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...

Ubiquitin-proteasome system (UPS) mediated proteolysis is responsible for the degradation of majority of cellular proteins, thereby playing essential roles in maintaining cellular homeostasis and regulating a number of cellular functions. UPS dysfunction was implicated in the pathogenesis of numerous disorders, including neurodegenerative disease, muscular dystrophy, and a subset of cardiomyopathies. However, monitoring in vivo functional changes of the UPS remains a challenge, which hinders the elucidation of UPS pathophysiology. We have recently created a novel transgenic mouse model that ubiquitously expresses a surrogate protein substrate for the UPS. The present study validates its suitability to monitor in vivo changes of UPS proteolytic function in virtually all major organs. Primary culture of cells derived from the adult transgenic mice was also developed and tested for their applications in probing UPS involvement in pathogenesis. Applying these newly established in vivo and in vitro approaches, we have proven in the present study that doxorubicin enhances UPS function in the heart and in cultured cardiomyocytes, suggesting that UPS hyper-function may play an important role in the acute cardiotoxicity of doxorubicin therapy.

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. Key Words: COP9 signalosome Ⅲ ubiquitin E3 ligases Ⅲ proteasome Ⅲ cell death Ⅲ heart failure T he ubiquitin-proteasome system (UPS) mediates the main proteolytic pathway of targeted protein degradation in the cell. UPS dysfunction has been observed in animal models of a variety of cardiac disorders, 1-4 including ischemic heart disease, pressure-overload cardiac hypertrophy and failure, diabetic cardiomyopathy, familial hypertrophic and dilated cardiomyopathy, 5,6 desmin-related cardiomyopathy, and doxorubicin cardiotoxicity. 7,8 UPS dysfunction has also been implicated in failing human hearts. 6,9,10 Both clinical observation and some animal experiments have suggested that proteasome functional insufficiency may play an important role in the genesis of congestive heart failure (CHF). [11][12][13][14] Therefore, a better understanding of the molecular mechanisms by which the UPS function is regulated in the heart may provide critical information for developing novel therapeutic strategies to treat the related cardiac disorders or to protect against cardiotoxicity from chemotherapies. To this end, a few recent reports have excitingly begun to decipher the regulation of proteasome activities in the heart under the baseline and certain pathological conditions by factors including posttranslational modifications. 14 -20 Meanwhile, currently, proteasome inhibition in intact animals can only be Original received August 16, 2010; revision received Octo...