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OBJECTIVE: Proteasome activation by the cAMP-dependent protein kinase (PKA) was long suggested and recent studies using both cell cultures and genetically engineered mice have established that direct phosphorylation of RPN6/PSMD11 at Serine14 (pS14-RPN6) mediates the activation of 26S proteasomes by PKA. Genetic mimicry of pS14-RPN6 has been shown to be benign at baseline and capable of protecting against cardiac proteinopathy in mice. Here we report the results from a comprehensive baseline characterization of the Rpn6S14A mice (S14A), the first animal model of genetic blockade of the activation of 26S proteasomes by PKA. METHOD: Wild type and homozygous S14A littermate mice were subjected to serial M-mode echocardiography at 1 through 7 months of age, to left ventricular (LV) catheterization via the carotid artery for assessment of LV mechanical performance, and to cardiac gravimetric analyses at 26 weeks of age. Mouse mortality and morbidity were monitored daily for up to one year. Males and females were studied in parallel. RESULTS: Mice homozygous for S14A were viable and fertile and did not show discernible developmental abnormalities or increased mortality or morbidity compared with their Rpn6 wild type littermates by at least one year of age, the longest cohort observed thus far. Neither serial echocardiography nor hemodynamic assessments detected a remarkable difference in cardiac morphometry and function between S14A and wild type littermate mice. No cardiac gravimetric difference was observed. CONCLUSION: The findings of the present study indicate that genetic blockade of the activation of 26S proteasomes by PKA is well tolerated by mice at baseline. Therefore, the S14A mouse provides a desirable genetic tool for further investigating the in vivo pathophysiological and pharmacological significance of pS14-RPN6.
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Previous studies revealed a controversial role of mechanistic target of rapamycin complex 1 (mTORC1) and mTORC1-regulated macroautophagy in isoproterenol (ISO)-induced cardiac injury. Here we investigated the role of mTORC1 and potential underlying mechanisms in ISO-induced cardiomyocyte necrosis. Two consecutive daily injections of ISO (85 mg/kg, s.c.) or vehicle control (CTL) were administered to C57BL/6J mice with or without rapamycin (RAP, 5 mg/kg, i.p.) pretreatment. Western blot analyses showed that myocardial mTORC1 signaling and the RIPK1-RIPK3-MLKL necroptotic pathway were activated, mRNA expression analyses revealed downregulation of representative TFEB target genes, and Evan's blue dye uptake assays detected increased cardiomyocyte necrosis in ISO-treated mice. However, RAP pretreatment prevented or significantly attenuated the ISO-induced cardiomyocyte necrosis, myocardial inflammation, downregulation of TFEB target genes, and activation of the RIPK1-RIPK3-MLKL pathway. LC3-II flux assays confirmed the impairment of myocardial autophagic flux in the ISO-treated mice. In cultured neonatal rat cardiomyocytes, mTORC1 signaling was also activated by ISO, and inhibition of mTORC1 by RAP attenuated ISO-induced cytotoxicity. These findings suggest that mTORC1 hyperactivation and resultant suppression of macroautophagy play a major role in the induction of cardiomyocyte necroptosis by catecholamine surges, identifying mTORC1 inhibition as a potential strategy to treat heart diseases with catecholamine surges.
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Catecolaminas , Miocitos Cardíacos , Animales , Ratones , Ratas , Ratones Endogámicos C57BL , Macroautofagia , Necroptosis , Isoproterenol/toxicidad , Diana Mecanicista del Complejo 1 de la Rapamicina , NecrosisRESUMEN
BACKGROUND: Calcium-free (Ca-free) solutions are theoretically the most ideal for regional citrate anticoagulation (RCA) in continuous renal replacement therapy (CRRT). However, the majority of medical centers in China had to make a compromise of using commercially available calcium-containing (Ca-containing) solutions instead of Ca-free ones due to their scarcity. This study was designed to probe into the potential of Ca-containing solution as a secure and efficient substitution for Ca-free solutions. METHODS: In this prospective, randomized single-center trial, 99 patients scheduled for CRRT were randomly assigned in a 1:1:1 ratio to one of three treatment groups: continuous veno-venous hemodialysis Ca-free dialysate (CVVHD Ca-free) group, continuous veno-venous hemodiafiltration calcium-free dialysate (CVVHDF Ca-free) group, and continuous veno-venous hemodiafiltration Ca-containing dialysate (CVVHDF Ca-containing) group at cardiac intensive care unit (CICU). The primary endpoint was the incidence of metabolic complications. The secondary endpoints included premature termination of treatment, thrombus of filter, and bubble trap after the process. RESULTS: The incidence of citrate accumulation (18.2% vs. 12.1% vs. 21.2%) and metabolic alkalosis (12.1% vs. 0% vs. 9.1%) did not significantly differ among three groups (p > 0.05 for both). The incidence of premature termination was comparable among the groups (18.2% vs. 9.1% vs. 9.1%, p = 0.582). The thrombus level of the filter and bubble trap was similar in the three groups (p > 0.05 for all). CONCLUSIONS: In RCA-CRRT for CICU population, RCA-CVVHDF with Ca-containing solutions and traditional RCA with Ca-free solutions had a comparable safety and feasibility. TRIAL REGISTRATION: ChiCTR2100048238 in the Chinese Clinical Trial Registry.
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Anticoagulantes , Ácido Cítrico , Terapia de Reemplazo Renal Continuo , Soluciones para Diálisis , Estudios de Factibilidad , Humanos , Femenino , Masculino , Terapia de Reemplazo Renal Continuo/métodos , Persona de Mediana Edad , Anticoagulantes/administración & dosificación , Estudios Prospectivos , Ácido Cítrico/administración & dosificación , Soluciones para Diálisis/administración & dosificación , Soluciones para Diálisis/química , Anciano , China , Calcio/sangre , Calcio/administración & dosificación , Lesión Renal Aguda/terapiaRESUMEN
INTRODUCTION: Acute mitral regurgitation (MR) due to papillary muscle rupture (PMR) is a rare but lethal mechanical complication of acute myocardial infarction (MI). The treatment of patients with post-MI PMR, especially those with cardiogenic shock, presents great challenges due to the high surgical risk. PATIENT CONCERNS: We report an 80-year-old woman with a history of hypertension and diabetes mellitus, presented with chest pain. Despite an early percutaneous coronary intervention and transfer to the intensive care unit, her general condition and hemodynamic parameters continued to deteriorate rapidly. DIAGNOSIS: Evidenced by electrocardiogram, echocardiogram and coronary angiography, the patient was diagnosed with acute lateral and posterior ST-segment elevation MI, cardiogenic shock, PMR, severe MR, and pulmonary edema. INTERVENTIONS: The patient received percutaneous mitral valve repair with MitraClip (Abbott Vascular, Santa Clara, CA, USA) supported by extracorporeal membranous oxygenation and intra-aortic balloon pump. OUTCOMES: The patient was discharged with relief of heart failure symptoms, reduced MR, and recovery of cardiac function, remaining in a stable condition in New York Heart Association class I after 15-month outpatient follow up. CONCLUSION: Transcatheter edge-to-edge repair with MitraClip can serve as a viable alternative to surgery in reducing MR in post-MI PMR patients at high surgical risk.
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Procedimientos Quirúrgicos Cardíacos , Implantación de Prótesis de Válvulas Cardíacas , Insuficiencia de la Válvula Mitral , Infarto del Miocardio , Humanos , Femenino , Anciano de 80 o más Años , Insuficiencia de la Válvula Mitral/etiología , Insuficiencia de la Válvula Mitral/cirugía , Choque Cardiogénico/etiología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/cirugía , Infarto del Miocardio/diagnóstico , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Ecocardiografía , Resultado del Tratamiento , Implantación de Prótesis de Válvulas Cardíacas/efectos adversosRESUMEN
BACKGROUND: A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that PKA (cAMP-dependent protein kinase or protein kinase A) activates the 26S proteasome by pS14-Rpn6 (serine14-phosphorylated Rpn6), but this discovery and its physiological significance remain to be established in vivo. METHODS: Male and female mice with Ser14 of Rpn6 (regulatory particle non-ATPase 6) mutated to Ala (S14A [Rpn6/Psmd11S14A]) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system functioning was evaluated with the GFPdgn (green fluorescence protein with carboxyl fusion of the CL1 degron) reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G [arginine120 to glycine missense mutant alpha B-crystallin]). RESULTS: PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type but not S14A embryonic fibroblasts (mouse embryonic fibroblasts), adult cardiomyocytes, and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and adult mouse cardiomyocytes than in wild-type counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with nontransgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than wild-type neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates, and of aberrant CryAB (alpha B-crystallin) protein aggregates, less fetal gene reactivation, and cardiac hypertrophy, and delays in cardiac malfunction. CONCLUSIONS: This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifying a new therapeutic target to reduce cardiac proteotoxicity.
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Complejo de la Endopetidasa Proteasomal , Cadena B de alfa-Cristalina , Femenino , Masculino , Animales , Ratones , Fibroblastos , Miocitos Cardíacos , Proteínas Quinasas Dependientes de AMP Cíclico , UbiquitinasRESUMEN
Background: A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that cAMP-dependent protein kinase (PKA) activates the 26S proteasome by phosphorylating Ser14 of RPN6 (pS14-RPN6), but this discovery and its physiological significance remain to be established in vivo . Methods: Male and female mice with Ser14 of Rpn6 mutated to Ala (S14A) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system (UPS) functioning was evaluated with the GFPdgn reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G). Results: PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type (WT) but not S14A embryonic fibroblasts (MEFs), adult cardiomyocytes (AMCMs), and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and AMCMs than in WT counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with non- transgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than WT neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates and of aberrant CryAB protein aggregates, less reactivation of fetal genes and cardiac hypertrophy, and delays in cardiac malfunction. Conclusions: This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifies a new therapeutic target to reduce cardiac proteotoxicity.
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Dyslipidemia is correlated with diverse cardiovascular problems, such as obesity, hypertension, and atherosclerosis, which are summarized as metabolic syndrome. Bicuspid aortic valve (BAV), as one of the congenital heart defects, is shown to influence approximately 2.2% of the general population worldwide, inducing the severe pathological development of aortic valve stenosis (AVS) or aortic valve regurgitation (AVR), and also to aortic dilatation. Notably, emerging evidence showed that BAV was correlated with not only the aortic valve and wall diseases but also the dyslipidemic related cardiovascular disorders. Recent results also proposed that multiple potential molecular mechanisms inducing the progression of dyslipidemia played important roles in BAV and the progression of AVS. Several altered serum biomarkers under dyslipidemic condition, including higher low-density lipoprotein cholesterol (LDL-C), higher lipoprotein (a) [Lp(a)], lower high-density lipoprotein cholesterol (HDL-C), and different pro-inflammatory signaling pathways, have proposed to embrace a vital function in the development of BAV correlated cardiovascular diseases. In this review, different molecular mechanisms which embrace an important role in personalized prognosis in the subjects with BAV was summarized. The illustration of those mechanisms might facilitate an accurate follow-up for patients with BAV and give new pharmacological strategies to improve development of dyslipidemia and BAV.
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Aterosclerosis , Enfermedad de la Válvula Aórtica Bicúspide , Enfermedades Cardiovasculares , Dislipidemias , Humanos , HDL-Colesterol , Dislipidemias/genética , Lipoproteína(a)RESUMEN
As is shown in previous reports, arginine vasopressin (AVP), as one of the most important hormones within circulation in human beings, is of great clinically significance given that it could maintain the body fluid balance and vascular tone. However, the laboratory measurements AVP in daily clinical practice are shown to be difficult and with low accuracy. Concerning on this notion, it is unpractical to use the serum levels of AVP in diagnosing multiple diseases. On the other hand, another key serum biomarker, copeptin, is confirmed as the C-terminal of the AVP precursor which could be released in equal amounts with AVP, resultantly making it as a sensitive marker of arginine vasopressin release. Notably, emerging recent evidence has demonstrated the critical function of copeptin as a clinical indicator, especially in the diagnosis and prognosis of several diseases in diverse organs, such as cardiovascular disease, kidney disease, and pulmonary disease. In addition, copeptin was recently verified to play an important role in diagnosing multiple acute diseases when combined it with other gold standard serum biomarkers, indicating that copeptin could be recognized as a vital disease marker. Herein, in the current review, the functions of copeptin as a new predictive diagnostic and prognostic biomarker of various diseases, according to the most recent studies, are well summarized. Furthermore, the importance of using copeptin as a serum biomarker in diverse medical departments and the impact of this on improving healthcare service is also summarized in the current review.
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Arginina Vasopresina , Glicopéptidos , Humanos , Pronóstico , BiomarcadoresRESUMEN
According to the previous studies, chronic low-grade systemic inflammatory response has been shown to be significantly associated with the pathological development of cardio-metabolic disorder diseases, including atherosclerosis, type 2 diabetes mellitus, and non-alcoholic fatty liver disease (NAFLD). On the other hand, auto-immunity process could also facilitate the pathogenesis of type 1 diabetes mellitus importantly. Concerning on this notion, the anti-inflammatory therapeutic strategy is demonstrated to embrace an essential function in those cardio-metabolic disorders in clinical practice. The α-1 anti-trypsin, also named Serpin-A1 and as an acute phase endogenous protein, has been verified to have several modulatory effects such as anti-inflammatory response, anti-apoptosis, and immunomodulatory functions. In addition, it is also used for therapeutic strategy of a rare genetic disease caused by the deficiency of α-1 anti-trypsin. Recent emerging evidence has indicated that the serum concentrations of α-1 anti-trypsin levels and its biological activity are significantly changed in those inflammatory and immune related cardio-metabolic disorder diseases. Nevertheless, the underlying mechanism is still not elucidated. In the current review, the basic experiments and clinical trials which provided the evidence revealing the potential therapeutic function of the α-1 anti-trypsin in cardio-metabolic disorder diseases were well-summarized. Furthermore, the results which indicated that the α-1 anti-trypsin presented the possibility as a novel serum biomarker in humans to predict those cardio-metabolic disorder diseases were also elucidated.
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Diabetes Mellitus Tipo 2 , Enfermedades Metabólicas , Síndrome Metabólico , Enfermedad del Hígado Graso no Alcohólico , Diabetes Mellitus Tipo 2/complicaciones , Humanos , Inflamación/complicaciones , Síndrome Metabólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicacionesRESUMEN
Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinase known to play essential roles in the nervous tissue. Myocardial upregulation of UCHL1 was observed in human dilated cardiomyopathy and several animal models of heart disease, but the (patho)physiological significance of UCHL1 in cardiomyocytes remains undefined. Hence, we conducted this study to fill this critical gap. We produced cardiomyocyte-restricted Uchl1 knockout (CKO) by coupling the Uchl1-floxed allele with transgenic Myh6-Cre in C57B/6J inbred mice. Mice transgenic for Myh6-Cre were used as controls (CTL). Myocardial Uchl1 proteins were markedly reduced in CKO mice but they did not display discernible abnormal phenotype. Ten-week old CTL or CKO mice were subjected to left anterior descending artery ligation (myocardial infarction, MI) or sham surgery (Sham) and characterized at 7- and 28-day after surgery. Compared with Sham mice, significant increases in myocardial UCHL1 proteins were detected in CTL MI but not in CKO MI mice. MI-induced left ventricular (LV) chamber dilation, reduction of ejection fraction (EF) and fractional shortening (FS), and LV anterior wall thinning detected by echocardiography were comparable between the CTL MI and CKO MI groups 7-day post-MI. However, by 28-day post-MI, MI-induced LV chamber dilatation, EF and FS reduction, increases of myocardial ubiquitin conjugates, and increases in the heart weight to body weight ratio and the ventricular weight to body weight ratio were significantly more pronounced in CKO MI than CTL MI mice. As further revealed by LV pressure-volume relationship analyses, CKO MI mice but not CTL MI mice displayed significant decreases in stroke volume, cardiac output, and the maximum rates of LV pressure rising or declining and of LV volume declining, as well as significant increases in LV end-diastolic pressure and Tau, compared with their respective Sham controls. LC3-II flux assays reveal that autophagic flux is decreased in CKO mouse myocardium as well as in cultured Uchl1-deficient cardiomyocytes. In conclusion, UCHL1 of cardiomyocytes is dispensable for development but promotes macroautophagy in cardiomyocytes. Upregulation of UCHL1 in post-MI hearts occurs primarily in the cardiomyocytes and protects against post-MI cardiac remodeling and malfunction likely through supporting autophagic flux and proteostasis during a stress condition.
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Heme oxygenase-1 (HO-1) has been reported to protect against oxidation and inflammation in atherosclerosis. It remains unclear how the immune system participates in the cytoprotective function of HO-1 in the context of atherosclerosis. In this study, we attempted to investigate the potential effect of a HO-1 inducer, hemin, and a HO-1 inhibitor, Tin-protoporphyrin IX (SnPP), on the progression of atherosclerosis in ApoE deficient mice. Using mass cytometry, 15 immune cell populations and 29 T cell sub-clusters in spleen and peripheral blood were thoroughly analyzed after hemin or SnPP treatment. SnPP elevated risk factors of atherosclerosis, whereas hemin reduced them. In-depth analysis showed that hemin significantly modified the immune system in both spleen and peripheral blood. Hemin increased dendritic (DC) and myeloid-derived suppressor cells (MDSCs), but decreased natural killer (NK) cells. An opposite effect was observed with SnPP treatment in terms of NK cells. NK cells and MDSCs were positively and negatively correlated with total cholesterol and low-density lipoprotein, respectively. Moreover, the T cell profiles were significantly reshaped by hemin, whereas only minor changes were observed with SnPP. Several hemin-modulated T cell clusters associated with atherosclerosis were also identified. In summary, we have unraveled an important regulatory role for HO-1 pathway in immune cell regulation and atherosclerosis. Our finding suggests that modulating HO-1 signaling represents a potential therapeutic strategy against atherosclerosis.
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Background: Catecholamine surges and resultant excessive ß-adrenergic stimulation occur in a broad spectrum of diseases. Excessive ß-adrenergic stimulation causes cardiomyocyte necrosis, but the underlying mechanism remains obscure. Necroptosis, a major form of regulated necrosis mediated by RIPK3-centered pathways, is implicated in heart failure; however, it remains unknown whether excessive ß-adrenergic stimulation-induced cardiac injury involves necroptosis. Hence, we conducted the present study to address these critical gaps. Methods and Results: Two consecutive daily injections of isoproterenol (ISO; 85 mg/kg, s.c.) or saline were administered to adult mixed-sex mice. At 24 h after the second ISO injection, cardiac area with Evans blue dye (EBD) uptake and myocardial protein levels of CD45, RIPK1, Ser166-phosphorylated RIPK1, RIPK3, and Ser345-phosphorylated MLKL (p-MLKL) were significantly greater, while Ser321-phosphorylated RIPK1 was significantly lower, in the ISO-treated than in saline-treated wild-type (WT) mice. The ISO-induced increase of EBD uptake was markedly less in RIPK3 -/- mice compared with WT mice (p = 0.016). Pretreatment with the RIPK1-selective inhibitor necrostatin-1 diminished ISO-induced increases in RIPK3 and p-MLKL in WT mice and significantly attenuated ISO-induced increases of EBD uptake in WT but not RIPK3-/- mice. Conclusions: A large proportion of cardiomyocyte necrosis induced by excessive ß-adrenergic stimulation belongs to necroptosis and is mediated by a RIPK1-RIPK3-dependent pathway, identifying RIPK1 and RIPK3 as potential therapeutic targets for catecholamine surges.
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Ubiquitin C-terminal Hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally identified in neurons. Our recent study showed that UCHL1 was expressed in C2C12 myoblast cells and mouse skeletal muscle. Here we report that in mouse skeletal muscle, UCHL1 is primarily expressed in oxidative muscle fibers. Skeletal muscle specific gene knockout (smKO) of UCHL1 in mice reduced oxidative activity in skeletal muscle measured by SDH staining. The in situ muscle contraction test revealed that gastrocnemius muscle from UCHL1 smKO mice was more prone to fatigue in response to the repetitive stimulation. This data suggests that UCHL1 plays a role in maintenance of muscle oxidative metabolism. Moreover, UCHL1 smKO caused a significant reduction in key proteins that are involved in mitochondrial oxidative phosphorylation in soleus muscles, suggesting that UCHL1 may be involved in regulation of mitochondrial content and function. Immunostaining showed the co-localization of UCHL1 and mitochondrial marker VDAC in skeletal muscle. Mitochondrial fractionation assay revealed that, although UCHL1 was primarily present in the cytosolic fraction, a low level of UCHL1 protein was present in mitochondrial fraction. The level of phosphorylation of AMPKα, a master regulator of mitochondrial biogenesis, were unchanged in UCHL1 smKO muscle. On the other hand, immunoprecipitation from soleus muscle sample indicated the interaction between UCHL1 and HSP60, a chaperon protein that is involved in mitochondrial protein transport. There was a trend of downregulation of HSP60 in UCHL1 smKO muscle. Overall, our data suggests UCHL1 is a novel regulator of mitochondrial function and oxidative activity in skeletal muscle.
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Músculo Esquelético/metabolismo , Estrés Oxidativo , Ubiquitina Tiolesterasa/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Chaperonina 60/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Contracción Muscular , Mioblastos/citología , Mioblastos/metabolismo , Fosforilación Oxidativa , Ubiquitina Tiolesterasa/deficiencia , Ubiquitina Tiolesterasa/genéticaRESUMEN
BACKGROUND: Mechanisms governing the induction of heart failure by the impairment of autophagy and the ubiquitin-proteasome system and the molecular pathways to cardiomyocyte necrosis remain incompletely understood. COPS8 is an essential subunit of the COP9 (COnstitutive Photomorphogenesis 9) signalosome, a key regulator of ubiquitination. Mice with cardiomyocyte-restricted knockout of Cops8 (Cops8-cko) show autophagic and ubiquitin-proteasome system malfunction and massive cardiomyocyte necrosis followed by acute heart failure and premature death, providing an excellent animal model to address the mechanistic gaps specified above. This study was conducted to determine the nature and underlying mechanisms of the cardiomyocyte necrosis in Cops8-cko mice. METHODS AND RESULTS: Compared with littermate control mice, myocardial protein levels of key factors in the necroptotic pathway (RIPK1 [receptor-interacting protein kinase 1], RIPK3, MLKL [mixed lineage kinase-like], the RIPK1-bound RIPK3), protein carbonyls, full-length Casp8 (caspase 8), and BCL2, as well as histochemical staining of superoxide anions were significantly higher but the cleaved Casp8 and the Casp8 activity were significantly lower in Cops8-cko mice. In vivo cardiomyocyte uptake of Evan's blue dye was used as an indicator of necrosis. Cops8-cko mice treated with a RIPK1 kinase inhibitor (Nec-1 [Necrostatin-1]) showed less Evans blue dye uptake (0.005% versus 0.20%; P<0.0001) and longer median lifespan (32.5 versus 27 days; P<0.01) than those treated with vehicle control. RIPK3 haploinsufficiency showed similar rescuing effects on Cops8-cko but Cyclophilin D deficiency did the opposite. CONCLUSIONS: Cardiac Cops8/COP9 signalosome malfunction causes RIPK1-RIPK3 dependent, but mitochondrial permeability transition pore independent, cardiomyocyte necroptosis in mice and the COP9 signalosome plays an indispensable role in suppressing cardiomyocyte necroptosis.
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Complejo del Señalosoma COP9/fisiología , Proteínas Portadoras/fisiología , Miocitos Cardíacos/metabolismo , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Caspasa 8/metabolismo , Imidazoles/farmacología , Indoles/farmacología , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , UbiquitinaciónRESUMEN
RATIONALE: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway 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 autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 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 autophagic-lysosomal pathway. METHODS AND RESULTS: Myocardial macroautophagy, TFEB (transcription factor EB) 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 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 wild type 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 wild type 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 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Calcineurina/metabolismo , Macroautofagia/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , ATPasas Asociadas con Actividades Celulares Diversas/genética , Animales , Antineoplásicos/farmacología , Bortezomib/farmacología , Calcineurina/genética , Inhibidores de la Calcineurina , Hipertrofia Ventricular Izquierda/inducido químicamente , Lisosomas/metabolismo , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Proteasoma , Proteostasis , ARN Interferente Pequeño , Ratas , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/fisiología , Canales de Potencial de Receptor Transitorio/metabolismo , Ubiquitina/metabolismo , Regulación hacia ArribaRESUMEN
AIMS: Skeletal muscle wasting is associated with many chronic diseases. Effective prevention and treatment of muscle wasting remain as a challenging task due to incomplete understanding of mechanisms by which muscle mass is maintained and regulated. This study investigated the functional role of Ubiquitin C-terminal hydrolase L1 (UCHL1) in skeletal muscle. MAIN METHODS: Mice with skeletal muscle specific gene knockout of UCHL1 and C2C12 myoblast cells with UCHL1 knockdown were used. Muscle fiber types and size were measured using tissue or cell staining. The mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 activities were assessed with the phosphorylation of their downstream targets. KEY FINDINGS: In mouse skeletal muscle, UCHL1 was primarily expressed in slow twitch muscle fibers. Mice with skeletal muscle specific knockout (skmKO) of UCHL1 exhibited enlarged muscle fiber sizes in slow twitch soleus but not fast twitch extensor digitorum longus (EDL) muscle. Meanwhile, UCHL1 skmKO enhanced mTORC1 activity and reduced mTORC2 activity in soleus but not in EDL. Consistently, in C2C12 cells, UCHL1 knockdown increased the myotube size, enhanced mTORC1 activity, and reduced mTORC2 activities as compared with control cells. UCHL1 knockdown did not change the major proteins of mTOR complex but decreased the protein turnover of PRAS40, an inhibitory factor of mTORC1. SIGNIFICANCE: These data revealed a novel function of UCHL1 in regulation of mTORC1 activity and skeletal muscle growth in slow twitch skeletal muscle. Given the upregulation of UCHL1 in denervation and spinal muscle atrophy, our finding advances understanding of regulators that are involved in muscle wasting.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/fisiología , Atrofia Muscular/fisiopatología , Mioblastos/fisiología , Ubiquitina Tiolesterasa/fisiología , Animales , Células Cultivadas , Femenino , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Ratones Noqueados , Fibras Musculares de Contracción Lenta/citología , Músculo Esquelético/citología , Atrofia Muscular/metabolismo , Mioblastos/citología , Fosforilación , Ubiquitina Tiolesterasa/antagonistas & inhibidoresRESUMEN
Macroautophagy (referred to as autophagy hereafter) plays an important role in the quality control of cellular proteins and organelles. Transcription Factor EB (TFEB) globally activates the expression of genes in the autophagic-lysosomal pathway (ALP) to replenish lysosomes and ALP machineries. We previously reported that myocardial TFEB signaling was impaired in advanced cardiac proteinopathy; however, myocardial ALP status and TFEB activity at earlier stages of cardiac proteinopathy remain uncharacterized. Here a stable line of CryABR120G transgenic (R120G) and non-transgenic (NTG) littermate mice with cardiomyocyte-restricted overexpression of CryABR120G were used at 1, 3, and 6 months of age. At 1 month when no cardiac phenotypes other than aberrant protein aggregation are discernible, R120G mice displayed a 5-fold increase in myocardial LC3-II flux. Interestingly, the LC3-II flux increase co-existed with increases in mTOR complex 1 (mTORC1) activities as well as cytoplasmic, but not nuclear, TFEB proteins. This increase in cytoplasmic TFEB proteins occurred without any discernible alteration in TFEB activity as reflected by unchanged mRNA levels of representative TFEB target genes (Mcoln1, M6pr, Sqstm1, Vps18, and Uvrag). At 3 months of age when hypertrophy and diastolic malfunction start to develop, the LC3-II flux remained significantly increased but to a lesser degree (2-fold) than at 1 month. The LC3-II flux increase was associated with decreased mTORC1 activities and with increased nuclear TFEB proteins and TFEB activities. At 6 months of age when congestive heart failure is apparent in R120G mice, both LC3-II flux and TFEB activities were severely suppressed, while mTORC1 activity increased. We conclude that changes in both autophagy and TFEB signaling are highly dynamic during the progression of cardiac proteinopathy. Increases in autophagy occur before increases in TFEB activities but both increase in the compensatory stage of cardiac proteinopathy. Once congestive heart failure develops, both autophagy and TFEB signaling become impaired. Our results suggest that TFEB signaling is regulated by both mTORC1-dependent and -independent mechanisms in hearts subjected to increased proteotoxic stress. For therapeutic exploration, it will be important to test the effect of TFEB stimulation at the early, intermediate, and late stages of cardiac proteinopathy.
RESUMEN
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.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Insuficiencia Cardíaca/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Deficiencias en la Proteostasis/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Densitometría , Ecocardiografía , Femenino , Genotipo , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/metabolismo , Hemodinámica , Humanos , Hidrólisis , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Fosforilación , Desnaturalización Proteica , Deficiencias en la Proteostasis/fisiopatología , RatasRESUMEN
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.