Aberrant protein aggregates in cardiomyocytes are frequently observed in many forms of cardiomyopathies and are often associated with impairment of proteolytic function of the ubiquitin-proteasome system (UPS). However, a causal relationship between mutant desmin (MTdes) induced aberrant protein aggregation and UPS impairment has not been established. The present study has tested the causal relationship. In cultured neonatal rat ventricular myocytes, modest overexpression of a human (cardio)myopathy-linked MT-des protein led to formation of desmin-positive aggregates and inhibited UPS proteolytic function in cardiomyocytes in a dose-dependent manner. Prevention or reduction of aberrant protein aggregation by co-expression of a heat shock protein (Hsp), αB-crystallin or inducible Hsp70, or by treatment of Congo red prevented and/or significantly attenuated the induction of UPS malfunction by MT-des. These findings prove for the first time that aberrant protein aggregation is not only sufficient but also required for MT-des to impair UPS proteolytic function in cardiomyocytes.

Protein misfolding and aberrant aggregation are associated with many severe disorders, such as neural degenerative diseases, desmin-related myopathy (DRM), and congestive heart failure. Intrasarcoplasmic amyloidosis and increased ubiquitinated proteins are observed in human failing hearts. The pathogenic roles of these derangements in the heart remain unknown. The ubiquitin-proteasome system (UPS) plays a central role in intracellular proteolysis and regulates critical cellular processes. In cultured cells, aberrant aggregation by a mutant (MT) or misfolded protein impairs the UPS. However, this has not been demonstrated in intact animals, and it is unclear how the UPS is impaired. Cross-breeding UPS reporter mice with a transgenic mouse model of DRM featured by aberrant protein aggregation in cardiomyocytes, we found that overexpression of MT-desmin but not normal desmin protein impairs UPS proteolytic function in the heart. The primary defect does not appear to be in the ubiquitination or the proteolytic activity of the 20S proteasome, because ubiquitinated proteins and the peptidase activities of 20S proteasomes were significantly increased rather than decreased in the DRM heart. Therefore, the defect resides apparently in the entry of ubiquitinated proteins into the 20S proteasome. Consistent with this notion, key components (Rpt3 and Rpt5) of 19S proteasomes were markedly decreased, while major components of 20S proteasomes were increased. Additional experiments with HEK cells suggest that proteasomal malfunction observed in MT-desmin hearts is not secondary to cardiac malfunction or to disruption of desmin filaments. Thus, UPS impairment may represent an important pathogenic mechanism underlying cardiac disorders with abnormal protein aggregation.

Dong, Xin, Jinbao Liu, Hanqiao Zheng, Joseph W. Glasford, Wei Huang, Quan Hai Chen, Niels R. Harden, Faqian Li, A. Martin Gerdes, and Xuejun Wang. In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes. Am J Physiol Heart Circ Physiol 287: H1417–H1425, 2004. First published April 22, 2004; 10.1152/ajpheart. 01233.2003.—The ubiquitin-proteasome system (UPS) is responsible for turnover of most cellular proteins in eukaryotes. Protein degradation by the UPS serves quality control and regulatory functions. Proteasome inhibition showed great promise in effectively treating cancer and restenosis. UPS dysfunction in cardiac hypertrophy and failure has recently been suspected but remains to be investigated. A system capable of monitoring dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ would no doubt benefit significantly efforts to decipher the pathogenic significance of UPS dysfunction in the heart and to evaluate the effect of proteasome inhibition on cardiac myocytes. We successfully established such a system in cultured cardiac myocytes by delivering and expressing a modified green fluorescence protein (GFPu) gene using recombinant adenoviruses. GFPu contains a ubiquitination signal sequence fused to the COOH terminus. Fluorescence microscopy and Western blots revealed that protein abundance of modified green fluorescent protein (GFPu), but not wild-type green fluorescent protein, in cultured cardiac myocytes was incrementally increased when function of the proteasomes was inhibited in various degrees by specific inhibitors. The increase in GFPu protein levels and fluorescence intensity is paralleled by a decrease in the in vitro peptidase activity of the proteasomes. Our results demonstrate that GFPu can be used as a surrogate marker to monitor dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ. Application of this novel system reveals that moderate levels of H2O2, a reactive oxygen species generator, impair proteolytic function of the UPS in cultured cardiac myocytes.

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.

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 (CryABR120G). 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 CryABR120G-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. (Circ Res. 2005; 97: 1018-1026.)