophy. Echocardiographic analysis indicated the presence of thickened ventricular free wall without alteration of the systolic function, showing that transgenic mice have compensated hypertrophy. Electron microscopic analysis revealed structural abnormalities including mitochondrial proliferation, as also manifested by histological staining. Transgenic mouse heart displayed a specific reduction in the expression levels of the adenine nucleotide translocator associated to increase in the succinate dehydrogenase and cytochrome C oxidase mitochondrial activities. Conclusions—Our results constitute the first genetic evidence that overexpression of the 5-HT2B receptor in the heart leads to compensated hypertrophic cardiomyopathy associated with proliferation of the mitochondria. This observation suggests a role for mitochondria in the hypertrophic signaling that is regulated by serotonin. These transgenic mice provide a new genetic model for hypertrophic heart disease.

Involvement of the serotonin 5-HT2B receptor in cardiac hypertrophy linked to sympathetic stimulation: control of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokine production by ventricular fibroblasts.

Background—The serotonergic 5-HT2B receptor regulates cardiomyocyte development and growth. A putative contribution of this receptor to fibroblast-dependent cardiac function has not been identified. Methods and Results—By mimicking sympathetic stimulation with chronic isoproterenol perfusion in vivo, we found that mice developed a cardiac hypertrophy, which was prevented by exposure to the 5-HT2B receptor antagonists SB206553 or SB215505 or in 5-HT2B receptor–knockout mice. The isoproterenol-induced hypertrophy was associated with an increase in the plasma levels of interleukin-1&bgr; and tumor necrosis factor-&agr; but not interleukin-6. In contrast, the plasma isoproterenol-induced cytokine increase was not observed in either 5-HT2B receptor–mutant or wild-type mice perfused with isoproterenol+SB206553. We demonstrated that stimulation of wild-type cardiac fibroblasts by isoproterenol markedly increased the production of the interleukin-6, interleukin-1&bgr;, and tumor necrosis factor-&agr; cytokines. Strikingly, we found that this isoproterenol-induced cytokine production was abolished by SB206553 or in 5-HT2B receptor–knockout fibroblasts. Serotonin also stimulated production of the 3 cytokines in wild-type fibroblasts, which was effectively reduced in 5-HT2B receptor–knockout fibroblasts. Conclusions—Our results demonstrate for the first time that 5-HT2B receptors are essential for isoproterenol-induced cardiac hypertrophy, which involves the regulation of interleukin-6, interleukin-1&bgr;, and tumor necrosis factor-&agr; cytokine production by cardiac fibroblasts.

Serotonin and Angiotensin Receptors in Cardiac Fibroblasts Coregulate Adrenergic-Dependent Cardiac Hypertrophy

By mimicking sympathetic stimulation in vivo, we previously reported that mice globally lacking serotonin 5-HT2B receptors did not develop isoproterenol-induced left ventricular hypertrophy. However, the exact cardiac cell type(s) expressing 5-HT2B receptors (cardiomyocytes versus noncardiomyocytes) involved in pathological heart hypertrophy was never addressed in vivo. We report here that mice expressing the 5-HT2B receptor solely in cardiomyocytes, like global 5-HT2B receptor–null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in cytokine plasma-levels. These data reveal a key role of noncardiomyocytes in isoproterenol-induced hypertrophy in vivo. Interestingly, we show that primary cultures of angiotensinogen null adult cardiac fibroblasts are releasing cytokines on stimulation with either angiotensin II or serotonin, but not in response to isoproterenol stimulation, demonstrating a critical role of angiotensinogen in adrenergic-dependent cytokine production. We then show a functional interdependence between AT1Rs and 5-HT2B receptors in fibroblasts by revealing a transinhibition mechanism that may involve heterodimeric receptor complexes. Both serotonin- and angiotensin II–dependent cytokine production occur via a Src/heparin-binding epidermal growth factor–dependent transactivation of epidermal growth factor receptors in cardiac fibroblasts, supporting a common signaling pathway. Finally, we demonstrate that 5-HT2B receptors are overexpressed in hearts from patients with congestive heart failure, this overexpression being positively correlated with cytokine and norepinephrine plasma levels. Collectively, these results reveal for the first time that interactions between AT1 and 5-HT2B receptors coexpressed by noncardiomyocytes are limiting key events in adrenergic agonist-induced, angiotensin-dependent cardiac hypertrophy. Accordingly, antagonists of 5-HT2B receptors might represent novel therapeutics for sympathetic overstimulation-dependent heart failure.

Cyclophilin A Promotes Cardiac Hypertrophy in Apolipoprotein E–Deficient Mice

Objective—Cyclophilin A (CyPA, encoded by Ppia) is a proinflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (Ang II)–induced reactive oxygen species (ROS) production in the aortas of apolipoprotein E (Apoe)−/− mice. In this study, we sought to evaluate the role of CyPA in Ang II–induced cardiac hypertrophy. Methods and Results—Cardiac hypertrophy was not significantly different between Ppia+/+ and Ppia−/− mice infused with Ang II (1000 ng/min per kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe−/− mice. In contrast to Apoe−/− mice, Apoe−/−Ppia−/− mice exhibited significantly less Ang II–induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. Ang II–induced ROS production, cardiac fibroblast proliferation, and cardiac fibroblast migration were markedly decreased in Apoe−/−Ppia−/− cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. Conclusion—CyPA is required for Ang II–mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.

β-Adrenergic receptor stimulation transactivates protease-activated receptor 1 via matrix metalloproteinase 13 in cardiac cells

Background— Chronic &bgr;-adrenergic receptor (&bgr;-AR) overstimulation, a hallmark of heart failure, is associated with increased cardiac expression of matrix metalloproteinases (MMPs). MMP-1 has been shown to cleave and activate the protease-activated receptor 1 (PAR1) in noncardiac cells. In the present study, we hypothesized that &bgr;-AR stimulation would result in MMP-dependent PAR1 transactivation in cardiac cells. Methods and Results— &bgr;-AR stimulation of neonatal rat ventricular myocytes (NRVMs) or cardiac fibroblasts with isoproterenol transduced with an alkaline phosphatase–tagged PAR1 elicited a significant increase in alkaline phosphatase–PAR1 cleavage. This isoproterenol-dependent cleavage was significantly reduced by the broad-spectrum MMP inhibitor GM6001. Importantly, specific MMP-13 inhibitors also decreased alkaline phosphatase–PAR1 cleavage in isoproterenol-stimulated NRVMs, as well as in NRVMs stimulated with conditioned medium from isoproterenol-stimulated cardiac fibroblasts. Moreover, we found that recombinant MMP-13 stimulation cleaved alkaline phosphatase–PAR1 in NRVMs at DPRS42↓43FLLRN. This also led to the activation of the ERK1/2 pathway through G&agr;q in NRVMs and via the G&agr;q/ErbB receptor pathways in cardiac fibroblasts. MMP-13 elicited similar levels of ERK1/2 activation but lower levels of generation of inositol phosphates in comparison to thrombin. Finally, we demonstrated that either PAR1 genetic ablation or pharmacological inhibition of MMP-13 prevented isoproterenol-dependent cardiac dysfunction in mice. Conclusions— In this study, we demonstrate that &bgr;-AR stimulation leads to MMP-13 transactivation of PAR1 in both cardiac fibroblasts and cardiomyocytes and that this likely contributes to pathological activation of G&agr;q and ErbB receptor–dependent pathways in the heart. We propose that this mechanism may underlie the development of &bgr;-AR overstimulation–dependent cardiac dysfunction.

Serotonin 5-HT(2B) receptor blockade prevents reactive oxygen species-induced cardiac hypertrophy in mice.

We established previously that 5-HT2B receptors are involved in cardiac hypertrophy through the regulation of hypertrophic cytokines in cardiac fibroblasts. Moreover, the generation of reactive oxygen species and tumor necrosis factor-α through the activation of reduced nicotinamide-adenine dinucleotide phosphate [NAD(P)H] oxidase has been implicated in cardiac hypertrophy. In this study, we investigated whether 5-HT2B receptors could be involved in the development of cardiac hypertrophy associated with superoxide anion production. Therefore, we measured the effects of serotonergic 5-HT2B receptor blockade on left-ventricular superoxide anion generation in 2 established pharmacological models of cardiac hypertrophy, ie, angiotensin II and isoproterenol infusions in mice. Angiotensin II infusion for 14 days increased superoxide anion concentration (+32%), NAD(P)H oxidase maximal activity (+84%), and p47phox NAD(P)H oxidase subunit expression in the left ventricle together with hypertension (+37 mm Hg) and cardiac hypertrophy (+17% for heart weight:body weight). The 5-HT2B receptor blockade by a selective antagonist (SB215505) prevented the increase in cardiac superoxide generation and hypertrophy. Similarly, infusion for 5 days of isoproterenol increased left-ventricular NAD(P)H oxidase activity (+48%) and cardiac hypertrophy (+31%) that were prevented by the 5-HT2B receptor blockade. Finally, in the primary culture of left-ventricular cardiac fibroblasts, angiotensin II and isoproterenol stimulated NAD(P)H oxidase activity. This activation was prevented by SB215505. These findings suggest that the 5-HT2B receptor may represent a new target to reduce cardiac hypertrophy and oxidative stress. Its blockade affects both angiotensin II and β-adrenergic trophic responses without significant hemodynamic alteration.

Beta-Adrenergic Receptor Stimulation Transactivates Protease-Activated Receptor 1 via MMP-13 in Cardiac Cells

Background— Chronic &bgr;-adrenergic receptor (&bgr;-AR) overstimulation, a hallmark of heart failure, is associated with increased cardiac expression of matrix metalloproteinases (MMPs). MMP-1 has been shown to cleave and activate the protease-activated receptor 1 (PAR1) in noncardiac cells. In the present study, we hypothesized that &bgr;-AR stimulation would result in MMP-dependent PAR1 transactivation in cardiac cells. Methods and Results— &bgr;-AR stimulation of neonatal rat ventricular myocytes (NRVMs) or cardiac fibroblasts with isoproterenol transduced with an alkaline phosphatase–tagged PAR1 elicited a significant increase in alkaline phosphatase–PAR1 cleavage. This isoproterenol-dependent cleavage was significantly reduced by the broad-spectrum MMP inhibitor GM6001. Importantly, specific MMP-13 inhibitors also decreased alkaline phosphatase–PAR1 cleavage in isoproterenol-stimulated NRVMs, as well as in NRVMs stimulated with conditioned medium from isoproterenol-stimulated cardiac fibroblasts. Moreover, we found that recombinant MMP-13 stimulation cleaved alkaline phosphatase–PAR1 in NRVMs at DPRS42↓43FLLRN. This also led to the activation of the ERK1/2 pathway through G&agr;q in NRVMs and via the G&agr;q/ErbB receptor pathways in cardiac fibroblasts. MMP-13 elicited similar levels of ERK1/2 activation but lower levels of generation of inositol phosphates in comparison to thrombin. Finally, we demonstrated that either PAR1 genetic ablation or pharmacological inhibition of MMP-13 prevented isoproterenol-dependent cardiac dysfunction in mice. Conclusions— In this study, we demonstrate that &bgr;-AR stimulation leads to MMP-13 transactivation of PAR1 in both cardiac fibroblasts and cardiomyocytes and that this likely contributes to pathological activation of G&agr;q and ErbB receptor–dependent pathways in the heart. We propose that this mechanism may underlie the development of &bgr;-AR overstimulation–dependent cardiac dysfunction.

β-Adrenergic Receptor Stimulation Transactivates Protease-Activated Receptor 1 via Matrix Metalloproteinase 13 in Cardiac CellsClinical Perspective

Background— Chronic &bgr;-adrenergic receptor (&bgr;-AR) overstimulation, a hallmark of heart failure, is associated with increased cardiac expression of matrix metalloproteinases (MMPs). MMP-1 has been shown to cleave and activate the protease-activated receptor 1 (PAR1) in noncardiac cells. In the present study, we hypothesized that &bgr;-AR stimulation would result in MMP-dependent PAR1 transactivation in cardiac cells. Methods and Results— &bgr;-AR stimulation of neonatal rat ventricular myocytes (NRVMs) or cardiac fibroblasts with isoproterenol transduced with an alkaline phosphatase–tagged PAR1 elicited a significant increase in alkaline phosphatase–PAR1 cleavage. This isoproterenol-dependent cleavage was significantly reduced by the broad-spectrum MMP inhibitor GM6001. Importantly, specific MMP-13 inhibitors also decreased alkaline phosphatase–PAR1 cleavage in isoproterenol-stimulated NRVMs, as well as in NRVMs stimulated with conditioned medium from isoproterenol-stimulated cardiac fibroblasts. Moreover, we found that recombinant MMP-13 stimulation cleaved alkaline phosphatase–PAR1 in NRVMs at DPRS42↓43FLLRN. This also led to the activation of the ERK1/2 pathway through G&agr;q in NRVMs and via the G&agr;q/ErbB receptor pathways in cardiac fibroblasts. MMP-13 elicited similar levels of ERK1/2 activation but lower levels of generation of inositol phosphates in comparison to thrombin. Finally, we demonstrated that either PAR1 genetic ablation or pharmacological inhibition of MMP-13 prevented isoproterenol-dependent cardiac dysfunction in mice. Conclusions— In this study, we demonstrate that &bgr;-AR stimulation leads to MMP-13 transactivation of PAR1 in both cardiac fibroblasts and cardiomyocytes and that this likely contributes to pathological activation of G&agr;q and ErbB receptor–dependent pathways in the heart. We propose that this mechanism may underlie the development of &bgr;-AR overstimulation–dependent cardiac dysfunction.