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The erythrocyte S1P transporter Mfsd2b is also expressed in the heart. We hypothesized that S1P transport by Mfsd2b is involved in cardiac function. Hypertension-induced cardiac remodeling was induced by 4-weeks Angiotensin II (AngII) administration and assessed by echocardiography. Ca2+ transients and sarcomere shortening were examined in adult cardiomyocytes (ACM) from Mfsd2b+/+ and Mfsd2b-/- mice. Tension and force development were measured in skinned cardiac fibers. Myocardial gene expression was determined by real-time PCR, Protein Phosphatase 2A (PP2A) by enzymatic assay, and S1P by LC/MS, respectively. Msfd2b was expressed in the murine and human heart, and its deficiency led to higher cardiac S1P. Mfsd2b-/- mice had regular basal cardiac function but were protected against AngII-induced deterioration of left-ventricular function as evidenced by ~ 30% better stroke volume and cardiac index, and preserved ejection fraction despite similar increases in blood pressure. Mfsd2b-/- ACM exhibited attenuated Ca2+ mobilization in response to isoprenaline whereas contractility was unchanged. Mfsd2b-/- ACM showed no changes in proteins responsible for Ca2+ homeostasis, and skinned cardiac fibers exhibited reduced passive tension generation with preserved contractility. Verapamil abolished the differences in Ca2+ mobilization between Mfsd2b+/+ and Mfsd2b-/- ACM suggesting that S1P inhibits L-type-Ca2+ channels (LTCC). In agreement, intracellular S1P activated the inhibitory LTCC phosphatase PP2A in ACM and PP2A activity was increased in Mfsd2b-/- hearts. We suggest that myocardial S1P protects from hypertension-induced left-ventricular remodeling by inhibiting LTCC through PP2A activation. Pharmacologic inhibition of Mfsd2b may thus offer a novel approach to heart failure.
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Miocardio , Humanos , Animales , Miocardio/metabolismo , Contracción Miocárdica/fisiología , Corazón/fisiologíaRESUMEN
The sharing and documentation of cardiovascular research data are essential for efficient use and reuse of data, thereby aiding scientific transparency, accelerating the progress of cardiovascular research and healthcare, and contributing to the reproducibility of research results. However, challenges remain. This position paper, written on behalf of and approved by the German Cardiac Society and German Centre for Cardiovascular Research, summarizes our current understanding of the challenges in cardiovascular research data management (RDM). These challenges include lack of time, awareness, incentives, and funding for implementing effective RDM; lack of standardization in RDM processes; a need to better identify meaningful and actionable data among the increasing volume and complexity of data being acquired; and a lack of understanding of the legal aspects of data sharing. While several tools exist to increase the degree to which data are findable, accessible, interoperable, and reusable (FAIR), more work is needed to lower the threshold for effective RDM not just in cardiovascular research but in all biomedical research, with data sharing and reuse being factored in at every stage of the scientific process. A culture of open science with FAIR research data should be fostered through education and training of early-career and established research professionals. Ultimately, FAIR RDM requires permanent, long-term effort at all levels. If outcomes can be shown to be superior and to promote better (and better value) science, modern RDM will make a positive difference to cardiovascular science and practice. The full position paper is available in the supplementary materials.
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Investigación Biomédica , Sistema Cardiovascular , Humanos , Manejo de Datos , Reproducibilidad de los Resultados , CorazónRESUMEN
Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.
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Cardiomiopatía Hipertrófica , Síndrome de Noonan , Proteínas Proto-Oncogénicas c-raf , Humanos , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Mutación de Línea Germinal , Miocitos Cardíacos/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/complicaciones , Síndrome de Noonan/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-raf/genéticaRESUMEN
BACKGROUND: ST-segment elevation myocardial infarction (STEMI) still causes significant mortality and morbidity despite best-practice revascularization and adjunct medical strategies. Within the STEMI population, there is a spectrum of higher and lower risk patients with respect to major adverse cardiovascular and cerebral events (MACCE) or re-hospitalization due to heart failure. Myocardial and systemic metabolic disorders modulate patient risk in STEMI. Systematic cardiocirculatory and metabolic phenotyping to assess the bidirectional interaction of cardiac and systemic metabolism in myocardial ischemia is lacking. METHODS: Systemic organ communication in STEMI (SYSTEMI) is an all-comer open-end prospective study in STEMI patients > 18 years of age to assess the interaction of cardiac and systemic metabolism in STEMI by systematically collecting data on a regional and systemic level. Primary endpoint will be myocardial function, left ventricular remodelling, myocardial texture and coronary patency at 6 month after STEMI. Secondary endpoint will be all-cause death, MACCE, and re-hospitalisation due to heart failure or revascularisation assessed 12 month after STEMI. The objective of SYSTEMI is to identify metabolic systemic and myocardial master switches that determine primary and secondary endpoints. In SYSTEMI 150-200 patients are expected to be recruited per year. Patient data will be collected at the index event, within 24 h, 5 days as well as 6 and 12 months after STEMI. Data acquisition will be performed in multilayer approaches. Myocardial function will be assessed by using serial cardiac imaging with cineventriculography, echocardiography and cardiovascular magnetic resonance. Myocardial metabolism will be analysed by multi-nuclei magnetic resonance spectroscopy. Systemic metabolism will be approached by serial liquid biopsies and analysed with respect to glucose and lipid metabolism as well as oxygen transport. In summary, SYSTEMI enables a comprehensive data analysis on the levels of organ structure and function alongside hemodynamic, genomic and transcriptomic information to assess cardiac and systemic metabolism. DISCUSSION: SYSTEMI aims to identify novel metabolic patterns and master-switches in the interaction of cardiac and systemic metabolism to improve diagnostic and therapeutic algorithms in myocardial ischemia for patient-risk assessment and tailored therapy. TRIAL REGISTRATION: Trial Registration Number: NCT03539133.
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Enfermedad de la Arteria Coronaria , Insuficiencia Cardíaca , Intervención Coronaria Percutánea , Infarto del Miocardio con Elevación del ST , Humanos , Infarto del Miocardio con Elevación del ST/diagnóstico por imagen , Infarto del Miocardio con Elevación del ST/terapia , Estudios de Cohortes , Estudios Prospectivos , Intervención Coronaria Percutánea/efectos adversos , Enfermedad de la Arteria Coronaria/complicaciones , Insuficiencia Cardíaca/etiología , Resultado del TratamientoRESUMEN
Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to ß-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon ß-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.
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Diabetes Mellitus Experimental , Infarto del Miocardio , Ratones , Animales , Conectina/metabolismo , Calcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Infarto del Miocardio/metabolismo , Reperfusión , Adrenérgicos , Contracción MiocárdicaRESUMEN
Sarcomeres are mainly composed of filament and signaling proteins and are the smallest molecular units of muscle contraction and relaxation. The sarcomere protein titin serves as a molecular spring whose stiffness mediates myofilament extensibility in skeletal and cardiac muscle. Due to the enormous size of titin and its tight integration into the sarcomere, the incorporation and degradation of the titin filament is a highly complex task. The details of the molecular processes involved in titin turnover are not fully understood, but the involvement of different intracellular degradation mechanisms has recently been described. This review summarizes the current state of research with particular emphasis on the relationship between titin and protein quality control. We highlight the involvement of the proteasome, autophagy, heat shock proteins, and proteases in the protection and degradation of titin in heart and skeletal muscle. Because the fine-tuned balance of degradation and protein expression can be disrupted under pathological conditions, the review also provides an overview of previously known perturbations in protein quality control and discusses how these affect sarcomeric proteins, and titin in particular, in various disease states.
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BACKGROUND: In the European Union proteins for food are largely animal based, consisting of meat and dairy products. Almost all soy but also a larger part of pulses and cereals consumed in the European Union are used for animal nutrition. While livestock is an important source of proteins, it also creates substantial environmental impacts. The food and feed system is closely linked to the planetary and health boundaries and a transformation to healthy diets will require substantial dietary shifts towards healthy foods, such as nuts, fruits, vegetables and legumes. RESULTS: Extrudated vegetable meat alternatives consisting of protein combined with amaranth or buckwheat flour and a vegetable milk alternative made from lentil proteins were shown to have the potential to generate significantly less environmental impact than their animal-based counterparts in most of the environmental indicators examined, taking into account both functional units (mass and protein content). The underlying field-to-fork life cycle assessment models include several variants for both plant and animal foods. The optimized plant-based foods show a clear potential for improvement in the environmental footprints. CONCLUSIONS: Development of higher processed and therefore higher performing products is crucial for appealing to potential user groups beyond dedicated vegetarians and vegans and ultimately achieving market expansion. The Protein2Food project showed that prototypes made from European-grown legumes and pseudocereals are a valuable source for high-quality protein foods, and despite being substantially processed they could help reduce the environmental impact of food consumption. © 2021 Society of Chemical Industry.
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Dieta , Proteínas de Plantas , Alimentación Animal , Animales , Productos Lácteos , Estadios del Ciclo de Vida , Carne , VerdurasRESUMEN
The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.
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Autofagia , Conectina , Complejo de la Endopetidasa Proteasomal , Proteolisis , Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Conectina/genética , Conectina/metabolismo , Técnicas de Silenciamiento del Gen , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Ratas Wistar , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.
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Miocardio/química , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Proteínas Quinasas/metabolismo , Animales , Elasticidad , Masculino , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocitos Cardíacos/química , Oxidación-Reducción , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genéticaRESUMEN
Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. The anthraquinone Rhein was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration during hypoxia, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein acts inhibitory on HDAC classes I/II as enzymatic inhibitor. Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent protein stabilization of p53 under normoxic but not hypoxic conditions. Functionally, Rhein inhibited collagen contraction, indicating anti-fibrotic property in cardiac remodeling. This was accompanied by increased abundance of SMAD7, but not SMAD2/3, and consistently SMAD-specific E3 ubiquitin ligase SMURF2. In conclusion, this study identifies Rhein as a novel potent direct HDAC inhibitor that may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.
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Antraquinonas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Adulto , Western Blotting , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Transcriptoma/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Cardiac aging affects the heart on the functional, structural, and molecular level and shares characteristic hallmarks with the development of chronic heart failure. Apart from age-dependent left ventricular hypertrophy and fibrosis that impairs diastolic function, diminished activity of cardiac protein-quality-control systems increases the risk of cytotoxic accumulation of defective proteins. Here, we studied the impact of cardiac aging on the sarcomeric protein titin by analyzing titin-based cardiomyocyte passive tension, titin modification and proteasomal titin turnover. We analyzed left ventricular samples from young (6â¯months) and old (20â¯months) wild-type mice and healthy human donor patients grouped according to age in young (17-50â¯years) and aged hearts (51-73â¯years). We found no age-dependent differences in titin isoform composition of mouse or human hearts. In aged hearts from mice and human we determined altered titin phosphorylation at serine residues S4010 and S4099 in the elastic N2B domain, but no significant changes in phosphorylation of S11878 and S12022 in the elastic PEVK region. Importantly, overall titin-based cardiomyocyte passive tension remained unchanged. In aged hearts, the calcium-activated protease calpain-1, which provides accessibility to ubiquitination by releasing titin from the sarcomere, showed decreased proteolytic activity. In addition, we observed a reduction in the proteasomal activities. Taken together, our data indicate that cardiac aging does not affect titin-based passive properties of the cardiomyocytes, but impairs protein-quality control, including titin, which may result in a diminished adaptive capacity of the aged myocardium.
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Envejecimiento/genética , Conectina/genética , Hipertrofia Ventricular Izquierda/genética , Miocitos Cardíacos/metabolismo , Adolescente , Adulto , Anciano , Envejecimiento/patología , Animales , Conectina/química , Femenino , Voluntarios Sanos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Hipertrofia Ventricular Izquierda/patología , Masculino , Ratones , Persona de Mediana Edad , Proteínas Musculares/química , Proteínas Musculares/genética , Miocardio/química , Miocardio/metabolismo , Miocitos Cardíacos/química , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Adulto JovenRESUMEN
RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.
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Matriz Extracelular/fisiología , Hialuronano Sintasas/deficiencia , Ácido Hialurónico/biosíntesis , Macrófagos/fisiología , Daño por Reperfusión Miocárdica/fisiopatología , Cicatrización de Heridas/fisiología , Actinas/metabolismo , Animales , Apoptosis , Comunicación Celular/fisiología , Supervivencia Celular , Microambiente Celular/fisiología , Matriz Extracelular/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/antagonistas & inhibidores , Imagen por Resonancia Magnética/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Monocitos/fisiología , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/citología , Miofibroblastos/metabolismo , Miofibroblastos/fisiologíaRESUMEN
Ischemic conditioning induces cardioprotection; the final infarct size following a myocardial ischemic event is reduced. However, whether ischemic conditioning has long-term beneficial effects on myocardial contractile function following such an ischemic event needs further elucidation. To date, ex vivo studies have shown that ischemic conditioning improves the contractile recovery of isolated ventricular papillary muscle or atrial trabeculae following simulated ischemia. However, in vivo animal studies and studies in patients undergoing elective cardiac surgery show conflicting results. At the subcellular level, it is known that ischemic conditioning improved energy metabolism, preserved mitochondrial respiration, ATP production, and Ca2+ homeostasis in isolated mitochondria from the myocardium. Ischemic conditioning also presents with post-translational modifications of proteins in the contractile machinery of the myocardium. The beneficial effects on myocardial contractile function need further elucidation. This article is part of a Special Issue entitled: The power of metabolism: Linking energy supply and demand to contractile function edited by Torsten Doenst, Michael Schwarzer and Christine Des Rosiers.
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Poscondicionamiento Isquémico/métodos , Contracción Miocárdica , Daño por Reperfusión Miocárdica/fisiopatología , Animales , Calcio/metabolismo , Humanos , Poscondicionamiento Isquémico/efectos adversos , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/terapiaRESUMEN
BACKGROUND: One of the first clinically detectable alterations in heart function in hypertrophic cardiomyopathy (HCM) is a decline in diastolic function. Diastolic dysfunction is caused by changes in intrinsic properties of cardiomyocytes or an increase in fibrosis. We investigated whether clinical and cellular parameters of diastolic function are different between male and female patients with HCM at the time of myectomy. METHODS AND RESULTS: Cardiac tissue from the interventricular septum of patients with HCM (27 women and 44 men) was obtained during myectomy preceded by echocardiography. At myectomy, female patients were 7 years older than male patients and showed more advanced diastolic dysfunction than men evident from significantly higher values for E/e' ratio, left ventricular filling pattern, tricuspid regurgitation velocity, and left atrial diameter indexed for body surface. Whereas most male patients (56%) showed mild (grade I) diastolic dysfunction, 50% of female patients showed grade III diastolic dysfunction. Passive tension in HCM cardiomyocytes was comparable with controls, and myofilament calcium sensitivity was higher in HCM compared with controls, but no sex differences were observed in myofilament function. In female patients with HCM, titin was more compliant, and more fibrosis was present compared with men. Differences between female and male patients with HCM remained significant after correction for age. CONCLUSIONS: Female patients with HCM are older at the time of myectomy and show greater impairment of diastolic function. Furthermore, left ventricular and left atrial remodeling is increased in women when corrected for body surface area. At a cellular level, HCM women showed increased compliant titin and a larger degree of interstitial fibrosis.
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Cardiomiopatía Hipertrófica/patología , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/patología , Caracteres Sexuales , Adolescente , Adulto , Anciano , Cardiomiopatías/fisiopatología , Cardiomiopatía Hipertrófica/fisiopatología , Niño , Ecocardiografía/métodos , Femenino , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Adulto JovenRESUMEN
RATIONALE: Increased titin-dependent cardiomyocyte tension is a hallmark of heart failure with preserved ejection fraction associated with type-2 diabetes mellitus. However, the insulin-related signaling pathways that modify titin-based cardiomyocyte tension, thereby contributing to modulation of diastolic function, are largely unknown. OBJECTIVE: We aimed to determine how impaired insulin signaling affects titin expression and phosphorylation and thus increases passive cardiomyocyte tension, and whether metformin or neuregulin-1 (NRG-1) can correct disturbed titin modifications and increased titin-based stiffness. METHODS AND RESULTS: We used cardiac biopsies from human diabetic (n=23) and nondiabetic patients (n=19), cultured rat cardiomyocytes, left ventricular tissue from apolipoprotein E-deficient mice with streptozotocin-induced diabetes mellitus (n=12-22), and ZSF1 (obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid) rats (n=5-6) and analyzed insulin-dependent signaling pathways that modulate titin phosphorylation. Titin-based passive tension was measured using permeabilized cardiomyocytes. In human diabetic hearts, we detected titin hypophosphorylation at S4099 and hyperphosphorylation at S11878, suggesting altered activity of protein kinases; cardiomyocyte passive tension was significantly increased. When applied to cultured cardiomyocytes, insulin and metformin increased titin phosphorylation at S4010, S4099, and S11878 via enhanced ERK1/2 (extracellular signal regulated kinase 1/2) and PKCα (protein kinase Cα) activity; NRG-1 application enhanced ERK1/2 activity but reduced PKCα activity. In apolipoprotein E-deficient mice, chronic treatment of streptozotocin-induced diabetes mellitus with NRG-1 corrected titin phosphorylation via increased PKG (protein kinase G) and ERK1/2 activity and reduced PKCα activity, which reversed the diabetes mellitus-associated changes in titin-based passive tension. Acute application of NRG-1 to obese ZSF1 rats with type-2 diabetes mellitus reduced end-diastolic pressure. CONCLUSIONS: Mechanistically, we found that impaired cGMP-PKG signaling and elevated PKCα activity are key modulators of titin-based cardiomyocyte stiffening in diabetic hearts. We conclude that by restoring normal kinase activities of PKG, ERK1/2, and PKCα, and by reducing cardiomyocyte passive tension, chronic NRG-1 application is a promising approach to modulate titin properties in heart failure with preserved ejection fraction associated with type-2 diabetes mellitus.
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Conectina/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Insulina/farmacología , Miocitos Cardíacos/metabolismo , Neurregulina-1/farmacología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Animales , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Humanos , Hipoglucemiantes/farmacología , Metformina/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Fosforilación , Proteína Quinasa C-alfa/metabolismo , Ratas , Ratas ZuckerRESUMEN
Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca2+ handling in the remote myocardium 24â¯h after 60â¯min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca2+ handling. The amplitude of the Ca2+ transients as well as the kinetics of Ca2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9⯱â¯0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca2+ ATPase SERCA2a (Pâ¯≤â¯0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (Pâ¯≤â¯0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (Pâ¯≤â¯0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca2+ cycling in the remote myocardium 24â¯h after I/R and contributes to contractile dysfunction.