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Aims: Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective., Methods and Results: Mice were subjected to transverse aortic constriction (TAC) or sham surgery and sacrificed 2 weeks, 4 weeks, or 6 weeks after the procedure. Samples from plasma, liver, skeletal muscle, and heart were collected and analysed using metabolomics. Cardiac samples were also analysed by transcriptional profiling. Progressive alterations of key cardiac metabolic pathways and gene expression patterns indicated impaired mitochondrial function and a metabolic switch during transition to heart failure. Similar to the heart, liver, and skeletal muscle revealed significant metabolic alterations such as depletion of essential fatty acids and glycerolipids in late stages of heart failure. Circulating metabolites, particularly fatty acids, reflected cardiac metabolic defects, and deteriorating heart function. For example, inverse correlation was found between plasma and the heart levels of triacylglycerol (C18:1, C18:2, C18:3), and sphingomyelin (d18:1, C23:0) already at an early stage of heart failure. Interestingly, combining metabolic and transcriptional data from cardiac tissue revealed that decreased carnitine shuttling and transportation preceded mitochondrial dysfunction. We, thus, studied the therapeutic potential of OCTN2 (Organic Cation/Carnitine Transporter 2), an important factor for carnitine transportation. Cardiac overexpression of OCTN2 using an adeno-associated viral vector significantly improved ejection fraction and reduced interstitial fibrosis in mice subjected to TAC., Conclusion: Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in heart failure, which can be used for identification of novel biomarkers and potential therapeutic targets., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X(L) proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.