Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice.

BACKGROUND: The ubiquitin-proteasome pathway (UPP) is a major protein degradation pathway that is activated during sepsis and has been proposed as a therapeutic target for preventing skeletal muscle loss due to cachexia. Although several studies have investigated the modulation of proteasome activity in response to LPS administration, none have characterized the overall UPP response to LPS administration in the fate of proteasome inhibition. METHODS: Here, we determined the modulation pattern of the main key components of the UPP in the gastrocnemius (GAS) of mice during the acute phase of lipopolysaccharide (LPS)-mediated endotoxemia (7.5 mg/kg - 8 h) by measuring all three ß1, ß2 and ß5 activites of the 20S and 26S proteasomes, the levels of steady state polyubiquitinated proteins, mRNA levels of muscle ligases, as well as signaling pathways regulating the UPP. Another goal was to assess the effects of administration of a specific proteasome inhibitor (epoxomicin, 0.5 mg/kg) on UPP response to sepsis. RESULTS: The acute phase of LPS-induced endotoxemia lowered GAS/body weight ratio and increased MuRF1 and MAFbx mRNA concomitantly to an activation of the pathways known to regulate their expression. Unexpectedly, we observed a decrease in all 20S and 26S proteasome activities measured in GAS, which might be related to oxidative stress, as oxidized proteins (carbonyl levels) increase with LPS. While significantly inhibiting 20S and 26S proteasome ß5 activities in heart and liver, epoxomicin did not lower proteasome activity in GAS. However, the increase in mRNA expression of the muscle ligases MuRF1 and MAFbx were partially rescued without affecting the other investigated signaling pathways. LPS also strongly activated autophagy, which could explain the observed GAS atrophy with LPS-induced reduction of proteasome activity. CONCLUSIONS: Our results highlight an opposite regulation of UPP in the early hours of LPS-induced muscle atrophy by showing reduced proteasome activities and increased mRNA expression of muscle specific ligases. Furthermore, our data do not support any preventive effect of epoxomicin in muscle atrophy due to acute cachexia since proteasome activities are not further repressed.

Proteomic analysis of hearts from Akita mice suggests that increases in soluble epoxide hydrolase and antioxidative programming are key changes in early stages of diabetic cardiomyopathy.

Cardiovascular disease is the leading cause of diabetic morbidity with more than 10% of type 1 diabetes mellitus (T1DM) patients dying before they are 40 years old. This study utilized Akita mice, a murine model with T1DM progression analogous to that of humans. Diabetic cardiomyopathy in Akita mice presents as cardiac atrophy and diastolic impairment at 3 months of age, but we observed cardiac atrophy in hearts from recently diabetic mice (5 weeks old). Hearts from 5 week old mice were analyzed with a rigorous label-free quantitative proteomic approach to identify proteins that may play a critical role in the early pathophysiology of diabetic cardiomyopathy. Eleven proteins were differentially expressed in diabetic hearts: products of GANC, PLEKHN1, COL1A1, GSTK1, ATP1A3, RAP1A, ACADS, EEF1A1, HRC, EPHX2, and PKP2 (gene names). These proteins are active in cellular defense, metabolism, insulin signaling, and calcium handling. Further analysis of Akita hearts using biochemical assays showed that the cellular defenses against oxidative stress were increased, including antioxidant capacity (2-3-fold) and glutathione levels (20%). Immunoblots of five and twelve week old Akita heart homogenates showed 30% and 145% increases in expression of soluble epoxide hydrolase (sEH (gene name EPHX2)), respectively, and an approximate 100% increase in sEH was seen in gastrocnemius tissue of 12 week old Akita mice. In contrast, 12 week old Akita livers showed no change in sEH expression. Our results suggest that increases in sEH and antioxidative programming are key factors in the development of type 1 diabetic cardiomyopathy in Akita mice and reveal several other proteins whose expression may be important in this complex pathophysiology.

Altered ubiquitin-proteasome signaling in right ventricular hypertrophy and failure.

Alterations in the ubiquitin-proteasome system (UPS) have been described in left ventricular hypertrophy and failure, although results have been inconsistent. The role of the UPS in right ventricular (RV) hypertrophy (RVH) and RV failure (RVF) is unknown. Given the greater percent increase in RV mass associated with RV afterload stress, as present in many congenital heart lesions, we hypothesized that alterations in the UPS could play an important role in RVH/RVF. UPS expression and activity were measured in the RV from mice with RVH/RVF secondary to pulmonary artery constriction (PAC). Epoxomicin and MG132 were used to inhibit the proteasome, and overexpression of the 11S PA28α subunit was used to activate the proteasome. PAC mice developed RVH (109.3% increase in RV weight to body weight), RV dilation with septal shift, RV dysfunction, and clinical RVF. Proteasomal function (26S ß5 chymotrypsin-like activity) was decreased 26% (P < 0.05). Protein expression of 19S subunit Rpt5 (P < 0.05), UCHL1 deubiquitinase (P < 0.0001), and Smurf1 E3 ubiquitin ligase (P < 0.01) were increased, as were polyubiquitinated proteins (P < 0.05) and free-ubiquitins (P = 0.05). Pro-apoptotic Bax was increased (P < 0.0001), whereas anti-apoptotic Bcl-2 decreased (P < 0.05), resulting in a sixfold increase in the Bax/Bcl-2 ratio. Proteasomal inhibition did not accelerate RVF. However, proteasome enhancement by cardiac-specific proteasome overexpression partially improved survival. Proteasome activity is decreased in RVH/RVF, associated with upregulation of key UPS regulators and pro-apoptotic signaling. Enhancement of proteasome function partially attenuates RVF, suggesting that UPS dysfunction contributes to RVF.

Upregulation of proteasome activity in muscle RING finger 1-null mice following denervation.

Deletion of muscle RING finger 1 (MuRF1), an E3 ubiquitin ligase, leads to sparing of muscle mass following denervation. The purpose of this study was to test the hypothesis that muscle sparing in mice with a deletion of MuRF1 is due to the selective inhibition of the ubiquitin proteasome system. Activities of the 20S and 26S proteasomes, calpain and cathepsin L, were measured in the triceps surae muscles of wild-type (WT) and MuRF1-knockout (KO) mice at 3 and 14 d following denervation. In addition, fractional protein synthesis rates and differential gene expression were measured in WT and KO muscle. The major finding was that 20S and 26S proteasome activities were significantly elevated (1.5- to 2.5-fold) after 14 d of denervation in both WT and KO mice relative to control, but interestingly, the activities of both the 20S and 26S proteasome were significantly higher in KO than WT mice. Further, mRNA expression of MAFbx was elevated after 14 d of denervation in KO, but not WT, mice. These data challenge the conventional dogma that MuRF1 is controlling the degradation of only contractile proteins and suggest a role for MuRF1 in the global control of the ubiquitin proteasome system and protein turnover.

Malignant and benign mutations in familial cardiomyopathies: insights into mutations linked to complex cardiovascular phenotypes.

Cardiomyopathies, familial or sporadic, have become recognized as one of the leading cardiac threats. Hypertrophic cardiomyopathy (HCM) affects 0.2% of the population and is the leading cause of sudden death in young adults. Dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM) are associated with sudden death as well as heart transplantations. Ventricular noncompaction cardiomyopathy (VNCM) is associated with heart failure and arrhythmias. Currently, more than 630 mutations in 10 sarcomeric genes associated with cardiomyopathy have been identified. HCM is associated with more than 550 mutations, whereas DCM, RCM and VNCM are associated with 52, 14 and 17 mutations, respectively. In many cases, the genes affected present a varying range of phenotypic and pathological severity. Recent data suggest that at least two main genetic determinants are involved in the pathogenesis and phenotypic variability within families afflicted by the same disease-linked gene. Individuals that are homozygous for a mutation or heterozygous for two or more mutations often show more severe phenotypes. Secondly, genetic modifiers are present in some cardiomyopathy patients and are associated with a poorer prognosis. At the protein level, changes in protein-protein interactions may also be important in determining the type of cardiomyopathy caused by different mutations. This review provides insight into the complex cardiovascular phenotypes and genetic variability associated with HCM, DCM, RCM and VNCM.

Amino Acid Changes at Arginine 204 of Troponin I Result in Increased Calcium Sensitivity of Force Development.

Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated, and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI associated with familial hypertrophic cardiomyopathy (FHC) is arginine (R), which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at cTnI arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. The R204H mutation has also been associated with restrictive cardiomyopathy (RCM). To characterize the effects of different mutations at the same residue (R204) on the physiological function of cTnI, six mutations at R204 (C, G, H, P, Q, W) were investigated in skinned fiber studies. Skinned fiber studies showed that all tested mutations at R204 caused significant increases in Ca2+ sensitivity of force development (ΔpCa50 = 0.22-0.35) when compared to wild-type (WT) cTnI. Investigation of the interactions between the cTnI mutants and WT cardiac troponin C (cTnC) or WT cardiac troponin T (cTnT) showed that all the mutations investigated, except R204G, affected either or both cTnI:cTnT and cTnI:cTnC interactions. The R204H mutation affected both cTnI:cTnT and cTnI:cTnC interactions while the R204C mutation affected only the cTnI:cTnC interaction. These results suggest that different mutations at the same site on cTnI could have varying effects on thin filament interactions. A mutation in fast skeletal TnI (R174Q, homologous to cTnI R204Q) also significantly increased Ca2+ sensitivity of force development (ΔpCa50 = 0.16). Our studies indicate that known cTnI mutations associated with poor prognosis (R204C and R204H) exhibit large increases in Ca2+ sensitivity of force development. Therefore, other R204 mutations that cause similar increases in Ca2+ sensitivity are also likely to have poor prognoses.

Subnormothermic Perfusion in the Isolated Rat Liver Preserves the Antioxidant Glutathione and Enhances the Function of the Ubiquitin Proteasome System.

The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated rat livers after 15 min perfusion at temperatures ranging from normothermia (37°C) to subnormothermia (26°C and 22°C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22°C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22°C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like (ß5) proteasome activity was significantly increased in the 26°C (46%) and 22°C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus.

Pregnancy is associated with decreased cardiac proteasome activity and oxidative stress in mice.

During pregnancy, the heart develops physiological hypertrophy. Proteasomal degradation has been shown to be altered in various models of pathological cardiac hypertrophy. Since the molecular signature of pregnancy-induced heart hypertrophy differs significantly from that of pathological heart hypertrophy, we investigated whether the cardiac proteasomal proteolytic pathway is affected by pregnancy in mice. We measured the proteasome activity, expression of proteasome subunits, ubiquitination levels and reactive oxygen production in the hearts of four groups of female mice: i) non pregnant (NP) at diestrus stage, ii) late pregnant (LP), iii) one day post-partum (PP1) and iv) 7 days post-partum (PP7). The activities of the 26 S proteasome subunits ß1 (caspase-like), and ß2 (trypsin-like) were significantly decreased in LP (ß1∶83.26 ± 1.96%; ß2∶74.74 ± 1.7%, normalized to NP) whereas ß5 (chymotrypsin-like) activity was not altered by pregnancy but significantly decreased 1 day post-partum. Interestingly, all three proteolytic activities of the proteasome were restored to normal levels 7 days post-partum. The decrease in proteasome activity in LP was not due to the surge of estrogen as estrogen treatment of ovariectomized mice did not alter the 26 S proteasome activity. The transcript and protein levels of RPN2 and RPT4 (subunits of 19 S), ß2 and α7 (subunits of 20 S) as well as PA28α and ß5i (protein only) were not significantly different among the four groups. High resolution confocal microscopy revealed that nuclear localization of both core (20S) and RPT4 in LP is increased ∼2-fold and is fully reversed in PP7. Pregnancy was also associated with decreased production of reactive oxygen species and ubiquitinated protein levels, while the de-ubiquitination activity was not altered by pregnancy or parturition. These results indicate that late pregnancy is associated with decreased ubiquitin-proteasome proteolytic activity and oxidative stress.

Cardioproteomics: advancing the discovery of signaling mechanisms involved in cardiovascular diseases.

Cardioproteomics (Cardiovascular proteomics) is fast becoming an indispensible technique in deciphering changes in signaling pathways that occur in cardiovascular diseases (CVDs). The quality and availability of the instruments and bioinformatics software used for cardioproteomics continues to improve, and these techniques are now available to most cardiovascular researchers either directly or indirectly via university core centers. The heart and aorta are specialized tissues which present unique challenges to investigate. Currently, the diverse range of proteomic techniques available for cardiovascular research makes the choice of the best method or best combination of methods for the disease parameter(s) being investigated as important as the equipment used. This review focuses on proteomic techniques and their applications which have advanced our understanding of the signaling mechanisms involved in CVDs at the levels of protein complex/protein-protein interaction, post-translational modifications and signaling induced protein changes.