The leading cause of morbidity and mortality worldwide is cardiovascular disease [1], frequently accompanied by the dysregulation of fatty acid metabolism associated with diabetes mellitus (DM). In the presence of DM, the morbidity and mortality of cardiovascular disease is increased and represents an important independent mechanism by which heart disease is exacerbated [2, 3]. The characteristic disturbances in myocardial energy and fatty acid homeostasis found in DM are mediated primarily by a network of peroxisome proliferator-activated receptor (PPAR) transcription factors that direct the energy substrates and determine the myocardial homeostasis [4, 5]. Chronic activation of PPARs in DM leads to an increase in free fatty acid uptake/oxidation corresponding to the level of insulin resistance in cardiomyocytes [6]. The increased reliance on fatty acid metabolism decreases the efficiency of the heart by increasing the amount of oxygen needed to create the needed energy, resulting in lipotoxicity [7]. The ligand (fatty acid)-driven activation of PPAR transcription factors regulate the expression of target genes, which control the uptake, utilization, oxidation, and storage of fatty acids [8]. In the heart, all three PPAR receptors have been identified (PPARα, PPARδ/β, and PPARγ) and implicated in cardiovascular disease [9].

Insulin resistance is a risk factor for left ventricular (LV) dysfunction and heart failure and is one of the hallmarks of type 2 DM [10]. Despite hyperinsulinemia and hyperglycemia, the diabetic heart relies almost exclusively on fatty acid utilization [11] in both rodent models and humans with excessive fat intake [12]. The resulting increase in fatty acid increases reaction oxygen species (ROS) production and accumulation of lipid intermediates [e.g. diacylglycerol (DAG)], which have a profound impact on insulin signaling [13]. The c-JUN NH 2-terminal kinase (JNK) and inhibitor κB kinase (IKK), activated by ROS [14, 15], parallel activation of protein kinase C (PKC) by DAG, all of which act to down-regulate insulin action by preventing insulin receptor substrate-1 (IRS-1) phosphorylation [13]. High systemic fatty acid uptake also inhibits Akt signaling, resulting in the downregulation of forkhead box O (FOXO) transcription factors [16, 17], while the increased ROS activate nuclear factor kappa B (NF-κB) [18], both of which contribute to the development of cardiac hypertrophy [12, 19].

The muscle ring finger (MuRF) family of ubiquitin ligases, including MuRF2 (Trim55), was identified in 2001 as a highly homologous group of proteins that homo- and hetero-dimerize through their coiled-coil domains [20]. This family of proteins is found in striated muscle, including skeletal and cardiac myocytes and was originally found to be a critical regulator of microtubule assembly during models of skeletal muscle development [21, 22]. Recent studies have detailed the importance of MuRF2 in the earliest stages of skeletal muscle differentiation and myofibrillogenesis in vivo [23]. In the present study, we identify that endogenous cardiomyocyte MuRF2 inhibits multiple PPAR isoforms, primarily PPARγ (but to a lesser extent PPARδ/β and PPARα). Given the relative importance of PPARs in the development of diabetic cardiomyopathy and the downstream pathophysiology, we challenged MuRF2−/− mice to a 60% fat diet-induced cardiomyopathy recently described [24, 25]. With PPAR signaling at the center of regulating fatty acid oxidation and mediating the pathogenesis of type 2 DM induced cardiomyopathy, we hypothesized that if MuRF2−/− hearts had enhanced PPAR signaling, they would undergo an accelerated cardiomyopathy due to MuRF2’s direct regulation of PPAR activity. We identified that MuRF2−/− hearts undergo an exaggerated diabetic cardiomyopathy, resulting from MuRF2’s multi-ubiquitination of PPARα and PPARγ1 in a proteasome-independent (non-degradatory) mechanism. These studies identify the first ubiquitin ligase to regulate PPAR via post-translational ubiquitination.

We have recently identified that MuRF2, a muscle-specific ubiquitin ligase, is a critical factor that regulates cardiomyocyte size during development in concert with MuRF1 [40]. MuRF2 has also been described as the effector protein in the Titin-nbr1-p62 complex that responds to mechanical changes in the sarcomere to inhibit transactivation of the nuclear transcription factor serum response factor (SRF) [41]. MuRF2’s regulation of the nuclear specific SRF was the first indication that MuRF2, found primarily in the cytoplasm, could regulate the activity of nuclear receptors, presumably through direct interaction, ubiquitination, and apparent nuclear export [41]. These findings led us to hypothesize that MuRF2 similarly regulates other nuclear receptors critical to cardiomyocytes. To test this, we used MuRF2−/− mice previously characterized without a cardiac or skeletal muscle phenotype [26]. However, we recently identified that MuRF2−/− hearts exhibited changes in metabolomics signatures, indicating that changes in metabolism are present despite any functional effect at baseline [37]. We initially assayed isolated nuclei from MuRF2−/− hearts for their DNA-binding activity contributed by PPARα, PPARβ, and PPARγ as the PPARs have been best described in altering cardiac metabolism [42] and have been reported to be regulated by ubiquitination [43]. To our surprise, we found that MuRF2−/− hearts had significantly increased PPAR activities, with increases in PPARα (+twofold), PPARβ (~1.6 fold), and PPARγ (over +fourfold) activities compared with sibling MuRF2+/+ control mice (Fig. 1a). These findings suggested that endogenous MuRF2 attenuated the activity of all three PPAR transcription factors found in cardiomyocytes. Since MuRF2 is an ubiquitin ligase and PPAR transcription factors have been described with post-translational modification by ubiquitin, we hypothesized that MuRF2 may regulate these PPAR transcription factors in a ubiquitination-dependent manner.

The pathogenesis of diabetic cardiomyopathy involves the enhanced activation of PPAR transcription factors [44]. Diabetic cardiomyopathy is characterized by increased free fatty acid oxidation in parallel with cardiomyocyte insulin resistance [6]. Both the resulting increased fatty acid oxidation and insulin contribute to the decreased ability for the heart to switch away from fatty acid utilization to glucose [45]. Since cardiac MuRF2−/− mice exhibited enhanced PPAR activity, we hypothesized that the induction of diabetic cardiomyopathy would result in both enhanced PPAR activity, resulting in significant cardiac dysfunction compared with wildtype mice. To test this hypothesis, we challenged the MuRF2−/− mice to a 60% high-fat diet, which reproducibly induces insulin resistance and diabetic cardiomyopathy (Fig. 1b) [24].

After 26 weeks of high fat diet challenge, MuRF2−/− mice had significantly lower blood glucose compared with sibling wildtype controls but no differences in serum insulin levels (Additional file 1: Figure S1a). Serum triglycerides were similarly elevated in MuRF2−/− and wildtype controls after 26 weeks of high fat diet (Additional file 1: Figure S1b). Increased cardiac MuRF2 protein levels were identified after high fat diet (Fig. 1c), paralleling MuRF2 increases identified in human inflammatory dilated cardiomyopathy (http://www.ncbi.nlm.nih.gov/geoprofiles/26614376) and coronary artery atherosclerosis (http://www.ncbi.nlm.nih.gov/geoprofiles/16462729). MuRF2−/− hearts increased total weight more than wildtype controls in high fat diet challenge (Fig. 1d) in addition to having significant increases in overall body weight at any 15, 22, and 25 weeks of HFD (Fig. 1e). However, no significant changes were identified in gastrocnemius, soleus, or tibialis anterior muscles weights (Additional file 1: Figure S1c).

Echocardiographic analysis of MuRF2−/− hearts at baseline found no deficits in function or differences in measurements (Fig. 2a; Table 1) as previously described [40, 46]. Significant deficits in heart function were identified in the MuRF2−/− hearts in as little as 6 weeks after the initiation of high fat diet (Fig. 2a, upper left panel). MuRF2−/− hearts were significantly thinner than MuRF2+/+ hearts from 15–26 weeks of high fat diet feeding (Fig. 2a, upper middle panels, Fig. 2b, d). Both MuRF2−/− and wildtype mice experienced an equal progressive dilation over time on a high fat diet, evidenced by increases in LVESD (Fig. 2a, far right panel). MuRF2−/− hearts exhibited significant dysfunction as early as 6 weeks of HFD compared to MuRF2+/+ hearts (Fig. 2b). MuRF2−/− hearts were significantly larger than MuRF2+/+ hearts after 26 weeks high fat diet (Fig. 2b–d). Diabetic cardiomyocyte-related changes in myosin heavy chain gene expression were next investigated to determine differences between groups. Comparable increases in βMHC were seen in MuRF2−/− and wildtype hearts (Fig. 2e), consistent with previous studies identifying these increases [47]. MuRF2−/− cardiac expression of skeletal muscle α-actin and αMHC were increased in chow control hearts compared to wild type mice, and MuRF2−/− skeletal muscle α-actin was significantly increased as compared to wild type mice after 26 weeks high fat diet (Fig. 2e). No difference existed in αMHC after 26 weeks of high fat diet in either MuRF2−/− or MuRF2 +/+ mice (Fig. 2e), although this is reported in other models of diabetic cardiomyopathy [48, 49]. Brain natriuretic protein (BNP) mRNA was decreased in both MuRF2−/− and controls after 26 weeks high fat diet feeding (Fig. 2e). Taken together, these studies identified that MuRF2−/− hearts failed sooner than MuRF2+/+ hearts, resulting in larger hearts, including LV wall thickness and heart weights after 26 weeks high fat diet challenge.

	MuRF2+/+ Baseline, N = 10 (1)	MuRF2−/− Baseline, N = 11 (2)	MuRF2+/+ 6 weeks High Fat Diet N = 11 (3)	MuRF2−/− 6 weeks High Fat Diet N = 11 (4)	MuRF2+/+ 12 weeks High Fat Diet N = 11 (5)	MuRF2−/− 12 weeks High Fat Diet N = 12 (6)	MuRF2+/+ 15 weeks High Fat Diet N = 11 (7)	MuRF2−/− 15 weeks High Fat Diet N = 12 (8)	MuRF2+/+ 22 weeks High Fat Diet N = 10 (9)	MuRF2−/− 22 weeks High Fat Diet N = 12 (10)	MuRF2 +/+ 26 weeks High Fat Diet N = 10 (11)	MuRF2 −/− 26 weeks High Fat Diet N = 12 (12)
AWTS (mm)§	1.79 ± 0.04	1.77 ± 0.03	1.82 ± 0.07	1.69 ± 0.05	1.88 ± 0.08	1.63 ± 0.06$	1.89 ± 0.05	1.68 ± 0.06$	1.87 ± 0.07	1.72 ± 0.07	1.85 ± 0.09	1.61 ± 0.04$
LVEDD (mm)	2.85 ± 0.17	3.27 ± 0.16	3.07 ± 0.12	3.05 ± 0.08	2.97 ± 0.09	3.02 ± 0.09	3.08 ± 0.14	2.94 ± 0.12	2.99 ± 0.10	3.05 ± 0.12	3.12 ± 0.10	3.34 ± 0.05
PWTS (mm)§	1.65 ± 0.04	1.59 ± 0.03	1.66 ± 0.05	1.42 ± 0.04$	1.71 ± 0.08	1.42 ± 0.02$	1.72 ± 0.09	1.65 ± 0.04	1.65 ± 0.04	1.65 ± 0.06	1.65 ± 0.12	1.65 ± 0.05
LV Mass (mg)§	105.1 ± 8.1	123.7 ± 7.6	121.4 ± 9.4	111.4 ± 44.2	120.5 ± 7.2	103.9 ± 5.5	153.6 ± 14.5¶	102.7 ± 4.9$	153.6 ± 15.4¶	130.2 ± 7.7¶	156.6 ± 12.5¶	125.7 ± 4.1$
LV Vol;d (μl)	32.5 ± 4.7	44.6 ± 4.8	37.9 ± 3.8	37.0 ± 2.4	34.6 ± 2.6	36.2 ± 2.5	38.7 ± 4.0	34.40 ± 3.6	37.1 ± 3.7	35.4 ± 2.9	39.1 ± 3.2	45.5 ± 1.7
LV Vol;s (μl)§	4.0 ± 0.9	6.5 ± 0.8	5.5 ± 0.8	9.6 ± 0.9¶,$	5.6 ± 0.7	9.7 ± 0.8¶,$	7.3 ± 1.4	9.1 ± 1.4¶	6.7 ± 1.1	9.8 ± 0.7¶,$	12.9 ± 1.9¶	14.6 ± 1.1¶
BW (g)§	20.9 ± 1.6	22.9 ± 1.2	24.5 ± 1.2	26.2 ± 1.4	29.1 ± 1.5¶	32.0 ± 1.8¶	31.7 ± 1.4¶	34.3 ± 1.7¶	33.4 ± 1.9¶	36.9 ± 2.3¶	36.2 ± 2.1¶	39.1 ± 2.3¶
HR (bpm)§	609 ± 19	590 ± 18	671 ± 8¶	636 ± 8$	658 ± 13¶	654 ± 14¶	681 ± 1¶	666 ± 13¶	665 ± 11¶	648 ± 13	667 ± 12¶	668 ± 9¶

High-resolution transthoracic echocardiography performed on conscious MuRF2−/− and age-matched wild type mice at baseline, 6, 12, 15, 22, and 26 weeks high fat diet. Data represent mean ± SEM. A One Way ANOVA was performed between all groups, followed by Holm-Sidak Multiple Comparisons vs. MuRF2+/+ baseline. A Student’s t test was then run to compare MuRF2−/− to wildtype control at the same time point.

HR heart rate, ExLVD external left ventricular diameter, bpm heart beats per minute, AWTD anterior wall thickness in diastole, AWTS anterior wall thickness in systole, PWTD posterior wall thickness in diastole, PWTS posterior wall thickness in systole, LVEDD left ventricular end-diastolic dimension, LVESD left ventricular end-systolic dimension, FS fractional shortening, calculated as (LVEDD-LVESD)/LVEDD × 100, EF% ejection fraction calculated as (end Simpson’s diastolic volume − end Simpson’s systolic volume)/end Simpson’s diastolic volume × 100, ND not determined.

^§ p < 0.05 by One Way ANOVA.

^¶ p < 0.05 vs. MuRF2+/+ Baseline by Multiple Comparisons.

^$ p < 0.05 vs. time-matched MuRF2+/+ (Student’s t test).

LV remodeling is a distinctive finding in the pathogenesis of diabetic cardiomyopathy. These changes include the development of fibrosis, resulting from the accumulation of extracellular collagen [50, 51]. Reduced MMP2 activity [52] and O-GlcNAcylation stimulated cardiac fibroblast collagen synthesis has been reported [53]. In this particular model, less than 2% fibrosis was identified throughout the heart in MuRF2−/− and wildtype controls (Fig. 3a). However, MuRF2−/− hearts revealed a parallel reduction in vimentin-positive fibroblasts (Fig. 3b). Throughout the course of the study, only one mouse died at 21 weeks of high fat diet. This wildtype mouse, interestingly, revealed almost 4% fibrosis (Additional file 2: Figure S2c) with amorphous waxy infiltrates and leukocyte infiltrates (Additional file 2: Figure S2b) not seen in either MuRF2−/− or wildtype hearts after 26 weeks high fat diet (Additional file 2: Figure S2a). Overall, while MuRF2−/− hearts have significant increases in fibrosis, the total fibrosis is minimal and does not account for the large changes in cardiac size, dysfunction, and suggests other non-structural signaling pathways likely are involved in the MuRF2−/− exaggerated cardiac dysfunction in diabetic cardiomyopathy.

Cardiac PPARα, PPARβ, and PPARγ1 have pivotal roles in the pathophysiology of diabetic cardiomyopathy [44]. Therefore, we next investigated the expression of cardiac PPAR isoform regulated genes previously described in vivo [54–56]. Gene expression of the cardiac PPARα target genes (not regulated by cardiac PPARβ i.e. glut1 and cd36) (Fig. 4a), cardiac PPARβ target genes associated with glucose metabolism (not regulated by cardiac PPARα, i.e. glut4, pfk, acc1, mcad, and lcad) (Fig. 4b, c), and cardiac PPARγ1-regulated cardiac genes (i.e. acox1, adrp, cpt1b, and pdk4) (Fig. 4d) were evaluated in MuRF2−/− mouse hearts. Notably, MuRF2−/− hearts challenged with high fat diet exhibited significantly increased levels of PPARα-regulated genes (Fig. 4a), PPARβ-regulated genes associated with fatty acid metabolism (Fig. 4c), and PPARγ1-regulated genes (Fig. 4d). MuRF2−/− hearts did not differ from MuRF2+/+ hearts in PPARβ-regulated target genes associated with glucose metabolism (glut4 and pfk, Fig. 4c). Like the PPAR isoform activities assays of the MuRF2−/− heart nuclei demonstrated, MuRF2−/− hearts exhibited enhanced PPAR activities. At the mRNA level, MuRF2−/− hearts exhibited significant increases in PPARα compared with wildtype mice, but no differences in PPARβ or PPARγ1 (Additional file 3: Figure S3).

Fatty acids are the primary fuel of the heart in addition to being ligands for the PPAR transcription factors. High fat diets have been reported to increase cardiac triglyceride content [57]. The increased storage fat (as myocardial triglyceride) that occurs in the development of type 2 diabetic cardiomyopathy has been hypothesized as one mechanism that free fatty acids are toxic to the heart [58–60]. The mishandling of cardiac glycogen is also a frequent manifestation of diabetic cardiomyopathy [61]. We hypothesized that increased levels of fatty acid in the MuRF2−/− hearts could contribute to the enhanced heart failure they demonstrated in diabetic cardiomyopathy. Since MuRF2 has been reported in the heart and skeletal muscle, in addition to the liver, we measured cardiac triglycerides and glycogen after 26 weeks of high fat diet to determine if alterations in these storage forms of fat and glucose could be contributing to the increased heart weight or dysfunction in the MuRF2−/− hearts (Fig. 5). Compared to the control feeding, both MuRF2−/− and MuRF2+/+ hearts had increased cardiac triglyceride levels (Fig. 5a). However, MuRF2−/− hearts did not have significantly different triglyceride levels compared with wildtype after 26 weeks high fat diet feeding. MuRF2−/− liver and skeletal muscle after 26 weeks high fat diet feeding was similarly not significantly different from wildtype controls (Fig. 5a). MuRF2−/− hearts from dietary controls (chow) had significantly decreased cardiac glycogen compared with wildtype hearts (Fig. 5b). While MuRF2−/− hearts accumulated significantly increased glycogen after 26 weeks high fat diet, the increases MuRF2−/− liver and skeletal muscle accumulated did not reach significance (Fig. 5b). Together, these studies illustrate that the MuRF2−/− hearts are able to store fat (as triglyceride), but have alterations in glycogen storage capacity both at steady state (baseline) conditions and after high fat diet challenges. Akt and glycogen synthase kinase (GSK)-3β are reported to be decreased in diabetic cardiomyopathy, along with increases in fibrosis and inflammation [48, 62].

Recent studies have demonstrated a role for PPAR activation in developing adiposity and weight gain in models of diabetes. In one study, treatment with rosiglitazone in mouse models of diabetes was shown to promote increases in cardiac size and enhanced fat volume [63]. Similarly, rosiglitazone side effects in patients have revealed increasing fat gain [64]. At baseline, MuRF2−/− mice had comparable fat and lean body mass as wildtype controls (Fig. 5c). However, during the development of insulin resistance, MuRF2−/− mice demonstrated significantly more fat mass at 6 and 12 weeks of high fat diet, but no changes in lean body mass (Fig. 5c). While the specific mechanisms by which rosiglitazone regulates fat mass is not completely clear, the enhanced PPAR activities seen in MuRF2−/− mice may be one contributing factor to the accumulation of fat mass during which cardiac function is significantly worse than wildtype mice challenged in parallel with a high fat diet.

The post-translational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) in diabetes is a result of the excess glucose that drives the reaction. O-GlcNAc, in concert with ubiquitin, mediates several aspects of diabetic cardiomyopathy [53, 65–68]. O-GlcNAc modified proteins impair cardiomyocyte calcium cycling via its direct effects on phospholamban [68, 69]. O-GlcNAcylation also blunts autophagy, down regulates Nkx2.5 expression, and stimulates cardiac fibroblast collagen synthesis to mediate cardiac dysfunction [53, 65, 66]. Therefore, we measured the amount of O-GlcNAc proteins in MuRF2−/− heart, hypothesizing that the loss of MuRF2 cleared fewer O-GlcNAc-modified proteins, to mediate the enhanced cardiomyopathy seen in vivo. Immunoblot analysis of O-GlcNAc-modified proteins in MuRF2−/− hearts demonstrated no differences from wildtype hearts when mice were fed a chow diet or 26 weeks of high fat diet (Additional file 4: Figure S4). While modest increases in O-GlcNAc levels were identified after 26 weeks of high fat diet, as expected with the observed hyperglycemia, differences in O-GlcNAc could did not appear to contribute to the exaggerated MuRF2−/− cardiac dysfunction.

Since NF-κB signaling, defective insulin signaling, JNK signaling, and alterations in autophagy have been implicated in the pathogenesis of diabetic cardiomyopathy [19, 70–72], we next determined if MuRF2−/− hearts had alterations in these processes that may explain their more severe phenotype. After 26 weeks of high fat diet, MuRF2−/− hearts did not exhibit enhanced NF-κB activity (determined by p-p65 western blot), decreased IRS-1 signaling (determined by p-IRS-1 western blot), or alternations in JNK signaling (determined by p-cJun) (Additional file 5: Figure S5a). Similarly, measures of cardiac autophagy in MuRF2−/− hearts after high fat diet did not differ from wildtype controls, including autophagy flux (LC3II/LC3I proteins ratio post-bafilomycin treatment by western blot), p62, or VPS34 protein levels by western blot (Additional file 5: Figure S5b). These studies demonstrated that the more severe MuRF2−/− phenotype was not due to alterations in NF-κB, insulin, or JNK signaling or reductions in autophagy that have been reported to result in more severe diabetic cardiomyopathy [19].

Evidence from a variety of cell culture studies have implicated ubiquitin as a post-translational modifier of PPAR transcription factors and their coreceptors/co-activators [43]. These have been in liver, lung, fibroblast, adipocytes, and macrophage (as recently reviewed [43]). These studies have found that the ubiquitin-mediated inhibition of PPAR isoforms PPARα, PPARβ, and PPARγ are: 1) ligand-dependent (ligand is required for ubiquitination and/or degradation to occur); and 2) the ratio of ubiquitin ligase (e.g. MDM2 [73]) determines activation (e.g. MDM2:PPARα ratio < 1) or inhibition (e.g. MDM2:PPARα > 1 [73]). Since considerable evidence shows that MuRF2−/− hearts enhance PPAR-activity suggesting that endogenous cardiac MuRF2 inhibits PPAR activities by nuclear PPRE-binding (Fig. 1a) and PPAR-regulated gene expression (Fig. 4), we next focused on how the muscle-specific ubiquitin ligase MuRF2 might exert its inhibitory effects based on our current knowledge of how ubiquitin regulates PPAR in cancer cells.

Like other ubiquitin ligases, MuRF2 interacts with a number of protein substrates. Notably, MuRF2 and MuRF1 redundantly interact with roponin-I (TnI), TnT, myosin light chain 2, and T-cap (telethonin) in yeast two-hybrid studies [74]. Unlike MuRF1, MuRF2 has not been shown to degrade any of these substrates (as recently reviewed [75] ). But critical regulation of microtubule, intermediate filament, and sarcomeric M-line stability during striated muscle development [22] and regulation of E2F activity [40]. Understanding that high fat diet induced MuRF2 expression, we next identified PPARα, PPARβ, and PPARγ1 (as the PPARγ2 isoform is restricted to adipocytes) (Fig. 6a). Interestingly, in steady state conditions, cardiac PPARα and PPARα protein levels in MuRF2−/− mice did not differ compared with wildtype controls. However, PPARγ1 levels were slightly (and significantly) increased at baseline (Fig. 6a, far right). After challenge with PPAR ligands (free fatty acids from high fat diet) for 26 weeks, no differences in MuRF2−/− cardiac PPARα and PPARγ1 were identified by immunoblot analysis, but a significant increase in PPARβ protein expression was identified (Fig. 6a). Taken together, these studies illustrate that the steady state levels of cardiac PPARα and PPARγ1 isoforms are not affected by the presence of MuRF2 or its increase (Fig. 1c) after high fat diet challenge. Moreover, these results suggest that MuRF2’s changes in PPARα and PPARγ1 activities could be due to one of the multiple non-canonical post-translational modifications by ubiquitin (e.g. mono-ubiquitination) that are not associated with proteasome dependent and degradation. How MuRF2 is regulating PPARβ without being able to ubiquitinate it directly (Fig. 6f) is unclear. But the mechanism would be indirect include the possibility that MuRF2 it targeting the inhibition of a yet to be determined ubiquitin ligase(s) that normally degrades PPARβ. For example, PPARβ in cancer cells (HEK293 and NIH3T3) is ubiquitinated and degraded in a ligand (GW501516)-dependent manner [76]. While the identification of the ubiquitin ligase targeting PPARβ is not known at this time, ubiquitin ligases degrading other isoforms (e.g. PPARγ) have been reported in adipocytes (MKRN1) [77]. Conversely, MuRF2 ubiquitination could be enhancing a de-ubiquitinase (DUB) that prevents proteasome-mediated degradation by this unidentified E3(s).

We next sought to determine the underlying mechanism by which endogenous MuRF2 exerted inhibition on PPAR-regulated genes (Fig. 4). Based in the limited work performed on PPAR ubiquitination (as recently reviewed [43]), we hypothesized that the ratio of MuRF2 to the substrate may regulate whether the protein was degraded in a proteasome-dependent manner, as previously reported in cancer with MDM2:PPARα ratios [73]. Increasing the MuRF2:PPARγ1 ratios resulted in a dose-dependent decrease in steady state protein levels, consistent with poly-ubiquitination and subsequent degradation (Fig. 6b). To determine the role of the proteasome in this process, we next repeated these experiments and found that the MuRF2-mediated decrease in PPARγ1 could be prevented by adding the proteasome inhibitor MG132 (Fig. 6d). Since previous studies have reported that ubiquitin ligase mediated proteasome degradation of PPARs is ligand dependent (as recently reviewed [43]), we next repeated these studies in the presence and absence of PPARγ ligand rosiglitazone, demonstrating that MuRF2’s dose-dependent degradation of PPARγ1 was ligand dependent (Fig. 6d). To establish that MuRF2 interacts with PPARγ1, we performed immunoprecipitation studies by co-transfecting cells with HA-MuRF2 or HA-MuRF2∆Ring (lacking the ubiquitin ligase region) and FLAG-PPARγ1 (Fig. 6c). Immunoprecipitating PPARγ1, we identified that MuRF2 bound PPARγ1 by immunoblots (Fig. 6c). Unexpectedly, MuRF2∆Ring did not bind PPARγ1 in parallel studies suggesting MuRF2’s Ring Finger domain has structural importance in the interaction with PPARγ1.

In vivo, the cardiac MuRF2 protein levels increased ~30% in wild type mice (Fig. 1c), while steady state levels of PPARα, PPARβ, and PPARγ1 were either increased (PPARα, PPARγ1) or unchanged (PPARβ) in wildtype mice in response to 26 weeks of high fat diet compared to chow-fed wildtype controls (Fig. 6a). With no evidence that cardiac MuRF2 affected steady state PPARγ1 isoform protein levels yet inhibited PPARγ1 activity in vivo (MuRF2−/− hearts had enhanced PPARγ1 activities), we next tested how MuRF2 may be inhibiting PPARγ1 mechanistically. Specifically, we wanted to determine why the physiological relevance of MuRF2-mediated degradation (with MuRF2:PPARg1 at levels 10:1) in vivo did not appear relevant in the context of diabetic cardiomyopathy. The experimental studies indicating that high MuRF2:PPARγ1 ratios resulted in ligand-dependent proteasome degradation may be relevant in other disease processes where MuRF2 levels are increased more in vivo. However, such a disease process has not been described to date. Therefore, we focused our studies of MuRF2-mediated ubiquitination of PPAR isoforms using ratios of 2:1 (Fig. 6e–g). Whereas, MuRF2 did not appear to add poly-ubiquitination leading to degradation, MuRF2 drove multi-mono-ubiquitination on PPARα and PPARγ1. Specifically, MuRF2 di-mono-ubiquitinated PPARα, while adding ~four ubiquitin moieties to PPARγ1 (Fig. 6e, g, respectively). PPARβ, however, was unexpectedly not modified by ubiquitin at all in vitro. These studies add perspective to our initial findings that endogenous cardiac MuRF2 had the greatest regulation of PPARγ, with MuRF2−/− hearts exhibiting 400%+ PPARγ activity found in the sibling wildtype hearts (Fig. 1a, far right). Endogenous MuRF2 similarly had the next most inhibition of PPARα, with MuRF2−/− hearts exhibiting ~250% PPARα activity found in sibling wildtype hearts (Fig. 1a, far left). Quite surprising was the finding that MuRF2 did not ubiquitinate PPARβ (Fig. 6f), despite MuRF2 hearts exhibiting 80% more activity than wildtype controls (Fig. 1a, middle).

To gain more insight on how MuRF2 may be inhibiting transcriptional activity by ubiquitination, we next performed nuclear localization studies using confocal microscopy (Fig. 7). In control cells, we found that PPARγ1 could be found in both the nucleus and cytosol, with most cells having primarily nuclear localization (81%) (Fig. 7a). Increasing MuRF2 (2:1 ratio of PPARγ1 transfected) interestingly resulted in an increase in the “perinuclear” localization of PPARγ1 (Fig. 7b). Notably, MuRF2 co-localized to these perinuclear regions (Fig. 7b). Parallel studies using the MuRF2 without its ubiquitin ligase activity (∆RING-MuRF2) abrogated the perinuclear targeting of PPARγ and colocalization with MuRF2 (Fig. 7c). Since we demonstrated that MuRF2, but not MuRF2∆Ring, bound to PPARγ1 (Fig. 6c), these studies indicates that MuRF2 regulation of PPARγ1 location may lie in its ubiquitin ligase activity and/or through some structural role required for interaction since MuRF2’s Ring Finger domain is required to bind PPARγ1 (Fig. 6c). Taken together, these studies suggested that MuRF2 targets an ubiquitin-mediated regulation of PPARγ1 activity by altering its localization within the nucleus, paralleling recent studies demonstrating autophagic sequestration of receptors in the endoplasmic reticulum and nucleus [78].

By non-targeted metabolomics analysis, MuRF2 hearts had significant decreases in taurine, myoinositol, and four metabolites involved in malate-aspartate shuttle (glycerol-1phosphate, urea, malic acid, and phosphoric acid) [37]. In the present study, we similarly analyzed MuRF2−/− hearts using non-targeted metabolomics analysis after 26 weeks of high fat diet (Fig. 8). The separation of MuRF2−/− hearts from wildtype was clear using Principal Components Analysis (PCA) (Fig. 8a) as well as Partial Least Squares Discriminant Analysis (PLS-DA) and Variable Interdependent Parameters (VIP) analysis (Fig. 8b). Among all of the annotated metabolites (Fig. 8c), the VIP significant analytes detected were taurine, sucrose, glyceric acid, 3-hydroxyflavone, pantothenic acid, and glutamic acid, among others (Fig. 8b). Enrichment analysis identified the (1) urea cycle; (2) aspartate metabolism; and (3) taurine and hypotaurine metabolism to be the highest fold enriched by metabolite sets (Fig. 8d). Based on location, the mitochondria, peroxisome, and lysosome were the most enriched (Fig. 8e). Pathway analysis identified (1) taurine and Hypotaurine metabolism; (2) glycine, serine, and threonine metabolism; and (3) alanine, aspartate, and glutamate metabolism as the pathways most significantly affected when both t test and VIP significant metabolites were analyzed (Additional file 6: Figure S6).

While the present study demonstrates a role for the muscle specific ubiquitin ligase in regulating PPAR transcription factors in the context of diabetes, the clinical implications of these findings are broader. Recent studies have identified reductions in MuRF2 in patients with diabetic ischemic heart failure (GEO ID: 87403976, http://www.ncbi.nlm.nih.gov/geoprofiles?term=87403976). Rare MuRF2 mutations in patients with familial hypertrophic cardiomyopathy have also been identified that are associated with a greater LV wall thickness than those without MuRF2 mutations [79]. Genome wide identification of SARS-CoV susceptibility loci using the collaborative cross has identified MuRF2 as a susceptibility factor to SARS infection, including evidence that MuRF2−/− mice are more susceptible (R.S. Baric, personal communication and GEO accession no. GSE64660, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64660). Taken together, these studies suggest a role for MuRF2 in an increased susceptibility to disease. Given the growing evidence that PPARs have anti-inflammatory activity by competitively inhibiting NF-κB and AP1 (cJun/cFos) experimentally (recently reviewed by Lockyer et al. [80]), it is possible that reduced (or ablated) MuRF2 in patients may increase their susceptibility to infection in addition to diabetes as we demonstrate in the present study.

The dynamic regulation of PPAR transcription factor activity reflects the complexity found routinely in biology. PPAR family transcription factors routinely partner with RXR to regulate transcription of target genes, but co-factors such as PGC-1a enhance PPAR activity (induced by exercise) and mitochondrial biogenesis, which nuclear receptor corepressor 1 (NCOR1) antagonizes these activity (as recently reviewed by Fan and Evans) [81]. PPAR activity is also regulated by ligands which bind the ligand binding domain and include endogenously free fatty acids, eicosinoids, and environmental compounds [82]. In addition to the various co-complex partners that regulate PPARs, there are other transcription factors that compete with PPARs to regulate their activity, notably NF-κB, making enhanced PPAR activity an anti-inflammatory state [80]. It is on top of this complexity that post-translational modification by ubiquitin and SUMO has been reported primarily in cancer cells, and recently by our group in cardiomyocytes for the first time [83]. In our recent review on this topic, few ubiquitin ligases have been described, but ubiquitination and proteasome-dependent degradation of PPAR as well as SUMOylation has been reported in PPARα, PPARβ, and PPARγ1 in primarily non-physiological cultured conditions. The report here of the first ubiquitin ligase regulating PPARγ1 by ubiquitination in a physiological process and cardiac pathophysiological process (diabetes) adds detail to a dimension of regulation we’ve just begun to understand.

These layers of complexity are apparent in the present study. While our initial studies revealed that MuRF2−/− hearts had enhanced PPARα, PPARβ, and PPARγ1 (most prominently PPARγ1) activity measured by binding of PPRE DNA (Fig. 1a). However, these enhanced activities were clearly not due to increased total protein levels of PPARα and PPARγ1 (Fig. 6a). Similarly, PPARα and PPARγ1 were multi-mono-ubiquitinated as a mechanism to explain their inhibition (Fig. 6e, g), while PPARβ was not ubiquitinated by MuRF2 (Fig. 6f). Like previous studies from our laboratory, the nuclear localization after MuRF protein post-translational ubiquitination may be regulating this process with PPARγ1 (Fig. 7) [83]. However, the indirect regulation of PPARβ protein levels (enhanced in MuRF2−/− hearts) is complex. The increase in PPARβ protein levels is not transcriptionally regulated (Additional file 3: Figure S3b), leading to our hypothesis that MuRF2 regulates PPARβ through complex post-translational mechanism(s) as described above (e.g. MuRF2 degradation of E3(s) targeting PPARβ or MuRF2 enhancing DUBs targeting PPARβ). Similarly complexity may lie with MuRF2’s regulation of PPARα as PPARα mRNA levels are enhanced in MuRF2−/− hearts throughout this study (Fig. 3a). The regulatory elements in the promoter region of PPAR include activator protein-1 (AP-1) and how MuRF2 may inhibit these proteins is not clear [84, 85]. MuRF2−/− mice have equivalent PPARγ1 protein (Fig. 6a) and PPARγ1 mRNA (Additional file 3: Figure S3c). However, MuRF2−/− hearts also have the most highly activated PPARγ1 activity by several measures (Figs. 1a, 4d), indicating that the strong post-translational multi-mono-ubiquitination in vitro (Fig. 6g) may be MuRF2’s primary mechanism of regulating PPARγ1 in vivo.

MuRF2 has previously been shown to regulate two nuclear transcription factors found in myocytes, paralleling its role in regulating nuclear PPAR isoforms in the present study. Initial studies investigating the role of MuRF2 in differentiated myocytes identified that MuRF2 binds critical signaling regions of the giant protein titin (via titin kinase region), to interact and regulate the activity and localization of the nuclear transcription factor SRF [41]. However, cardiac MuRF2 did not appear to regulate SRF activity in vivo, when stimulated by known SRF activating pressure overload-induced cardiac hypertrophy [26], suggesting this regulation is disease context specific. Subsequently, MuRF2 has been implicated in redundantly regulating (with MuRF1) other nuclear transcription factors implicated in developmental physiological hypertrophy [40]. In both cases, no evidence of MuRF2-mediated degradation was identified, consistent with the degradation-independent regulation of PPAR isoforms in the present study. The most commonly reported regulation of PPAR isoforms from the cancer literature has identified that post-translational modification with ubiquitin and SUMO appears to regulate PPAR most commonly to inhibit their regulation.

The post-translational regulation of PPAR by ubiquitin has previously been identified in cancer cells. However, ubiquitin-regulation of PPARs in the heart has not. Similarly, specific ubiquitin ligases have not been identified in these processes prior to this identification of MuRF2. In contrast to prior studies where ubiquitination was ligand dependent and resulted in PPAR degradation (as recently reviewed [43]), here we identified that the MuRF2:PPARγ1 ratio determined if degradation occurred (Fig. 6b) and that the physiologically relevant non-degradation of PPARα, PPARβ, and PPARγ1 (Fig. 6a) resulted from multi-mono-ubiquitination of PPAR isoform substrates (Fig. 6e, g).

The ubiquitin ligase:substrate ratio effects on ubiquitination chain type has been studied extensively in cancer with MDM2’s regulation of p53 [86–90]. In these series of elegant studies, investigators identified that low levels of MDM2 induced mono-ubiquitination and nuclear export of p53, whereas high levels of MDM2 promoted poly-ubiquitination and nuclear degradation of p53 [90]. In the context of stress, Li et al. endorsed the notion that non-stressed cells regulated p53 by mono-ubiquitination to circumvent the more extensive investment in energy the poly-ubiquitination and degradation require [89]. Conceptually, this increased energy expenditure may be worthwhile during stress considering that the bigger degradation response leads to apoptosis, whereas mono-ubiquitnation does not [89]. These studies also highlight the highly dynamic process of transcription factor regulation at the post-translational level [87]. Subsequent studies identified that SUMOylation, a process paralleling ubiquitination, can regulate the strength of the MDM2-p53 interaction and participates in the nuclear export [88]. This process appears to involve the stepwise interplay between SUMOylation and ubiquitination of p53 [86]. Very much like p53, all three PPAR isoforms are both SUMOylated and ubiquitinated, so future studies investigating the possible role of MuRF2-regulation of PPAR isoform ubiquitination may take this into consideration. The requirement of SUMOylation for ubiquitination to occur may also explain MuRF2’s apparent regulation of PPARβ activity (Fig. 1a, middle frame) in vivo, but absence of PPARβ ubiquitination in vitro (Fig. 6f) with demonstrable MuRF2 activity but lack of SUMO and/or other interacting proteins.

Another unexpected finding in the present study is the multi-mono-ubiquitination of PPARα and PPARγ1 proteins identified in the in vitro ubiquitination assays (Fig. 6e, g, respectively). In cancer cells, the ubiquitin ligase 14ARF has been reported to di-ubiquitinate p53 in a manner which inhibits MDM2, another 14ARF substrate [91]. Like previous reports of multi-ubiquitinated (e.g. mono- and di-ubiquitination) substrates [92–94], di- or tri-ubiquitination of PPAR does not lead to its degradation in the physiological conditions. Interestingly, 14ARF induces p53-dependent SUMOylation in its target substrates, including MDM2 and NPM, in addition to ubiquitinating the protein [91]. MuRF2’s multi-ubiquitination may offer additional clues into the complex regulation of cardiac PPAR isoforms previously unknown.

We previously identified that MuRF2−/− hearts exhibited alterations in taurine, aspartic acid, and d-malic acid in vivo compared to strain-matched wildtype hearts [37]. In the present study, we expanded these findings in MuRF2−/− hearts after 26 weeks high fat diet to illustrate that differences in taurine, sucrose, glyceric acid, 3-hydroxyflavone, pantothenic acid, and glutamic acid (Fig. 8b). Alternations in taurine and hypotaurine metabolite sets (Fig. 8d) in the MuRF2−/− hearts are interesting given the emerging role of taurine on chronic heart failure. Taurine is an abundant amino acid that influences the heart’s response to stress. It is one of the most abundant amino acids in the left ventricle, acting as an osmoregulator to trigger osmotic preconditioning, a process that activates Akt-dependent cytoprotective signaling [95]. The loss of taurine can depress protein synthesis and reduce energy reserves after cardiac surgery and has been found to be preserved [96]. Specifically, taurine has been shown to attenuate oxidative stress and alleviate heart failure in diabetic rates [97]. Supplementation of taurine in patients with heart failure has been used clinically [98, 99]. Our understanding of cardiac taurine biology is limited, but regulation of taurine by the taurine/Na+ symport is believed to play an important functional role in heart failure and replacement an emerging practice in Japan. It’s role in diabetic cardiomyopathy, in particular, has been found to reduce AGE, oxidized LDL by scavenging malondialdehyde, and hypochlorous acid and downstream HClO-dependent NO reduction [100].

At least three ubiquitin ligases, namely RNF5, TRAF6, and Nedd4 have been described as regulators of autophagy components ATG4B and Beclin1 in non-cardiovascular systems [101]. With the growing appreciation of E3s in regulating autophagy and MuRF2’s interactions with the autophagy-related Nbr1, p62, and LC3 proteins during cardiac myofibril assembly and turnover [41, 102], it was surprising that MuRF2−/− cardiac autophagy was not affected differentially after the high fat diet challenge (Additional file 5: Figure S5b). Despite these provocative parallels, no previous studies of MuRF2’s regulation of autophagic flux have been described, and in the present studies, the lack of endogenous MuRF2 did not affect autophagic flux after high fat diet challenge (Additional file 5: Figure S5b). Similarly, steady state cardiac p62 protein levels were unaffected by the lack of endogenous MuRF2 (Additional file 5: Figure S5b), indicating that cardiac MuRF2 may not have a role in cardiac autophagy or that other MuRF family proteins, such as MuRF1 which has been described in multiple processes [37, 40], are functionally redundant and is compensating in the MuRF2−/− model. As emerging evidence that autophagy plays a role in the pathogenesis of diabetic cardiomyopathy by clearing post-translationally modified proteins, such as advanced glycation end products and the severity of disease [19], targeting autophagy may offer one therapeutic pathway [103]. In the present study, we did not identify that endogenous MuRF2 was protective via this pathway, however.

A host of changes have been described in diabetic cardiomyopathy, characterized by cardiac hypertrophy, inflammation, fibrosis, and apoptosis due to altered insulin signaling and calcium handling [104]. The MuRF family ubiquitin ligases, including MuRF1 and MuRF2 have shown to be critical regulators of cardiomyocyte growth and atrophy. Specifically, both physiological and pathological growth has been attributed to MuRF1 and MuRF2 in the heart [40, 46, 105] and skeletal muscle [106], while MuRF1 regulation of cardiac [106] and skeletal muscle atrophy [107, 108]. While the changes seen in diabetic cardiomyopathy are vast, including alternations in metabolism, structural proteins, signal transduction, and ion channels [109], the crucial role of enhanced PPAR signaling has been central to the pathogenesis of this disease downstream of altered insulin resistance [12, 44]. Regulation of PPAR activity, including post-translational modification-mediated regulation, is a process little understood in any cell type, including the cardiomyocyte. The findings of the current study implicate the first cardiac specific ubiquitin ligase that functionally regulates PPAR isoform signaling, by ubiquitination, inhibiting a central pathway in the pathogenesis of disease. Since the regulation of PPARs are dynamic during the course of diabetic cardiomyopathy [110–113], these studies identify the role of MuRF2 in the pathogenesis of diabetic cardiomyopathy and its regulation of PPAR isoforms, including the post-translational inhibition of PPARγ1 that is cardioprotective in vivo.

We describe the first mechanism by which an ubiquitin ligase inhibits multiple cardiac PPAR isoforms, to protect against high fat diet-induced diabetic cardiomyopathy. We identified that MuRF2 protein levels increase ~20% during the development of diabetic cardiomyopathy induced by high fat diet. Compared to littermate wildtype hearts, MuRF2−/− hearts exhibit an exaggerated diabetic cardiomyopathy, characterized by an early onset systolic dysfunction, larger left ventricular mass, and higher heart weight. MuRF2−/− hearts had significantly increased PPARα- and PPARγ1-regulated gene expression by RT-qPCR, consistent with MuRF2’s regulation of these transcription factors in vivo. Recent studies have described MuRF2 mutations to contribute to the severity of familial hypertrophic cardiomyopathy. The present study suggests that the lack of MuRF2 activity, as found in these patients, can result in an exaggerated diabetic cardiomyopathy. These present studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation and may represent a novel potential therapeutic target against heart failure in diabetes.