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BACKGROUND: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. METHODS: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RESULTS: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. CONCLUSIONS: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.
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Sulfuro de Hidrógeno , Telomerasa , Animales , Humanos , Ratones , Senescencia Celular , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Células Endoteliales/metabolismo , Sulfuro de Hidrógeno/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Sitios de Unión , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Células Cultivadas , Células HEK293 , Humanos , Miocitos Cardíacos/metabolismo , Unión Proteica , Ratas , Ratas WistarRESUMEN
Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE-/- or LDL-R-/- mice transgenic models. A new AAV-PCSK9DYDY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9DY (0.5, 1 or 5 × 1011 VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 1011 VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.
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Aterosclerosis , Proproteína Convertasa 9 , Animales , Aterosclerosis/patología , Colesterol , Modelos Animales de Enfermedad , Células Endoteliales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía de Fuerza Atómica , Proproteína Convertasa 9/genética , TriglicéridosRESUMEN
Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.
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Células Madre Pluripotentes Inducidas , Edición de ARN , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , ARN/química , ARN/genética , ARN/metabolismo , ARN Circular/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
RATIONALE: Secreted and membrane-bound proteins, which account for 1/3 of all proteins, play critical roles in heart health and disease. The endoplasmic reticulum (ER) is the site for synthesis, folding, and quality control of these proteins. Loss of ER homeostasis and function underlies the pathogenesis of many forms of heart disease. OBJECTIVE: To investigate mechanisms responsible for regulating cardiac ER function, and to explore therapeutic potentials of strengthening ER function to treat heart disease. METHODS AND RESULTS: Screening a range of signaling molecules led to the discovery that Pak (p21-activated kinase)2 is a stress-responsive kinase localized in close proximity to the ER membrane in cardiomyocytes. We found that Pak2 cardiac deleted mice (Pak2-CKO) under tunicamycin stress or pressure overload manifested a defective ER response, cardiac dysfunction, and profound cell death. Small chemical chaperone tauroursodeoxycholic acid treatment of Pak2-CKO mice substantiated that Pak2 loss-induced cardiac damage is an ER-dependent pathology. Gene array analysis prompted a detailed mechanistic study, which revealed that Pak2 regulation of protective ER function was via the IRE (inositol-requiring enzyme)-1/XBP (X-box-binding protein)-1-dependent pathway. We further discovered that this regulation was conferred by Pak2 inhibition of PP2A (protein phosphatase 2A) activity. Moreover, IRE-1 activator, Quercetin, and adeno-associated virus serotype-9-delivered XBP-1s were able to relieve ER dysfunction in Pak2-CKO hearts. This provides functional evidence, which supports the mechanism underlying Pak2 regulation of IRE-1/XBP-1s signaling. Therapeutically, inducing Pak2 activation by genetic overexpression or adeno-associated virus serotype-9-based gene delivery was capable of strengthening ER function, improving cardiac performance, and diminishing apoptosis, thus protecting the heart from failure. CONCLUSIONS: Our findings uncover a new cardioprotective mechanism, which promotes a protective ER stress response via the modulation of Pak2. This novel therapeutic strategy may present as a promising option for treating cardiac disease and heart failure.
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Estrés del Retículo Endoplásmico , Insuficiencia Cardíaca/enzimología , Miocitos Cardíacos/enzimología , Quinasas p21 Activadas/metabolismo , Animales , Apoptosis , Línea Celular , Modelos Animales de Enfermedad , Terapia Genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/terapia , Células Madre Pluripotentes Inducidas/enzimología , Macaca mulatta , Masculino , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Miocitos Cardíacos/patología , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Transducción de Señal , Proteína 1 de Unión a la X-Box/metabolismo , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
The z-disc is a structural component at the lateral borders of the sarcomere and is important for mechanical stability and contractility of both cardiac and skeletal muscles. Of note, the sarcomeric z-disc also represents a nodal point in cardiomyocyte function and signaling. Mutations of numerous z-disc proteins are associated with cardiomyopathies and muscle diseases. To identify additional z-disc proteins that might contribute to cardiac disease, we employed an in silico screen for cardiac-enriched cDNAs. This screen yielded a previously uncharacterized protein named cardiac-enriched FHL2-interacting protein (CEFIP), which exhibited a heart- and skeletal muscle-specific expression profile. Importantly, CEFIP was located at the z-disc and was up-regulated in several models of cardiomyopathy. We also found that CEFIP overexpression induced the fetal gene program and cardiomyocyte hypertrophy. Yeast two-hybrid screens revealed that CEFIP interacts with the calcineurin-binding protein four and a half LIM domains 2 (FHL2). Because FHL2 binds calcineurin, a phosphatase controlling hypertrophic signaling, we examined the effects of CEFIP on the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. These experiments revealed that CEFIP overexpression further enhances calcineurin-dependent hypertrophic signal transduction, and its knockdown repressed hypertrophy and calcineurin/NFAT activity. In summary, we report on a previously uncharacterized protein CEFIP that modulates calcineurin/NFAT signaling in cardiomyocytes, a finding with possible implications for the pathogenesis of cardiomyopathy.
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Calcineurina/metabolismo , Proteínas Portadoras/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Sarcómeros/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Cardiomegalia/metabolismo , Cardiomegalia/patología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular Transformada , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/patología , Transporte de Proteínas , Interferencia de ARN , Ratas Wistar , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismoRESUMEN
AIMS: Despite massive efforts, we remain far behind in our attempts to identify effective therapies to treat heart failure with preserved ejection fraction (HFpEF). Diastolic function is critically regulated by sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase 2a (SERCA2a), which forms a functional cardiomyocyte (CM) microdomain where 3',5'-cyclic adenosine monophosphate (cAMP) produced upon ß-adrenergic receptor (ß-AR) stimulation leads to phospholamban (PLN) phosphorylation and facilitated Ca2+ re-uptake. METHODS AND RESULTS: To visualize real-time cAMP dynamics in the direct vicinity of SERCA2a in healthy and diseased myocytes, we generated a novel mouse model on the leprdb background that stably expresses the Epac1-PLN Förster resonance energy transfer biosensor. Mice homozygous for the leprdb mutation (db/db) developed obesity and type 2 diabetes and presented with a HFpEF phenotype, evident by mild left ventricular hypertrophy and elevated left atria filling pressures. Live cell imaging uncovered a substantial ß2-AR subtype stimulated cAMP response within the PLN/SERCA2a microdomain of db/db but not healthy control (db/+) CMs, which was accompanied by increased PLN phosphorylation and accelerated calcium re-uptake. Importantly, db/db CMs also exhibited a desensitization of ß1-AR stimulated cAMP pools within the PLN/SERCA2a microdomain, which was accompanied by a blunted lusitropic effect, suggesting that the increased ß2-AR control is an intrinsic compensatory mechanism to maintain PLN/SERCA2a-mediated calcium dynamics and cardiac relaxation. Mechanistically, this was due to a local loss of cAMP-degrading phosphodiesterase 4 associated specifically with the PLN/SERCA2a complex. CONCLUSION: These newly identified alterations of cAMP dynamics at the subcellular level in HFpEF should provide mechanistic understanding of microdomain remodelling and pave the way towards new therapies.
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Diabetes Mellitus Tipo 2 , Insuficiencia Cardíaca , Animales , Ratones , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , AMP Cíclico , Diabetes Mellitus Tipo 2/complicaciones , Insuficiencia Cardíaca/etiología , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Volumen SistólicoRESUMEN
Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells in vitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.
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Introduction: The development of cognitive dysfunction is not necessarily associated with diet-induced obesity. We hypothesized that cognitive dysfunction might require additional vascular damage, for example, in atherosclerotic mice. Methods: We induced atherosclerosis in male C57BL/6N mice by injecting AAV-PCSK9DY (2x1011 VG) and feeding them a cholesterol-rich Western diet. After 3 months, mice were examined for cognition using Barnes maze procedure and for cerebral blood flow. Cerebral vascular morphology was examined by immunehistology. Results: In AAV-PCSK9DY-treated mice, plaque burden, plasma cholesterol, and triglycerides are elevated. RNAseq analyses followed by KEGG annotation show increased expression of genes linked to inflammatory processes in the aortas of these mice. In AAV-PCSK9DY-treated mice learning was delayed and long-term memory impaired. Blood flow was reduced in the cingulate cortex (-17%), caudate putamen (-15%), and hippocampus (-10%). Immunohistological studies also show an increased incidence of string vessels and pericytes (CD31/Col IV staining) in the hippocampus accompanied by patchy blood-brain barrier leaks (IgG staining) and increased macrophage infiltrations (CD68 staining). Discussion: We conclude that the hyperlipidemic PCSK9DY mouse model can serve as an appropriate approach to induce microvascular dysfunction that leads to reduced blood flow in the hippocampus, which could explain the cognitive dysfunction in these mice.
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Aterosclerosis , Hiperlipidemias , Masculino , Ratones , Animales , Proproteína Convertasa 9/genética , Incidencia , Ratones Endogámicos C57BL , Hiperlipidemias/patología , Aterosclerosis/metabolismo , Colesterol , Circulación Cerebrovascular/fisiologíaRESUMEN
Obesity-induced lipid overload in cardiomyocytes contributes to profound oxidative stress and cardiomyopathy, culminating in heart failure. In this study, we investigate a novel mechanism whereby lipids accumulate in cardiomyocytes and seek the relevant treatment strategies. P21-activated kinase 3 (PAK3) was elevated in obese human myocardium, and the murine hearts and cardiomyocytes upon diet- or fatty acid-induced stress, respectively. Mice with cardiac-specific overexpression of PAK3 were more susceptible to the development of cardiac dysfunction upon diet stress, at least partially, due to increased deposition of toxic lipids within the myocardium. Mechanistically, PAK3 promoted the nuclear expression of sterol regulatory element binding protein 1c (SREBP1c) through activation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase beta-1 (S6K1) pathway in cardiomyocytes, resulting in abnormal lipid genes profile, accumulation of excessive lipids, and oxidative stress. More importantly, PAK3 knockdown attenuated fatty acid-induced lipotoxicity and cell death in rat and human cardiomyocytes. More importantly, the S6K1 or SREBP1c inhibitor alleviated PAK3-triggered intracellular lipid overload and cardiac dysfunction under obese stress. Collectively, we have demonstrated that PAK3 impairs myocardial lipid homeostasis, while inhibition of cardiac lipotoxicity mitigates cardiac dysfunction. Our study provides a promising therapeutic strategy for ameliorating obesity cardiomyopathy.
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Despite the identification of numerous molecular pathways modulating cardiac hypertrophy its pathogenesis is not completely understood. In this study we define an unexpected role for Fibin ("fin bud initiation factor homolog") in cardiomyocyte hypertrophy. Via gene expression profiling in hypertrophic murine hearts after transverse aortic constriction we found a significant induction of Fibin. Moreover, Fibin was upregulated in another mouse model of cardiac hypertrophy (calcineurin-transgenics) as well as in patients with dilated cardiomyopathy. Immunoflourescence microscopy revealed subcellular localization of Fibin at the sarcomeric z-disc. Overexpression of Fibin in neonatal rat ventricular cardiomyocytes revealed a strong anti-hypertrophic effect through inhibiting both, NFAT- and SRF-dependent signalling. In contrast, transgenic mice with cardiac-restricted overexpression of Fibin developed dilated cardiomyopathy, accompanied by induction of hypertrophy-associated genes. Moreover, Fibin overexpression accelerated the progression to heart failure in the presence of prohypertrophic stimuli such as pressure overload and calcineurin overexpression. Histological and ultrastructural analyses surprisingly showed large protein aggregates containing Fibin. On the molecular level, aggregate formation was accompanied by an induction of the unfolded protein response subsequent UPR-mediated apoptosis and autophagy. Taken together, we identified Fibin as a novel potent negative regulator of cardiomyocyte hypertrophy in vitro. Yet, heart-specific Fibin overexpression in vivo causes development of a protein-aggregate-associated cardiomyopathy. Because of close similarities to myofibrillar myopathies, Fibin represents a candidate gene for cardiomyopathy and Fibin transgenic mice may provide additional mechanistic insight into aggregate formation in these diseases.
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(1) Background: Diabetic cardiomyopathy is a major health problem worldwide. CTRP9, a secreted glycoprotein, is mainly expressed in cardiac endothelial cells and becomes downregulated in mouse models of diabetes mellitus; (2) Methods: In this study, we investigated the impact of CTRP9 on early stages of diabetic cardiomyopathy induced by 12 weeks of high-fat diet; (3) Results: While the lack of CTRP9 in knock-out mice aggravated insulin resistance and triggered diastolic left ventricular dysfunction, AAV9-mediated cardiac CTRP9 overexpression ameliorated cardiomyopathy under these conditions. At this early disease state upon high-fat diet, no fibrosis, no oxidative damage and no lipid deposition were identified in the myocardium of any of the experimental groups. Mechanistically, we found that CTRP9 is required for insulin-dependent signaling, cardiac glucose uptake in vivo and oxidative energy production in cardiomyocytes. Extensive RNA sequencing from myocardial tissue of CTRP9-overexpressing and knock-out as well as respective control mice revealed that CTRP9 acts as an anti-inflammatory mediator in the myocardium. Hence, CTRP9 knock-out exerted more, while CTRP9-overexpressing mice showed less leukocytes accumulation in the heart during high-fat diet; (4) Conclusions: In summary, endothelial-derived CTRP9 plays a prominent paracrine role to protect against diabetic cardiomyopathy and might constitute a therapeutic target.
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Diabetes Mellitus , Cardiomiopatías Diabéticas , Resistencia a la Insulina , Ratones , Animales , Cardiomiopatías Diabéticas/metabolismo , Complemento C1q/metabolismo , Células Endoteliales/metabolismo , Adiponectina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Miocitos Cardíacos/metabolismo , Inflamación/patología , Ratones Noqueados , Diabetes Mellitus/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismoRESUMEN
Despite the development of clinical treatments, heart failure remains the leading cause of mortality. We observed that p21-activated kinase 3 (PAK3) was augmented in failing human and mouse hearts. Furthermore, mice with cardiac-specific PAK3 overexpression exhibited exacerbated pathological remodeling and deteriorated cardiac function. Myocardium with PAK3 overexpression displayed hypertrophic growth, excessive fibrosis, and aggravated apoptosis following isoprenaline stimulation as early as two days. Mechanistically, using cultured cardiomyocytes and human-relevant samples under distinct stimulations, we, for the first time, demonstrated that PAK3 acts as a suppressor of autophagy through hyper-activation of the mechanistic target of rapamycin complex 1 (mTORC1). Defective autophagy in the myocardium contributes to the progression of heart failure. More importantly, PAK3-provoked cardiac dysfunction was mitigated by administering an autophagic inducer. Our study illustrates a unique role of PAK3 in autophagy regulation and the therapeutic potential of targeting this axis for heart failure.
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BACKGROUND: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-ß) is involved in both processes. We uncovered an increased expression of the TGF-ß receptor II (TßRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TßRII signalling impairs myogenic differentiation in response to inflammation. METHODS: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-ß/TßRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. RESULTS: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1ß (IL-1ß), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1ß-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TßRII, resulting in TßRII ubiquitination and destabilization. SPSB1 impaired TßRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. CONCLUSIONS: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TßRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.
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Interleucina-6 , Proteínas Proto-Oncogénicas c-akt , Animales , Ratones , Citocinas , Inflamación , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Miogenina/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfaRESUMEN
Background Prolonged activation of angiotensin II is the main mediator that contributes to the development of heart diseases, so converting angiotensin II into angiotensin 1-7 has emerged as a new strategy to attenuate detrimental effects of angiotensin II. Prolylcarboxypeptidase is a lysosomal pro-X carboxypeptidase that is able to cleave angiotensin II at a preferential acidic pH optimum. However, insufficient attention has been given to the cardioprotective functions of prolylcarboxylpeptidase. Methods and Results We established a CRISPR/CRISPR-associated protein 9-mediated global prolylcarboxylpeptidase-knockout and adeno-associated virus serotype 9-mediated cardiac prolylcarboxylpeptidase overexpression mouse models, which were challenged with the angiotensin II infusion (2 mg/kg per day) for 4 weeks, aiming to investigate the cardioprotective effect of prolylcarboxylpeptidase against hypertensive cardiac hypertrophy. Prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated afterward in wild-type mouse myocardium, suggesting its compensatory function against angiotensin II stress. Moreover, angiotensin II-treated prolylcarboxylpeptidase-knockout mice showed aggravated cardiac remodeling and dampened cardiac contractility independent of hypertension. We also found that prolylcarboxylpeptidase localizes in cardiomyocyte lysosomes, and loss of prolylcarboxylpeptidase led to excessive angiotensin II levels in myocardial tissue. Further screening demonstrated that hypertrophic prolylcarboxylpeptidase-knockout hearts showed upregulated extracellular signal-regulated kinases 1/2 and downregulated protein kinase B activities. Importantly, adeno-associated virus serotype 9-mediated restoration of prolylcarboxylpeptidase expression in prolylcarboxylpeptidase-knockout hearts alleviated angiotensin II-induced hypertrophy, fibrosis, and cell death. Interestingly, the combination of adeno-associated virus serotype 9-mediated prolylcarboxylpeptidase overexpression and an antihypertensive drug, losartan, likely conferred more effective protection than a single treatment protocol to mitigate angiotensin II-induced cardiac dysfunction. Conclusions Our data demonstrate that prolylcarboxylpeptidase protects the heart from angiotensin II-induced hypertrophic remodeling by controlling myocardial angiotensin II levels.
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Angiotensina II , Hipertensión , Ratones , Animales , Angiotensina II/metabolismo , Remodelación Ventricular/fisiología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Ratones Noqueados , Fibrosis , Ratones Endogámicos C57BLRESUMEN
Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.
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Envejecimiento , Senescencia Celular , Corazón , MicroARNs , Densidad Microvascular , Miocardio , Semaforina-3A , Corazón/inervación , Microcirculación , MicroARNs/genética , MicroARNs/metabolismo , Semaforina-3A/genética , Animales , Ratones , Envejecimiento/genética , Envejecimiento/patología , Masculino , Ratones Endogámicos C57BL , Senescencia Celular/genética , Miocardio/patología , AxonesRESUMEN
Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of heart failure. The previously used technique is based on a threaded suture around the aortic arch tied over a blunted 27 G needle to create stenosis. This method depends on the surgeon manually tightening the thread and, thus, leads to high variance in the diameter size. A newly refined method described by Melleby et al. promises less variance and more reproducibility after surgery. The new technique, o-ring- aortic banding (ORAB), uses a non-slip rubber ring instead of a suture with a thread, resulting in reduced variation in pressure overload and reproducible phenotypes of cardiac hypertrophy. During surgery, the o-ring is placed between the brachiocephalic and left carotid arteries. Successful constriction is confirmed by echocardiography. After 1 day, correct placement of the ring results in an increased flow velocity in the transverse aorta over the o-ring-induced stenosis. After 2 weeks, impaired cardiac function is proven by decreased ejection fraction and increased wall thickness. Importantly, besides less variance in the diameter size, ORAB is associated with lower intra- and post-operative mortality rates compared with transverse aortic constriction (TAC). Thus, ORAB represents a superior method to the commonly used TAC surgery, resulting in more reproducible results and a possible reduction in the number of animals needed.
Asunto(s)
Estenosis de la Válvula Aórtica , Goma , Ratones , Animales , Ratones Endogámicos C57BL , Constricción , Constricción Patológica/etiología , Constricción Patológica/cirugía , Reproducibilidad de los Resultados , Modelos Animales de Enfermedad , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/etiología , Aorta/diagnóstico por imagen , Aorta/cirugía , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/cirugíaRESUMEN
Endoplasmic Reticulum (ER) stress and oxidative stress have been highly implicated in the pathogenesis of cardiac hypertrophy and heart failure (HF). However, the mechanisms involved in the interplay between these processes in the heart are not fully understood. The present study sought to determine a causative link between Pak2-dependent UPR activation and oxidative stress via Nrf2 regulation under pathological ER stress. We report that sustained ER stress and Pak2 deletion in cardiomyocytes enhance Nrf2 expression. Conversely, AAV9 mediated Pak2 delivery in the heart leads to a significant decrease in Nrf2 levels. Pak2 overexpression enhances the XBP1-Hrd1 UPR axis and ameliorates tunicamycin induced cardiac apoptosis and dysfunction in mice. We found that Pak2 deletion and altered proteostasis render Nrf2 detrimental by switching from its antioxidant role to renin-angiotensin aldosterone system (RAAS) gene regulator. Mechanistically, Pak2 mediated Hrd1 expression targets Nrf2 for ubiquitination and degradation thus preventing its aberrant activation. Moreover, we find a significant increase in Nrf2 with a decrease in Pak2 in human myocardium of dilated heart disease. Using human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we find that Pak2 is able to ameliorate Nrf2 induced RAAS activation under ER stress. These findings demonstrate that Pak2 is a novel Nrf2 regulator in the stressed heart. Activation of XBP1-Hrd1 is attributed to prevent ER stress-induced Nrf2 RAAS component upregulation. This mechanism explains the functional dichotomy of Nrf2 in the stressed heart. Thus, Pak2 regulation of Nrf2 homeostasis may present as a potential therapeutic route to alleviate detrimental ER stress and heart failure.
RESUMEN
Myocardial inflammation contributes to cardiomyopathy in diabetic patients through incompletely defined underlying mechanisms. In both human and time-course experimental samples, diabetic hearts exhibited abnormal ER, with a maladaptive shift over time in rodents. Furthermore, as a cardiac ER dysfunction model, mice with cardiac-specific p21-activated kinase 2 (PAK2) deletion exhibited heightened myocardial inflammatory response in diabetes. Mechanistically, maladaptive ER stress-induced CCAAT/enhancer-binding protein homologous protein (CHOP) is a novel transcriptional regulator of cardiac high-mobility group box-1 (HMGB1). Cardiac stress-induced release of HMGB1 facilitates M1 macrophage polarization, aggravating myocardial inflammation. Therapeutically, sequestering the extracellular HMGB1 using glycyrrhizin conferred cardioprotection through its anti-inflammatory action. Our findings also indicated that an intact cardiac ER function and protective effects of the antidiabetic drug interdependently attenuated the cardiac inflammation-induced dysfunction. Collectively, we introduce an ER stress-mediated cardiomyocyte-macrophage link, altering the macrophage response, thereby providing insight into therapeutic prospects for diabetes-associated cardiac dysfunction.
RESUMEN
Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors-AAVMYO2 and AAVMYO3-prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.