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The ubiquitin proteasome system (UPS) degrades abnormal proteins and most unneeded normal proteins, thereby playing a critical role in protein homeostasis in the cell. Proteasome inhibition is effective in treating certain forms of cancer, while UPS dysfunction is increasingly implicated in the pathogenesis of many severe and yet common diseases. It has been previously shown that doxorubicin (Dox) enhances the degradation of a UPS surrogate substrate in mouse hearts. To address the underlying mechanism, in the present study, we report that 1) Dox not only enhances the degradation of an exogenous UPS reporter (GFPu) but also antagonizes the proteasome inhibitor-induced accumulation of endogenous substrates (e.g., ~-catenin and c-Jun) of the UPS in cultured NIH 3T3 cells and cardiomyocytes; 2) Dox facilitates the in vitro degradation of GFPu and c-Jun by the reconstituted UPS via the enhancement of proteasomal function; 3) Dox at a therapeutically relevant dose directly stimulates the peptidase activities of purified 20S proteasomes; and 4) Dox increases, whereas proteasome inhibition decreases, E3 ligase COOH-terminus of heat shock protein cognate 70 in 3T3 cells via a posttranscriptional mechanism. These new findings suggest that Dox activates the UPS by acting directly on both the ubiquitination apparatus and proteasome. [ABSTRACT FROM AUTHOR]

The ubiquitin proteasome system (UPS) degrades all abnormal proteins and most normal proteins that are no longer needed, thereby playing critical roles in protein quality control and homeostasis in the cell. Proteasome inhibition has proven to be effective in treating certain forms of cancer while UPS impairment is increasingly implicated in the pathogenesis of many severe and yet common diseases. To establish the pathogenic role of UPS malfunction a UPS activator is required. We previously reported that doxorubicin (Dox) enhances the degradation of a UPS reporter in mouse hearts. To address the underlying mechanism, here we report that: (1) Dox not only enhances the degradation of an exogenous UPS reporter (GFPu) but also antagonizes proteasome inhibitor induced accumulation of endogenous substrates (e.g., β-catenin, c-Jun) of the UPS in cultured NIH3T3 cells and cardiomyocytes; (2) Dox facilitates in vitro degradation of GFPu and c-Jun by either 3T3 cell lysates or the purified UPS; (3) Dox directly stimulates the peptidase activities of purified 20S proteasomes at a low dose (1µM) but inhibits them at a higher dose (10µM); (4) Dox attenuates MG- 132-induced accumulation of neddylated cullin1; and (5) Dox stabilizes E3 ligase CHIP while proteasome inhibition depletes CHIP in 3T3 cells. These new findings suggest that Dox activates the UPS by acting directly on both ubiquitination apparatuses and the proteasome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood, The transcription factor GATA4 is essential for cardiac homeostasis, and its protein levels are dramatically reduced in the heart in response to diverse pathologic stresses. In this study, we investigated if hyperglycemia affects GATA4 expression in cardiomyocytes and if enhancing GATA4 signaling could attenuate hyperglycemia-induced cardiomyocyte injury. In cultured rat cardiomyocytes, high glucose (HG, 25 or 40 mM) markedly reduced GATA4 protein levels as compared with normal glucose (NG, 5.5 mM). Equal amount of mannitol did not affect GATA4 protein expression (NG, 100 ± 12%; mannitol, 97 ± 8%, versus HG, 43 ± 16%, p < 0.05). The GATA4 mRNA content, either steady-state or polysome-associated, remained unchanged. HG-induced GATA4 reduction was reversed by MG262, a specific proteasome inhibitor. HG did not activate the ubiquitin proteasome system (UPS) in cardiomyocytes as indicated by a UPS reporter, nor did it increase the peptidase activities or protein expression of the proteasomal subunits. However, the mRNA levels of ubiquitin-protein isopeptide ligase (E3) carboxyl terminus of Hsp70-interacting protein (CHIP) were markedly increased in HG-treated cardiomyocytes. CHIP overexpression promoted GATA4 protein degradation, whereas small interfering RNA-mediated CHIP knockdown prevented HG-induced GATA4 depletion. Moreover, overexpression of GATA4 blocked HG-induced cardiomyocyte death. Also, GATA4 protein levels were diminished in the hearts of streptozotocin and db/db diabetic mice (44 ± 7% and 67 ± 13% of control, p < 0.05), which correlated with increased CHIP mRNA abundance. In summary, increased GATA4 protein degradation may be an important mechanism that contributes to hyperglycemic cardiotoxicity. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) is responsible for turnover of most cellular proteins in eukaryotes. Protein degradation by the UPS serves quality control and regulatory functions. Proteasome inhibition showed great promise in effectively treating cancer and restenosis. UPS dysfunction in cardiac hypertrophy and failure has recently been suspected but remains to be investigated. A system capable of monitoring dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ would no doubt benefit significantly efforts to decipher the pathogenic significance of UPS dysfunction in the heart and to evaluate the effect of proteasome inhibition on cardiac myocytes. We successfully established such a system in cultured cardiac myocytes by delivering and expressing a modified green fluorescence protein (GFPu) gene using recombinant adenoviruses. GFPu contains a ubiquitination signal sequence fused to the COOH terminus. Fluorescence microscopy and Western blots revealed that protein abundance of modified green fluorescent protein (GFPu), but not wild-type green fluorescent protein, in cultured cardiac myocytes was incrementally increased when function of the proteasomes was inhibited in various degrees by specific inhibitors. The increase in GFPu protein levels and fluorescence intensity is paralleled by a decrease in the in vitro peptidase activity of the proteasomes. Our results demonstrate that GFPu can be used as a surrogate marker to monitor dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ. Application of this novel system reveals that moderate levels of H2O2, a reactive oxygen species generator, impair proteolytic function of the UPS in cultured cardiac myocytes. [ABSTRACT FROM AUTHOR]

Presents a response by A. R. K. Kumarapeli et al to a letter to the editor about their article "A Novel Transgenic Mouse Model Reveals Deregulation of the Ubiquitin-Proteasome System in the Heart by Doxorubicin.

Background: Short-term dietary restriction (DR) without malnutrition preconditions against surgical stress in rodents; however, the nutritional basis and underlying nutrient/energy-sensing pathways remain poorly understood. Objectives: We investigated the relative contribution of protein restriction (PR) vs. calorie restriction (CR) to protection from renal ischemia reperfusion injury (IRI) and changes in organ-autonomous nutrient/energy-sensing pathways and hormones underlying beneficial effects. Methods: Mice were preconditioned on experimental diets lacking total calories (0-50% CR) or protein/essential amino acids (EAAs) vs. complete diets consumed ad libitum (AL) for 1 wk before IRI. Renal outcome was assessed by serum markers and histology and integrated over a 2-dimensional protein/energy landscape by geometric framework analysis. Changes in renal nutrient/energy-sensing signal transduction and systemic hormones leptin and adiponectin were also measured. The genetic requirement for amino acid sensing via general control non-derepressible 2 (GCN2) was tested with knockout vs. control mice. The involvement of the hormone leptin was tested by injection of recombinant protein vs. vehicle during the preconditioning period. Results: CR-mediated protection was dose dependent up to 50% with maximal 2-fold effect sizes. PR benefits were abrogated by EAA re-addition and additive with CR, with maximal benefits at any given amount of CR occurring with a protein-free diet. GCN2 was not required for functional benefits of PR. Activation and repression of nutrient/energy-sensing kinases, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1), respectively, on PR reflected a state of negative energy balance, paralleled by 13% weight loss and an 87% decrease in leptin, independent of calorie intake. Recombinant leptin administration partially abrogated benefits of dietary preconditioning against renal IRI. Conclusions: In male mice, PR and CR both contributed to the benefits of short-term DR against renal IRI independent of GCN2 but partially dependent on reduced circulating leptin and coincident with AMPK activation and mTORC1 repression. [ABSTRACT FROM AUTHOR]

Background--Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca2+ release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein-coupled receptor stimulation, affecting Ca2+ cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. Methods and Results--IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein-coupled receptor activation, Ca2+ mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca2+ transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca2+ elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein-coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca2+ load by IP3Rs promote Ca2+ extrusion by forward-mode Na+/Ca2+ exchange, an important mechanism of arrhythmic events. Conclusions--The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias. [ABSTRACT FROM AUTHOR]

Background: Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. Methods: Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. Results: Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. Conclusions: Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.) N Engl J Med 2011;364:1795-806. [ABSTRACT FROM AUTHOR]

The Notch receptor mediates cell fate decision in multiple organs. In the current work we tested the hypothesis that Nkx2.5 is a target gene of Notchi and raised the possibility that Notchi regulates myocyte commitment in the adult heart. Cardiac progenitor cells (CPC5) in the niches express Notch 1 receptor, and the supporting cells exhibit the Notch ligand Jaggedi. The nuclear translocation of Notchi intracellular domain (N1ICD) up-regulates Nkx2.5 in CPC5 and promotes the formation of cycling myocytes in vitro. N1ICD and RBP-Jk form a protein complex, which in turn binds to the Nkx2.5 promoter initiating transcription and myocyte differentiation. In contrast, transcription factors of vascular cells are down-regulated by Jaggedi activation of the Notch1 pathway. Importantly, inhibition of Notchi in infarcted mice impairs the commitment of resident CPC5 to the myocyte lineage opposing cardiomyogenesis. These observations indicate that Notchl favors the early specification of CPC5 to the myocyte phenotype but maintains the newly formed cells in a highly proliferative state. Dividing Nkx2.5-positive myocytes correspond to transit amplifying cells, which condition the replicative capacity of the heart. In conclusion, Notchi may have critical implications in the control of heart homeostasis and its adaptation to pathologic states. [ABSTRACT FROM AUTHOR]

Presents a study that examined impairment of the ubiquitin-proteosome system (UPS) in desminopathy mouse hearts. Reason for the impairment of the UPS; Impairment of UPS in cultured non-muscle cells by mutant-des; Conclusions and significance of the study.

The article presents a study which developed transgenic (TG) mouse model that ubiquitously expresses a surrogate protein substrate for the ubiquitin-proteasome system (UPS) and validated its suitability to monitor in vivo changes of UPS proteolytic function in all major organs or tissues. The article also developed and examined primary culture of cardiomyocytes derived from adult TG mice to probe UPS involvement in pathogenesis. Principal findings are given. Conclusions and significance of the study are discussed.

A response from the authors of the article "Inositol 1,4,5-Trisphosphate Receptors and Human Left Ventricular Myocytes" is presented.