RESUMEN
Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Estrés Nitrosativo , Volumen Sistólico , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Endorribonucleasas/metabolismo , Insuficiencia Cardíaca/prevención & control , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3ß) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3-9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10-13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Cardiopatías/prevención & control , Cardiopatías/fisiopatología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/química , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Animales , Autofagia , Células Cultivadas , Progresión de la Enfermedad , Activación Enzimática , Everolimus/farmacología , Femenino , Técnicas de Sustitución del Gen , Células HEK293 , Cardiopatías/genética , Cardiopatías/patología , Humanos , Hipertrofia/tratamiento farmacológico , Hipertrofia/patología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones , Mutación , Miocitos Cardíacos/patología , Fosforilación , Fosfoserina/metabolismo , Presión , Ratas , Ratas Wistar , Serina/genética , Serina/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
BACKGROUND: The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown. METHODS: Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning. RESULTS: Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m2 versus 26.1 kg/m2; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites. CONCLUSIONS: Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.
Asunto(s)
Diabetes Mellitus , Insuficiencia Cardíaca , Humanos , Insuficiencia Cardíaca/metabolismo , Volumen Sistólico , Miocardio/metabolismo , Diabetes Mellitus/patología , Obesidad/patología , Ácidos GrasosRESUMEN
BACKGROUND: Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. We thus sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms. METHODS: Resting, calcium-, and load-dependent mechanics were prospectively studied in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 patients with HFrEF-PH undergoing cardiac transplantation and 9 organ donor controls. RESULTS: Unsupervised machine learning using myocyte mechanical data with the highest variance yielded 2 HFrEF-PH subgroups that in turn mapped to patients with decompensated or compensated clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in decompensated clinical RV function, whereas surprisingly, many other major myocyte contractile measures including peak power and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick filament defects, myofibrillar structure was assessed by x-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in decompensated clinical RV function, but not compensated clinical RV function, as compared with controls. This corresponded to reduced myosin ATP turnover in decompensated clinical RV function myocytes, indicating less myosin in a crossbridge-ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Last, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human heart failure. CONCLUSIONS: Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.
Asunto(s)
Insuficiencia Cardíaca , Hipertensión Pulmonar , Disfunción Ventricular Derecha , Humanos , Sarcómeros , Calcio , Depresión , Volumen Sistólico , Miocitos Cardíacos , Función Ventricular Derecha/fisiologíaRESUMEN
[Figure: see text].
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/antagonistas & inhibidores , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Animales , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Inhibidores Enzimáticos/farmacología , Acoplamiento Excitación-Contracción , Cobayas , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiologíaRESUMEN
[Figure: see text].
Asunto(s)
Insuficiencia Cardíaca Diastólica/terapia , Mitocondrias Cardíacas/metabolismo , Miopatías Mitocondriales/terapia , NAD/biosíntesis , Niacinamida/análogos & derivados , Compuestos de Piridinio/administración & dosificación , Acetilación , Acil-CoA Deshidrogenasa/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Ácidos Grasos/metabolismo , Humanos , Cetona Oxidorreductasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miopatías Mitocondriales/metabolismo , NAD/análisis , NAD/deficiencia , NAD/genética , Niacinamida/administración & dosificación , Oxidación-Reducción , Consumo de Oxígeno , Sirtuina 3/metabolismoRESUMEN
RATIONALE: The mTORC1 (mechanistic target of rapamycin complex-1) controls metabolism and protein homeostasis and is activated following ischemia reperfusion (IR) injury and by ischemic preconditioning (IPC). However, studies vary as to whether this activation is beneficial or detrimental, and its influence on metabolism after IR is little reported. A limitation of prior investigations is their use of broad gain/loss of mTORC1 function, mostly applied before ischemic stress. This can be circumvented by regulating one serine (S1365) on TSC2 (tuberous sclerosis complex) to achieve bidirectional mTORC1 modulation but only with TCS2-regulated costimulation. OBJECTIVE: We tested the hypothesis that reduced TSC2 S1365 phosphorylation protects the myocardium against IR and is required for IPC by amplifying mTORC1 activity to favor glycolytic metabolism. METHODS AND RESULTS: Mice with either S1365A (TSC2SA; phospho-null) or S1365E (TSC2SE; phosphomimetic) knockin mutations were studied ex vivo and in vivo. In response to IR, hearts from TSC2SA mice had amplified mTORC1 activation and improved heart function compared with wild-type and TSC2SE hearts. The magnitude of protection matched IPC. IPC requited less S1365 phosphorylation, as TSC2SE hearts gained no benefit and failed to activate mTORC1 with IPC. IR metabolism was altered in TSC2SA, with increased mitochondrial oxygen consumption rate and glycolytic capacity (stressed/maximal extracellular acidification) after myocyte hypoxia-reperfusion. In whole heart, lactate increased and long-chain acylcarnitine levels declined during ischemia. The relative IR protection in TSC2SA was lost by lowering glucose in the perfusate by 36%. Adding fatty acid (palmitate) compensated for reduced glucose in wild type and TSC2SE but not TSC2SA which had the worst post-IR function under these conditions. CONCLUSIONS: TSC2-S1365 phosphorylation status regulates myocardial substrate utilization, and its decline activates mTORC1 biasing metabolism away from fatty acid oxidation to glycolysis to confer protection against IR. This pathway is also engaged and reduced TSC2 S1365 phosphorylation required for effective IPC. Graphic Abstract: A graphic abstract is available for this article.
Asunto(s)
Glucólisis , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Carnitina/análogos & derivados , Carnitina/metabolismo , Células Cultivadas , Glucosa/metabolismo , Precondicionamiento Isquémico , Ácido Láctico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/metabolismo , Mutación , Daño por Reperfusión Miocárdica/terapia , Oxígeno/metabolismo , Fosforilación , Ratas , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
BACKGROUND: Heart failure (HF) with preserved ejection fraction (HFpEF) constitutes half of all HF but lacks effective therapy. Understanding of its myocardial biology remains limited because of a paucity of heart tissue molecular analysis. METHODS: We performed RNA sequencing on right ventricular septal endomyocardial biopsies prospectively obtained from patients meeting consensus criteria for HFpEF (n=41) contrasted with right ventricular septal tissue from patients with HF with reduced ejection fraction (HFrEF, n=30) and donor controls (n=24). Principal component analysis and hierarchical clustering tested for transcriptomic distinctiveness between groups, effect of comorbidities, and differential gene expression with pathway enrichment contrasted HF groups and donor controls. Within HFpEF, non-negative matrix factorization and weighted gene coexpression analysis identified molecular subgroups, and the resulting clusters were correlated with hemodynamic and clinical data. RESULTS: Patients with HFpEF were more often women (59%), African American (68%), obese (median body mass index 41), and hypertensive (98%), with clinical HF characterized by 65% New York Heart Association Class III or IV, nearly all on a loop diuretic, and 70% with a HF hospitalization in the previous year. Principal component analysis separated HFpEF from HFrEF and donor controls with minimal overlap, and this persisted after adjusting for primary comorbidities: body mass index, sex, age, diabetes, and renal function. Hierarchical clustering confirmed group separation. Nearly half the significantly altered genes in HFpEF versus donor controls (1882 up, 2593 down) changed in the same direction in HFrEF; however, 5745 genes were uniquely altered between HF groups. Compared with controls, uniquely upregulated genes in HFpEF were enriched in mitochondrial adenosine triphosphate synthesis/electron transport, pathways downregulated in HFrEF. HFpEF-specific downregulated genes engaged endoplasmic reticulum stress, autophagy, and angiogenesis. Body mass index differences largely accounted for HFpEF upregulated genes, whereas neither this nor broader comorbidity adjustment altered pathways enriched in downregulated genes. Non-negative matrix factorization identified 3 HFpEF transcriptomic subgroups with distinctive pathways and clinical correlates, including a group closest to HFrEF with higher mortality, and a mostly female group with smaller hearts and proinflammatory signaling. These groupings remained after sex adjustment. Weighted gene coexpression analysis yielded analogous gene clusters and clinical groupings. CONCLUSIONS: HFpEF exhibits distinctive broad transcriptomic signatures and molecular subgroupings with particular clinical features and outcomes. The data reveal new signaling targets to consider for precision therapeutics.
Asunto(s)
Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Volumen Sistólico/fisiología , Transcriptoma/fisiología , Anciano , Cateterismo Cardíaco/métodos , Femenino , Insuficiencia Cardíaca/patología , Humanos , Masculino , Persona de Mediana Edad , Miocardio/patología , Estudios ProspectivosRESUMEN
BACKGROUND: Therapy for heart failure with preserved ejection fraction (HFpEF) remains an unmet need with lack of a consensus definition of HFpEF for inclusion into clinical trials. We evaluated whether hemodynamically characterized patients from a HFpEF referral center met inclusion criteria for 4 major HFpEF trials. METHODS AND RESULTS: Patients were assessed for theoretical inclusion into 4 major clinical trials (I-PRESERVE, RELAX, TOPCAT, and PARAGON-HF). Clinical, echocardiographic and hemodynamic characteristics and cardiovascular outcomes were compared between patients who met the inclusion criteria vs those who did not for each trial. Of 131 patients with HFpEF, 23% of patients met the enrollment criteria for I-PRESERVE, 38% for RELAX, 18% for TOPCAT, and 13% for PARAGON-HF. The top criteria that excluded patients included low natriuretic peptide level, obesity, uncontrolled hypertension, young age, and low hemoglobin. There was no difference in the probability of HF hospitalization or death in patients included or excluded into each clinical trial. CONCLUSIONS: In a cohort with hemodynamic evidence of HFpEF, a low proportion of patients met inclusion criteria for major HFpEF clinical trials, with no difference in outcomes in patients who did or did not meet inclusion criteria. Given relative the lack of proven therapies in HFpEF, consideration should be given to modifying clinical trial enrollment criteria to better represent contemporary patients with HFpEF in future clinical trials.
Asunto(s)
Insuficiencia Cardíaca , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/terapia , Humanos , Péptidos Natriuréticos , Obesidad/complicaciones , Obesidad/epidemiología , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.
Asunto(s)
Cardiomegalia/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Guanilato Ciclasa/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Aorta , Autofagia/fisiología , Benzoatos/metabolismo , Compuestos de Bifenilo/metabolismo , Constricción Patológica , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Cisteína/metabolismo , Endotelina-1/farmacología , Activación Enzimática , Everolimus/farmacología , Técnicas de Sustitución del Gen , Hidrocarburos Fluorados/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Presión , Proteostasis , Ratas , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration â¼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Nefroesclerosis/tratamiento farmacológico , Canal Catiónico TRPC6/antagonistas & inhibidores , Animales , Evaluación Preclínica de Medicamentos , Fibrosis , Células HEK293 , Corazón/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , RatonesRESUMEN
Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as "likely" or "real." This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.
Asunto(s)
Fibras Musculares de Contracción Lenta , Enfermedades Musculares , Humanos , Fibras Musculares de Contracción Rápida , Músculo Esquelético , ProteómicaRESUMEN
Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.
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Insuficiencia Cardíaca/epidemiología , Investigación/normas , Humanos , National Heart, Lung, and Blood Institute (U.S.) , Volumen Sistólico , Estados UnidosRESUMEN
Advances in the treatment of heart failure with reduced ejection fraction due to systolic dysfunction are engaging an ever-expanding compendium of molecular signaling targets. Well established approaches modifying hemodynamics and cell biology by neurohumoral receptor blockade are evolving, exploring the role and impact of modulating intracellular signaling pathways with more direct myocardial effects. Even well-tread avenues are being reconsidered with new insights into the signaling engaged and thus opportunity to treat underlying myocardial disease. This review explores therapies that have proven successful, those that have not, those that are moving into the clinic but whose utility remains to be confirmed, and those that remain in the experimental realm. The emphasis is on signaling pathways that are tractable for therapeutic manipulation. Of the approaches yet to be tested in humans, we chose those with a well-established experimental history, where clinical translation may be around the corner. The breadth of opportunities bodes well for the next generation of heart failure therapeutics.
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Fármacos Cardiovasculares/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Miocardio/metabolismo , Transducción de Señal/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacos , Animales , Fármacos Cardiovasculares/efectos adversos , Diseño de Fármacos , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Terapia Molecular Dirigida , Recuperación de la Función , Resultado del TratamientoRESUMEN
Purpose: Cyclic guanosine monophosphate (cGMP) is a second messenger for natriuretic peptide (NP) and nitric oxide pathways; its enhancement a target for heart failure and cardiovascular disease (CVD). We evaluated whether plasma cGMP was associated with change in left ventricular mass (LVM) among individuals free of CVD and if this differed by sex.Methods and Results: In 611 men and 612 women aged 45-84 years with plasma cGMP measured at baseline and cardiac MRI performed at baseline and 10 years later, we tested associations of cGMP [log-transformed, per 1 SD increment] with LVM, adjusting for CVD risk factors and N-terminal pro-B-type-NP (NT-proBNP). Participants had mean (SD) age of 63.1(8.5) years and cGMP 4.8(2.6) pmol/mL. Cross-sectionally, higher cGMP was associated with lesser LVM, non-lin- early. In contrast, longitudinally, higher cGMP was associated with increase in LVM [1.70g (0.61, 2.78)] over 10 years. Higher cGMP was associated with greater LVM change in men [2.68g (1.57, 3.79)] but not women [0.24g ((-0.92, 1.39); p-interaction < 0.001].Conclusion: In conclusion, in a community-based cohort, higher cGMP levels were associated with increase in LVM over 10 years independent of CVD risk factors and NT-proBNP in men, perhaps reflecting compensatory changes. Further studies are needed to understand mechanistic roles of cGMP in LV remodelling and associated sex differences.
Asunto(s)
Aterosclerosis/sangre , Enfermedades Cardiovasculares/sangre , GMP Cíclico/sangre , Remodelación Ventricular , Negro o Afroamericano/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Asiático/estadística & datos numéricos , Aterosclerosis/diagnóstico , Aterosclerosis/etnología , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/etnología , Estudios de Cohortes , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/fisiopatología , Hispánicos o Latinos/estadística & datos numéricos , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Factores de Riesgo , Factores de Tiempo , Población Blanca/estadística & datos numéricosRESUMEN
Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Cardiomegalia/enzimología , Cardiomegalia/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/deficiencia , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Animales , Estenosis de la Válvula Aórtica/complicaciones , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/etiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Musculares/enzimología , Miocardio/enzimología , Péptidos Natriuréticos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Presión , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico , Regulación hacia ArribaRESUMEN
Cyclic nucleotide phosphodiesterases (PDEs) form an 11-member superfamily comprising 100 different isoforms that regulate the second messengers cyclic adenosine or guanosine 3',5'-monophosphate (cAMP or cGMP). These PDE isoforms differ with respect to substrate selectivity and their localized control of cAMP and cGMP within nanodomains that target specific cellular pools and synthesis pathways for the cyclic nucleotides. Seven PDE family members are physiologically relevant to regulating cardiac function, disease remodeling of the heart, or both: PDE1 and PDE2, both dual-substrate (cAMP and cGMP) esterases; PDE3, PDE4, and PDE8, which principally hydrolyze cAMP; and PDE5A and PDE9A, which target cGMP. New insights regarding the different roles of PDEs in health and disease and their local signaling control are broadening the potential therapeutic utility for PDE-selective inhibitors. In this review, we discuss these PDEs, focusing on the different mechanisms by which they control cardiac function in health and disease by regulating intracellular nanodomains.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Fenómenos Fisiológicos Cardiovasculares , Cardiopatías/metabolismo , Nanopartículas/metabolismo , Inhibidores de Fosfodiesterasa/uso terapéutico , Dominios Proteicos/fisiología , Transducción de Señal/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/fisiología , GMP Cíclico/antagonistas & inhibidores , GMP Cíclico/fisiología , Cardiopatías/tratamiento farmacológico , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/fisiología , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: GSK-3ß (glycogen synthase kinase 3ß) is a multifunctional and constitutively active kinase known to regulate a myriad of cellular processes. The primary mechanism to regulate its function is through phosphorylation-dependent inhibition at serine-9 residue. Emerging evidence indicates that there may be alternative mechanisms that control GSK-3ß for certain functions. OBJECTIVES: Here, we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK-3ß. SNO-dependent modulation of the localization of GSK-3ß and its ability to phosphorylate downstream targets was investigated in vitro, and the network of proteins differentially impacted by phospho- or SNO-dependent GSK-3ß regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied. METHODS AND RESULTS: We found that GSK-3ß undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes, as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. S-nitrosylation of GSK-3ß significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK-3ß promotes its nuclear translocation and access to novel downstream phosphosubstrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phosphoproteomics pathway analysis reveals that nuclear GSK-3ß plays a central role in cell cycle control, RNA splicing, and DNA damage response. CONCLUSIONS: The results indicate that SNO has a differential effect on the location and activity of GSK-3ß in the cytoplasm versus the nucleus. SNO modification of GSK-3ß occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.
Asunto(s)
Muerte Súbita Cardíaca , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Proteína S/metabolismo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Cobayas , Óxido Nítrico/metabolismo , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
Cyclic nucleotide phosphodiesterases comprise an 11-member superfamily yielding near 100 isoform variants that hydrolyze cAMP or cGMP to their respective 5'-monophosphate form. Each plays a role in compartmentalized cyclic nucleotide signaling, with varying selectivity for each substrate, and conveying cell and intracellular-specific localized control. This review focuses on the 5 phosphodiesterases (PDEs) expressed in the cardiac myocyte capable of hydrolyzing cGMP and that have been shown to play a role in cardiac physiological and pathological processes. PDE1, PDE2, and PDE3 catabolize cAMP as well, whereas PDE5 and PDE9 are cGMP selective. PDE3 and PDE5 are already in clinical use, the former for heart failure, and PDE1, PDE9, and PDE5 are all being actively studied for this indication in patients. Research in just the past few years has revealed many novel cardiac influences of each isoform, expanding the therapeutic potential from their selective pharmacological blockade or in some instances, activation. PDE1C inhibition was found to confer cell survival protection and enhance cardiac contractility, whereas PDE2 inhibition or activation induces beneficial effects in hypertrophied or failing hearts, respectively. PDE3 inhibition is already clinically used to treat acute decompensated heart failure, although toxicity has precluded its long-term use. However, newer approaches including isoform-specific allosteric modulation may change this. Finally, inhibition of PDE5A and PDE9A counter pathological remodeling of the heart and are both being pursued in clinical trials. Here, we discuss recent research advances in each of these PDEs, their impact on the myocardium, and cardiac therapeutic potential.