Age influences inflammatory responses, hemodynamics, and cardiac proteasome activation during acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a significant source of morbidity and mortality in critically ill patients. Age is a major determinant of clinical outcome in ALI. The increased ALI-associated mortality in the older population suggests that there are age-dependent alterations in the responses to pulmonary challenge. The objective of this observational study was to evaluate age-dependent differences in the acute (within 6 hours) immunological and physiological responses of the heart and lung, to pulmonary challenge, that could result in increased severity. METHODS: Male C57Bl/6 mice (young: 2-3 months, old: 18-20 months) were challenged intratracheally with cell wall components from Gram-positive bacteria (lipoteichoic acid and peptidoglycan). After 6 hours, both biochemical and physiological consequences of the challenge were assessed. Alveolar infiltration of inflammatory cells and protein, airspace and blood cytokines, cardiac function and myocardial proteasome activity were determined. RESULTS: In young mice, there was a dose-dependent response to pulmonary challenge resulting in increased airspace neutrophil counts, lung permeability, and concentrations of cytokines in bronchoalveolar lavage fluid and plasma. A midrange dose was then selected to compare the responses in young and old animals. In comparison, the old animals displayed increased neutrophil accumulation in the airspaces, decreased arterial oxygen saturation, body temperatures, plasma cytokine concentrations, and a lack of myocardial proteasome response, following challenge. CONCLUSIONS: Age-dependent differences in the onset of systemic response and in maintenance of vital functions, including temperature control, oxygen saturation, and myocardial proteasome activation, are evident. We believe a better understanding of these age-related consequences of ALI can lead to more appropriate treatments in the elderly patient population.

The ubiquitin proteasome system and myocardial ischemia.

The ubiquitin proteasome system (UPS) has been the subject of intensive research over the past 20 years to define its role in normal physiology and in pathophysiology. Many of these studies have focused in on the cardiovascular system and have determined that the UPS becomes dysfunctional in several pathologies such as familial and idiopathic cardiomyopathies, atherosclerosis, and myocardial ischemia. This review presents a synopsis of the literature as it relates to the role of the UPS in myocardial ischemia. Studies have shown that the UPS is dysfunctional during myocardial ischemia, and recent studies have shed some light on possible mechanisms. Other studies have defined a role for the UPS in ischemic preconditioning which is best associated with myocardial ischemia and is thus presented here. Very recent studies have started to define roles for specific proteasome subunits and components of the ubiquitination machinery in various aspects of myocardial ischemia. Lastly, despite the evidence linking myocardial ischemia and proteasome dysfunction, there are continuing suggestions that proteasome inhibitors may be useful to mitigate ischemic injury. This review presents the rationale behind this and discusses both supportive and nonsupportive studies and presents possible future directions that may help in clarifying this controversy.

Enhancement of proteasome function by PA28α overexpression protects against oxidative stress.

The principal function of the proteasome is targeted degradation of intracellular proteins. Proteasome dysfunction has been observed in experimental cardiomyopathies and implicated in human congestive heart failure. Measures to enhance proteasome proteolytic function are currently lacking but would be beneficial in testing the pathogenic role of proteasome dysfunction and could have significant therapeutic potential. The association of proteasome activator 28 (PA28) with the 20S proteasome may play a role in antigen processing. It is unclear, however, whether the PA28 plays any important role outside of antigen presentation, although up-regulation of PA28 has been observed in certain types of cardiomyopathy. Here, we show that PA28α overexpression (PA28αOE) stabilized PA28ß, increased 11S proteasomes, and enhanced the degradation of a previously validated proteasome surrogate substrate (GFPu) in cultured neonatal rat cardiomyocytes. PA28αOE significantly attenuated H(2)O(2)-induced increases in the protein carbonyls and markedly suppressed apoptosis in cultured cardiomyocytes under basal conditions or when stressed by H(2)O(2). We conclude that PA28αOE is sufficient to up-regulate 11S proteasomes, enhance proteasome-mediated removal of misfolded and oxidized proteins, and protect against oxidative stress in cardiomyocytes, providing a highly sought means to increase proteasomal degradation of abnormal cellular proteins.

Myocardial ischemic preconditioning preserves postischemic function of the 26S proteasome through diminished oxidative damage to 19S regulatory particle subunits.

RATIONALE: The ubiquitin proteasome system (UPS) becomes dysfunctional as a result of ischemia/reperfusion (I/R), which may lead to dysregulation of signaling pathways. Ischemic preconditioning (IPC) may prevent dysregulation by preventing UPS dysfunction through inhibition of oxidative damage. OBJECTIVE: Examine the hypothesis that early IPC preserves postischemic UPS function thus facilitating prosurvival signaling events. METHODS AND RESULTS: I/R decreased proteasome chymotryptic activity by 50% in isolated rat heart and an in vivo murine left anterior descending coronary artery occlusion model. Following IPC, proteasome activity was decreased 25% (P<0.05) in isolated heart and not different from baseline in the murine model. Enriched 26S proteasome was prepared and analyzed for protein carbonyl content. Increased (P<0.05) carbonylation in a 53-kDa band following I/R was diminished by IPC. Immunoprecipitation studies indicated that the 53-kDa carbonylation signal was of proteasomal origin. Two-dimensional gel electrophoresis resolved the 53-kDa band into spots analyzed by liquid chromatography/tandem mass spectrometry containing Rpt3/Rpt5 both of which could be immunoprecipitated conjugated to dinitrophenylhydrazine (DNPH). Higher amounts of DNPH-tagged Rpt5 were immunoprecipitated from the I/R samples and less from the IPC samples. I/R increased Bax levels by 63% (P<0.05) which was decreased by IPC. Lactacystin (lac) pretreatment of preconditioned hearts increased Bax by 140% (P<0.05) and also increased ubiquitinated proteins. Pretreatment of hearts with a proteasome inhibitor reversed the effects of IPC on postischemic Rpt5 carbonylation, cardiac function, morphology and morphometry, and ubiquitinated and signaling proteins. CONCLUSIONS: These studies suggest that IPC protects function of the UPS by diminishing oxidative damage to 19S regulatory particle subunits allowing this complex to facilitate degradation of proapoptotic proteins.

Ubiquitin proteasome dysfunction in human hypertrophic and dilated cardiomyopathies.

BACKGROUND: The ubiquitin proteasome system maintains a dynamic equilibrium of proteins and prevents accumulation of damaged and misfolded proteins, yet its role in human cardiac dysfunction is not well understood. The present study evaluated ubiquitin proteasome system function in human heart failure and hypertrophic cardiomyopathy (HCM). METHODS AND RESULTS: Proteasome function was studied in human nonfailing donor hearts, explanted failing hearts, and myectomy samples from patients with HCM. Proteasome proteolytic activities were markedly reduced in failing and HCM hearts compared with nonfailing hearts (P<0.01). This activity was partially restored after mechanical unloading in failing hearts (P<0.01) and was significantly lower in HCM hearts with pathogenic sarcomere mutations than in those lacking these mutations (P<0.05). There were no changes in the protein content of ubiquitin proteasome system subunits (ie, 11S, 20S, and 19S) or in active-site labeling of the 20S proteolytic subunit beta-5 among groups to explain decreased ubiquitin proteasome system activity in HCM and failing hearts. Examination of protein oxidation revealed that total protein carbonyls, 4-hydroxynonenylated proteins, and oxidative modification to 19S ATPase subunit Rpt 5 were increased in failing compared with nonfailing hearts. CONCLUSIONS: Proteasome activity in HCM and failing human hearts is impaired in the absence of changes in proteasome protein content or availability of proteolytic active sites. These data provide strong evidence that posttranslational modifications to the proteasome may account for defective protein degradation in human cardiomyopathies.

Proteasome functional insufficiency in cardiac pathogenesis.

The ubiquitin-proteasome system (UPS) is responsible for the degradation of most cellular proteins. Alterations in cardiac UPS, including changes in the degradation of regulatory proteins and proteasome functional insufficiency, are observed in many forms of heart disease and have been shown to play an important role in cardiac pathogenesis. In the past several years, remarkable progress in understanding the mechanisms that regulate UPS-mediated protein degradation has been achieved. A transgenic mouse model of benign enhancement of cardiac proteasome proteolytic function has been created. This has led to the first demonstration of the necessity of proteasome functional insufficiency in the genesis of important pathological processes. Cardiomyocyte-restricted enhancement of proteasome proteolytic function by overexpression of proteasome activator 28α protects against cardiac proteinopathy and myocardial ischemia-reperfusion injury. Additionally, exciting advances have recently been achieved in the search for a pharmacological agent to activate the proteasome. These breakthroughs are expected to serve as an impetus to further investigation into the involvement of UPS dysfunction in molecular pathogenesis and to the development of new therapeutic strategies for combating heart disease. An interplay between the UPS and macroautophagy is increasingly suggested in noncardiac systems but is not well understood in the cardiac system. Further investigations into the interplay are expected to provide a more comprehensive picture of cardiac protein quality control and degradation.

Upregulation of myocardial 11S-activated proteasome in experimental hyperglycemia.

This study examined the hypothesis that the ubiquitin proteasome system (UPS) degrades proteins damaged by exposure to hyperglycemia. Experimental hyperglycemia was induced in male rats by treatment with streptozotocin. After 30 days, echocardiography confirmed the presence of cardiomyopathy as ejection fraction, fractional shortening, and diastolic function (E/A ratio) were decreased, and chamber diameter was increased in hyperglycemic animals. Proteasome non-ATP-dependent chymotryptic activity was increased over 2-fold in hyperglycemic hearts, but the ATP-dependent activity was decreased and levels of ubiquitinated proteins were increased. Protein levels of the PA28alpha of the 11S-activator ring were increased by 128% and the PA28beta subunit increased by 58% in the hyperglycemic hearts. The alpha3 subunit of the 20S-proteasome was increased by 82% while the catalytic beta5 subunit was increased by 68% in hyperglycemic hearts. Protein oxidation as indicated by protein carbonyls was significantly higher in hyperglycemic hearts. These studies support the conclusion that the UPS becomes dysfunctional during long term hyperglycemia. However, 11S-activated proteasome was increased suggesting a response to oxidative protein damage and a potential role for this form of the proteasome in a cardiac pathophysiology.

Caveolin and proteasome in tocotrienol mediated myocardial protection.

The effect of different isomers of tocotrienol was tested on myocardial ischemia reperfusion injury. Although all of the tocotrienol isomers offered some degree of cardioprotection, gamma-tocotrienol was the most protective as evident from the result of myocardial apoptosis. To study the mechanism of tocotrienol mediated cardioprotection, we examined the interaction and/or translocation of different signaling components to caveolins and activity of proteasome. The results suggest that differential interaction of MAP kinases with caveolin 1/3 in conjuncture with proteasome stabilization play a unique role in tocotrienol mediated cardioprotection possibly by altering the availability of pro-survival and anti-survival proteins.

Proteasome mediates removal of proteins oxidized during myocardial ischemia.

Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.

Interferon-dependent immunoproteasome activity during mouse adenovirus type 1 infection.

The immunoproteasome is an inducible host mechanism that aids in the clearance of damaged proteins. The immunoproteasome also influences immune function by enhancing peptide presentation by MHC class I and promotes inflammation via IκB degradation and activation of NF-κB. We used mouse adenovirus type 1 (MAV-1) to characterize the role of the immunoproteasome in adenovirus pathogenesis. Following intranasal infection of mice, immunoproteasome activity in the heart and lung was significantly increased in an IFN-γ-dependent manner. Absence of the ß5i immunoproteasome subunit and pharmacological inhibition of ß5i activity had minimal effects on viral replication, virus-induced cellular inflammation, or induction of cytokine expression. Likewise, the establishment of protective immunity following primary infection was not significantly altered by ß5i deficiency. Thus, although immunoproteasome activity is robustly induced during acute infection with MAV-1, our data suggest that other mechanisms are capable of compensating for immunoproteasome activity to maintain antiviral immunity and appropriate inflammatory responses.