Background-Cellular hypertrophy requires coordinated regulation of progrowth and antigrowth mechanisms. In cultured neonatal cardiomyocytes, Foxo transcription factors trigger an atrophy-related gene program that counters hypertrophic growth. However, downstream molecular events are not yet well defined. Methods and Results-Here, we report that expression of either Foxo1 or Foxo3 in cardiomyocytes attenuates calcineurin phosphatase activity and inhibits agonist-induced hypertrophic growth. Consistent with these results, Foxo proteins decrease calcineurin phosphatase activity and repress both basal and hypertrophic agonist-induced expression of MCIP1.4, a direct downstream target of the calcineurin/NFAT pathway. Furthermore, hearts from Foxo3-null mice exhibit increased MCIP1.4 abundance and a hypertrophic phenotype with normal systolic function at baseline. Together, these results suggest that Foxo proteins repress cardiac growth at least in part through inhibition of the calcineurin/NFAT pathway. Given that hypertrophic growth of the heart occurs in multiple contexts, our findings also suggest that certain hypertrophic signals are capable of overriding the antigrowth program induced by Foxo. Consistent with this, multiple hypertrophic agonists triggered inactivation of Foxo proteins in cardiomyocytes through a mechanism requiring the PI3K/Akt pathway. In addition, both Foxo1 and Foxo3 are phosphorylated and consequently inactivated in hearts undergoing hypertrophic growth induced by hemodynamic stress. Key Words: angiotensin Ⅲ calcineurin Ⅲ hypertrophy I n response to stress from neurohumoral activation, hypertension, or other myocardial injury, the heart initially compensates with an adaptive hypertrophic increase in mass. The resulting growth and remodeling response alters the balance between protein synthesis and protein degradation. In skeletal muscle, activation of progrowth signaling pathways is accompanied by deactivation of pathways that promote proteolysis. Prominent among the atrophy-inducing pathways are those governed by Forkhead box transcription factors, O subfamily (Foxo). Conclusions-This Clinical Perspective p 1168Foxo transcription factors regulate key physiological functions, including responses to stress, cell-cycle progression, protein degradation, and apoptosis. 1,2 There are 4 mammalian Foxo genes: Foxo1 (FKHR), Foxo3 (FKHRL1), Foxo4 (AFX), and Foxo6. The transcriptional activities of Foxo proteins are governed by posttranslational modifications such as phosphorylation and acetylation. With respect to myocyte growth and remodeling, Foxo proteins induce ubiquitin ligases and promote proteolysis in skeletal muscle. 3,4 In heart, a number of signaling cascades involving transcription factors, kinases, and G-protein-coupled receptors are implicated in the regulation of muscle growth (see reviews 5-7 ). Among these, the calcineurin/nuclear factor of activated T cells (NFAT) pathway has been shown to be a key signaling cascade that promotes cardiac hypertrophy. 8 It has been reported recently...

There is considerable interest in developing nonpeptidic, small molecule α-helix mimetics to disrupt α-helix-mediated protein-protein interactions. Herein, we report the design of a novel pyrrolopyrimidine-based scaffold for such α-helix mimetics with increased conformational rigidity. We also developed a facile solid phase synthetic route, which is amenable to divergent synthesis of a large library. Using a fluorescence polarization-based assay, we identified cell permeable, dual MDMX/MDM2 inhibitors, demonstrating that the designed molecules can act as α-helix mimetics.α-Helices represent one of the most common protein secondary structures and are involved in various protein-protein interactions (PPIs). In many PPIs, short helical peptides play an important role as a recognition motif, where side chains at i, i+3 or i+4, and i+7 positions often become a critical determinant for PPIs ( Figure 1A).1 Since α-helix-mediated PPIs are involved in a wide array of cellular signaling pathways, inhibition of these interactions could be promising therapeutic targets. While peptide-and peptidomimetic-based approaches have shown successful applications to such targets,2 non-peptidic, small molecules have advantages in terms of desirable bioavailability and cell permeability. Thus, there is considerable interest in developing non-peptidic, small molecule α-helix mimetics that can disrupt such PPIs. Hamilton and co-workers demonstrated that rationally designed terphenyl 1 and similar scaffolds can serve as α-helix mimetics ( Figure 1B).1e Following their pioneering work, a number of terphenyl-inspired structures have been reported.1e , 3 While some of these compounds have been shown to effectively disrupt certain PPIs, there are several important issues associated with terphenyl-related structures, such as low aqueous solubility, relatively flexible scaffold structure, and long synthetic routes. Herein, we report design of a novel class of small molecule α-helix mimetics, development of a facile solidphase synthetic pathway, and a subsequent high-throughput screen, which led to the identification of potent inhibitors that disrupt the interaction between p53 and MDMX/ MDM2.On the basis of the Hamilton's terephthalamide 2 ( Figure 1B),4 we designed a pyrrolopyrimidine-based scaffold 3 ( Figure 1C) with a hypothesis that the bicyclic ring in 3 could replace a pseudo-bicyclic structure formed by an intramolecular hydrogenbond in the terephthalamide 2. Thus, the planar heterocyclic framework could provide a pre-organized * hualu@iupui.edu .* limhyun@iupui.edu . structure with increased conformational rigidity. An energy-minimization study suggests that three functional groups on R 1 , R 2 , and R 3 of this scaffold can mimic the spatial orientation of the side chains of i, i+3 or i+4, and i+7 amino acids in an α-helix ( Figure 1A,C). In addition, the pyrrolopyrimidine template is expected to possess favorable physical properties including water solubility and cell permeability, similar to its structurally related purine ...

Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/ Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.Adult mammalian skeletal muscle is composed of multinucleated myofibers that can be classified based upon expression of one of four adult myosin heavy chain (MyHC) genes that contribute specific contractile properties (23). Muscle fiber types are identified as either type 1 (slow) or type 2 (fast) based upon velocity of contraction and rate of fatigue. Type 1 slow fibers express MyHC-1, are highly oxidative, and are rich in both mitochondria and myoglobin, which gives them their red color. Type 2 fast fibers can be further subdivided as 2A, 2X (also denoted 2D), and 2B based upon expression of MyHC-2A, MyHC-2X, and MyHC-2B, respectively. Type 2A fibers are oxidative and are rich in both mitochondria and myoglobin. In contrast, type 2B fibers are glycolytic and lack myoglobin. Type 2X fibers have an intermediate phenotype. In the mouse embryo, a pattern of fast and slow muscle fibers is established during several waves of myoblast fusion. Primary myofibers are formed during embryonic day 12 (E12) to E14 (18). A second wave of myotube formation occurs during E16 to E18. MyHC-1 is expressed both in the embryo and adult, whereas embryo-specific fast fiber isoforms, MyHC-emb and MyHCneo, are replaced by adult isoforms after birth.Postnatally, skeletal fiber-type composition is highly plastic and remodels in response to neuronal input and motor function in order to meet physiological demand (26). For instance, inactivity results in a general shift in MyHC expression and metabolic properties along the progression of 132A3 2X32B. Endurance training promotes a shift in the opposite direction, 2B32X32A31 (28). The calcium-activated protein phosphatase calcineurin has been proposed as an important regulator of muscle fiber type that activates the transcription f...

BackgroundMechanical assist device therapy has emerged recently as an important and rapidly expanding therapy in advanced heart failure, triggering in some patients a beneficial reverse remodeling response. However, mechanisms underlying this benefit are unclear.Methods and ResultsIn a model of mechanical unloading of the left ventricle, we observed progressive myocyte atrophy, autophagy, and robust activation of the transcription factor FoxO3, an established regulator of catabolic processes in other cell types. Evidence for FoxO3 activation was similarly detected in unloaded failing human myocardium. To determine the role of FoxO3 activation in cardiac muscle in vivo, we engineered transgenic mice harboring a cardiomyocyte‐specific constitutively active FoxO3 mutant (caFoxO3flox;αMHC‐Mer‐Cre‐Mer). Expression of caFoxO3 triggered dramatic and progressive loss of cardiac mass, robust increases in cardiomyocyte autophagy, declines in mitochondrial biomass and function, and early mortality. Whereas increases in cardiomyocyte apoptosis were not apparent, we detected robust increases in Bnip3 (Bcl2/adenovirus E1B 19‐kDa interacting protein 3), an established downstream target of FoxO3. To test the role of Bnip3, we crossed the caFoxO3flox;αMHC‐Mer‐Cre‐Mer mice with Bnip3‐null animals. Remarkably, the atrophy and autophagy phenotypes were significantly blunted, yet the early mortality triggered by FoxO3 activation persisted. Rather, declines in cardiac performance were attenuated by proteasome inhibitors. Consistent with involvement of FoxO3‐driven activation of the ubiquitin‐proteasome system, we detected time‐dependent activation of the atrogenes program and sarcomere protein breakdown.ConclusionsIn aggregate, these data point to FoxO3, a protein activated by mechanical unloading, as a master regulator that governs both the autophagy‐lysosomal and ubiquitin‐proteasomal pathways to orchestrate cardiac muscle atrophy.