Published online before print June 4, 2009, doi: 10.1161/CIRCRESAHA.109.194928
© 2009 American Heart Association, Inc.
Integrative Physiology |
Cardiac Muscle Ring Finger-1 Increases Susceptibility to Heart Failure In Vivo
From the Carolina Cardiovascular Biology Center (M.S.W., J.C.S., E.G.H., P.C.C., C.P.) and Departments of Pathology and Laboratory Medicine (M.S.W., L.L., J.E.R.), Medicine (P.C.C., C.P.), Cell and Developmental Biology (C.P.), and Pharmacology (C.P.), University of North Carolina, Chapel Hill.
Correspondence to Monte S. Willis, MD, PhD, Carolina Cardiovascular Biology Center, University of North Carolina, 2340B Medical Biomolecular Research Bldg, 103 Mason Farm Rd, Chapel Hill, NC 27599-7525. E-mail monte_willis{at}med.unc.edu
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Abstract |
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Muscle ring finger (MuRF)1 is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates and heterodimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal, whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy, indicating cooperative control of muscle mass by MuRF1 and MuRF2. To identify the unique role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a nonprogressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by transaortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild-type mice despite the depressed contractility following TAC. In comparing the level and activity of creatine kinase (CK) between wild-type and MuRF1 Tg+ hearts, we found that mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts; however, total CK activity was significantly inhibited. We conclude that increased expression of cardiac MuRF1 results in a broad disruption of primary metabolic functions, including alterations in CK activity that leads to increased susceptibility to heart failure following TAC. This study demonstrates for the first time a role for MuRF1 in the regulation of cardiac energetics in vivo.
Key Words: muscle ring finger-1 • MuRF1 • ubiquitin ligase • cardiac hypertrophy • heart failure • creatine kinase
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Introduction |
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The muscle ring finger (MuRF) proteins are striated muscle–specific proteins that have been implicated in various aspects of contractile regulation and myogenic responses.1 MuRF1 is a well-characterized RING finger–dependent ubiquitin ligase that targets sarcomere proteins, such as cardiac troponin (cTn)I, during the process of skeletal muscle atrophy.2,3 MuRF1 has also been implicated in the regulation of cardiac myocyte size and contractility3–5 and inhibits the development of cardiac hypertrophy,6,7 a dynamic process commonly thought of as a precursor to heart failure.8 To date, the study of the regulation of cardiac muscle mass by MuRF1 has centered around its involvement in the regulation of sarcomere protein degradation. Although this is certainly an important function, in this report, we propose that MuRF1 operates in a broader capacity that encompasses both protein turnover as well as control of cardiac metabolism.
Soon after the discovery of MuRF1, the related proteins MuRF2 and MuRF3 were identified as interacting proteins capable of forming heterodimers with MuRF1.9 Subsequently, functional redundancy between the MuRF1 and MuRF2 proteins was suggested by yeast 2-hybrid studies that identified that MuRF1 and MuRF2 interact with many of the same sarcomeric proteins.10 The idea of functional redundancy was strengthened by the finding that mice lacking either MuRF1 or MuRF2 appear relatively normal, whereas mice lacking both MuRF1 and MuRF2 develop both cardiac and skeletal muscle hypertrophy.11 Although the studies involving these double knockouts clarified the importance of MuRF1 and MuRF2 in cardiac regulation, the individual roles that these 2 proteins play in vivo have yet to be ascertained because of a lack of helpful transgenic models (ie, single knockouts of these proteins have no cardiac phenotype at baseline, whereas double knockouts are lethal). To overcome this issue, we created transgenic mice with increased cardiac expression of MuRF1 to dissect out the functions of MuRF1 in the heart. Using this animal model, we confirmed that MuRF1 regulates cardiac function during the development of cardiac hypertrophy in vivo. More importantly, we identified that MuRF1 alters cardiac energy metabolism in vivo, in part, by inhibiting creatine kinase (CK) activity, a rate-limiting step in the phospho-creatine ATP shuttle in the heart. These results shed new light on the contributions of MuRF1 to cardiac function in vivo via the regulation of key enzymes necessary to distribute ATP throughout the cell.
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Materials and Methods |
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Experimental Design
MuRF1 Tg+ and littermate wild-type controls (50% male/50% female) at 10 to 12 weeks of age underwent transaortic constriction (TAC) to induce cardiac hypertrophy as previously described.7 No significant differences between sexes were noted throughout the study. MuRF1 Tg+ and wild-type controls underwent conscious echocardiographic analysis at baseline (before TAC) and then weekly for 4 weeks following the TAC procedure (10 mice/group). Additional mice (5/group) underwent conscious echocardiographic analysis at baseline and subsequently every 2 months for 18 months. M-mode and 2D echocardiography was performed using the Vevo 660 ultrasound system as previously described.7 Hearts were dissected from the body and perfused with 4% paraformaldehyde. Paraffin sections were stained with hematoxylin and eosin, Masson’s trichrome, or fluorescently labeled lectin as previously described.7 For cross-sectional area analysis of cardiomyocytes, TRITC-conjugated lectin (Triticum vulgaris) staining was performed and measured as previously described and examined by fluorescence microscopy.7
An expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
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Results |
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Mild Baseline Cardiac Changes Identified in MuRF1 Tg+Mice
In our first series of studies, MuRF1 Tg+ and wild-type littermates were monitored for survival and cardiac function by echocardiography for up to 18 months. Equal numbers of male and female MuRF1 Tg+ and wild-type offspring were identified at birth, indicating that the increased expression of cardiac MuRF1 did not affect developmental viability. No differences in overall cardiac mass were identified between adult MuRF1 Tg+ and wild-type littermate controls (heart weight to body weight ratio 5.8±0.2 versus 5.6±0.1 mg/g, respectively; Online Table I). However, by echocardiography, MuRF1 Tg+ mice displayed thinner anterior and posterior left ventricular wall thickness (19.3% and 19.8% less than wild-type mice, respectively, in diastole; Figure 1A, left and middle), an increase in left ventricular end diastolic dimension (9.9% greater than wild-type mice; Figure 1A, right), and a decrease in function as measured by fractional shortening percentage (FS%) (39.5±1.3 versus 54.5±0.5 in wild-type mice; Figure 1B, left). Serial echocardiography revealed that this mild phenotype did not progress with age. Functional assessment by cardiac catheterization revealed no functional differences between MuRF1 Tg+ and wild-type hearts (Online Table III), supporting the possibility that cardiac dysfunction did not precede decreases in CK activity in MuRF1 Tg+ hearts.
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Increased Cardiac MuRF1 Enhances Susceptibility to Heart Failure in Response to Pressure Overload
In our second series of studies, MuRF1 Tg+ and wild-type littermates underwent surgery to induce pressure overload by transaortic constriction (TAC) and were monitored for survival daily, with cardiac function analyzed weekly by echocardiography. Following TAC, no differences in survival between MuRF1 Tg+ and wild-type mice were identified for the duration of the 4-week study. MuRF1, but not MuRF2, mRNA levels significantly decreased to 40% of baseline in both MuRF1 Tg+ and wild-type mice during the progression of cardiac hypertrophy (Online Figure I, C and D). Although MuRF1 Tg+ mouse survival following TAC did not differ from littermate wild-type controls, MuRF1 Tg+ mice rapidly progressed to heart failure during this time (Figure 1A and 1B), while maintaining 20-fold levels of endogenous cardiac MuRF1 (Online Figure I, B). The mild baseline anterior and posterior wall thickness deficits seen in MuRF1 Tg+ mice become exaggerated (27.6% and 32.5% less than wild-type mice, respectively, in diastole) as did left ventricular dilation, with interventricular diameter increasing by 36% (Figure 1A, right) after TAC. The anatomic changes seen in MuRF1 Tg+ mice following TAC were accompanied by a rapid functional decompensation, with the degree of FS decreasing by 70% in these mice.
MuRF1 Tg+ Mice Develop an Eccentric Cardiac Hypertrophy After TAC
Typically, in response to pressure overload, a heart will develop "concentric" hypertrophy, characterized by an increase in cardiac mass attributable to a corresponding increase in cardiomyocyte cross-sectional area and wall thickness. Importantly, maintenance of function is preserved in this form of hypertrophy. Conversely, in "eccentric" hypertrophy, pressure overload causes an increase in cardiac mass accompanied by cardiomyocyte elongation, rather than increasing cross-sectional area. In the present study, following TAC, both MuRF1 Tg+ and wild-type mice developed cardiac hypertrophy as evidenced by increases in cardiac mass (heart weight/body weight, 8.3±0.5 versus 8.4±0.8 mg/g, respectively; Online Table I) and the upregulation of "fetal" genes associated with cardiac hypertrophy (brain natriuretic peptide, smooth muscle 
-actin, and β-myosin heavy chain) (Figure 1C). In wild-type mice, TAC induced a typical concentric hypertrophy with increased wall thickness and cardiomyocyte area (Figure 2A and 2B). However, MuRF1 Tg+ mice did not develop increases in wall thickness (Figure 2A) or cardiomyocyte cross-sectional area (Figure 2B) and had the characteristic loss of function associated with eccentric cardiac hypertrophy (FS% in Figure 1B; ejection fraction percentage in Online Table I). Apoptosis of cardiomyocytes often accompanies eccentric hypertrophy, adding to the overall deficit in contractility and heart function. However, analysis of TUNEL staining of sections of cardiac muscle taken from wild type and MuRF1 Tg+ determined no difference in the level of apoptosis between MuRF1 Tg+ and wild-type mice (Figure 2C). Therefore, increased loss of cardiac cells was not an apparent contributing factor to the development of heart failure in the MuRF1 Tg+ mice.
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MuRF1 Tg+ Mice Have Alterations in Cardiac Sarcomere M-Line
MuRF1 has been localized to the M-line and, to a lesser extent, the Z-disc of the sarcomere.9 Therefore, we sought to determine whether the loss of cardiac function observed in the MuRF1 Tg+ mice could be the result of defects in the structure of the cardiomyocyte contractile apparatus. Low-power transmission electron microscopy revealed no gross defects in sarcomeric or mitochondrial structure in MuRF1 Tg+ cardiomyocytes (Online Figure II, B). However, under high-power evaluation, MuRF1 Tg+ cardiomyocytes displayed a mild disruption in the M-line, which was exaggerated following TAC (Figure 2D). The dramatic deficits in cardiac function we observed in the MuRF1 Tg+ mice seem out of proportion to this relatively mild sarcomeric defect, leading us to consider additional causative factors leading to the heart failure in these mice.
Increased Cardiac MuRF1 Expression Is Not Accompanied by Increased Ubiquitin-Dependent Degradation of Sarcomeric Proteins In Vivo
We have previously demonstrated in vitro that MuRF1 recognizes and targets cTnI for degradation3 and that the degree of degradation is directly proportional to the amount of MuRF1 present in the system. It is possible that elevated levels of MuRF1 could affect steady-state levels of cTnI in the MuRF1 Tg+ mice, thereby leading to disruption of sarcomeric function and subsequent heart failure. On investigation, however, we found that steady-state levels of cTnI did not differ between MuRF1 Tg+ and wild-type hearts either at baseline or after TAC (Online Figure III, A). Similarly, steady-state levels of other sarcomere proteins reported to interact with MuRF1 (including cTnT, cTnC, telethonin, and myosin light chain [MLC]210) were also unaffected (Online Figure III, A and B). Furthermore, we detected no difference in cTnI mRNA levels between MuRF1 Tg+ and wild-type mice (Online Figure III, C), ruling out transcriptional compensation as a means by which protein levels of cTnI were maintained in MuRF1 Tg+ mice. Although these findings contrast sharply with our previous in vitro studies,3 the apparent lack of effect of increased MuRF1 levels on these sarcomeric proteins is consistent with our findings in the hearts of MuRF1–/– mice,7 where steady-state levels of these proteins also did not differ from their strain-matched wild-type controls, either at baseline (Online Figure IV) or after TAC (data not shown). These results suggest that, in the hearts of MuRF1 Tg+ mice, MuRF1 is not obviously triggering ubiquitin-dependent degradation of sarcomeric proteins. This surprising finding is the first indication that MuRF1 may be playing a novel, and as yet unidentified, role in regulating cardiac function, independent of its role as a ubiquitin ligase acting on sarcomeric proteins.
Genes Involved in Cardiac Energy Metabolism Are Differentially Expressed in MuRF1 Tg+ Hearts
Our physiological data indicate that MuRF1 Tg+ mice have only minor functional cardiac deficits at baseline (including a small decrease in both fractional shortening and ventricular wall thickness) but upon the induction of cardiac hypertrophy via TAC, an eccentric cardiac hypertrophy rapidly develops and leads to heart failure. Examination of the levels of apoptosis and sarcomere protein degradation in the MuRF1 Tg+ mice did not reveal any mechanisms underlying the observed heart failure. Therefore, to gain a more global view of how increased MuRF1 expression affects cardiac physiology, we used microarray analysis to identify changes in gene expression in MuRF1 Tg+ versus wild-type mouse hearts, both at baseline and during the development of cardiac hypertrophy (1 and 4 weeks following TAC). We identified 3 major patterns of differentially expressed genes between MuRF1 Tg+ and wild-type mice, designated cluster 1, 2, and 3 (Online Figure V and Online Table II).
Clusters 1 and 2 represent genes that were differentially expressed between the MuRF1 Tg+ and wild-type mice ONLY at 4 weeks after TAC (Online Figure V, A, outline in blue). The genes comprising cluster 1 exhibited decreased expression levels in both MuRF1 Tg+ and wild-type mice (red) after 1 week of TAC. However, whereas the expression level of these genes returned to baseline in wild-type mice by 4 weeks of TAC, in MuRF1 Tg+ mice, these genes continued to exhibit decreased expression levels. Of the 3 clusters identified, only cluster 1 yielded a distinct pattern to the signaling pathways represented by the genes in this group. Cluster 1 genes were comprised mainly of members of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontologies (GO) associated with generation of energy from oxidative phosphorylation and mitochondrial genes (Mrlpl28, Slc25a10, Mrps28, Dci, Mrpl46, Ndufb8, Ndufs4, Cox4l1, Ndufb5, Ndufv2, Sdha, Dlat, Atp5j, and Bcl2l11), as outlined in more detail in Online Figure V, B. The pathways represented by these genes include the MSigDB categories of mitochondria, oxidative phosphorylation, and peroxisome proliferator-activated receptor 
coactivator (PGC); the KEGG categories of oxidative phosphorylation and TCA cycle; and the GO categories of precursor metabolites and energy, coenzyme metabolism, and cofactor metabolism (see Online Figure V, B). Interestingly, the PGC pathway represents genes involved in PGC-1 signaling, which serve as inducible coregulators of nuclear receptors that control cellular energy metabolic processes.12
The genes identified in cluster 2 of our analysis were characterized by an increase in expression in both MuRF1 Tg+ and wild-type mice (red) after 1 week of TAC, which at 4 weeks of TAC decreased to baseline levels in the hearts of wild-type mice but remained elevated in MuRF1 Tg+ hearts (Online Figure V, A). Only a few gene pathway signals were identified in cluster 2, and these included genes in the MSigDB category PGC and lvad_heartfailure_up, consistent and overlapping with the metabolic pathways seen in cluster 1. Finally, cluster 3 was comprised of those genes that displayed increased expression in MuRF1 Tg+ mouse hearts compared to wild-type mouse hearts at any time point investigated (Online Figure V, A). Because these genes were consistently increased over time, they likely represent fundamental changes of the heart transcriptome brought about by increased MuRF1 expression, independent of the underlying cardiac hypertrophy seen in these mice. Analysis of cluster 3 genes using MSigBD identified genes in the category of mitochondria, PGC, and lvad_heartfailure_up and the GO categories of precursor metabolites and energy and energy from oxidation of organic compounds. The results of pathway analysis of the genes found to be differentially expressed in wild-type and MuRF1 Tg+ mice over the course of TAC strongly suggest that MuRF1 wields an effect on cardiac energy metabolism, both at baseline and after the development of cardiac hypertrophy. This realization led us to investigate next the ability of MuRF1 Tg+ hearts to forge metabolic adaptations after TAC.
Cardiac MuRF1 Does Not Affect Cardiac Glucose and Fatty Acid Utilization
Our microarray data supported the hypothesis that cardiac MuRF1 regulates key metabolic processes involved in oxidative phosphorylation, which is closely linked to ATP production. In addition, recent studies have identified that MuRF1 interacts with several proteins involved in glucose oxidation, including pyruvate kinase, aldolase A, and pyruvate dehydrogenase kinase.10 This prompted us to investigate the possibility that MuRF1 was involved in the regulation of glucose oxidation, because maladaptive shifts in glucose oxidation during the development of cardiac hypertrophy could explain the functional deficits seen in MuRF1 Tg+ hearts. Using 14C-labeled glucose or 14C-labeled oleate (a long chain fatty acid), we were able to quantify the efficiency with which whole heart homogenates from MuRF1 Tg+ and wild-type mice oxidized glucose and oleate at baseline and 4 weeks following TAC (Figure 3A and 3B). As expected, the hearts of wild-type mice used more oleate (fatty acids) than glucose at baseline, and shifted toward glucose utilization after TAC (Figure 3C). Surprisingly, however, MuRF1 Tg+ mice also used oleate and glucose to a similar extent (Figure 3A and 3B). This lack of differences in cardiac glucose and fatty acid utilization suggested that MuRF1 regulation of glucose oxidation is not a causative factor in the cardiac dysfunction seen in the MuRF1 Tg+ mice in response to pressure overload.
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MuRF1 Tg+ Hearts Maintain Total ATP Concentrations
Data obtained from our microarray analysis indicated that genes found in mitochondria and involved in oxidative phosphorylation including Ndufb8, Ndufs4, Ndufb5, Ndufv2, and Atp5j were significantly downregulated in the hearts of MuRF1 Tg+ mice. A decrease in ATP production could account for the deficient contractility exhibited by MuRF1 Tg+ hearts. However, analysis of cardiac ATP levels revealed that total ATP did not differ significantly between MuRF1 Tg+ and wild-type mice at baseline or 4 weeks following TAC (Figure 3D), indicating that MuRF1 Tg+ hearts progressively failed during pressure overload despite having adequate levels of ATP, suggesting that the deficit may in fact lie in the ability of cardiac MuRF1 hearts to use ATP. This led us to investigate whether or not MuRF1 Tg+ hearts displayed any maladaptations in pathways necessary for effective ATP usage.
Increased Cardiac MuRF1 Inhibits CK Activity In Vivo
In the heart, the phospho-creatine-ATP shuttle transports ATP throughout the cell to maintain ATP-dependent processes such as ion (ie, Ca2+, K+) transport through channels necessary for contractility. Vital to this ATP transport is the creatine kinase (CK) enzyme, which is responsible for the transport of ATP from the mitochondria to many areas of the cell through a phospho-creatine intermediate.13 A recent in vitro study suggested that CK is a MuRF1 substrate for ubiquitination,14 prompting us to investigate whether CK expression or activity was altered in the MuRF1 Tg+ mice. Indeed, we found that total CK activity was significantly depressed in MuRF1 Tg+ hearts compared to wild-type hearts at baseline. In addition, the normal increase in enzyme activity seen in wild-type mice following the induction of hypertrophy was considerably attenuated in the MuRF1 Tg+ hearts (Figure 4A).
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Several CK isoforms exist in the heart, which are able to form dimers, including CK-MM (
70% of total cardiac CK) and CK-MB (25% to 30% of total cardiac CK). A mitochondrial isoform (mtCK) also exists which is essential for the transport of ATP out of the mitochondria. Using antibodies that recognize each of the CK isoforms, we determined that MuRF1 Tg+ heart CK-MM/CK-MB and mtCK steady-state protein levels did not differ from wild-type hearts (Figure 4C and 4D), although increased levels of CK-M/B modification in MuRF1 Tg+ hearts was evident (Online Figure VI, C). The lack of decrease in CK isoform protein levels in MuRF1 Tg+ hearts was not attributable to compensatory increases in CK mRNA, because measurements of CK-M, CK-B mRNA levels of were equal in both MuRF1 Tg+ hearts and wild-type controls (Online Figure VI, A). Interestingly, however, quantitative analysis of 3 mitochondrial DNA components in MuRF1 Tg+ and wild-type mice identified a nearly 2- to 3-fold increase in the number of mitochondria in the MuRF1 Tg+ mice (Figure 4B and Online Figure VII). This finding of increased mitochondrial numbers in the MuRF1 Tg+ hearts without a concomitant increase in mtCK levels might suggest that mtCK is preferentially degraded by MuRF1, although further studies would be needed to validate this. Regardless, the fact that CK protein levels did not differ between MuRF1 Tg+ and wild-type hearts was a surprising finding given that we had detected a decrease in CK activity in these mice and previous studies had identified (in vitro) that CK is a target for MuRF1 ubiquitin ligase activity.3,14 These results prompted us to investigate how CK activity is differentially regulated in MuRF1 Tg+ hearts compared to wild-type hearts.
Our knowledge of the mechanisms by which CK activity is regulated is limited. Recent studies have identified that posttranslational modification of CK by AMP-activated protein kinase (AMPK) phosphorylation supports CK activity.15,16 In an effort to determine whether MuRF1 was possibly affecting CK activity by its affects on AMPK, we quantified the amount of phosphorylated AMPK in MuRF1 Tg+ and wild-type hearts at baseline and after 4 weeks of TAC (Online Figure VI, B). We identified that MuRF1 Tg+ hearts had a small, but significant, decrease in phosphorylation AMPK compared to wild-type mouse hearts. Although it remains to be determined whether or not this decrease in AMPK phosphorylation is attributable to imbalances in AMP/ATP levels secondary to heart failure, or attributable to other direct or indirect effects related to the increase in cardiac MuRF1 expression, these results suggest that MuRF1 regulates myocardial CK activity, possibly through its ubiquitin-mediated degradation and turnover of mtCK or other non–degradation pathways in which ubiquitin can regulate enzyme activity.
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Discussion |
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In this report, we demonstrate that MuRF1 in vivo is able to manipulate cardiac function through its effects on the regulation of cardiac metabolism. We identify for the first time that increased cardiac MuRF1 predisposes the heart to failure, possibly because of its attenuation of CK activity, the enzyme responsible for the transfer of ATP from the mitochondria to cellular compartments throughout the cell.
CK has recently been identified as a target substrate of MuRF1 ubiquitination.14,17 However, whereas these reports demonstrated the ability of MuRF1 to ubiquitinate CK, the in vitro platform on which these studies were performed did not allow investigations into whether or not this ubiquitination was preemptive of actual degradation of CK. In our study, we observed a decrease in CK activity in MuRF1 Tg+ hearts in the absence of any change in steady-state CK enzyme protein levels or compensatory increases in CK mRNA. However, there was evidence of increased mitochondrial number, consistent with our microarray findings of regulation of the PGC-1 pathways in the MuRF1 Tg+ mice. Although these results do not prove a direct causal relationship between increased MuRF1 expression and decreased CK activity, it is possible that MuRF1 is degrading the possible increased mtCK or that MuRF1 is regulating CK activity in a manner that is independent of a degradation pathway. The ubiquitination of substrates by ubiquitin ligases, such as MuRF1, can result in outcomes other than degradation, such as protein relocalization, protein repair/refolding, and cellular signaling (reviewed by Willis and Patterson18). Our laboratory recently identified that the E3 ligase atrogin-1 enhances the activity of the transcription factor FOXO by placing ubiquitin chains on it.19 Alternatively, the lack of degradation of CK proteins in the MuRF1 Tg+ mice could be attributable to a compensatory increase in deubiquitinating proteins. In mammalian genomes, there are more than 100 genes that encode putative deubiquitinating proteins, consistent with the diverse and specific functions they regulate.20–22 Our studies demonstrate that MuRF1 regulation of cardiac CK activity in vivo is more complex than the simple ubiquitin mediated degradation of a target. MuRF1 may play a role in regulating CK in a degradation-independent manner, or it is possible compensatory mechanisms are preventing the identification of MuRF1-mediated CK degradation.
Although further studies will be needed to identify the exact mechanisms involved, our results clearly demonstrate that MuRF1 plays a role in the largely unchartered area of the regulation of CK activity in vivo. Because CK is the key component of the ATP/phospho-creatine system,23 decreases in CK results in impaired transfer of ATP from the mitochondria to transporters, pumps, and enzymes in the cytosol and sarcomeres necessary to maintain cardiac function.23–25 In the present study, we identified that MuRF1 Tg+ hearts did have a decreased level of phospho (Thr172)-AMPK, which opens the possibility that MuRF1 may be influencing CK activity indirectly through AMPK. Although a complete understanding of how CK phosphorylation is necessary for CK activity is not yet known, phosphorylation by AMPK may be necessary for optimal activity. Hence, the effects of MuRF1 on CK-MM/MB and/or mtCK activity could, in part, underlie the functional deficits seen in MuRF1 Tg+ mice that are amplified on the induction of cardiac hypertrophy.
The decrease in cardiac CK activity, as seen in MuRF1 Tg+ mice in this study, has been associated with the development of heart failure. The effect of decreased CK activity in the heart has not been studied directly; however, cardiac function following the complete loss of cardiac CK isoforms has been reported. CK-deficient mice have a close to normal cardiac function at low or moderate workloads26 and have been reported to develop a spontaneous hypertrophy on a mixed but not pure background.27 However, with increased workloads, fiber kinetics were found to be impaired.26 By echocardiography, mice deficient in CK have normal cardiac function at baseline, but their response to β-adrenergic stimulation is blunted.28 These phenotypes parallel in many ways the near normal baseline cardiac function (determined by our catheter studies; Online Table III) we see in MuRF1 Tg+ hearts, which quickly decompensates on stress brought on by TAC. The attenuation of CK by MuRF1, therefore, may be largely responsible for the functional impairment of cardiac contractility of MuRF1 Tg+ mice. Although it is possible that MuRF1 effects on CK activity are not a result of direct interaction, the interaction of MuRF1 and CK has been identified in multiple in vitro studies.10,14,17 It cannot be assumed that MuRF1 effects on CK activity results from the cardiac dysfunction identified, because abnormal phospho-creatine energetics have been shown to precede, rather than are a consequence, of cardiac hypertrophy.29
Because we previously identified that MuRF1 interacts directly with troponin I and posttranslationally modifies it with ubiquitin to target it for degradation by the proteasome in vitro,3 it was surprising to identify that cardiac MuRF1 levels in vivo did not affect steady-state levels of cTnI in the present study. This finding might be explained by recent studies that have identified regulatory systems that prevent the ubiquitin–proteasome system from interacting with sarcomeric proteins.30,31 Briefly, activation of the calcium-activated proteins (calpains) have been implicated in the homeostatic turnover of sarcomere proteins.30,31 Increased cardiac calpain-1 in mice resulted in increased substrate-specific proteolytic activity and hyperubiquitination of cardiac proteins with increased 26S proteasome activity and accelerated protein turnover.30 Similarly, forced overexpression of cardiac calpastatin (a naturally occurring calpain inhibitor) had opposite effects.30 This study demonstrated that adenovirus transfection of cardiomyocytes activated physiological calpain-1,30 which would explain why our adenoviral mediated expression of MuRF1 resulted in degraded cTnI in vitro.3 The ubiquitin ligase–directed degradation of sarcomere proteins is part of a larger program of protein quality control just now being elucidated (recently reviewed by Willis et al32).
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Acknowledgments |
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We thank Robert Bagnell and Vicky Madden (Microscopy Services Laboratory of the University of North Carolina Department of Pathology & Laboratory Medicine) for assistance with the electron microscopy experiments. We thank Janice Weaver (University of North Carolina Animal Histopathology Laboratory) for assistance in preparing histological specimens.
Sources of Funding
This work was supported by National Heart, Lung, and Blood Institute grant R01HL065619 (to C.P.); the University of North Carolina Research Council (to M.W.); the University of North Carolina Foundation’s R. J. Reynolds Faculty Development Award (to M.W.); the Children’s Cardiomyopathy Foundation (to M.W.); and an American Heart Association Scientist Development Grant (to M.W.).
Disclosures
None.
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Footnotes |
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Original received June 5, 2008; resubmission received January 30, 2009; revised resubmission received May 22, 2009; accepted May 26, 2009.
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References |
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