Comprehensive Analysis of Proteasomal Complexes in Mouse Brain Regions Detects ENO2 as a Potential Partner of the Proteasome in the Striatum.

Defects in the activity of the proteasome or its regulators are linked to several pathologies, including neurodegenerative diseases. We hypothesize that proteasome heterogeneity and its selective partners vary across brain regions and have a significant impact on proteasomal catalytic activities. Using neuronal cell cultures and brain tissues obtained from mice, we compared proteasomal activities from two distinct brain regions affected in neurodegenerative diseases, the striatum and the hippocampus. The results indicated that proteasome activities and their responses to proteasome inhibitors are determined by their subcellular localizations and their brain regions. Using an iodixanol gradient fractionation method, proteasome complexes were isolated, followed by proteomic analysis for proteasomal interaction partners. Proteomic results revealed brain region-specific non-proteasomal partners, including gamma-enolase (ENO2). ENO2 showed more association to proteasome complexes purified from the striatum than to those from the hippocampus. These results highlight a potential key role for non-proteasomal partners of proteasomes regarding the diverse activities of the proteasome complex recorded in several brain regions.

Posttranslational modification and quality control.

Protein quality control functions to minimize the level and toxicity of misfolded proteins in the cell. Protein quality control is performed by intricate collaboration among chaperones and target protein degradation. The latter is performed primarily by the ubiquitin-proteasome system and perhaps autophagy. Terminally misfolded proteins that are not timely removed tend to form aggregates. Their clearance requires macroautophagy. Macroautophagy serves in intracellular quality control also by selectively segregating defective organelles (eg, mitochondria) and targeting them for degradation by the lysosome. Inadequate protein quality control is observed in a large subset of failing human hearts with a variety of causes, and its pathogenic role has been experimentally demonstrated. Multiple posttranslational modifications can occur to substrate proteins and protein quality control machineries, promoting or hindering the removal of the misfolded proteins. This article highlights recent advances in posttranslational modification-mediated regulation of intracellular quality control mechanisms and its known involvement in cardiac pathology.

Atg7 induces basal autophagy and rescues autophagic deficiency in CryABR120G cardiomyocytes.

RATIONALE: Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. Several cardiomyopathies, including the αB-crystallin R120G mutation (CryAB(R120G)) model of desmin-related cardiomyopathy, accumulate cytotoxic misfolded proteins in the form of preamyloid oligomers and aggresomes. Impaired autophagic function is a potential cause of misfolded protein accumulations, cytoplasmic aggregate loads, and cardiac disease. Atg7, a mediator of autophagosomal biogenesis, is a putative regulator of autophagic function. OBJECTIVE: To determine whether autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes. METHODS AND RESULTS: To define the gain and loss of function effects of Atg7 expression on CryAB(R120G) protein misfolding and aggregates, neonatal rat cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB or CryAB(R120G) and coinfected with Atg7 adenovirus or with Atg7 silencing siRNAs to produce gain-of or loss-of Atg7 function. Atg7 overexpression effectively induced basal autophagy with no detrimental effects on cell survival, suggesting that Atg7 can activate autophagy with no apparent cytotoxic effects. Autophagic flux assays on CryAB(R120G)-expressing cardiomyocytes revealed reduced autophagic function, which probably contributed to the failure of misfolded proteins and aggregates to be cleared. Coexpression of Atg7 and CryAB(R120G) significantly reduced preamyloid oligomer staining, aggregate content, and cardiomyocyte cytotoxicity. Conversely, Atg7 silencing in the CryAB(R120G) background significantly inhibited the already reduced rate of autophagy and increased CryAB(R120G) aggregate content and cytotoxicity. CONCLUSIONS: Atg7 induces basal autophagy, rescues the CryAB(R120G) autophagic deficiency, and attenuates the accumulation of misfolded proteins and aggregates in cardiomyocytes.

Cardiomyocyte expression of a polyglutamine preamyloid oligomer causes heart failure.

BACKGROUND: To determine whether soluble preamyloid oligomers (PAOs) are toxic when expressed internally in the cardiomyocyte, we tested the hypothesis that cardiomyocyte-restricted expression and accumulation of a known PAO is cytotoxic and sufficient to cause heart failure. METHODS AND RESULTS: Intracellular PAOs, the entities believed to cause toxicity in many neurodegenerative diseases, have been observed in cardiomyocytes derived from mouse and human heart failure samples. Long (>50) polyglutamine (PQ) repeats form PAOs and cause neurotoxicity in Huntington disease and other neurodegenerative diseases, whereas shorter PQ peptides are benign. We created transgenic mice in which cardiomyocyte-autonomous expression of an 83 residue-long PQ repeat (PQ83) or a non-amyloid-forming peptide of 19 PQ repeats (PQ19) as a nonpathological control was expressed. A PQ83 line with relatively low levels of expression was generated, along with a PQ19 line that expressed approximately 9-fold the levels observed in the PQ83 line. Hearts expressing PQ83 exhibited reduced cardiac function and dilation by 5 months, and all mice died by 8 months, whereas PQ19 mice had normal cardiac function, morphology, and life span. PQ83 protein accumulated within aggresomes with PAO-specific staining. The PQ83 hearts showed increased autophagosomal and lysosomal content but also showed markers of necrotic death, including inflammatory cell infiltration and increased sarcolemmal permeability. CONCLUSIONS: The data confirm the hypothesis that expression of an exogenous PAO-forming peptide is toxic to cardiomyocytes and is sufficient to cause cardiomyocyte loss and heart failure in a murine model.

Macroautophagy and Chaperone-Mediated Autophagy in Heart Failure: The Known and the Unknown.

Cardiac diseases including hypertrophic and ischemic cardiomyopathies are increasingly being reported to accumulate misfolded proteins and damaged organelles. These findings have led to an increasing interest in protein degradation pathways, like autophagy, which are essential not only for normal protein turnover but also in the removal of misfolded and damaged proteins. Emerging evidence suggests a previously unprecedented role for autophagic processes in cardiac physiology and pathology. This review focuses on the major types of autophagic processes, the genes and protein complexes involved, and their regulation. It discusses the key similarities and differences between macroautophagy, chaperone-mediated autophagy, and selective mitophagy structures and functions. The genetic models available to study loss and gain of macroautophagy, mitophagy, and CMA are discussed. It defines the markers of autophagic processes, methods for measuring autophagic activities, and their interpretations. This review then summarizes the major studies of autophagy in the heart and their contribution to cardiac pathology. Some reports suggest macroautophagy imparts cardioprotection from heart failure pathology. Meanwhile, other studies find macroautophagy activation may be detrimental in cardiac pathology. An improved understanding of autophagic processes and their regulation may lead to a new genre of treatments for cardiac diseases.

Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle.

The direction of change in skeletal muscle mass differs between young and old individuals, growing in young animals and atrophying in old animals. The purpose of the experiment was to develop a statistically conservative list of genes whose expression differed significantly between young growing and old atrophying (sarcopenic) skeletal muscles, which may be contributing to physical frailty. Gene expression levels of >24,000 transcripts were determined in soleus muscle samples from young (3-4 mo) and old (30-31 mo) rats. Age-related differences were determined using a Student's t-test (alpha of 0.05) with a Bonferroni adjustment, which yielded 682 probe sets that differed significantly between young (n = 25) and old (n = 20) animals. Of 347 total decreases in aged/sarcopenic muscle relative to young muscles, 199 were functionally identified; the major theme being that 24% had a biological role in the extracellular matrix and cell adhesion. Three themes were observed from 213 of the 335 total increases in sarcopenic muscles whose functions were documented in databases: 1) 14% are involved in immune response; 2) 9% play a role in proteolysis, ubiquitin-dependent degradation, and proteasome components; and 3) 7% act in stress/antioxidant responses. A total of 270 differentially expressed genes and ESTs had unknown/unclear functions. By decreasing the sample sizes of young and old animals from 25 x 20 to 15 x 15, 10 x 10, and 5 x 5 observations, we observed 682, 331, 73, and 3 statistically different mRNAs, respectively. Use of large sample size and a Bonferroni multiple testing adjustment in combination yielded increased statistical power, while protecting against false positives. Finally, multiple mRNAs that differ between young growing and old, sarcopenic muscles were identified and may highlight new candidate mechanisms that regulate skeletal muscle mass during sarcopenia.

Selected Contribution: Identification of differentially expressed genes between young and old rat soleus muscle during recovery from immobilization-induced atrophy.

After cessation of hindlimb immobilization, which resulted in a 27-37% loss in soleus mass, the atrophied soleus muscle of young but not old rats regrows to its mass before treatment. We hypothesized that during remobilization the mRNA levels of growth potentiating factor(s) would be present in the soleus muscle of young (3- to 4-mo-old) but absent in old (30- to 31-mo-old) Fischer 344 x Brown Norway rats or that mRNAs for growth inhibitory factor(s) would be absent in young but present in old. Gene expression levels of >24,000 transcripts were determined by using Affymetrix RGU34A-C high-density oligonucleotide microarrays in soleus muscles at 3, 6, 10, and 30 days of remobilization after cessation of a 10-day period of hindlimb immobilization. Each muscle sample was applied to an independent set of arrays. Recovery-related differences were determined by using a three-factor ANOVA with a false discovery rate-adjustment of P = 0.01, which yielded 64 significantly different probe sets. Elfin, amphiregulin, and clusterin mRNAs were selected for further confirmation by real-time PCR. Elfin mRNA levels were less in old than in young rats at 6, 10, and 30 days of remobilization. Amphiregulin expression exhibited a unique spike on the 10th day of successful regrowth in young rats but remained unchanged old. Clusterin mRNA was unchanged in young muscles but was elevated on the 3rd, 6th, and 10th days of recovery in old soleus muscles. The mRNAs identified as differentially expressed between young and old recovery may modulate muscle growth that could highlight new candidate mechanisms to explain the failure of old soleus muscle to recover lost muscle mass.

Enhanced autophagy ameliorates cardiac proteinopathy.

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein. Hearts from these mice had enhanced autophagy, but normal morphology and function. We crossed these mice with CryABR120G mice, a model of DRC in which autophagy is significantly attenuated in the heart, to test the functional significance of autophagy activation in a proteotoxic model of heart failure. Sustained Atg7-induced autophagy in the CryABR120G hearts decreased interstitial fibrosis, ameliorated ventricular dysfunction, decreased cardiac hypertrophy, reduced intracellular aggregates and prolonged survival. To determine whether different methods of autophagy upregulation have additive or even synergistic benefits, we subjected the autophagy-deficient CryABR120G mice and the Atg7-crossed CryABR120G mice to voluntary exercise, which also upregulates autophagy. The entire exercised Atg7-crossed CryABR120G cohort survived to 7 months. These findings suggest that activating autophagy may be a viable therapeutic strategy for improving cardiac performance under proteotoxic conditions.

Macrophage autophagy plays a protective role in advanced atherosclerosis.

In advanced atherosclerosis, macrophage apoptosis coupled with defective phagocytic clearance of the apoptotic cells (efferocytosis) promotes plaque necrosis, which precipitates acute atherothrombotic cardiovascular events. Oxidative and endoplasmic reticulum (ER) stress in macrophages are important causes of advanced lesional macrophage apoptosis. We now show that proapoptotic oxidative/ER stress inducers trigger another stress reaction in macrophages, autophagy. Inhibition of autophagy by silencing ATG5 or other autophagy mediators enhances apoptosis and NADPH oxidase-mediated oxidative stress while at the same time rendering the apoptotic cells less well recognized by efferocytes. Most importantly, macrophage ATG5 deficiency in fat-fed Ldlr(-/-) mice increases apoptosis and oxidative stress in advanced lesional macrophages, promotes plaque necrosis, and worsens lesional efferocytosis. These findings reveal a protective process in oxidatively stressed macrophages relevant to plaque necrosis, suggesting a mechanism-based strategy to therapeutically suppress atherosclerosis progression and its clinical sequelae.

Autophagy and proteotoxicity in cardiomyocytes.

Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. We wished to determine if autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes. We used loss- and gain-of-function approaches in cultured cardiomyocytes to determine the effects of ATG7 knockdown and Atg7 overexpression in protein conformation-based toxicity induced by expression of a mutant aB crystallin (CryAB (R120G) ) known to cause human heart disease. We show that Atg7 induces basal autophagy and rescues the CryAB accumulation of misfolded proteins and aggregates in cardiomyocytes.

Protein misfolding and cardiac disease: establishing cause and effect.

Numerous neurodegenerative diseases are characterized by the accumulation of misfolded amyloidogenic proteins. Recent data indicate that a soluble pre-amyloid oligomer (PAO) may be the toxic entity in these diseases and the visible amyloid plaques, rather than causing the disease, may simply mark the terminal pathology. In prior studies, we observed PAO in the cardiomyocytes of many human heart failure samples. To test the hypothesis that cardiomyocyte-restricted expression of a known PAO is sufficient to cause heart failure, transgenic mice were created expressing polyglutamine repeats of 83 (PQ83) or 19 (PQ19). Long PQ repeats (>50) form PAOs and result in neurotoxicity in Huntington's disease, whereas shorter PQ repeats are benign. PQ83 expression caused the intracellular accumulation of PAOs and aggregates leading to cardiomyocyte death and heart failure. Evidence of increased autophagy and necrosis accompanied the PQ83 cardiomyocyte pathology. The data confirm that protein misfolding resulting in intracellular PAO accumulation is sufficient to cause cardiomyocyte death and heart failure.

Skeletal muscle IGF-binding protein-3 and -5 expressions are age, muscle, and load dependent.

The purpose of the current study was to examine IGFBP-3, -4, and -5 mRNA and protein expression levels as a function of muscle type, age, and regrowth from an immobilization-induced atrophy in Fischer 344 x Brown Norway rats. IGFBP-3 mRNA expression in the 4-mo-old animals was significantly higher in the red and white portions of the gastrocnemius muscle compared with the soleus muscle. However, there were no significant differences in IGFBP-3 mRNA expression among any of the muscle groups in the 30-mo-old animals. There were no significant differences in IGFBP-5 mRNA expression in any of the muscle groups, whereas in the 30-mo-old animals there was significantly less IGFBP-5 mRNA expression in the white gastrocnemius compared with the red gastrocnemius muscles. Although IGFBP-3 and -5 proteins were detected in the type I soleus muscle with Western blot analyses, no detection was observed in the type II red and white portions of the gastrocnemius muscle. Aging from adult (18 mo) to old animals (30 mo) was associated with decreases in IGFBP-3 mRNA and protein and IGFBP-5 protein only in the soleus muscle. After 10 days of recovery from 10 days of hindlimb immobilization, IGFBP-3 mRNA and protein increased in soleus muscles from young (4-mo) rats; however, only IGFBP-3 protein increased in the old (30-mo) rats. Whereas there were no changes in IGFBP-5 mRNA expression during recovery, IGFBP-5 protein in the 10-day-recovery soleus muscle did increase in the young, but not in the old, rats. Because one of the functions of IGFBPs is to modulate IGF-I action on muscle size and phenotype, it is hypothesized that IGFBP-3 and -5 proteins may have potential modulatory roles in type I fiber-dominated muscles, aging, and regrowth from atrophy.

Expression profiling identifies dysregulation of myosin heavy chains IIb and IIx during limb immobilization in the soleus muscles of old rats.

Aged individuals suffer from multiple dysfunctions during skeletal muscle atrophy. The purpose of this study was to determine differential changes in gene expression in atrophied soleus muscle induced by hindlimb immobilization in young (3-4 months) and old (30-31 months) rats. The hypothesis was that differentially expressed mRNAs with age-atrophy interactions would reveal candidates that induce loss of function responses in aged animals. Each muscle was applied to an independent set of Affymetrix micoarrays, with 385 differentially expressed mRNAs with atrophy and 354 age-atrophy interactions detected by two-factor ANOVA (alpha of 0.05 with a Bonferroni adjustment). Functional trends were observed for 23 and 15 probe sets involved in electron transport and the extracellular matrix, respectively, decreasing more in the young than in the old. Other functional categories with atrophy in both ages included chaperones, glutathione-S-transferases, the tricarboxylic acid cycle, reductions in Z-line-associated proteins and increases in probe sets for protein degradation. Surprisingly, myosin heavy chain IIb and IIx mRNAs were suppressed in the atrophied soleus muscle of old rats as opposed to the large increases in the young animals (16- and 25-fold, respectively, with microarrays, and 61- and 68-fold, respectively, with real-time PCR). No significant changes were observed in myosin heavy chain IIb and IIx mRNA with micoarrays in the atrophied soleus muscles of old rats, but they were found to increase six- and fivefold, respectively, with real-time PCR. Therefore, deficiencies in pre-translational signals that normally upregulate myosin heavy chain IIb and IIx mRNAs during atrophy may exist in the soleus muscle of old animals.