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Our prior study (Tarasov et al., 2022) discovered that numerous adaptive mechanisms emerge in response to cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) which included overexpression of a large number of proteins. Here, we conducted an unbiased phosphoproteomics analysis in order to determine the role of altered protein phosphorylation in the adaptive heart performance and protection profile of adult TGAC8 left ventricle (LV) at 3-4 months of age, and integrated the phosphoproteome with transcriptome and proteome. Based on differentially regulated phosphoproteins by genotype, numerous stress-response pathways within reprogrammed TGAC8 LV, including PKA, PI3K, and AMPK signaling pathways, predicted upstream regulators (e.g. PDPK1, PAK1, and PTK2B), and downstream functions (e.g. cell viability, protein quality control), and metabolism were enriched. In addition to PKA, numerous other kinases and phosphatases were hyper-phosphorylated in TGAC8 vs. WT. Hyper-phosphorylated transcriptional factors in TGAC8 were associated with increased mRNA transcription, immune responses, and metabolic pathways. Combination of the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome enabled the elucidation of cardiac performance and adaptive protection profiles coordinately regulated at post-translational modification (PTM) (phosphorylation), translational, and transcriptional levels. Many stress-response signaling pathways, i.e., PI3K/AKT, ERK/MAPK, and ubiquitin labeling, were consistently enriched and activated in the TGAC8 LV at transcriptional, translational, and PTM levels. Thus, reprogramming of the cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. We identified numerous pathways/function predictions via gene sets, phosphopeptides, and phosphoproteins, which may point to potential novel therapeutic targets to enhance heart adaptivity, maintaining heart performance while avoiding cardiac dysfunction.
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Proteoma , Resiliencia Psicológica , Adulto , Humanos , Adenilil Ciclasas/genética , Transcriptoma , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/genética , Proteínas Quinasas Dependientes de 3-FosfoinosítidoRESUMEN
Advancing age is the most important risk factor for cardiovascular diseases (CVDs). Two types of cells, within the heart pacemaker, sinoatrial node (SAN), and within the left ventricle (LV), control two crucial characteristics of heart function, heart beat rate and contraction strength. As age advances, the heart's structure becomes remodeled, and SAN and LV cell functions deteriorate, thus increasing the risk for CVDs. However, the different molecular features of age-associated changes in SAN and LV cells have never been compared in omics scale in the context of aging. We applied deep RNA sequencing to four groups of samples, young LV, old LV, young SAN and old SAN, followed by numerous bioinformatic analyses. In addition to profiling the differences in gene expression patterns between the two heart chambers (LV vs. SAN), we also identified the chamber-specific concordant or discordant age-associated changes in: (1) genes linked to energy production related to cardiomyocyte contraction, (2) genes related to post-transcriptional processing, (3) genes involved in KEGG longevity regulating pathway, (4) prolongevity and antilongevity genes recorded and curated in the GenAge database, and (5) CVD marker genes. Our bioinformatic analysis also predicted the regulation activities and mapped the expression of upstream regulators including transcription regulators and post-transcriptional regulator miRNAs. This comprehensive analysis promotes our understanding of regulation of heart functions and will enable discovery of gene-specific therapeutic targets of CVDs in advanced age.
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BACKGROUND: Photobiomodulation (PBM) therapy, a form of low-dose light therapy, has been noted to be effective in several age-associated chronic diseases such as hypertension and atherosclerosis. Here, we examined the effects of PBM therapy on age-associated cardiovascular changes in a mouse model of accelerated cardiac aging. METHODS: Fourteen months old Adenylyl cyclase type VIII (AC8) overexpressing transgenic mice (n = 8) and their wild-type (WT) littermates (n = 8) were treated with daily exposure to Near-Infrared Light (850 nm) at 25 mW/cm2 for 2 min each weekday for a total dose of 1 Einstein (4.5 p.J/cm2 or fluence 3 J/cm2 ) and compared to untreated controls over an 8-month period. PBM therapy was administered for 3.5 months (Early Treatment period), paused, due to Covid-19 restrictions for the following 3 months, and restarted again for 1.5 months. Serial echocardiography and gait analyses were performed at monthly intervals, and serum TGF-ß1 levels were assessed following sacrifice. RESULTS: During the Early Treatment period PBM treatments: reduced the age-associated increases in left ventricular (LV) mass in both genotypes (p = 0.0003), reduced the LV end-diastolic volume (EDV) in AC8 (p = 0.04); and reduced the left atrial dimension in both genotypes (p = 0.02). PBM treatments substantially increased the LV ejection fraction (p = 0.03), reduced the aortic wall stiffness (p = 0.001), and improved gait symmetry, an index of neuro-muscular coordination (p = 0.005). The effects of PBM treatments, measured following the pause, persisted. Total TGF-ß1 levels were significantly increased in circulation (serum) in AC8 following PBM treatments (p = 0.01). We observed a striking increase in cumulative survival in PBM-treated AC8 mice (100%; p = 0.01) compared to untreated AC8 mice (43%). CONCLUSION: PBM treatment mitigated age-associated cardiovascular remodeling and reduced cardiac function, improved neuromuscular coordination, and increased longevity in an experimental animal model. These responses correlate with increased TGF-ß1 in circulation. Future mechanistic and dose optimization studies are necessary to assess these anti-aging effects of PBM, and validation in future controlled human studies is required for effective clinical translation.
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COVID-19 , Terapia por Luz de Baja Intensidad , Humanos , Ratones , Animales , Lactante , Factor de Crecimiento Transformador beta1 , Terapia por Luz de Baja Intensidad/métodos , Envejecimiento , CorazónRESUMEN
Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.
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Adaptación Fisiológica , Miocitos Cardíacos , Estrés Fisiológico , Animales , Ratones , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Hipertrofia/fisiopatología , Ratones Transgénicos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , HumanosRESUMEN
Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein-protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.
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Proteínas 14-3-3 , Proteómica , Ratones , Animales , Proteínas 14-3-3/metabolismo , Mitocondrias/metabolismo , Corazón , InmunoprecipitaciónRESUMEN
Heart rate (HR) and HR variability (HRV), predictors of over-all organism health, are widely believed to be driven by autonomic input to the sinoatrial node (SAN), with sympathetic input increasing HR and reducing HRV. However, variability in spontaneous beating intervals in isolated SAN tissue and single SAN cells, devoid of autonomic neural input, suggests that clocks intrinsic to SAN cells may also contribute to HR and HRV in vivo. We assessed contributions of both intrinsic and autonomic neuronal input mechanisms of SAN cell function on HR and HRV via in vivo, telemetric EKG recordings. This was done in both wild type (WT) mice, and those in which adenylyl cyclase type 8 (ADCY8), a main driver of intrinsic cAMP-PKA-Ca2+ mediated pacemaker function, was overexpressed exclusively in the heart (TGAC8). We hypothesized that TGAC8 mice would: (1) manifest a more coherent pattern of HRV in vivo, i.e., a reduced HRV driven by mechanisms intrinsic to SAN cells, and less so to modulation by autonomic input and (2) utilize unique adaptations to limit sympathetic input to a heart with high levels of intrinsic cAMP-Ca2+ signaling. Increased adenylyl cyclase (AC) activity in TGAC8 SAN tissue was accompanied by a marked increase in HR and a concurrent marked reduction in HRV, both in the absence or presence of dual autonomic blockade. The marked increase in intrinsic HR and coherence of HRV in TGAC8 mice occurred in the context of: (1) reduced HR and HRV responses to ß-adrenergic receptor (ß-AR) stimulation; (2) increased transcription of genes and expression of proteins [ß-Arrestin, G Protein-Coupled Receptor Kinase 5 (GRK5) and Clathrin Adaptor Protein (Dab2)] that desensitize ß-AR signaling within SAN tissue, (3) reduced transcripts or protein levels of enzymes [dopamine beta-hydorxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)] required for catecholamine production in intrinsic cardiac adrenergic cells, and (4) substantially reduced plasma catecholamine levels. Thus, mechanisms driven by cAMP-PKA-Ca2+ signaling intrinsic to SAN cells underlie the marked coherence of TGAC8 mice HRV. Adaptations to limit additional activation of AC signaling, via decreased neuronal sympathetic input, are utilized to ensure the hearts survival and prevent Ca2+ overload.
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BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.
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Potenciales de Acción , Relojes Biológicos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Frecuencia Cardíaca , Nodo Sinoatrial/enzimología , Potenciales de Acción/efectos de los fármacos , Animales , Señalización del Calcio , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Activación Enzimática , Frecuencia Cardíaca/efectos de los fármacos , Cinética , Inhibidores de Fosfodiesterasa 3/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacosRESUMEN
Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases.
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Cardiomiopatías/terapia , Diferenciación Celular , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Trasplante de Células Madre , Envejecimiento , Animales , Animales Recién Nacidos , Calcio/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Forma de la Célula , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/ultraestructura , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Contracción Miocárdica , Fenotipo , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Cyclic guanosine monophosphate-protein kinase G-phosphodiesterase 5 signaling may be disturbed in heart failure (HF) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl-peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular (LV) function. METHODS AND RESULTS: We assessed LV hypertrophy and function at the organ and cellular level in aortic-banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end-systolic pressure-volume relationship, preload recruitable SW) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil-treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening-frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium-retention capacity and Complex II-dependent respiratory control, was present in both HF and tadalafil-treated animals. CONCLUSIONS: Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function.
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Adamantano/análogos & derivados , GMP Cíclico/fisiología , Dipéptidos/farmacología , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Transducción de Señal/efectos de los fármacos , Tadalafilo/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Adamantano/farmacología , Animales , Factor Natriurético Atrial/sangre , Modelos Animales de Enfermedad , Ecocardiografía , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Masculino , Péptido Natriurético Encefálico/sangre , Neuropéptido Y/sangre , Porcinos , Porcinos EnanosRESUMEN
Uncoordinated contraction from electromechanical delay worsens heart failure pathophysiology and prognosis, but restoring coordination with biventricular pacing, known as cardiac resynchronization therapy (CRT), improves both. However, not every patient qualifies for CRT. We show that heart failure with synchronous contraction is improved by inducing dyssynchrony for 6 hours daily by right ventricular pacing using an intracardiac pacing device, in a process we call pacemaker-induced transient asynchrony (PITA). In dogs with heart failure induced by 6 weeks of atrial tachypacing, PITA (starting on week 3) suppressed progressive cardiac dilation as well as chamber and myocyte dysfunction. PITA enhanced ß-adrenergic responsiveness in vivo and normalized it in myocytes. Myofilament calcium response declined in dogs with synchronous heart failure, which was accompanied by sarcomere disarray and generation of myofibers with severely reduced function, and these changes were absent in PITA-treated hearts. The benefits of PITA were not replicated when the same number of right ventricular paced beats was randomly distributed throughout the day, indicating that continuity of dyssynchrony exposure is necessary to trigger the beneficial biological response upon resynchronization. These results suggest that PITA could bring the benefits of CRT to the many heart failure patients with synchronous contraction who are not CRT candidates.
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Progresión de la Enfermedad , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/terapia , Marcapaso Artificial , Animales , Calcio/metabolismo , Perros , Insuficiencia Cardíaca/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miofibrillas/metabolismo , Proteómica , Receptores Adrenérgicos beta/metabolismo , Sarcómeros/metabolismoRESUMEN
BACKGROUND: Currently there is no reliable medical treatment for aortic regurgitation (AR). METHODS: Thirty-nine Sprague-Dawley rats underwent creation of AR or sham operation. Treated rats were assigned to early or late institution of sildenafil therapy (100 mg/kg/day) for a total of 10 weeks. Treatment-effects were measured by serial echocardiography, invasive hemodynamic measurements, and tissue analysis. RESULTS: Rats assigned to early treatment developed less remodeling than untreated rats. Thus, left ventricular (LV) dilation was blunted by sildenafil with end-systolic diameter being significantly smaller (6.6 ± 0.4 vs. 7.7 ± 0.4 mm, respectively, p < 0.05). Also, LV wall thickness was significantly decreased in treated rats compared to controls (2.23 ± 0.08 vs. 2.16 ± 0.05 mm, p < 0.01). Fractional shortening was improved by treatment (p < 0.05). Myocardial fibrosis was borderline decreased by treatment (p = 0.09). Akt was increased in treated compared to controls (p < 0.05). CONCLUSION: Sildenafil slightly inhibits LV remodeling and improves fractional shortening in rats with AR when treatment is initiated early.
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Contractile dysfunction and increased deposition of O-linked ß-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined ß-elimination/Michael addition with tandem mass tags (TMT)-labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca(2+) sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca(2+) in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.
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Acetilglucosamina/análogos & derivados , Calcio/metabolismo , Diabetes Mellitus Experimental/complicaciones , Cardiomiopatías Diabéticas/metabolismo , Acetilglucosamina/metabolismo , Animales , Diabetes Mellitus Experimental/patología , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Miocardio/patología , Miocardio/ultraestructura , Miofibrillas/metabolismo , Ratas , Ratas Sprague-Dawley , Sarcómeros/enzimología , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
BACKGROUND: Biomarkers that predict response to cardiac resynchronization therapy (CRT) in heart failure patients with dyssynchrony (HFDYS) would be clinically important. Circulating extracellular microRNAs (miRNAs) have emerged as novel biomarkers that may also play important functional roles, but their relevance as markers for CRT response has not been examined. METHODS AND RESULTS: Comprehensive miRNA polymerase chain reaction arrays were used to assess baseline levels of 766 plasma miRNAs in patients undergoing clinically indicated CRT in an initial discovery set (n=12) with and without subsequent echocardiographic improvement at 6 months after CRT. Validation of candidate miRNAs in 61 additional patients confirmed that baseline plasma miR-30d was associated with CRT response (defined as an increase in left ventricular ejection fraction ≥10%). MiR-30d was enriched in coronary sinus blood and increased in late-contracting myocardium in a canine model of HFDYS, indicating cardiac origin with maximal expression in areas of high mechanical stress. We examined the functional effects of miR-30d in cultured cardiomyocytes and determined that miR-30d is expressed in cardiomyocytes and released in vesicles in response to mechanical stress. Overexpression of miR-30d in cultured cardiomyocytes led to cardiomyocyte growth and protected against apoptosis by targeting the mitogen-associated kinase 4, a downstream effector of tumor necrosis factor. In HFDYS patients, miR-30d plasma levels inversely correlated with high-sensitivity troponin T, a marker of myocardial necrosis. CONCLUSIONS: Baseline plasma miR-30d level is associated with response to CRT in HFDYS in this translational pilot study. MiR-30d increase in cardiomyocytes correlates with areas of increased wall stress in HFDYS and is protective against deleterious tumor necrosis factor signaling.
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Apoptosis/fisiología , Terapia de Resincronización Cardíaca , Insuficiencia Cardíaca/sangre , MicroARNs/sangre , Miocitos Cardíacos/fisiología , Investigación Biomédica Traslacional , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/sangre , Terapia de Resincronización Cardíaca/tendencias , Perros , Femenino , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Ratas , Ratas Sprague-Dawley , Investigación Biomédica Traslacional/tendencias , Resultado del TratamientoRESUMEN
AIMS: The ultimate cause of heart failure (HF) is not known to date. The cytoskeletal protein desmin is differentially modified and forms amyloid-like oligomers in HF. We postulated that desmin post-translational modifications (PTMs) could drive aberrant desmin aggregation in HF. Therefore, we identified these PTMs and investigated their impact on desmin amyloidogenicity in human and experimental HF. METHODS AND RESULTS: We detected increased levels of selectively phosphorylated and cleaved desmin in a canine pacing model of dyssynchronous HF (DHF) compared with either controls or animals treated with cardiac resynchronization therapy (CRT). This unique animal model combines clinically relevant features with the possibility of a partly rescued phenotype. We confirmed analogous changes in desmin modifications in human HF and identified two phosphorylation sites within a glycogen synthase kinase 3 (GSK3) consensus sequence. Desmin-positive oligomers were also increased in DHF hearts compared with controls. Their amyloid properties were decreased by treatment with CRT or an anti-amyloid small molecule. Finally, we confirmed GSK3's involvement with desmin phosphorylation using an in vitro model. CONCLUSIONS: Based on these findings, we postulate a new mechanism of cardiac toxicity based on the PTM-driven accumulation of desmin amyloid-like oligomers. Phosphorylation and cleavage as well as oligomers formation are reduced by treatment (CRT) indicating a relationship between the three. Finally, the decrease of desmin amyloid-like oligomers with CRT or small molecules points both to a general mechanism of HF based on desmin toxicity that is independent of protein mutations and to novel potential therapies.
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Desmina/metabolismo , Insuficiencia Cardíaca/metabolismo , Agregado de Proteínas , Animales , Terapia de Resincronización Cardíaca , Perros , Glucógeno Sintasa Quinasa 3/metabolismo , Insuficiencia Cardíaca/etiología , Mutación/genética , Fosforilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteómica/métodosRESUMEN
The development of left ventricular hypertrophy and dysfunction in aortic regurgitation (AR) has only been sparsely studied experimentally. In a new model of chronic AR in rats, we examined activation of molecular pathways involved in myocardial hypertrophy. Chronic AR was produced by damaging one or two valve cusps, resulting in eccentric remodeling and left ventricular dysfunction, with no increase in overall fibrosis. Western blotting showed increased activation of Akt and p38 at 12 weeks and of c-Jun amino-terminal kinase at 2 weeks, decreased activation of extracellular regulated kinase 5 at both 2 and 12 weeks, while activation of calcium/calmodulin-dependent protein kinase II and extracellular regulated kinase 1/2 was unchanged. Expression of calcineurin and ANF was also unchanged. Eccentric hypertrophy and early cardiac dysfunction in experimental AR are associated with a pattern of activation of intracellular pathways different from that seen with pathological hypertrophy in pressure overload, and more similar to that associated with benign physiological hypertrophy.
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Insuficiencia de la Válvula Aórtica/complicaciones , Hipertrofia Ventricular Izquierda/etiología , Miocardio/metabolismo , Transducción de Señal , Disfunción Ventricular Izquierda/etiología , Animales , Insuficiencia de la Válvula Aórtica/diagnóstico por imagen , Insuficiencia de la Válvula Aórtica/metabolismo , Insuficiencia de la Válvula Aórtica/fisiopatología , Factor Natriurético Atrial/metabolismo , Calcineurina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Ecocardiografía Doppler en Color , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Miocardio/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/fisiopatología , Función Ventricular Izquierda , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Slowed Na⺠current (INa) decay and enhanced late INa (INa-L) prolong the action potential duration (APD) and contribute to early afterdepolarizations. Cardiac resynchronization therapy (CRT) shortens APD compared with dyssynchronous heart failure (DHF); however, the role of altered Na⺠channel gating in CRT remains unexplored. METHODS AND RESULTS: Adult dogs underwent left-bundle branch ablation and right atrial pacing (200 beats/min) for 6 weeks (DHF) or 3 weeks followed by 3 weeks of biventricular pacing at the same rate (CRT). INa and INa-L were measured in left ventricular myocytes from nonfailing, DHF, and CRT dogs. DHF shifted voltage-dependence of INa availability by -3 mV compared with nonfailing, enhanced intermediate inactivation, and slowed recovery from inactivation. CRT reversed the DHF-induced voltage shift of availability, partially reversed enhanced intermediate inactivation but did not affect DHF-induced slowed recovery. DHF markedly increased INa-L compared with nonfailing. CRT dramatically reduced DHF-induced enhanced INa-L, abbreviated the APD, and suppressed early afterdepolarizations. CRT was associated with a global reduction in phosphorylated Ca²âº/Calmodulin protein kinase II, which has distinct effects on inactivation of cardiac Na⺠channels. In a canine AP model, alterations of INa-L are sufficient to reproduce the effects on APD observed in DHF and CRT myocytes. CONCLUSIONS: CRT improves DHF-induced alterations of Na⺠channel function, especially suppression of INa-L, thus, abbreviating the APD and reducing the frequency of early afterdepolarizations. Changes in the levels of phosphorylated Ca²âº/Calmodulin protein kinase II suggest a molecular pathway for regulation of INa by biventricular pacing of the failing heart.
Asunto(s)
Terapia de Resincronización Cardíaca/métodos , Insuficiencia Cardíaca/terapia , Canales de Sodio/metabolismo , Remodelación Ventricular/fisiología , Animales , Simulación por Computador , Modelos Animales de Enfermedad , Perros , Sistema de Conducción Cardíaco/metabolismo , Sistema de Conducción Cardíaco/fisiopatología , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/mortalidad , Activación del Canal Iónico/fisiología , Masculino , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , Medición de Riesgo , Sensibilidad y Especificidad , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Cardiac resynchronization therapy reduces morbidity and mortality in patients with symptomatic systolic heart failure (New York Heart Association class III or IV) and ventricular conduction delay. The current review focuses on how high-throughput technologies including gene expression profiling and proteomics have helped in our understanding of the pathophysiology of electromechanical dyssynchrony and the molecular mechanisms by which cardiac resynchronization therapy (CRT) exerts its beneficial effects. Comparing gene expression changes in early-activated anterior vs. late-activated lateral left ventricular myocardium in a large animal model of dyssynchronous heart failure, we demonstrated a profound effect of electromechanical dyssynchrony on the regional cardiac transcriptome, as changes in gene expression were primarily observed in the early-activated anterior left ventricular myocardium. This increase in regional heterogeneity of gene expression within the left ventricle was reversed by CRT. Specifically, CRT remodeled transcripts with metabolic and cell signaling function, which was corroborated by protein data. In addition, high-throughput or "omic" techniques also hold great promise to identify key pathways and biomarkers that are regulated differentially in CRT responders vs. nonresponders.
Asunto(s)
Terapia de Resincronización Cardíaca , Insuficiencia Cardíaca , Metaboloma/genética , Proteoma/genética , Transcriptoma/genética , Disfunción Ventricular Izquierda , Remodelación Ventricular/genética , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/terapia , Humanos , Miocardio/metabolismo , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatología , Disfunción Ventricular Izquierda/terapiaRESUMEN
RATIONALE: Phosphorylation of ß(2)-adrenergic receptor (ß(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. OBJECTIVE: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of ß(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. METHODS AND RESULTS: Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to ßAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant ß(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type ß(2)AR (WT-TG) or a mutant ß(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased ß(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of ß(2)AR by GRK and resultant increase in G(i)-biased ß(2)AR signaling play an important role in the development of heart failure. CONCLUSIONS: Our data show that enhanced ß(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased ß(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Insuficiencia Cardíaca/enzimología , Miocitos Cardíacos/enzimología , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Agonistas Adrenérgicos beta/farmacología , Animales , Cardiomegalia/enzimología , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Contracción Miocárdica , Miocitos Cardíacos/efectos de los fármacos , Toxina del Pertussis/farmacología , Fosforilación , Receptores Adrenérgicos beta 2/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Regulación hacia Arriba , Función Ventricular Izquierda , Presión Ventricular , Remodelación VentricularRESUMEN
Cardiac resynchronization therapy (CRT), in which both ventricles are paced to recoordinate contraction in hearts that are dyssynchronous from conduction delay, is the only heart failure (HF) therapy to date to clinically improve acute and chronic function while also lowering mortality. CRT acutely enhances chamber mechanical efficiency but chronically alters myocyte signaling, including improving ß-adrenergic receptor reserve. We speculated that the latter would identify unique CRT effects that might themselves be effective for HF more generally. HF was induced in dogs by 6 weeks of atrial rapid pacing with (HFdys, left bundle ablated) or without (HFsyn) dyssynchrony. We used dyssynchronous followed by resynchronized tachypacing (each 3 weeks) for CRT. Both HFdys and HFsyn myocytes had similarly depressed rest and ß-adrenergic receptor sarcomere and calcium responses, particularly the ß2-adrenergic response, whereas cells subjected to CRT behaved similarly to those from healthy controls. CRT myocytes exhibited suppressed Gαi signaling linked to increased regulator of G protein (heterotrimeric guanine nucleotide-binding protein) signaling (RGS2, RGS3), yielding Gαs-biased ß2-adrenergic responses. This included increased adenosine cyclic AMP responsiveness and activation of sarcoplasmic reticulum-localized protein kinase A. Human CRT responders also showed up-regulated myocardial RGS2 and RGS3. Inhibition of Gαi (with pertussis toxin, RGS3, or RGS2 transfection), stimulation with a Gαs-biased ß2 agonist (fenoterol), or transient (2-week) exposure to dyssynchrony restored ß-adrenergic receptor responses in HFsyn to the values obtained after CRT. These results identify a key pathway that is triggered by restoring contractile synchrony and that may represent a new therapeutic approach for a broad population of HF patients.