Cardiac hypertrophy is a major cause of human morbidity and mortality. Although much is known about the pathways that promote hypertrophic responses, mechanisms that antagonize these pathways have not been as clearly defined. Atrogin-1, also known as muscle atrophy F-box, is an F-box protein that inhibits pathologic cardiac hypertrophy by participating in a ubiquitin ligase complex that triggers degradation of calcineurin, a factor involved in promotion of pathologic hypertrophy. Here we demonstrated that atrogin-1 also disrupted Akt-dependent pathways responsible for physiologic cardiac hypertrophy. Our results indicate that atrogin-1 does not affect the activity of Akt itself, but serves as a coactivator for members of the Forkhead family of transcription factors that function downstream of Akt. This coactivator function of atrogin-1 was dependent on its ubiquitin ligase activity and the deposition of polyubiquitin chains on lysine 63 of Foxo1 and Foxo3a. Transgenic mice expressing atrogin-1 in the heart displayed increased Foxo1 ubiquitylation and upregulation of known Forkhead target genes concomitant with suppression of cardiac hypertrophy, while mice lacking atrogin-1 displayed the opposite physiologic phenotype. These experiments define a role for lysine 63-linked ubiquitin chains in transcriptional coactivation and demonstrate that atrogin-1 uses this mechanism to disrupt physiologic cardiac hypertrophic signaling through its effects on Forkhead transcription factors. IntroductionFactors that increase LV afterload - such as hypertension, aortic stenosis, and age-related arterial stiffness - elicit cardiac hypertrophy as an adaptive mechanism to normalize wall stress. The shortterm hemodynamic benefits of hypertrophy occur at a cost: cardiac hypertrophy leads to diastolic dysfunction and heart failure and is a powerful predictor of cardiovascular mortality even in the absence of symptoms (1, 2). At the cellular level, cardiac hypertrophy is a consequence of increased cardiomyocyte cell volume (1, 2), a process that requires coordination of cellular signaling cascades, activation of fetal cardiac gene expression programs, increased protein synthesis, sarcomere assembly, and modulation of cellular energy sources. At the present time, no specific pharmacologic strategies to reverse cardiac hypertrophy have been approved for clinical use, so the delineation of hypertrophic mechanisms (especially those that prevent or reverse hypertrophy) remains a priority.Although complexity and redundancy exist in the signaling pathways that activate cardiac hypertrophy, 2 independent circuits that elicit distinct manifestations of hypertrophy are now recognized. Hypertrophy in response to stimuli such as pressure overload and adrenergic stimulation activates the calcineurin/ nuclear factor of activated T cell-dependent signaling pathway, resulting in so-called "pathological" hypertrophy that is associated with maladaptive features such as fibrosis, chamber dilatation,

During the course of biological aging, there is a gradual accumulation of damaged proteins and a concomitant functional decline in the protein degradation system. Protein quality control is normally ensured by the coordinated actions of molecular chaperones and the protein degradation system that collectively help to maintain protein homeostasis. The carboxyl terminus of Hsp70-interacting protein (CHIP), a ubiquitin ligase/ cochaperone, participates in protein quality control by targeting a broad range of chaperone substrates for proteasome degradation via the ubiquitin-proteasome system, demonstrating a broad involvement of CHIP in maintaining cytoplasmic protein quality control. In the present study, we have investigated the influence that protein quality control exerts on the aging process by using CHIP ؊/؊ mice. CHIP deficiency in mice leads to a markedly reduced life span, along with accelerated age-related pathophysiological phenotypes. These features were accompanied by indications of accelerated cellular senescence and increased indices of oxidative stress. In addition, CHIP ؊/؊ mice exhibit a deregulation of protein quality control, as indicated by elevated levels of toxic oligomer proteins and a decline in proteasome activity. Taken together, these data reveal that impaired protein quality control contributes to cellular senescence and implicates CHIP-dependent quality control mechanisms in the regulation of mammalian longevity in vivo.

Protein quality control and metabolic homeostasis are integral to maintaining cardiac function during stress; however, little is known about if or how these systems interact. Here we demonstrate that C terminus of HSC70-interacting protein (CHIP), a regulator of protein quality control, influences the metabolic response to pressure overload by direct regulation of the catalytic α subunit of AMPK. Induction of cardiac pressure overload in Chip -/-mice resulted in robust hypertrophy and decreased cardiac function and energy generation stemming from a failure to activate AMPK. Mechanistically, CHIP promoted LKB1-mediated phosphorylation of AMPK, increased the specific activity of AMPK, and was necessary and sufficient for stress-dependent activation of AMPK. CHIP-dependent effects on AMPK activity were accompanied by conformational changes specific to the α subunit, both in vitro and in vivo, identifying AMPK as the first physiological substrate for CHIP chaperone activity and establishing a link between cardiac proteolytic and metabolic pathways. IntroductionPathological cardiac hypertrophy (subsequently referred to as cardiac hypertrophy) is an adaptive response to increased afterload brought about by hypertension, decreased arterial compliance, and other cardiac stressors (1). During the development of cardiac hypertrophy, numerous cellular processes come into play, including protein synthesis and proteolysis to account for the structural changes that accompany hypertrophy, as well as changes in cardiac metabolism to cope with increased energy demands. Despite the well-known fact that protein turnover and metabolic changes accompany cardiac hypertrophy, surprisingly little is known about how stress-dependent protein quality control mechanisms and metabolic regulation are coordinated, if at all, in the heart. C terminus of HSC70-interacting protein (CHIP, also known as STUB1) is a 35-kDa cytosolic protein initially identified in a screen for proteins that interact with the mammalian chaperones HSC70, HSP70 (2), and HSP90 (3). CHIP is ubiquitously expressed in mammalian tissues but is expressed at highest levels in the adult heart (2). CHIP plays an important dual role as both a cochaperone and a ubiquitin ligase (2-5). More recently, however, an additional role for CHIP in protein quality control has been suggested. Evidence that CHIP can act as an autonomous chaperone, independent of its association with HSPs, has been reported (6). However, these observations were made with a nonphysiological substrate (luciferase), and, to date, verification of CHIP's chaperone activity and identification of a physiological substrate have not been made.Changes in cardiac metabolism, such as oxidative substrate preference, mitochondrial function, and biogenesis, play a key role in the adaptive response to cardiac stress (7). As cardiomyocyte hypertrophy progresses, energy demand increases and cellu-

Rationale Among the extracellular modulators of Bmp (bone morphogenetic protein) signaling, Bmper (Bmp endothelial cell precursor-derived regulator) both enhances and inhibits Bmp signaling. Recently we found that Bmper modulates Bmp4 activity via a concentration-dependent, endocytic trap-and-sink mechanism. Objective To investigate the molecular mechanisms required for endocytosis of the Bmper/Bmp4 and signaling complex and determine the mechanism of Bmper’s differential effects on Bmp4 signaling. Methods and Results Using an array of biochemical and cell biology techniques, we report that LRP1 (Low density lipoprotein receptor-related protein 1), a member of the LDL receptor family, acts as an endocytic receptor for Bmper and a co-receptor of Bmp4 to mediate the endocytosis of the Bmper/Bmp4 signaling complex. Furthermore, we demonstrate that LRP1-dependent Bmper/Bmp4 endocytosis is essential for Bmp4 signaling, as evidenced by the phenotype of lrp1-deficient zebrafish, which have abnormal cardiovascular development and decreased Smad1/5/8 activity in key vasculogenic structures. Conclusions Together, these data reveal a novel role for LRP1 in the regulation of Bmp4 signaling by regulating receptor complex endocytosis. In addition, these data introduce LRP1 as a critical regulator of vascular development. These observations demonstrate Bmper’s ability to fine-tune Bmp4 signaling at the single-cell level, unlike the spatial regulatory mechanisms applied by other Bmp modulators.