Abstract-Upregulation of ␣B-crystallin (CryAB), a small heat shock protein, is associated with a variety of diseases, including the desmin-related myopathies. CryAB, which binds to both desmin and cytoplasmic actin, may participate as a chaperone in intermediate filament formation and maintenance, but the physiological consequences of CryAB upregulation are unknown. A mutation in CryAB, R120G, has been linked to a familial desminopathy. However, it is unclear whether the mutation is directly causative. We created multiple transgenic mouse lines that overexpressed either murine wild-type CryAB or the R120G mutation in cardiomyocytes. Overexpression of wild-type CryAB was relatively benign, with no increases in mortality and no induction of desmin-related cardiomyopathy even in a line in which CryAB mRNA expression was increased Ϸ104-fold and the protein level increased by 11-fold. In contrast, lines expressing the R120G mutation were compromised, with a high-expressing line exhibiting 100% mortality by early adulthood. Modest expression levels resulted in a phenotype that was strikingly similar to that observed for the desmin-related cardiomyopathies. The desmin filaments in the cardiomyocytes were overtly affected, myofibril alignment was significantly impaired, and a hypertrophic response occurred at both the molecular and cellular levels. The data show that the R120G mutation causes a desminopathy, is dominant negative, and results in cardiac hypertrophy. Key Words: transgenic Ⅲ heart disease Ⅲ mouse Ⅲ cardiac Ⅲ genetics T he small heat shock-related protein ␣B-crystallin (CryAB) was originally discovered and classified as a lens protein. 1 CryAB is also found in nonlenticular tissues and is abundant in cardiac and skeletal muscle. 2,3 CryAB binds both desmin and cytoplasmic actin and possesses molecular chaperone function in vitro. 4 -6 When a cell is subjected to stress, CryAB transits from the cytosol onto the cytoskeleton. 7 Phosphorylation by mitogen-activated protein kinase, p38, and other kinases may regulate this translocation and presumably its chaperone function. 8,9 The upregulation of the gene and subsequent accumulation of CryAB occurs in a number of cardiac disorders including familial hypertrophic cardiomyopathy and desminopathy, 10 -12 as well as degenerative neural pathologies such as Alexander and Alzheimer diseases. 2,13 However, the pathophysiological significance, if any, of CryAB protein upregulation in muscle remains obscure.A missense mutation (R120G) of CryAB has recently been linked to familial desmin-related myopathy (DRM), a disease that is characterized by intrasarcoplasmic accumulation of desmin. 14 Restrictive, hypertrophic, and dilated cardiomyopathies have all been observed in the desminopathies and often result in death. 12,15 Overexpression of R120G-CryAB in a muscle cell line caused formation of electron-dense aggregates containing CryAB in the center and desmin at the periphery. 14 However, there is no direct in vivo evidence, outside of linkage analysis, proving that th...

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.

Abstract-The heart is constantly under mechanical, metabolic, and thermal stress, even at baseline physiological conditions, and cardiac stress may increase as a result of environmental or intrinsic pathological insults. Cardiomyocytes are continuously challenged to efficiently and properly fold nascent polypeptides, traffic them to their appropriate cellular locations, and keep them from denaturing in the face of normal and pathological stimuli. ecause of alternative splicing of primary transcripts, posttranslational modifications, and the ability to assume multiple conformations that differ in activity, the proteome, in terms of its informational content, is considerably more complex than the genome and transcriptome. Thus, it is not surprising that controlling the quality of this information is essential for cell survival and function. Multiple layers of quality control for protein production and maintenance exist. After their initial synthesis, proteins targeted for the membrane and secretory pathways are modified, folded, and assembled in the endoplasmic reticulum (ER), whereas other cellular proteins may be synthesized and processed independently of the ER in the cytosol. Accordingly, there exist both ER-associated protein quality control (PQC) and ERindependent PQC. In both cases, molecular chaperones and the ubiquitin/proteasome system (UPS) play essential roles. In general, chaperones are responsible for protecting unfolded or partially folded nascent or mature proteins, with many chaperones participating in protein repair, 1 whereas the UPS is largely responsible for removing terminally misfolded proteins permanently, thereby preventing misfolded proteins from accumulating in the cell. 2 Although the first lines of defense rest in the "proofreading" of the primary DNA and RNA sequences, the cell has evolved multiple layers of control at the posttranslational level as well, and nascent proteins are subject to rigorous surveillance as they are synthesized on the polysomes. Although small-and medium-sized proteins often can assume their correct tertiary and quaternary conformations spontaneously, the majority of proteins cannot and depend on the help of and interaction with other proteins to fold correctly. The complete sequence is often necessary for assuming the correct conformation, but the linear process of protein synthesis presents unfinished proteins to the cellular environ-

Abstract-The presence of increased ubiquitinated proteins and amyloid oligomers in failing human hearts strikingly resembles the characteristic pathology in the brain of many neurodegenerative diseases. The ubiquitin-proteasome system (UPS) is responsible for degradation of most cellular proteins and plays essential roles in virtually all cellular processes. UPS impairment by aberrant protein aggregation was previously shown in cell culture but remains to be demonstrated in intact animals. Mechanisms underlying the impairment are poorly understood. We report here that UPS proteolytic function is severely impaired in the heart of a mouse model of intrasarcoplasmic amyloidosis caused by cardiac-restricted expression of a human desmin-related myopathy-linked missense mutation of ␣B-crystallin (CryAB R120G ). The UPS impairment was detected before cardiac hypertrophy, and failure became discernible, suggesting that defective protein turnover likely contributes to cardiac remodeling and failure in this model. Further analyses reveal that the impairment is likely attributable to insufficient delivery of substrate proteins into the 20S proteasomes, and depletion of key components of the 19S subcomplex may be responsible. The derangement is likely caused by aberrant protein aggregation rather than loss of function of the CryAB gene because UPS malfunction was not evident in CryAB-null hearts and inhibition of aberrant protein aggregation by Congo red or a heat shock protein significantly attenuated CryAB R120G -induced UPS malfunction in cultured cardiomyocytes. Because of the central role of the UPS in cell regulation and the high intrasarcoplasmic amyloidosis prevalence in failing human hearts, our data suggest a novel pathogenic process in cardiac disorders with abnormal protein aggregation. Key Words: proteasome Ⅲ ubiquitin Ⅲ protein aggregation Ⅲ ␣B-crystallin Ⅲ desmin-related cardiomyopathy Ⅲ amyloidosis Ⅲ transgenic mice M ost cellular proteins are degraded through the ubiquitin-proteasome system (UPS). UPS-mediated proteolysis includes 2 major steps: attachment of a chain of ubiquitin to the target protein molecule through a process known as ubiquitination and degradation of the ubiquitinated proteins by the 26S proteasome. The latter consists of a barrel-shaped 20S core and the 19S cap on 1 or both ends of the 20S. The actual proteolytic activity resides in the interior of the 20S, whereas the 19S plays a critical role in channeling ubiquitinated protein molecules into the 20S. 1 Ubiquitinated proteins accumulate in the cell when the proteasome is inhibited. 1 Aberrant protein aggregation is a common process in many neural degenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. 2,3 Remaining to be demonstrated in intact animals, this process was shown in cell culture to impair UPS-mediated proteolysis. 4 Therefore, UPS malfunction is considered an important pathogenic mechanism in neurodegeneration. 5 Recent studies revealed that abnormal protein aggregation, intrasarcoplasmic amyl...