Insufficient lysosomal removal of autophagic cargoes in cardiomyocytes has been suggested as a main cause for the impairment of the autophagic-lysosomal pathway (ALP) in many forms of heart disease including cardiac proteinopathy and may play an important pathogenic role; however, the molecular basis and the correcting strategy for the cardiac ALP insufficiency require further investigation. The present study was sought to determine whether myocardial expression and activity of TFEB, the recently identified ALP master regulator, are impaired in a cardiac proteinopathy mouse model and to determine the effect of genetic manipulation of TFEB expression on autophagy and proteotoxicity in a cardiomyocyte model of proteinopathy. We found that increased myocardial TFEB mRNA levels and a TFEB protein isoform switch were associated with marked decreases in the mRNA levels of representative TFEB target genes and increased mTORC1 activation, in mice with cardiac transgenic expression of a missense (R120G) mutant αB-crystallin (CryABR120G), a well-established model of cardiac proteinopathy. Using neonatal rat ventricular cardiomyocyte cultures, we demonstrated that downregulation of TFEB decreased autophagic flux in cardiomyocytes both at baseline and during CryABR120G overexpression and increased CryABR120G protein aggregates. Conversely, forced TFEB overexpression increased autophagic flux and remarkably attenuated the CryABR120G overexpression- induced accumulation of ubiquitinated proteins, caspase 3 cleavage, LDH leakage, and decreases in cell viability. Moreover, these protective effects of TFEB were dramatically diminished by inhibiting autophagy. We conclude that myocardial TFEB signaling is impaired in cardiac proteinopathy and forced TFEB overexpression protects against proteotoxicity in cardiomyocytes through improving ALP activity.

No current treatment targets cardiac proteotoxicity or can reduce mortality of heart failure (HF) with preserved ejection fraction (HFpEF). Selective degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is vital to the cell. Proteasome impairment contributes to HF. Activation of cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG) facilitates proteasome functioning. Phosphodiesterase 1 (PDE1) hydrolyzes both cyclic nucleotides and accounts for most PDE activities in human myocardium. We report that PDE1 inhibition (IC86430) increases myocardial 26S proteasome activities and UPS proteolytic function in mice. Mice with CryABR120G-based proteinopathy develop HFpEF and show increased myocardial PDE1A expression. PDE1 inhibition markedly attenuates HFpEF, improves mouse survival, increases PKA-mediated proteasome phosphorylation, and reduces myocardial misfolded CryAB. Therefore, PDE1 inhibition induces PKA- and PKG-mediated promotion of proteasomal degradation of misfolded proteins and treats HFpEF caused by CryABR120G, representing a potentially new therapeutic strategy for HFpEF and heart disease with increased proteotoxic stress.

Rationale: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP) are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and ALP defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the two catabolic pathways will help advance cardiac pathophysiology and medicine. Objective: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac ALP. Methods and Results: Myocardial macroautophagy, transcription factor EB (TFEB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type (WT) mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from WT mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with WT mice. Conclusions: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac ALP activation during proteasome malfunction.

The ubiquitin-proteasome system (UPS) degrades a protein molecule via 2 main steps: ubiquitination and proteasomal degradation. Extraproteasomal ubiquitin receptors are thought to couple the 2 steps, but this proposition has not been tested in vivo with vertebrates. More importantly, impaired UPS performance plays a major role in cardiac pathogenesis, including myocardial ischemia-reperfusion injury (IRI), but the molecular basis of UPS impairment remains poorly understood. Ubiquilin1 is a bona fide extraproteasomal ubiquitin receptor. Here, we report that mice with a cardiomyocyte-restricted knockout of Ubiquilin1 (Ubqln1-CKO mice) accumulated a surrogate UPS substrate (GFPdgn) and increased myocardial ubiquitinated proteins without altering proteasome activities, resulting in late-onset cardiomyopathy and a markedly shortened life span. When subject to regional myocardial ischemia-reperfusion, young Ubqln1-CKO mice showed substantially exacerbated cardiac malfunction and enlarged infarct size, and conversely, mice with transgenic Ubqln1 overexpression displayed attenuated IRI. Furthermore, Ubqln1 overexpression facilitated proteasomal degradation of oxidized proteins and the degradation of a UPS surrogate substrate in cultured cardiomyocytes without increasing autophagic flux. These findings demonstrate that Ubiquilin1 is essential to cardiac ubiquitination-proteasome coupling and that an inadequacy in the coupling represents a major pathogenic factor for myocardial IRI; therefore, strategies to strengthen coupling have the potential to reduce IRI.

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors.