Huabo Su

Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice

The ubiquitin-proteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) has been observed in proteinopathies, such as desmin-related cardiomyopathy, and implicated in many common diseases, including dilated cardiomyopathy and ischemic heart disease. However, the pathogenic role of PFI has not been established. Here we created inducible Tg mice with cardiomyocyte-restricted overexpression of proteasome 28 subunit α (CR-PA28αOE) to investigate whether upregulation of the 11S proteasome enhances the proteolytic function of the proteasome in mice and, if so, whether the enhancement can rescue a bona fide proteinopathy and protect against ischemia/reperfusion (I/R) injury. We found that CR-PA28αOE did not alter the homeostasis of normal proteins and cardiac function, but did facilitate the degradation of a surrogate misfolded protein in the heart. By breeding mice with CR-PA28αOE with mice representing a well-established model of desmin-related cardiomyopathy, we demonstrated that CR-PA28αOE markedly reduced aberrant protein aggregation. Cardiac hypertrophy was decreased, and the lifespan of the animals was increased. Furthermore, PA28α knockdown promoted, whereas PA28α overexpression attenuated, accumulation of the mutant protein associated with desmin-related cardiomyopathy in cultured cardiomyocytes. Moreover, CR-PA28αOE limited infarct size and prevented postreperfusion cardiac dysfunction in mice with myocardial I/R injury. We therefore conclude that benign enhancement of cardiac proteasome proteolytic function can be achieved by CR-PA28αOE and that PFI plays a major pathogenic role in cardiac proteinopathy and myocardial I/R injury.

Autophagy and p62 in Cardiac Proteinopathy

Rationale: Recent studies suggest an important role of autophagy in protection against &agr;B-crystallin-based (CryABR120G) desmin-related cardiomyopathies (DRC), but this has not been demonstrated in a different model of cardiac proteinopathy. Mechanisms underlying the response of cardiomyocytes to proteotoxic stress remain incompletely understood. Objective: Our first objective was to determine whether and how the autophagic activity is changed in a mouse model of desminopathy. We also investigated the role of p62 in the protein quality control of cardiomyocytes. Methods and Results: Using an autophagosome reporter and determining changes in LC3-II protein levels in response to lysosomal inhibition, we found significantly increased autophagic flux in mouse hearts with transgenic overexpression of a DRC-linked mutant desmin. Similarly, autophagic flux was increased in cultured neonatal rat ventricular myocytes (NRVMs) expressing a mutant desmin. Suppression of autophagy by 3-methyladenine increased, whereas enhancement of autophagy by rapamycin reduced the ability of a comparable level of mutant desmin overexpression to accumulate ubiquitinated proteins in NRVMs. Furthermore, p62 mRNA and protein expression was significantly up-regulated in cardiomyocytes by transgenic overexpression of the mutant desmin or CryABR120G both in intact mice and in vitro. The p62 depletion impaired aggresome and autophagosome formation, exacerbated cell injury, and decreased cell viability in cultured NRVMs expressing the misfolded proteins. Conclusions: Autophagic flux is increased in desminopathic hearts, and as previously suggested in CryABR120G-based DRC, this increased autophagic flux serves as an adaptive response to overexpression of misfolded proteins. The p62 is up-regulated in mouse proteinopathic hearts. The p62 promotes aggresome formation and autophagy activation and protects cardiomyocytes against proteotoxic stress.

The ubiquitin-proteasome system in cardiac proteinopathy: A quality control perspective

Protein quality control (PQC) depends on elegant collaboration between molecular chaperones and targeted proteolysis in the cell. The latter is primarily carried out by the ubiquitin-proteasome system, but recent advances in this area of research suggest a supplementary role for the autophagy-lysosomal pathway in PQC-related proteolysis. The (patho)physiological significance of PQC in the heart is best illustrated in cardiac proteinopathy, which belongs to a family of cardiac diseases caused by expression of aggregation-prone proteins in cardiomyocytes. Cardiac proteasome functional insufficiency (PFI) is best studied in desmin-related cardiomyopathy, a bona fide cardiac proteinopathy. Emerging evidence suggests that many common forms of cardiomyopathy may belong to proteinopathy. This review focuses on examining current evidence, as it relates to the hypothesis that PFI impairs PQC in cardiomyocytes and contributes to the progression of cardiac proteinopathies to heart failure.

COP9 Signalosome Regulates Autophagosome Maturation

Background— Autophagy is essential to intracellular homeostasis and is involved in the pathophysiology of a variety of diseases. Mechanisms regulating selective autophagy remain poorly understood. The COP9 signalosome (CSN) is a conserved protein complex consisting of 8 subunits (CSN1 through CSN8), and is known to regulate the ubiquitin-proteasome system. However, it is unknown whether CSN plays a role in autophagy. Methods and Results— Marked increases in the LC3-II and p62 proteins were observed on Csn8 depletion in the cardiomyocytes of mouse hearts with cardiomyocyte-restricted knockout of the gene encoding CSN subunit 8 (CR-Csn8KO). The increases in autophagosomes were confirmed by probing with green fluorescent protein–LC3 and electron microscopy. Autophagic flux assessments revealed that defective autophagosome removal was the cause of autophagosome accumulation and occurred before a global ubiquitin-proteasome system impairment in Csn8-deficient hearts. Analyzing the prevalence of different stages of autophagic vacuoles revealed defective autophagosome maturation. Downregulation of Rab7 was found to colocalize strikingly with the autophagosome accumulation at the individual cardiomyocyte level. A significantly higher percent of cardiomyocytes with autophagosome accumulation underwent necrosis in CR-Csn8KO hearts. Long-term lysosomal inhibition with chloroquine induced cardiomyocyte necrosis in mice. Rab7 knockdown impaired autophagosome maturation of nonselective and selective autophagy and exacerbated cell death induced by proteasome inhibition in cultured cardiomyocytes. Conclusions— Csn8/CSN is a central regulator in not only the proteasomal proteolytic pathway, but also selective autophagy. Likely through regulating the expression of Rab7, Csn8/CSN plays a critical role in autophagosome maturation. Impaired autophagosome maturation causes cardiomyocytes to undergo necrosis.

Proteasome functional insufficiency activates the calcineurin-NFAT pathway in cardiomyocytes and promotes maladaptive remodelling of stressed mouse hearts

AIMS Proteasome functional insufficiency (PFI) may play an important role in the progression of congestive heart failure but the underlying molecular mechanism is poorly understood. Calcineurin and nuclear factor of activated T-cells (NFAT) are degraded by the proteasome, and the calcineurin-NFAT pathway mediates cardiac remodelling. The present study examined the hypothesis that PFI activates the calcineurin-NFAT pathway and promotes maladaptive remodelling of the heart. METHODS AND RESULTS Using a reporter gene assay, we found that pharmacological inhibition of 20S proteasomes stimulated NFAT transactivation in both mouse hearts and cultured adult mouse cardiomyocytes. Proteasome inhibition stimulated NFAT nuclear translocation in a calcineurin-dependent manner and led to a maladaptive cell shape change in cultured neonatal rat ventricular myocytes. Proteasome inhibition facilitated left ventricular dilatation and functional decompensation and increased fatality in mice with aortic constriction while causing cardiac hypertrophy in the sham surgery group. It was further revealed that both calcineurin protein levels and NFAT transactivation were markedly increased in the mouse hearts with desmin-related cardiomyopathy and severe PFI. Expression of an aggregation-prone mutant desmin also directly increased calcineurin protein levels in cultured cardiomyocytes. CONCLUSIONS The calcineurin-NFAT pathway in the heart can be activated by proteasome inhibition and is activated in the heart of a mouse model of desmin-related cardiomyopathy that is characterized by severe PFI. The interplay between PFI and the calcineurin-NFAT pathway may contribute to the pathological remodelling of cardiomyocytes characteristic of congestive heart failure.

Protein quality control and degradation in cardiomyocytes.

The heart is constantly under stress and cardiomyocytes face enormous challenges to correctly fold nascent polypeptides and keep mature proteins from denaturing. To meet the challenge, cardiomyocytes have developed multi-layered protein quality control (PQC) mechanisms which are carried out primarily by chaperones and ubiquitin-proteasome system mediated proteolysis. Autophagy may also participate in PQC in cardiomyocytes, especially under pathological conditions. Cardiac PQC often becomes inadequate in heart disease, which may play an important role in the development of congestive heart failure.

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 &bgr;5 subunit (T60A-&bgr;5) driven by an attenuated mouse mhc6 promoter. Overexpressed T60A-&bgr;5 can replace endogenous &bgr;5 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 C&dgr; (PKC&dgr;), decreased Akt activation, and reduced PKC[Latin Small Letter Open E]. Conclusions: Myocardial I/R causes proteasome functional insufficiency in cardiomyocytes and moderate CR-PSMI augments PTEN and PKC&dgr;, suppresses Akt and PKC[Latin Small Letter Open E], increases cardiomyocyte apoptosis, and aggravates I/R injury in mice.

Perturbation of Cullin Deneddylation via Conditional Csn8 Ablation Impairs the Ubiquitin–Proteasome System and Causes Cardiomyocyte Necrosis and Dilated Cardiomyopathy in Mice

Rationale: Ubiquitin–proteasome system (UPS) dysfunction has been implicated in cardiac pathogenesis. Understanding how cardiac UPS function is regulated will facilitate delineating the pathophysiological significance of UPS dysfunction and developing new therapeutic strategies. The COP9 (constitutive photomorphogenesis mutant 9) signalosome (CSN) may regulate the UPS, but this has not been tested in a critical vertebrate organ. Moreover, the role of CSN in a postmitotic organ and the impact of cardiomyocyte-restricted UPS dysfunction on the heart have not been reported. Objective: We sought to determine the role of CSN-mediated deneddylation in UPS function and postnatal cardiac development and function. Methods and Results: Cardiomyocyte-restricted Csn8 gene knockout (CR-Csn8KO) in mice was achieved using a Cre-LoxP system. CR-Csn8KO impaired CSN holocomplex formation and cullin deneddylation and resulted in decreases in F-box proteins. Probing with a surrogate misfolded protein revealed severe impairment of UPS function in CR-Csn8KO hearts. Consequently, CR-Csn8KO mice developed cardiac hypertrophy, which rapidly progressed to heart failure and premature death. Massive cardiomyocyte necrosis rather than apoptosis appears to be the primary cause of the heart failure. This is because (1) massive necrotic cell death and increased infiltration of leukocytes were observed before increased apoptosis; (2) increased apoptosis was not detectable until overt heart failure was observed; and (3) cardiac overexpression of Bcl2 failed to ameliorate CR-Csn8KO mouse premature death. Conclusions: Csn8/CSN plays an essential role in cullin deneddylation, UPS-mediated degradation of a subset of proteins, and the survival of cardiomyocytes and, therefore, is indispensable in postnatal development and function of the heart. Cardiomyocyte-restricted UPS malfunction can cause heart failure.

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.

Long Non-Coding RNAs as Master Regulators in Cardiovascular Diseases

Cardiovascular disease is the leading cause of death in the United States, accounting for nearly one in every seven deaths. Over the last decade, various targeted therapeutics have been introduced, but there has been no corresponding improvement in patient survival. Since the mortality rate of cardiovascular disease has not been significantly decreased, efforts have been made to understand the link between heart disease and novel therapeutic targets such as non-coding RNAs. Among multiple non-coding RNAs, long non-coding RNA (lncRNA) has emerged as a novel therapeutic in cardiovascular medicine. LncRNAs are endogenous RNAs that contain over 200 nucleotides and regulate gene expression. Recent studies suggest critical roles of lncRNAs in modulating the initiation and progression of cardiovascular diseases. For example, aberrant lncRNA expression has been associated with the pathogenesis of ischemic heart failure. In this article, we present a synopsis of recent discoveries that link the roles and molecular interactions of lncRNAs to cardiovascular diseases. Moreover, we describe the prevalence of circulating lncRNAs and assess their potential utilities as biomarkers for diagnosis and prognosis of heart disease.