Celine Latouche

Prevention of liver cancer cachexia-induced cardiac wasting and heart failure

AIMS Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130). METHODS AND RESULTS Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated. CONCLUSION Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the β-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel

Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up‐regulated in response to physio‐logical concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone‐responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone‐regulated sodium retention in the kidney. CCD cell lines stably overexpress‐ing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC‐mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.—Ziera, T., Irlbacher, H., Fromm, A., Latouche, C., Krug, S. M., Fromm, M., Jaisser, F., Borden, S. A. Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel. FASEB J. 23, 3936‐3946 (2009). www.fasebj.org

Neutrophil Gelatinase-Associated Lipocalin Is a Novel Mineralocorticoid Target in the Cardiovascular System

Mineralocorticoid receptor (MR) activation may be deleterious to the cardiovascular system, and MR antagonists improve morbidity and mortality of patients with heart failure. However, mineralocorticoid signaling in the heart remains largely unknown. Using a pan-genomic transcriptomic analysis, we identified neutrophil gelatinase-associated lipocalin (NGAL or lipocalin 2) as a strongly induced gene in the heart of mice with conditional and targeted MR overexpression in cardiomyocytes (whereas induction was low in glucocorticoid receptor–overexpressing mice). NGAL mRNA levels were enhanced after hormonal stimulation by the MR ligand aldosterone in cultured cardiac cells and in the heart of wild-type mice. Mineralocorticoid pathological challenge induced by nephrectomy/aldosterone/salt treatment upregulated NGAL expression in the heart and aorta and its plasma levels. We show evidence for MR binding to an NGAL promoter, providing a mechanism for NGAL regulation. We propose that NGAL may be a marker of mineralocorticoid-dependent injury in the cardiovascular system in mice.

Molecular Signature of Mineralocorticoid Receptor Signaling in Cardiomyocytes: From Cultured Cells to Mouse Heart

Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (alpha1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.

Neutrophil Gelatinase–Associated Lipocalin, a Novel Mineralocorticoid Biotarget, Mediates Vascular Profibrotic Effects of Mineralocorticoids

Activation of the mineralocorticoid receptor has been shown to be deleterious in cardiovascular diseases (CVDs). We have recently shown that lipocalin 2 (Lcn2), or neutrophil gelatinase–associated lipocalin (NGAL), is a primary target of aldosterone/mineralocorticoid receptor in the cardiovascular system. Lcn2 is a circulating protein, which binds matrix metalloproteinase 9 and modulates its stability. We hypothesized that Lcn2 could be a mediator of aldosterone/mineralocorticoid receptor profibrotic effects in the cardiovascular system. Correlations between aldosterone and profibrotic markers, such as procollagen type I N-terminal peptide, were investigated in healthy subjects and subjects with abdominal obesity. The implication of Lcn2 in the mineralocorticoid pathway was studied using Lcn2 knockout mice subjected to a nephrectomy/aldosterone/salt (NAS) challenge for 4 weeks. In human subjects, NGAL/matrix metalloproteinase 9 was positively correlated with plasma aldosterone and fibrosis biomarkers. In mice, loss of Lcn2 prevented the NAS-induced increase of plasma procollagen type I N-terminal peptide, as well as the increase of collagen fibers deposition and collagen I expression in the coronary vessels and the aorta. The lack of Lcn2 also blunted the NAS-induced increase in systolic blood pressure. Ex vivo, treatment of human fibroblasts with recombinant Lcn2 induced the expression of collagen I and the profibrotic galectin-3 and cardiotrophin-1 molecules. Our results showed that Lcn2 plays a key role in aldosterone/mineralocorticoid receptor–mediated vascular fibrosis. The clinical data indicate that this may translate in human patients. Lcn2 is, therefore, a new biotarget in cardiovascular fibrosis induced by mineralocorticoid activation.

Cardiac expression of neutrophil gelatinase-associated lipocalin in a model of cancer cachexia-induced cardiomyopathy: Cardiac expression of NGAL in cancer cachexia-induced cardiomyopathy

Cachexia is a severe consequence of cancer. Although cancer‐induced heart atrophy leads to cardiac dysfunction and heart failure (HF), biomarkers for their diagnosis have not been identified. Neutrophil gelatinase‐associated lipocalin (NGAL) is an aldosterone‐responsive gene increased in HF. We studied NGAL and its association with aldosterone levels in a model of cancer cachexia‐induced cardiomyopathy.

411 NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN IS A NOVEL MINERALOCORTICOID TARGET IN THE CARDIOVASCULAR SYSTEM

Background: Mineralocorticoid receptor (MR) activation may be deleterious to the cardiovascular system and MR antagonists improve morbidity and mortality of patients with heart failure. However mineralocorticoid signalling in the heart remains largely unknown. Methods and Results: Using genetically modified mouse models, pharmacological mineralocorticoid stress in rodents and cultured cells, we showed that Neutrophil Gelatinase-Associated Lipocalin (NGAL) expression is directly controlled by mineralocorticoid activation in the cardiovascular system. Cardiac NGAL expression is enhanced by cardiomyocyte-specific MR overexpression, short term (6 hours) and chronic (3 weeks) aldosterone treatment in mice. In cultured HL1 cells expressing MR, MR can bind to the NGAL promoter, providing a mechanism for NGAL induction. Expression of NGAL by aldo/MR activation is prevented by MR antagonists ex vivo and in vivo. Mineralocorticoid pathological challenge induced by nephrectomy/aldosterone/salt (NAS) treatment enhances NGAL expression in the heart and aorta as well as NGAL plasma levels. In NGAL KO mice, cardiac and vascular induction by NAS of several molecular markers of extracellular matrix remodelling is prevented, indicating NGAL is crucial for the occurrence of extracellular matrix remodelling induced by mineralocorticoid excess. Perivascular fibrosis is also largely blunted in the heart of these mice. Moreover, in asymptomatic obese subjects prone to develop heart failure, circulating levels of NGAL-MMP9 complexes are correlated with plasma aldosterone and with biomarkers of cardiovascular fibrosis. Conclusions: Based on these experimental and clinical data, we suggest that NGAL is a novel MR biotarget and may provide a link between mineralocorticoid stress and cardiovascular remodelling in humans.

L009 Changes in cardiac transcriptome induced by altered corticosteroid receptor signaling in the heart

Aldosterone plays a major role in the regulation of extracellular volume and blood pressure, through its renal action via the mineralocorticoid receptor (MR). The extra-renal roles of aldosterone/MR in non-epithelial cells (neurones, cardiomyocytes, vessels, adipocytes…) are now intensively investigated. Animal models (aldo/DOCA-salt) and clinical trials (RALES, EPHESUS) evidenced a major pathophysiological contribution of aldosterone/ MR in the cardiovascular system. To evaluate MR cardiac signaling, we analyzed the transcriptomic profile of 2 transgenic mouse models with conditional and inducible overexpression of the MR or of the glucocorticoid receptor (GR) in cardiomyocytes (MHCMR and MHC-GR). Using dedicated cardiochips, we identified 520 cardiac genes that are differentially expressed between control and MHC-MR mice (250 up, 270 down-regulated), and 1232 genes differentially expressed between control and MHC-GR mice (728 up, 504 down-regulated). Some of these genes were validated by real-time PCR, such as Periostin [MHC-MR vs control 10.56±2.2, p=0.02 ; MHC-GR vs control 4.36±1.72, NS], Troponin T3 [MHC-MR vs control 41.66±9.23, p=0.048 ; MHC-GR vs control 1.78±0.41, NS], Fkbp5 [MHC-MR vs control 34.51±6.16, p=0.01 ; MHC-GR vs control 3.33±0.59, p=0.006]. We have also compared the effects of aldosterone in the presence or not of different MR/GR antagonists on gene expression in a cardiomyocytes cell line (H9C2 cells) stably expressing the MR, in order to analyze hormone and antagonist specificity. Importantly, some of the MR-specific genes encode for secreted proteins that may represent novel markers of MR activation in circulating blood, including in human pathology.