Kathleen M. Horak

Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts

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.

Intrasarcoplasmic Amyloidosis Impairs Proteolytic Function of Proteasomes in Cardiomyocytes by Compromising Substrate Uptake

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 &agr;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.

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.

A novel transgenic mouse model reveals deregulation of the ubiquitin-proteasome system in the heart by doxorubicin

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.

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.

Doxycycline Attenuates Protein Aggregation in Cardiomyocytes and Improves Survival of a Mouse Model of Cardiac Proteinopathy

OBJECTIVES The goal of this pre-clinical study was to assess the therapeutic efficacy of doxycycline (Doxy) for desmin-related cardiomyopathy (DRC) and to elucidate the potential mechanisms involved. BACKGROUND DRC, exemplifying cardiac proteinopathy, is characterized by intrasarcoplasmic protein aggregation and cardiac insufficiency. No effective treatment for DRC is available presently. Doxy was shown to attenuate aberrant intranuclear aggregation and toxicity of misfolded proteins in noncardiac cells and animal models of other proteinopathies. METHODS Mice and cultured neonatal rat cardiomyocytes with transgenic (TG) expression of a human DRC-linked missense mutation R120G of αB-crystallin (CryAB(R120G)) were used for testing the effect of Doxy. Doxy was administered via drinking water (6 mg/ml) initiated at 8 or 16 weeks of age. RESULTS Doxy treatment initiated at 16 weeks of age significantly delayed the premature death of CryAB(R120G) TG mice, with a median lifespan of 30.4 weeks (placebo group, 25 weeks; p < 0.01). In another cohort of CryAB(R120G) TG mice, Doxy treatment initiated at 8 weeks of age significantly attenuated cardiac hypertrophy in 1 month. Further investigation revealed that Doxy significantly reduced the abundance of CryAB-positive microscopic aggregates, detergent-resistant CryAB oligomers, and total ubiquitinated proteins in CryAB(R120G) TG hearts. In cell culture, Doxy treatment dose-dependently suppressed the formation of both microscopic protein aggregates and detergent-resistant soluble CryAB(R120G) oligomers and reversed the up-regulation of p62 protein induced by adenovirus-mediated CryAB(R120G) expression. CONCLUSIONS Doxy suppresses CryAB(R120G)-induced aberrant protein aggregation in cardiomyocytes and prolongs CryAB(R120G)-based DRC mouse survival.