Multifaceted roles of Meg3 in cellular senescence and atherosclerosis.

BACKGROUND AND AIMS: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. METHODS AND RESULTS: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr-/- mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. CONCLUSIONS: Our data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.

BSCL2/Seipin deficiency in hearts causes cardiac energy deficit and dysfunction via inducing excessive lipid catabolism.

BACKGROUND: Heart failure (HF) is one of the leading causes of death worldwide and is associated with cardiac metabolic perturbations. Human Type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease is caused by mutations in the BSCL2 gene. Global lipodystrophic Bscl2-/- mice exhibit hypertrophic cardiomyopathy with reduced cardiac steatosis. Whether BSCL2 plays a direct role in regulating cardiac substrate metabolism and/or contractile function remains unknown. METHODS: We generated mice with cardiomyocyte-specific deletion of Bscl2 (Bscl2cKO ) and studied their cardiac substrate utilisation, bioenergetics, lipidomics and contractile function under baseline or after either a treatment regimen using fatty acid oxidation (FAO) inhibitor trimetazidine (TMZ) or a prevention regimen with high-fat diet (HFD) feeding. Mice with partial ATGL deletion and cardiac-specific deletion of Bscl2 were also generated followed by cardiac phenotyping. RESULTS: Different from hypertrophic cardiomyopathy in Bscl2-/- mice, mice with cardiac-specific deletion of Bscl2 developed systolic dysfunction with dilation. Myocardial BSCL2 deletion led to elevated ATGL expression and FAO along with reduced cardiac lipid contents. Cardiac dysfunction in Bscl2cKO mice was independent of mitochondrial dysfunction and oxidative stress, but associated with decreased metabolic reserve and ATP levels. Importantly, cardiac dysfunction in Bscl2cKO mice could be partially reversed by FAO inhibitor TMZ, or prevented by genetic abolishment of one ATGL allele or HFD feeding. Lipidomic analysis further identified markedly reduced glycerolipids, glycerophospholipids, NEFA and acylcarnitines in Bscl2cKO hearts, which were partially normalised by TMZ or HFD. CONCLUSIONS: We identified a new form of cardiac dysfunction with excessive lipid utilisation which ultimately causes cardiac substrate depletion and bioenergetics failure. Our findings also uncover a crucial role of BSCL2 in controlling cardiac lipid catabolism and contractile function and provide novel insights into metabolically treating energy-starved HF using FAO inhibitor or HFD.

Cysteine oxidation of copper transporter CTR1 drives VEGFR2 signalling and angiogenesis.

Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.

The Calcineurin-TFEB-p62 Pathway Mediates the Activation of Cardiac Macroautophagy by Proteasomal Malfunction.

RATIONALE: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 catabolic pathways will help advance cardiac pathophysiology and medicine. OBJECTIVE: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac autophagic-lysosomal pathway. METHODS AND RESULTS: Myocardial macroautophagy, TFEB (transcription factor EB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from wild type mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with wild type mice. CONCLUSIONS: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.

Adenosine kinase inhibition enhances microvascular dilator function and improves left ventricle diastolic dysfunction.

OBJECTIVE: Inhibition of adenosine kinase (ADK), via augmenting endogenous adenosine levels exerts cardiovascular protection. We tested the hypothesis that ADK inhibition improves microvascular dilator and left ventricle (LV) contractile function under metabolic or hemodynamic stress. METHODS AND RESULTS: In Obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid rats, treatment with the selective ADK inhibitor, ABT-702 (1.5 mg/kg, intraperitoneal injections for 8-week) restored acetylcholine-, sodium nitroprusside-, and adenosine-induced dilations in isolated coronary arterioles, an effect that was accompanied by normalized end-diastolic pressure (in mm Hg, Lean: 3.4 ± 0.6, Obese: 17.6 ± 4.2, Obese + ABT: 6.6 ± 1.4) and LV relaxation constant, Tau (in ms, Lean: 6.9 ± 1.5, Obese: 13.9 ± 1.7, Obese + ABT: 6.0 ± 1.1). Mice with vascular endothelium selective ADK deletion (ADKVEC KO) exhibited an enhanced dilation to acetylcholine in isolated gracilis muscle (lgEC50 WT: -8.2 ± 0.1, ADKVEC KO: -8.8 ± 0.1, P < .05) and mesenteric arterioles (lgEC50 WT: -7.4 ± 0.2, ADKVEC KO: -8.1 ± 1.2, P < .05) when compared to wild-type (WT) mice, whereas relaxation of the femoral artery and aorta (lgEC50 WT: -7.03 ± 0.6, ADKVEC KO: -7.05 ± 0.8) was similar in the two groups. Wild-type mice progressively developed LV systolic and diastolic dysfunction when they underwent transverse aortic constriction surgery, whereas ADKVEC -KO mice displayed a lesser degree in decline of LV function. CONCLUSIONS: Our results indicate that ADK inhibition selectively enhances microvascular vasodilator function, whereby it improves LV perfusion and LV contractile function under metabolic and hemodynamic stress.

Neddylation, an Emerging Mechanism Regulating Cardiac Development and Function.

Defects in protein quality control have been increasingly recognized as pathogenic factors in the development of heart failure, a persistent devastating disease lacking efficacious therapies. Ubiquitin and ubiquitin-like proteins, a family of post-translational modifying polypeptides, play important roles in controlling protein quality by maintaining the stability and functional diversity of the proteome. NEDD8 (neural precursor cell expressed, developmentally downregulated 8), a small ubiquitin-like protein, was discovered two decades ago but until recently the biological significance of NEDD8 modifications (neddylation) in the heart has not been appreciated. In this review, we summarize the current knowledge of the biology of neddylation, highlighting several mechanisms by which neddylation regulates the function of its downstream targets, and discuss the expanding roles for neddylation in cardiac physiology and disease, with an emphasis on cardiac protein quality control. Finally, we outline challenges linked to the study of neddylation in health and disease.

Adenosine Kinase Inhibition Augments Conducted Vasodilation and Prevents Left Ventricle Diastolic Dysfunction in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is often manifested as impaired cardiovascular reserve. We sought to determine if conducted vasodilation, which coordinates microvascular resistance longitudinally to match tissue metabolic demand, becomes compromised in HFpEF. We hypothesized that the metabolic vasodilator adenosine facilitates and that inhibition of ADK (adenosine kinase) augments conducted vasodilation for a more efficient myocardial perfusion and improved left ventricle (LV) diastolic function in HFpEF. METHODS AND RESULTS: We assessed conducted vasodilation in obese ZSF1 rats that develop LV diastolic dysfunction and is used to model human HFpEF. Additionally, conducted vasodilation was measured in arterioles isolated from the right atrial appendages of patients with HFpEF. We found a markedly reduced conducted vasodilation both in obese ZSF1 rats and in patients with HFpEF. Impaired conducted vasodilation was accompanied by increased vascular ADK expression. Isolated rat and human arterioles incubated with adenosine (10 nmol/L) or ADK inhibitor ABT-702 (0.1 µmol/L) both displayed augmented conducted vasodilation. Treatment of obese ZSF1 rats with ABT-702 (1.5 mg/kg, IP for 8 weeks) prevented LV diastolic dysfunction, and in a crossover design augmented conducted vasodilation and improved LV diastolic function. ABT-702 treated obese ZSF1 rats exhibited reduced expression of myocardial carbonic anhydrase 9 and collagen, surrogate markers of myocardial hypoxia. CONCLUSIONS: Upregulation of vascular ADK mitigates adenosine-facilitated conducted vasodilation in obese ZSF1 rats and in patients with HFpEF. We propose that pharmacological inhibition of ADK could be beneficial for therapeutic augmentation of conducted vasodilation, thereby improving tissue perfusion and LV diastolic function in HFpEF.

Targeting ATGL to rescue BSCL2 lipodystrophy and its associated cardiomyopathy.

Mutations in BSCL2 gene underlie human type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease. Global Bscl2-/- mice recapitulate human BSCL2 lipodystrophy and develop insulin resistance and hypertrophic cardiomyopathy. The pathological mechanisms underlying the development of lipodystrophy and cardiomyopathy in BSCL2 are controversial. Here we report that Bscl2-/- mice develop cardiac hypertrophy due to increased basal IGF1 receptor (IGF1R)-mediated PI3K/AKT signaling. Bscl2-/- hearts exhibited increased adipose triglyceride lipase (ATGL) protein stability and expression causing drastic reduction of glycerolipids. Excessive fatty acid oxidation was overt in Bscl2-/- hearts, partially attributing to the hyperacetylation of cardiac mitochondrial proteins. Intriguingly, pharmacological inhibition or genetic inactivation of ATGL could rescue adipocyte differentiation and lipodystrophy in Bscl2-/- cells and mice. Restoring a small portion of fat mass by ATGL partial deletion in Bscl2-/- mice not only reversed the systemic insulin resistance, but also ameliorated cardiac protein hyperacetylation, normalized cardiac substrate metabolism and improved contractile function. Collectively, our study uncovers novel pathways underlying lipodystrophy-induced cardiac hypertrophy and metabolic remodeling and pinpoints ATGL as a downstream target of BSCL2 in regulating the development of lipodystrophy and its associated cardiomyopathy.

A new gold(I) complex-Au(PPh3)PT is a deubiquitinase inhibitor and inhibits tumor growth.

BACKGROUND: Ubiquitin-proteasome system (UPS) is integral to cell survival by maintaining protein homeostasis, and its dysfunction has been linked to cancer and several other human diseases. Through counteracting ubiquitination, deubiquitinases (DUBs) can either positively or negatively regulate UPS function, thereby representing attractive targets of cancer therapies. Previous studies have shown that metal complexes can inhibit tumor growth through targeting the UPS; however, novel metal complexes with higher specificity for cancer therapy are still lacking. METHODS: We synthesized a new gold(I) complex, Au(PPh3)PT. The inhibitory activity of Au(PPh3)PT on the UPS and the growth of multiple cancer cell types were tested in vitro, ex vivo, and in vivo. Furthermore, we compared the efficacy of Au(PPh3)PT with other metal compounds in inhibition of UPS function and tumor growth. FINDINGS: Here we report that (i) a new gold(I) complex-pyrithione, i.e., Au(PPh3)PT, induced apoptosis in two lung cancer cell lines A549 and NCI-H1299; (ii) Au(PPh3)PT severely impaired UPS proteolytic function; (iii) Au(PPh3)PT selectively inhibited 19S proteasome-associated DUBs (UCHL5 and USP14) and other non-proteasomal DUBs with minimal effects on the function of 20S proteasome; (iv) Au(PPh3)PT induced apoptosis in cancer cells from acute myeloid leukemia patients; (v) Au(PPh3)PT effectively suppressed the growth of lung adenocarcinoma xenografts in nude mice; and (vi) Au (PPh3)PT elicited less cytotoxicity in normal cells than several other metal compounds. INTERPRETATION: Together, this study discovers a new gold(I) complex to be an effective inhibitor of the DUBs and a potential anti-cancer drug. FUND: The National High Technology Research and Development Program of China, the project of Guangdong Province Natural Science Foundation, the projects from Foundation for Higher Education of Guangdong, the project from Guangzhou Medical University for Doctor Scientists, the Medical Scientific Research Foundation of Guangdong Province, and the Guangzhou Key Medical Discipline Construction Project Fund.

Neddylation mediates ventricular chamber maturation through repression of Hippo signaling.

During development, ventricular chamber maturation is a crucial step in the formation of a functionally competent postnatal heart. Defects in this process can lead to left ventricular noncompaction cardiomyopathy and heart failure. However, molecular mechanisms underlying ventricular chamber development remain incompletely understood. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that neddylation is temporally regulated in the heart and plays a key role in cardiac development. Cardiomyocyte-specific knockout of NAE1, a subunit of the E1 neddylation activating enzyme, significantly decreased neddylated proteins in the heart. Mice lacking NAE1 developed myocardial hypoplasia, ventricular noncompaction, and heart failure at late gestation, which led to perinatal lethality. NAE1 deletion resulted in dysregulation of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro, which was accompanied by the accumulation of the Hippo kinases Mst1 and LATS1/2 and the inactivation of the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1-inactivated cardiomyocytes restored cell proliferation, and YAP-deficient hearts displayed a noncompaction phenotype, supporting an important role of Hippo-YAP signaling in NAE1-depleted hearts. Mechanistically, we found that neddylation regulates Mst1 and LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyocyte proliferation. Together, these findings demonstrate a role for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling.

Characterization of mice carrying a conditional TEAD1 allele.

The Hippo- yes-associated protein (YAP) pathway is essential for controlling organ size and tumorigenesis. Previous studies have demonstrated that the primary outcome of YAP signaling in the nucleus is achieved by interaction with the transcription factor TEA domain transcription factor (TEAD1). The YAP/TEAD1 complex binds to DNA element and regulates the expression of genes involved in cell growth. However, constitutive knockout of TEAD1 leads to early embryonic lethality in mice. Thus, generation of a floxed TEAD1 mouse becomes crucial for further understanding mid- to late-gestation and post-natal role of TEAD1. Herein, we created and characterized a mouse model that allows for conditional disruption of TEAD1. Embryonic fibroblasts derived from the floxed TEAD1 mice enabled the Cre-mediated deletion of TEAD1 in vitro using virally delivered Cre recombinase. Furthermore, crossing the floxed TEAD1 mouse with a ubiquitously expressing Cre mouse resulted in efficient ablation of the floxed allele in vivo, and the animals recapitulated early embryonic lethality defects. In conclusion, our data demonstrate an important role of TEAD1 in early development in mice, and the floxed TEAD1 mouse model will be a valuable genetic tool to determine the temporal and tissue-specific functions of TEAD1.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Crosstalk between Long Noncoding RNAs and MicroRNAs in Health and Disease.

Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert "sponge-like" effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation.

MicroRNA-150 deletion in mice protects kidney from myocardial infarction-induced acute kidney injury.

Despite greater understanding of acute kidney injury (AKI) in animal models, many of the preclinical studies are not translatable. Most of the data were derived from a bilateral renal pedicle clamping model with warm ischemia. However, ischemic injury of the kidney in humans is distinctly different and does not involve clamping of renal vessel. Permanent ligation of the left anterior descending coronary artery model was used to test the role of microRNA (miR)-150 in AKI. Myocardial infarction in this model causes AKI which is similar to human cardiac bypass surgery. Moreover, the time course of serum creatinine and biomarker elevation were also similar to human ischemic injury. Deletion of miR-150 suppressed AKI which was associated with suppression of inflammation and interstitial cell apoptosis. Immunofluorescence staining with endothelial marker and marker of apoptosis suggested that dying cells are mostly endothelial cells with minimal epithelial cell apoptosis in this model. Interestingly, deletion of miR-150 also suppressed interstitial fibrosis. Consistent with protection, miR-150 deletion causes induction of its target gene insulin-like growth factor-1 receptor (IGF-1R) and overexpression of miR-150 in endothelial cells downregulated IGF-1R, suggesting miR-150 may mediate its detrimental effects through suppression of IGF-1R pathways.

Neddylation and deneddylation in cardiac biology.

Neddylation is a post-translational protein modification that conjugates a ubiquitin-like protein NEDD8 to target proteins. Similar to ubiquitination, neddylation is mediated by a cascade of three NEDD8 specific enzymes, an E1 activating enzyme, an E2 conjugating enzyme and one of the several E3 ligases. Neddylation is countered by the action of deneddylases via a process termed deneddylation. By altering the substrate's conformation, stability, subcellular localization or binding affinity to DNA or proteins, neddylation regulates diverse cellular processes including the ubiquitin-proteasome system-mediated protein degradation, protein transcription, cell signaling etc. Dysregulation of neddylation has been linked to cancer, neurodegenerative disorders, and more recently, cardiac disease. Here we comprehensively overview the biochemistry, the proteome and the biological function of neddylation. We also summarize the recent progress in revealing the physiological and pathological role of neddylation and deneddylation in the heart.

Hepatic deficiency of COP9 signalosome subunit 8 induces ubiquitin-proteasome system impairment and Bim-mediated apoptosis in murine livers.

The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.

Genetically induced moderate inhibition of the proteasome in cardiomyocytes exacerbates myocardial ischemia-reperfusion injury in mice.

RATIONALE: Both cardiomyocyte-restricted proteasome functional enhancement and pharmacological proteasome inhibition (PSMI) were shown to attenuate myocardial ischemia/reperfusion (I/R) injury. The role of cardiac proteasome dysfunction during I/R and the perspective to diminish I/R injury by manipulating proteasome function remain unclear. OBJECTIVES: We sought to determine proteasome adequacy in I/R hearts, create a mouse model of cardiomyocyte-restricted PSMI (CR-PSMI), and test CR-PSMI impact on I/R injury. METHODS AND RESULTS: Myocardial I/R were modeled by ligation (30 minutes) and subsequent release of the left anterior descending artery in mice overexpressing GFPdgn, a validated surrogate proteasome substrate. At 24 hours of reperfusion, myocardial proteasome activities were significantly lower whereas total ubiquitin conjugates and GFPdgn protein levels were markedly higher in all regions of the I/R hearts than the sham controls, indicative of proteasome functional insufficiency. CR-PSMI in intact mice was achieved by transgenic (tg) overexpression of a peptidase-disabled mouse ß5 subunit (T60A-ß5) driven by an attenuated mouse mhc6 promoter. Overexpressed T60A-ß5 can replace endogenous ß5 and inhibits proteasome chymotrypsin-like activities in the heart. Mice with moderate CR-PSMI showed no abnormalities at the baseline but displayed markedly more pronounced structural and functional damage during I/R, compared with non-tg littermates. The exacerbation of I/R injury by moderate CR-PSMI was associated with significant increases in the protein level of PTEN and protein kinase Cδ (PKCδ), decreased Akt activation, and reduced PKCε. CONCLUSIONS: Myocardial I/R causes proteasome functional insufficiency in cardiomyocytes and moderate CR-PSMI augments PTEN and PKCδ, suppresses Akt and PKCε, increases cardiomyocyte apoptosis, and aggravates I/R injury in mice.

Proteasome malfunction activates macroautophagy in the heart.

Protein quality control (PQC) senses and repairs misfolded/unfolded proteins and, if the repair fails, degrades the terminally misfolded polypeptides through an intricate collaboration between molecular chaperones and targeted proteolysis. Proteolysis of damaged proteins is performed primarily by the ubiquitin-proteasome system (UPS). Macroautophagy (commonly known as autophagy) may also play a role in PQC-associated proteolysis, especially when UPS function becomes inadequate. The development of a range of heart diseases, including bona fide cardiac proteinopathies and various forms of cardiac dysfunction has been linked to proteasome functional insufficiency (PFI). Both PFI and activation of autophagy have been observed in the heart of well-established mouse models of cardiac proteinopathy. A causal relationship between PFI and autophagic activation was suggested by a study using cultured cardiomyocytes but has not been established in the heart of intact animals. Taking advantage of an autophagy reporter, we demonstrated here that pharmacologically induced proteasome inhibition is sufficient to activate autophagy in cardiomyocytes in both intact animals and cell cultures, unveiling a potential cross-talk between the two major degradation pathways in cardiac PQC.

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.