Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

Article20 December 2007Open Access Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2 Christian C Witt Christian C Witt Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, GermanyThese authors equally contributed to the work Search for more papers by this author Stephanie H Witt Stephanie H Witt Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, GermanyThese authors equally contributed to the work Search for more papers by this author Stefanie Lerche Stefanie Lerche Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Dietmar Labeit Dietmar Labeit Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Walter Back Walter Back Institute of Pathology, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Siegfried Labeit Corresponding Author Siegfried Labeit Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Christian C Witt Christian C Witt Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, GermanyThese authors equally contributed to the work Search for more papers by this author Stephanie H Witt Stephanie H Witt Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, GermanyThese authors equally contributed to the work Search for more papers by this author Stefanie Lerche Stefanie Lerche Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Dietmar Labeit Dietmar Labeit Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Walter Back Walter Back Institute of Pathology, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Siegfried Labeit Corresponding Author Siegfried Labeit Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany Search for more papers by this author Author Information Christian C Witt1, Stephanie H Witt1, Stefanie Lerche1, Dietmar Labeit1, Walter Back2 and Siegfried Labeit 1 1Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany 2Institute of Pathology, Universitätsklinikum Mannheim, Mannheim, Germany *Corresponding author. Institute of Anesthesiology and Intensive Care, University Clinic Mannheim, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany. Tel.: +49 621 383 1626; Fax: +49 621 383 1971; E-mail: [email protected] The EMBO Journal (2008)27:350-360https://doi.org/10.1038/sj.emboj.7601952 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4–18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing. Introduction Striated muscle cells respond to changing functional requirements by a coordinated set of adaptations. For example, resistance training effectively remodels myofibrils, fiber type compositions, mitochondrial content, and muscle cell sizes (Seynnes et al, 2007), leading to body and skeletal muscle mass increases of 2–5 kg and strength gains of 5–20% within one week in athletes after optimal exercise and nutrition (for review, see Hartgens and Kuipers, 2004). Conversely, acute muscle unloading or starvation induces muscle protein catabolism (about 0.3% of calf muscle mass is lost per day in bed-rested patients (Rittweger et al, 2005), and up to 35% muscle loss in rats after 1 week of space flight (Fitts et al, 2001)). The trophic pathways promoting muscle growth and protein synthesis are modulated by a plethora of factors, including hormones, exercise, and myocellular stretch (for review, see Bassel-Duby and Olson, 2006), that enhance muscle tissue and myocytic cell growth. In vivo, these must be balanced by anti-anabolic signals that are less understood. Pathophysiologically, dominance of catabolism causes muscle wasting syndromes, for example, critical illness myopathies (Latronico et al, 2005), or blunted anabolism causes sarcopenia during ageing (Solomon and Bouloux, 2006). Because of their clinical importance, muscle catabolism-promoting factors are receiving increasing attention (for review, see Bodine, 2006), in particular, the ubiquitin ligases MAFbx/atrogin1 and MuRF1, because of their consistent upregulation during a variety of catabolic muscle states, including denervation, long-term immobilization, and microgravity (Lecker et al, 2004; Nikawa et al, 2004). Consistent with a critical role of MAFbx/atrogin1 and MURF1 for muscle catabolism, MAFbx/atrogin1 and MuRF1 knockout (KO) mice develop muscle atrophy more slowly after denervation (Bodine et al, 2001). The shared binding of MuRF1 and MuRF2 to a set of seven structural muscle proteins (including titin; see Witt et al, 2005) prompted us to investigate the functional relationship between MuRF1 and MuRF2 by mouse genetics. For example, titin may regulate the E3-ligase activities of MuRF1 and MuRF2 in an activity-dependent fashion: titin is a giant intrasarcomeric protein that makes up a myofibrillar spanning system, confers elastic properties to the myofibrils, and associates with numerous signaling molecules whose expression is muscle-activity dependent (for review, see Miller et al, 2003; Granzier and Labeit, 2004; Lange et al, 2005; Peng et al, 2005). Thus we generated MuRF1 and MuRF2 KO mouse models for testing whether MuRF1 and MuRF2 functionally cooperate. Both MuRF1 and MuRF2 KO mice have normal fertility and life span (consistent with Bodine et al, 2001; Willis et al, 2007). In contrast, MuRF1/MuRF2 double knockout (dKO) mice develop an extreme and lifelong muscle hypertrophy. The molecular basis for the cooperative nature of MuRF1/2 signaling is likely their combined regulation of a large shared set of ligands, including proteins required for myofibrillar stretch sensing, translation, and transcription factors. Cooperative MuRF1/2 signaling is emerging as an important pathway that coordinates myofibrils, ribosomes, and nuclear gene expression in myocytes when being stressed metabolically or biomechanically. Results Increase of heart organ and cardiac myocyte size after deletion of MuRF1 and MuRF2 Our MuRF1 and MuRF2 KO mouse models correspond both to constitutive null models (for details on gene targeting, see Supplementary Figure 1B–D). The obtained MuRF1 and MuRF2 KO mice that are homozygous null for MuRF1 or MuRF2 have normal fertility and are viable (consistent with Bodine et al, 2001; Willis et al, 2007). Also, deletion of MuRF1 or MuRF2 has no effect on life span until month 24 (our oldest MuRF1 or MuRF2 KO mice so far). Fertility of single MuRF1 or MuRF2 KO mice allowed the generation of dKO strains by breeding them together. Newborn dKO mice have a severe phenotype: 74% of mice homozygous for both MuRF1 and MuRF2 KO alleles die within the first 7–16 days of life. Dissection of these young dKO mice revealed grossly enlarged hearts that filled the entire mediastinum (Figure 1A and B). Figure 1.Synergistic control of heart muscle mass and cardiac myocyte size by MuRF1 and MuRF2. (A) Dissection of 13-day-old MuRF1 and MuRF2-dKO mice revealed grossly enlarged hearts (h), causing caudally lung compression (lu); the liver appears hyperaemic (li). (B) Effect of MuRF1/MuRF2 genotypes on heart weights. Left: hearts isolated from two matched pairs of WT and dKO mice (13 days old). Right: dKO mouse hearts (ventricles only) had 231% (P=0.001) increased HW/BW ratios, whereas weights of MuRF1, MuRF2 KO, and WT hearts did not differ significantly (young mice between d8 and d24; MuRF1: 25.5% increase, P=0.1, MuRF2: 27%, P=0.2, heart ventricles weights, respectively; dKO n=18; MuRF1 KO n=6, MuRF2 KO n=7; WT n=7). (C) Left: hematoxylin/eosin (HE) sections indicated that cardiomyocytes from young dKO hearts were hypertrophic. Scale bar, 20 μm. Right: morphometric comparison of WT and dKO sections nuclei had 59% increased length and cardiomyocytes were also 58% larger/cell density was reduced by 58% (as indicated by the number of cells in 0.2 mm2 large sections). (D) HE (top) and Masson histology (bottom) of WT and dKO hearts indicated concentric-type physiological hypertrophy at month 12, with intact inner and outer circular fiber systems and absence of fibrosis. Scale bar, 1 mm. Download figure Download PowerPoint Histology of mice that died spontaneously revealed microthrombi in the heart, edema in the lung, microbleedings, and compression of the neighboring organs (Supplementary Figure 2). Taken together, these pathological findings are consistent with death from chronic heart insufficiency and acute cardiac decompensation with heart failure. Morphometric analysis of single cardiac myocytes demonstrated 59% enlarged myocytes and 58% enlarged nuclei, both indicating cellular hypertrophy and explaining muscle hypertrophy at least in part (see Figure 1C). The effects of deleting MuRF1 and MuRF2 alleles on heart to body weight (HW/BW) ratios were synergistic: young MuRF1 or MuRF2 KO mice had 10 and 8% HW/BW increases (P=0.1 and 0.2 respectively), whereas hearts of dKO mice had 231% increased HW/BW ratios (P=0.001, <1 month; Figure 1B right). The histological studies of older mice revealed for dKO myocardium a concentric-type hypertrophy. No fibrosis by Masson stainings or myofibrillar disarray was observed at light microscopic level (Figure 1D bottom). The inner and outer layers of the myocardium could still be distinguished (Figure 1D). Taken together, the severe phenotype of dKO mice contrasts the absence of a noticeable phenotype in MuRF1 or MuRF2 KO mice and therefore demonstrates cooperativity of MuRF1 and MuRF2 on the genetic level. dKO mice develop and maintain skeletal and cardiac muscle hypertrophy throughout their life span Those dKO mice that survived the first two postnatal weeks became long-term survivors: All dKO mice alive at week 3 (n=27; 26% of total dKO offspring) were still alive at month 18 (unless being killed for our studies) and were able to have offspring. Adult dKO mice maintain cardiac hypertrophy (dKO: 84% increase, P=0.001; see Figure 2B). Consistent with this, electrocardiography indicated intact excitation conduction in dKO hearts from aged mice (see Supplementary Figure 3; contrasting, for example, the conduction blocks observed in Nkx2.5 KO mice; see Pashmforoush et al, 2004). Next, we examined the physiological cardiac performance by MRI imaging in more detail. This indicated massive persistent hypertrophy (see Figure 2A). Next, we estimated ejection fractions (EF) and stroke volumes by time-resolved MRI. This showed grossly reduced EFs (dKO: 0.2–0.3; wild type (WT): 0.7; see Figure 2A), whereas stroke volumes were 26% reduced under basal conditions. Figure 2.Phenotype of dKO mice at 18 months of age. Perinatally, dKO mice have a high mortality of 74%. The 26% of dKO mice (n=27) that survived became long-term survivors and were all alive at month 18 (unless killed, see below), thus allowing phenotypic studies in aged dKO. (A) MRI scans detected hypertrophic hearts in adult dKO mice with reduced EFs and stroke volumes (for time-resolved MRI scans, see also Supplementary Videos 4, 5, 6 and 7). LV, left ventricle; RV, right ventricle; EDV, end-diastolic volume; ESV, end-systolic volume (total number of mice scanned: two mice of each genotype. (B) Left: effect of MuRF1/2 genotypes on heart (ventricles, left) and quadriceps skeletal muscle (right) to body weight ratios. dKO mice maintain cardiac hypertrophy during ageing (dKO, 84% increase, P=0.001; MuRF1, 24.5% increase; MuRF2, 19%). In addition, dKO mice have 38.1% increased QW/BW ratios (P=0.001), whereas MuRF1 KO and MuRF2 KO genotypes have moderate effects on skeletal muscle mass (MuRF1, 16% increase, P=0.05; MuRF2, 11% increase, P=0.08). Despite increasing body mass in WT during months 4–18, skeletal muscles remain more hypertrophic in dKO mice during ageing (4–18 months of age, dKO n=27; MuRF1 KO n=52; MuRF2 KO n=81; WT n=49). For absolute weights, please refer to Supplementary Figure 8. Right: hematoxylin/eosin sections indicated that skeletal myofibers from dKO muscle show hypertrophic fibers with slightly augmented cross-section areas alternating with normal-appearing fibers (mice aged 4 months). Scale bar, 100 μm. (C) Weight gain of WT and dKO mice during ageing. Left: between months 4–18, dKO mice gain less weight (red; P<0.001) than WT. Right: dissections revealed that aged dKO mice were leaner (mice aged 18 months). Download figure Download PowerPoint Because dKO hearts support physiological circulation at least under non-challenged laboratory conditions for up to month 18, the effects of the absence of both MuRF1 and MuRF2 could also be studied in muscles of adult and aged animals. Similar to sustaining cardiac hypertrophy up to month 18 (our oldest dKO mice), MuRF1 and MuRF2 had also synergistic lifelong effects on skeletal muscle mass: after deletion of all four alleles, we found 38% increased quadriceps to body weight (QW/BW) ratios in dKO mice when compared with WT (P=0.001), whereas single MuRF1 or MuRF2 KOs had 17 and 11% increased QW/BW ratios (P=0.05 and 0.08, respectively; Figure 2B left). Similar as in heart muscle, skeletal muscle hypertrophy correlated with the hypertrophy of individual fibers (see histology of quadriceps muscle; Figure 2B right). Intriguingly, during ageing, body weights of dKO and WT mice progressively diverged: whereas ageing WT mice substantially gained weight (64% increase when comparing months 5 and 18), weight gains were attenuated in dKO mice (17, 27, and 35% differences at 12, 15, and 18 months, respectively (P=0.001), Figure 2C left). Dissections of senescent WT and dKO mice indicated that a striking lack of body fat accumulation in senescent dKO mice accounted for their reduced weights (Figure 2C right). This was apparently not linked to a general cachexia, because skeletal muscle hypertrophy was maintained (Supplementary Figure 8 right). Identification of binding partners shared by MuRF1 and MuRF2 and their convergent signaling on CARP, FHL2, and SQSTM1 Because muscle hypertrophy and the lean phenotype develop only after inactivation of all four MuRF1 and MuRF2 alleles, we searched for binding partners that are recognized by both MuRF1 and MuRF2 in an attempt to find molecular explanations for the phenotypic synergistic effects of MuRF1 and MuRF2 on muscle protein and lipid/energy metabolism. A total of 87 genes were identified as MuRF1 or MuRF2 interacting prey clones that coded for myofibrillar proteins (18, including 11 Z-disk proteins), transcriptional regulators (11), translation factors (4), and component of the mitochondrial proteome (including ATP-synthesis (9)). Of these 87 genes, a set of 35 genes was fished with both MuRF1 and MuRF2 baits and was further confirmed by mating studies. The group of ligands shared by MuRF1 and MuRF2 included a set of four myofibrillar Z-disk proteins and the transcriptional regulators CARP, myozenin1/calsarcin2, FHL2 (also associated with Z-disk region; for review, see Clark et al, 2002) (Figure 3A). To further test whether the Yeast Two-Hybrid (YTH) prey clones code indeed for MuRF1 and MuRF2 binding proteins, we performed in vitro pull-down studies using expressed CARP, myozenin1/calsarcin2 (two molecules selected as known transcriptional regulators of muscle gene expression), MRP-L41/pig3 (selected as a member of the mitochondrial ribosomal group, also being implicated in growth control; see Yoo et al, 2005), and EEF1G/EF-1γ (selected as a sophisticatedly regulated component of the translation machinery; see Belle et al, 1995) as well as its mitochondrial counterpart GFM1. Results indicated that a central MuRF1 fragment that comprises the MuRF1 residues 109–315 ('MuRF1Bcc'; see Supplementary Figure 1A) is both sufficient and required for interaction with CARP, EEF1G, GFM1, myozenin1/calsarcin-2, and pig3/MRP-L41 (Figure 3B). Similarly, expressed MuRF2Bcc interacted in vitro with CARP, EEF1G, and GFM1 (Figure 3B). Finally, YTH mating suggested that MuRF3Bcc does not interact with CARP, myozenin-1/calsarcin-2, and pig3/MRP-L41 (data not shown). Figure 3.MuRF1 and MuRF2 interact with a shared set of myocellular proteins. (A) YTH screens with full-length MuRF1 and MuRF2 baits of both human cardiac ('heart') and skeletal cDNA libraries ('SKM') fished a total of 87 genes. The table summarizes those 35 prey clones identified independently in both MuRF1 and MuRF2 screens and thus predicted to interact with both MuRF1+2: 13 prey clone inserts code for sarcomeric proteins (4 of which are components of the Z-disk), 10 code for transcriptional regulators (2 of which are also associated with the Z-disk), 5 genes are involved in mitochondrial ATP production, and 6 genes participate in translation initiation and elongation. Numbers indicate independently identified prey clones in respective screens. M=interaction was found by mating. An SRF prey clone fished with the MuRF1 bait could not be confirmed by mating, as in our hands the 3′ UTR and not the coding sequence of SRF activated yeast growth during mating with MuRF1 and 2. (B) The interaction of selected proteins derived from the above-mentioned genes was studied in vitro by pull-downs using expressed MuRF1/MuRF2 Bcc (B-Box+coiled-coil domain) and MuRF1cc (coiled-coil domain) constructs (see also Supplementary Figure S1 and methods). MuRF1cc and MuRF1Bcc (arrows) co-eluted together with CARP, EEF1G, GFM1 MBP fusion proteins. Below: left—MuRF1cc co-eluted with myozenin-1/calsarcin-2, and MRP-L41/Pig3 MBP-fusion proteins; right—MuRF2Bcc co-eluted together with CARP, EEFG1, GFM1 MBP fusion proteins; controls—MBP plus MuRF1cc, Bcc, MuRF2Bcc, respectively, or fusion proteins only. Download figure Download PowerPoint MuRF3 was recently shown to interact also with FHL2 and suggested to regulate its expression as an E3-ubiquitin ligase (Fielitz et al, 2007). Therefore, we tested next whether the expression of MuRF3 and FHL2 are affected in dKO mice. MuRF3 was expressed at normal levels in dKO mice (Array Express E-MEXP-1321), whereas the FHL2 protein was highly upregulated in dKO mice deficient for both MuRF1 and MuRF2 (Figure 5A). Thus MuRF1/2 signaling on FHL2 is cooperative and cannot be substituted by the related ubiquitin ligase MuRF3. Intriguingly, other catabolic factors, such as atrogin1, are expressed at normal levels in dKO myocardium (see Supplementary Table 9), suggesting that MuRF1/MuRF2 and atrogins are functioning in different pathways. In contrast, CARP and SQSTM1 (Sequestosome1/p62) became strongly upregulated only after inactivation of all four MuRF1 and MuRF2 alleles (Figure 5A). Gene expression profiling with Affymetrix system indicated that FHL2 and SQSTM1 mRNA levels are normal in dKO myocardium, and CARP is moderately upregulated (Supplementary Table 9). Therefore, upregulation of CARP, FHL2, and SQSTM1 in dKO hearts are primarily caused by post-transcriptional mechanisms. Impaired mitochondrial ultrastructure and alteration of Z-disks after deletion of MuRF1 and MuRF2 Because MuRF1 and MuRF2 interact with multiple components of the Z-disk and of the mitochondrium (Figure 3A), we studied the ultrastructural effects of the absence of MuRF1 and MuRF2 on Z-disks and mitochondria in myocardium by electron microscopy. We were unable to detect differences between WT, MuRF1, and MuRF2-KO myocardium (Figure 4A and B). In contrast, myofibrils in dKO myocardium were abnormal: myofibrils had more electron-dense Z-disks and were less regular (Figure 4C and D). Occasionally, myofibrils assembled in dKO hearts had free ends, somewhat reminiscent of growth tips found in proliferating skeletal myotubes (Ojima et al, 1999), and projected into regions rich in unassembled free filaments (not shown). Figure 4.Altered Z-disks and mitochondrial ultrastructure in dKO myocardium. (A, B) At 18 months, we noted no ultrastructural abnormalities in myocardium from WT (A), MuRF1-KO (B), and MuRF2 KO (not shown) mice. (C, D) In dKO myocardium, sarcomere lengths and myofiber alignments are less regular (maximal variation is 2.5-fold larger than in WT sarcomeres). Z-disks have a denser appearance (arrows). Vacuoles (V) are frequently found between or embedded within mitochondria (M). Mitochondria are less regular in shape including abnormally small mitochondria and are, unlike in WT, not tightly packed together. Scale bar, 1 μm. Download figure Download PowerPoint Mitochondria were less regular in shape and less orderly packed together. dKO myocardium also contained vacuoles, often embedded into mitochondrial clusters (Figure 4C and D). Because of the mitochondrial defects, we tested the expression of PGC-1-α (as a master gene for mitochondrial biogenesis; see Rasbach and Schnellmann, 2007). PGC-1-α transcription was not dysregulated in dKO myocardium (see Supplementary Table S9). Future studies are required to determine the molecular basis of altered mitochondrial numbers and structures in dKO myocardium. These ultrastructural changes were only present after inactivation of all four MuRF1 and MuRF2 alleles, again demonstrating that the MuRF1 and MuRF2 loci are genetically a complementation group. Elevated ANP and MLP stretch signals in dKO muscles implicate MuRF1/MuRF2 in stretch signal inhibition To gain insights into the mechanisms causing cardiac hypertrophy in dKO mice, we analyzed their transcriptomes by gene expression profiling. The transcriptional changes we found in dKO ventricles resembled those present in pressure induced aortic constriction (Zhao et al, 2004), including the upregulation of skeletal-type alpha actin 1, myosin light chains, atrial natriuretic peptide (ANP; isoforms A and B), and thrombospondin (see Supplementary Figure 9 and ArrayExpress accession E-MEXP-1321). Consistent with elevated stretch signaling, ANP was strikingly upregulated in dKO ventricles (Figure 5B). Normal to moderately elevated expression of other markers for cardiac hypertrophy suggested that the ANP induction was not a secondary consequence of heart failure and calcium overload (Figure 5A). SERCA2a (a marker for calcium overload during heart failure), serum response factor (SRF, previously suggested to transmit titin kinase/MuRF2-dependent stretch signals; Lange et al, 2005), and p38 MAPK (activated by the ERK/Map kinase pathway) were not affected by the absence of MuRF1 or MuRF2. In dKO myocardium, neither microarrays nor western blots showed an upregulation of SRF (Figure 5B and Supplementary Table 9). Figure 5.Characterization of altered signaling pathways in dKO myocardium by western blot studies. (A) Upregulation of the MuRF1/MuRF2 binding proteins CARP, FHL2, and SQSTM1 in dKO myocardium. Striking upregulation required the deletion of all four MuRF1/2 alleles, suggesting that both MuRF1 and MuRF2 synergistically control the transcriptional regulators CARP, FHL2, and SQSTM1. Below, cTnI and total multi-ubiquitinated protein species were not affected by the inactivation of MuRF1 and MuRF2 alleles. Among SUMO family members, we noticed for SUMO4 differential reactivity in the 8–30 kDa region. (B, C) Hyperactive stretch signaling in dKO as suggested by chronic upregulation of stretch-dependent signaling markers. (B) In myocardium, ANP is barely detectable in WT, MuRF1-KO, and MuRF2-KO hearts. ANP is strikingly upregulated in dKO myocardium (ventricles, 12 months old). Other markers for cardiomyopathy/hypertrophy remain normal or are moderately upregulated: SERCA2a (used as a marker for heart failure/calcium overload), SRF (previously implicated in stretch-dependent MuRF2 signaling), p38 Map kinase (a marker for ERK signaling and heart failure). (C) In dKO quadriceps muscles, hyperactive stretch signaling was suggested by the effect of 72 h immobilization on the stretch marker MLP: abnormally high levels of MLP/Crsp3 are maintained after 72 h immobilization in dKO quadriceps (NT=no treatment, byc=bycast immobilization). Download figure Download PowerPoint Based on the elevated ANP levels in dKO ventricles, we hypothesized that stretch signaling is augmented in dKO muscles because of the failure to attenuate it. To test this hypothesis in skeletal muscle, we immobilized dKO skeletal muscles by a bycast. MLP/Csrp3 is required for stretch-regulated responses in myocardium and a binding partner of the Z-disk-associated protein TCap (Knoell et al, 2002) and is also expressed in skeletal muscle (in contrast to ANP). Therefore, we monitored MLP/Csrp3 as a marker protein for stretch signaling. MLP/Csrp3, already elevated at basal conditions, remained highly elevated after a 72 h bycast immobilization in dKO (see Figure 5C). Taken together, our data demonstrate that combined inactivation of MuRF1 and MuRF2 leads to chronic upregulation of stretch signals in both heart and skeletal muscle and failure to downregulate them. Synergistic control of translational regulatory components by MuRF1/2 Possibly, the massive hypertrophic phenotype of dKO mice might be caused by reduced multi-ubiquitination and degradation of total muscle proteins (linked to inactivation of MuRF1/2 E3-ubiquitin-ligase activities). To test for reduced ubiquitination/degradation, we determined the levels of multi-ubiquitinated proteins on western blot panels displaying the different MuRF1/2 genotypes. Total levels of multi-ubiquitinated proteins did not change upon MuRF1/2 inactivation (Figure 5A). Among the family of ubiquitin-related modifiers, we only noted for SUMO4 an upregulation of SUMO4ylated species after MuRF1/2 inactivation (Figure 5A). Next, we speculated that, as an alternative mechanism, MuRF1 and MuRF2 might regulate the translational machinery directly (e.g., via interaction with INT6, EEF1G, GFM1; see Figure 3). When testing the myocardial expression of MuRF1/2-associated translation factors, we found that EEF1G and INT6 (subunits of EF-1 and elF3a, respectively; see Belle et al, 1995; Morris et al, 2007) were upregulated specifically after deletion of all four MuRF1/2 alleles (Figure 6A). Consistent with a general translational activation, we found upregulation of p70S6K (Figures 6A) and its activated phosphorylated form phospho-p70S6K, as well as its substrate phospho-S6 (markers for an activated Akt/mTor pathway; see Figure 6B). Phospho-p70S6K was recruited to the nucleus (Figure 6B), thus mimicking changes observed in exercise-induced translational activation (Koopman et al, 2006). For SRF, we noted no nuclear recruitment after MuRF1/2 deletions (Figure 6C). Figure 6.Elevated muscle protein synthesis after inactivation of MuRF1 and MuRF2. (A) The translation elongation factor EEF1G (interacting with MuRF1 and MuRF2; see Figure 3), its binding protein elF3a subunit INT6, and the translation-promoting p70S6 kinase are upregulated in dKO myocardium. (B) Immunohistochemistry with phospho-specific antibodies demonstrated the upregulation of the activated forms of p70S6K and its substrate S6 (phospho-p70S6K and phospho-S6, respectively) in dKO myocardium, suggesting activation of the AKT/mTOR pathway. Scale bar, 20 μm. (C) Nuclear entry of phospho-p70S6K was stimulated in dKO myocardium, whereas cellular distribution of SRF did not change significantly (data represent counts of 200 nuclei on 4HPF sections). (D) Fractional synthesis rate (% per 48 h) of total cardiac muscle proteins was determined by injecting D5-F i.p. into mice of each genotype (n=6). Total cardiac muscle protein synthesis is elevated in dKO myocardium (P=0.05 in dKO versus WT, mice aged between 6 and 16 months). (E) Serum level of creatinine was increased in dKO mice (n=6 in each group, P=0.01 in dKO versus WT mice, mice aged between 6 and 16 months). Download figure Download PowerPoint To test more directly for translational activation, we injected deuterium-labeled phenylalanine (D5-F) into dKO and control mice, allowing the determination of fractional de novo muscle protein synthesis by comparing D5-F to phenylalanine contents in muscle protein lysates (see, e.g., Dardevet et al, 2002). Incorporation of D5-F into dKO myocardium after 2 days was 46.6% higher than in WT myocardium (Figure 6D),