Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor ␥). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart.Heart failure is a debilitating syndrome that involves insufficient cardiac performance. Multiple pathomechanisms have been elucidated, but treatment options remain insufficient, and hence the mortality of heart failure is high (1). The causes of heart failure are complex with ischemic heart disease being the most frequently associated condition (2). Co-existing disorders such as diabetes, hypertension, and obesity further deteriorate symptoms (3). Despite having a different etiology, late-stage heart failure is commonly characterized by severe changes in myocardial substrate metabolism, with a switch from fatty acid oxidation toward predominant glycolysis (4 -6). Conflicting evidence exists as to whether this substrate switch is beneficial or detrimental (7), but several previous studies have indicated that an increased availability of lipid substrates that counteract the substrate switch could improve cardiac function (7,8). Moreover, treatment options, which improve substrate availability, are attractive because the failing heart is often considered to be "an engine running out of fuel" (9).Following this concept, we aimed to investigate the impact of improved cardiac substrate availability by generating transgenic mice with myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. Such an approach is also supported by data obtained for myocardium-specific Fasn deficiency, which have revealed the cardioprotective potential of Fasn (10). Moreover, hearts from patients with heart failure showed an increased express...

Background: Mechanisms underlying the cardioprotective profile of G-protein-coupled receptor kinase 2 (GRK2) inhibitors are incompletely understood. Results: GRK2 inhibition activated the growth-promoting MAPK pathway, which contributed to cardioprotection by preventing cardiomyocyte death. Conclusion: Cardioprotective activity of GRK2 inhibitors overlaps with enhanced tumor growth. Significance: A promising class of kinase inhibitors for heart failure treatment shows overlapping of cardioprotective signaling with tumor growth promotion.

Many experimental and clinical studies suggest a relationship between enhanced angiotensin II release by the angiotensinconverting enzyme (ACE) and the pathophysiology of atherosclerosis. The atherosclerosis-enhancing effects of angiotensin II are complex and incompletely understood. To identify antiatherogenic target genes, we performed microarray gene expression profiling of the aorta during atherosclerosis prevention with the ACE inhibitor, captopril. Atherosclerosis-prone apolipoprotein E (apoE)-deficient mice were used as a model to decipher susceptible genes regulated during atherosclerosis prevention with captopril. Microarray gene expression profiling and immunohistology revealed that captopril treatment for 7 months strongly decreased the recruitment of pro-atherogenic immune cells into the aorta. Captopril-mediated inhibition of plaque-infiltrating immune cells involved down-regulation of the C-C chemokine receptor 9 (CCR9). Reduced cell migration correlated with decreased numbers of aorta-resident cells expressing the CCR9-specific chemoattractant factor, chemokine ligand 25 (CCL25). The CCL25-CCR9 axis was pro-atherogenic, because inhibition of CCR9 by RNA interference in hematopoietic progenitors of apoE-deficient mice significantly retarded the development of atherosclerosis. Analysis of coronary artery biopsy specimens of patients with coronary artery atherosclerosis undergoing bypass surgery also showed strong infiltrates of CCR9-positive cells in atherosclerotic lesions. Thus, the C-C chemokine receptor, CCR9, exerts a significant role in atherosclerosis.Several clinical studies show that pharmacological inhibition of the renin-angiotensin-aldosterone system could mediate atheroprotection (1-4). In agreement with those clinical data, experimental studies demonstrated a causal relationship between angiotensin II release by the angiotensin-converting enzyme (ACE), 2 the angiotensin II AT 1 receptor, and the development of atherosclerosis because genetic inactivation of either ACE or the AT 1 receptor prevents atherosclerosis development in various animal models of atherosclerosis (5, 6). Likewise, inhibition of angiotensin II activity by ACE inhibitors or AT 1 receptor antagonists is curative in such animal models of atherosclerosis (7,8).In view of the major anti-atherosclerotic effects of ACE inhibitors and AT 1 antagonists, many studies investigated mechanisms that could contribute to atheroprotection upon angiotensin II inhibition. The effects of angiotensin II in atherosclerosis are complex involving actions on circulating blood cells and arterial smooth muscle cells as major targets (9 -11). Established pro-atherogenic effects of angiotensin II on smooth muscle cells are related to a phenotype transformation from contractile to synthetic (11). The effects of angiotensin II on circulating immune cells are less clear, although, gene inactivation studies clearly demonstrated the involvement of circulating blood and progenitor cells in the atherosclerosis-enhancing activity of ACE and the AT 1 r...

Inhibition of the G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment approach for heart failure. Therefore, cardio-protective mechanisms induced by GRK2 inhibition are under investigation. We compared two different GRK2 inhibitors, i.e., (i) the dual-specific GRK2 and raf kinase inhibitor protein, RKIP, and (ii) the dominant-negative GRK2-K220R mutant. We found that RKIP induced a strong sensitization of Gq/11-dependent, heart failure-promoting angiotensin II AT1 receptor signaling. The AT1-sensitizing function of RKIP was mediated by the RKIP-GRK2 interaction because the RKIP-S153V mutant, which does not interact with GRK2, had no effect on AT1-stimulated signaling. In contrast, GRK2-K220R significantly inhibited the AT1-stimulated signal. The in vivo relevance of these major differences between two different approaches of GRK2 inhibition was analyzed by generation of transgenic mice with myocardium-specific expression of RKIP and GRK2-K220R. Our results showed that a moderately increased cardiac protein level of RKIP was sufficient to induce major symptoms of heart failure in aged, 8-months-old RKIP-transgenic mice in two different genetic backgrounds. In contrast, GRK2-K220R protected against chronic pressure overload-induced cardiac dysfunction. The AT1 receptor contributed to RKIP-induced heart failure because treatment with the AT1 receptor antagonist, losartan, retarded symptoms of heart failure in RKIP-transgenic mice. Thus, sensitization of the heart failure-promoting AT1 receptor by the RKIP-GRK2 interaction contributes to heart failure whereas dominant-negative GRK2-K220R is cardioprotective. Because RKIP is up-regulated on cardiac biopsy specimens of heart failure patients, the deduced heart failure-promoting mechanism of RKIP could also be relevant for the human disease.

Reactive oxygen species (ROS) is a significant feature of atherosclerosis but the impact of ROS on atherogenesis is not clear since antioxidants such as vitamin E have little effect on atherosclerosis development in vivo. To investigate the role of ROS in atherosclerosis, we used ApoE-deficient mice, and compared the treatment effect of the antioxidant vitamin E with that of the angiotensin-converting enzyme (ACE) inhibitor, captopril, because angiotensin II is a major source of ROS in the vasculature. Dihydroethidium (DHE) staining demonstrated that vitamin E and captopril both prevented the atherosclerosis-induced increase in aortic superoxide content. In contrast, seven months of vitamin E treatment retarded the development of atherosclerotic lesions by only 45.8 ± 11.5% whereas captopril reduced the aortic plaque area by 88.1 ± 7.5%. To discriminate between vitamin E-sensitive and -insensitive effects of ACE inhibition, we performed whole genome microarray gene expression profiling. Gene ontology (GO) and immunohistology analyses showed that vitamin E and captopril prevented atherosclerosis-related changes of aortic intima and media genes. However, vitamin E did not reduce the expression of probe sets detecting the aortic recruitment of pro-inflammatory immune cells while immune cell-specific genes were normalized by captopril treatment. Moreover, vitamin E did not prevent the atherosclerosis-dependent down-regulation of perivascular nerve-specific genes, which were preserved in captopril-treated aortas. Taken together, our study detected antioxidant vitamin E-like effects of angiotensin II inhibition in atherosclerosis treatment regarding preservation of aortic intima and media genes. Additional vitamin E-insensitive effects targeting atherosclerosis-enhancing aortic immune cell recruitment and perivascular nerve degeneration could account for the stronger anti-atherogenic activity of ACE inhibition compared to vitamin E.

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In individuals with diverse cardiovascular risk factors, signalling stimulated by the AT(1) receptor for the vasopressor angiotensin II is sensitized by heterodimerization with the receptor for the vasodepressor bradykinin, B(2). Signal sensitization and receptor heterodimerization rely on efficient maturation of the B(2) receptor protein. To assess functional features of that important cardiovascular receptor system, we established an in vivo model by using immunodeficient NOD.Scid mice for the expansion of transfected cells under physiological conditions. Compared to cultivated cells, the in vivo model strongly facilitated B(2) receptor maturation and heterodimerization. To elucidate the mechanisms underlying the enhancement of B(2) receptor protein maturation under in vivo conditions, we performed microarray gene expression profiling. Microarray analysis revealed a more than 1.7-fold up-regulation of the chaperone calreticulin upon in vivo cell expansion whereas other important members of the general chaperone system were only marginally altered. Down regulation of calreticulin expression by RNA interference confirmed the importance of calreticulin for efficient B(2) receptor maturation under in vivo conditions. Receptor proteins synthesized in the Nod.Scid cell expansion model were functionally active and sensitive to drug treatment as exemplified by treatment with the AT(1)-specific antagonist losartan. Thus, we established a model system that can be used to analyze functional features of proteins in vivo by expanding transfected cells in immunodeficient NOD.Scid mice.

Heart failure is a major cause of death worldwide with insufficient treatment options. In the search for pathomechanisms, we found up-regulation of an enzyme, stearoyl-CoA desaturase 1 (Scd1), in different experimental models of heart failure induced by advanced atherosclerosis, chronic pressure overload, and/or volume overload. Because the pathophysiological role of Scd1/SCD in heart failure is not clear, we investigated the impact of cardiac SCD upregulation through the generation of C57BL/6-Tg(MHCSCD)Sjaa mice with myocardium-specific expression of SCD. Echocardiographic examination showed that 4.9-fold-increased SCD levels triggered cardiac hypertrophy and symptoms of heart failure at an age of eight months. Tg-SCD mice had a significantly reduced left ventricular cardiac ejection fraction of 25.7 ± 2.9% compared to 54.3 ± 4.5% of non-transgenic B6 control mice. Whole-genome gene expression profiling identified up-regulated heart-failure-related genes such as resistin, adiponectin, and fatty acid synthase, and type 1 and 3 collagens. Tg-SCD mice were characterized by cardiac lipid accumulation with 1.6- and 1.7-fold-increased cardiac contents of saturated lipids, palmitate, and stearate, respectively. In contrast, unsaturated lipids were not changed. Together with saturated lipids, apoptosis-enhancing p53 protein contents were elevated. Imaging by autoradiography revealed that the heart-failure-promoting and membrane-spanning angiotensin II AT1 receptor protein of Tg-SCD hearts was significantly up-regulated. In transfected HEK cells, the expression of SCD increased the number of cell-surface angiotensin II AT1 receptor binding sites. In addition, increased AT1 receptor protein levels were detected by fluorescence spectroscopy of fluorescent protein-labeled AT1 receptor-Cerulean. Taken together, we found that SCD promotes cardiac dysfunction with overload of cardiotoxic saturated lipids and up-regulation of the heart-failure-promoting AT1 receptor protein.