RESUMEN
The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region of titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. In contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal ß-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins. Interestingly, while volume-overload resulted in FHL1 protein expression levels that were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload, FHL2 might act as a signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.
Asunto(s)
Conectina , Mecanotransducción Celular , Ratones Noqueados , Animales , Ratones , Conectina/metabolismo , Conectina/genética , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/genética , Miocardio/metabolismo , Miocardio/patología , Masculino , Condicionamiento Físico Animal , Proteínas con Homeodominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Modelos Animales de Enfermedad , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Proteínas Quinasas , Péptidos y Proteínas de Señalización IntracelularRESUMEN
KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65 evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction< 50%, 8% with atrial fibrillation, 9% with ventricular tachycardia, and 20% with repolarization abnormalities. Five patients received an implantable cardioverter defibrillator, three cases of sudden cardiac death were reported. Linkage analysis confirmed cosegregation of the KBTBD13 p.R408C variant with the cardiac phenotype. Mouse studies revealed that (1) mice harboring the Kbtbd13 p.R408C variant display mild diastolic dysfunction; (2) Kbtbd13-deficient mice have systolic dysfunction. Hence, (1) KBTBD13 is associated with cardiac dysfunction and cardiomyopathy; (2) KBTBD13 should be added to the cardiomyopathy gene panel; (3) NEM6 patients should be referred to the cardiologist.
Asunto(s)
Cardiomiopatías , Proteínas Musculares , Animales , Humanos , Ratones , Arritmias Cardíacas , Cardiomiopatías/genética , Muerte Súbita Cardíaca/etiología , Desfibriladores Implantables , Proteínas Musculares/genética , Volumen Sistólico/fisiología , Función Ventricular IzquierdaRESUMEN
Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.
Asunto(s)
Cardiomiopatía Dilatada , Insuficiencia Cardíaca , Animales , Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/genética , Humanos , Ratones , Mortalidad Prematura , Mutación , ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de RiesgoRESUMEN
Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1-2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1-2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1-2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.
Asunto(s)
Miofibrillas , Sarcómeros , Animales , Conectina/genética , Ratones , Contracción Muscular , Miocitos Cardíacos , Proteínas Quinasas/genética , Sarcómeros/fisiologíaRESUMEN
AIMS: Heart failure with preserved ejection fraction (HFpEF) is associated with reduced exercise capacity elicited by skeletal muscle (SM) alterations. Up to now, no clear medical treatment advice for HFpEF is available. Identification of the ideal animal model mimicking the human condition is a critical step in developing and testing treatment strategies. Several HFpEF animals have been described, but the most suitable in terms of comparability with SM alterations in HFpEF patients is unclear. The aim of the present study was to investigate molecular changes in SM of three different animal models and to compare them with alterations of muscle biopsies obtained from human HFpEF patients. METHODS AND RESULTS: Skeletal muscle tissue was obtained from HFpEF and control patients and from three different animal models including the respective controls-ZSF1 rat, Dahl salt-sensitive rat, and transverse aortic constriction surgery/deoxycorticosterone mouse. The development of HFpEF was verified by echocardiography. Protein expression and enzyme activity of selected markers were assessed in SM tissue homogenates. Protein expression between SM tissue obtained from HFpEF patients and the ZSF1 rats revealed similarities for protein markers involved in muscle atrophy (MuRF1 expression, protein ubiquitinylation, and LC3) and mitochondrial metabolism (succinate dehydrogenase and malate dehydrogenase activity, porin expression). The other two animal models exhibited far less similarities to the human samples. CONCLUSIONS: None of the three tested animal models mimics the condition in HFpEF patients completely, but among the animal models tested, the ZSF1 rat (ZSF1-lean vs. ZSF1-obese) shows the highest overlap to the human condition. Therefore, when studying therapeutic interventions to treat HFpEF and especially alterations in the SM, we suggest that the ZSF1 rat is a suitable model.
Asunto(s)
Insuficiencia Cardíaca , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Músculo Esquelético , Ratas , Ratas Endogámicas Dahl , Volumen SistólicoRESUMEN
BACKGROUND: Low myocardial cGMP-PKG (cyclic guanosine monophosphate-protein kinase G) activity has been associated with increased cardiomyocyte diastolic stiffness in heart failure with preserved ejection fraction. Cyclic guanosine monophosphate is mainly hydrolyzed by PDE (phosphodiesterases) 5a and 9a. Importantly, PDE9a expression has been reported to be upregulated in human heart failure with preserved ejection fraction myocardium and chronic administration of a PDE9a inhibitor reverses preestablished cardiac hypertrophy and systolic dysfunction in mice subjected to transverse aortic constriction (TAC). We hypothesized that inhibiting PDE9a activity ameliorates diastolic dysfunction. METHODS: To examine the effect of chronic PDE9a inhibition, 2 diastolic dysfunction mouse models were studied: (1) TAC-deoxycorticosterone acetate and (2) Leprdb/db. PDE9a inhibitor (5 and 8 mg/kg per day) was administered to the mice via subcutaneously implanted osmotic minipumps for 28 days. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from TAC-deoxycorticosterone acetate mice. Atrial natriuretic peptide together with PDE9a inhibitor were administered to the isolated intact cardiomyocytes through the cell perfusate. RESULTS: For acute inhibition, no cellular stiffness reduction was found, whereas chronic PDE9a inhibition resulted in reduced left ventricular chamber stiffness in TAC-deoxycorticosterone acetate, but not in Leprdb/db mice. Passive cardiomyocyte stiffness was reduced by chronic PDE9a inhibition, with no differences in myocardial fibrosis or cardiac morphometry. PDE9a inhibition increased the ventricular-arterial coupling ratio, reflecting impaired systolic function. CONCLUSIONS: Chronic PDE9a inhibition lowers left ventricular chamber stiffness in TAC-deoxycorticosterone acetate mice. However, the usefulness of PDE9a inhibition to treat high-diastolic stiffness may be limited as the required PDE9a inhibitor dose also impairs systolic function, observed as a decline in ventricular-arterial coordination, in this model.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Disfunción Ventricular Izquierda/tratamiento farmacológico , Función Ventricular Izquierda/efectos de los fármacos , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Diástole , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/enzimología , Inhibidores de Fosfodiesterasa/toxicidad , Disfunción Ventricular Izquierda/enzimología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
BACKGROUND: Patients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction, and kidney failure in Col4a3-/- mice, a model of human Alport syndrome. OBJECTIVES: The purpose of this study was to identify potential OPN targets in biopsies of HF patients, healthy control subjects, and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), and to characterize the cardiac phenotype of Col4a3-/- mice, relate this to HFpEF, and investigate possible causative roles for OPN in driving the cardiomyopathy. METHODS: OGDHL mRNA and protein were quantified in myocardial samples from patients with HFpEF, heart failure with reduced ejection fraction, and donor control subjects. OGDHL expression was quantified in hiPS-CMs treated with or without anti-OPN antibody. Cardiac parameters were evaluated in Col4a3-/- mice with and without global OPN knockout or AAV9-mediated delivery of 2-oxoglutarate dehydrogenase-like (Ogdhl) to the heart. RESULTS: OGDHL mRNA and protein displayed abnormal abundances in cardiac biopsies of HFpEF (n = 17) compared with donor control subjects (n = 12; p < 0.01) or heart failure with reduced ejection fraction patients (n = 12; p < 0.05). Blockade of OPN in hiPS-CMs conferred increased OGDHL expression. Col4a3-/- mice demonstrated cardiomyopathy with similarities to HFpEF, including diastolic dysfunction, cardiac hypertrophy and fibrosis, pulmonary edema, and impaired mitochondrial function. The cardiomyopathy was ameliorated by Opn-/- coincident with improved renal function and increased expression of Ogdhl. Heart-specific overexpression of Ogdhl in Col4a3-/- mice also improved cardiac function and cardiomyocyte energy state. CONCLUSIONS: Col4a3-/- mice present a model of HFpEF secondary to CKD wherein OPN and OGDHL are intermediates, and possibly therapeutic targets.
Asunto(s)
Modelos Animales de Enfermedad , Insuficiencia Cardíaca Diastólica/etiología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Osteopontina/metabolismo , Disfunción Ventricular Izquierda/etiología , Animales , Autoantígenos/genética , Colágeno Tipo IV/genética , Fibrosis , Terapia Genética , Insuficiencia Cardíaca Diastólica/metabolismo , Insuficiencia Cardíaca Diastólica/patología , Insuficiencia Cardíaca Diastólica/terapia , Complejo Cetoglutarato Deshidrogenasa/genética , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Miocardio/metabolismo , Miocardio/patología , Nefritis Hereditaria/complicaciones , Osteopontina/genética , Estrés Oxidativo , Disfunción Ventricular Izquierda/metabolismoRESUMEN
BACKGROUND: Titin is a giant elastic protein that spans the half-sarcomere from Z-disk to M-band. It acts as a molecular spring and mechanosensor and has been linked to striated muscle disease. The pathways that govern titin-dependent cardiac growth and contribute to disease are diverse and difficult to dissect. METHODS: To study titin deficiency versus dysfunction, the authors generated and compared striated muscle specific knockouts (KOs) with progressive postnatal loss of the complete titin protein by removing exon 2 (E2-KO) or an M-band truncation that eliminates proper sarcomeric integration, but retains all other functional domains (M-band exon 1/2 [M1/2]-KO). The authors evaluated cardiac function, cardiomyocyte mechanics, and the molecular basis of the phenotype. RESULTS: Skeletal muscle atrophy with reduced strength, severe sarcomere disassembly, and lethality from 2 weeks of age were shared between the models. Cardiac phenotypes differed considerably: loss of titin leads to dilated cardiomyopathy with combined systolic and diastolic dysfunction-the absence of M-band titin to cardiac atrophy and preserved function. The elastic properties of M1/2-KO cardiomyocytes are maintained, while passive stiffness is reduced in the E2-KO. In both KOs, we find an increased stress response and increased expression of proteins linked to titin-based mechanotransduction (CryAB, ANKRD1, muscle LIM protein, FHLs, p42, Camk2d, p62, and Nbr1). Among them, FHL2 and the M-band signaling proteins p62 and Nbr1 are exclusively upregulated in the E2-KO, suggesting a role in the differential pathology of titin truncation versus deficiency of the full-length protein. The differential stress response is consistent with truncated titin contributing to the mechanical properties in M1/2-KOs, while low titin levels in E2-KOs lead to reduced titin-based stiffness and increased strain on the remaining titin molecules. CONCLUSIONS: Progressive depletion of titin leads to sarcomere disassembly and atrophy in striated muscle. In the complete knockout, remaining titin molecules experience increased strain, resulting in mechanically induced trophic signaling and eventually dilated cardiomyopathy. The truncated titin in M1/2-KO helps maintain the passive properties and thus reduces mechanically induced signaling. Together, these findings contribute to the molecular understanding of why titin mutations differentially affect cardiac growth and have implications for genotype-phenotype relations that support a personalized medicine approach to the diverse titinopathies.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Mecanotransducción Celular , Miocitos Cardíacos/metabolismo , Proteínas Quinasas/deficiencia , Sarcómeros/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Derecha/metabolismo , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Eliminación de Gen , Masculino , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Miocitos Cardíacos/patología , Fenotipo , Proteínas Quinasas/genética , Sarcómeros/patología , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Disfunción Ventricular Derecha/genética , Disfunción Ventricular Derecha/patología , Disfunción Ventricular Derecha/fisiopatología , Función Ventricular Izquierda , Función Ventricular DerechaRESUMEN
Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca2+-induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin-actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca2+-induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca2+-troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length-dependent enhancement of troponin regulation with both Ca2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length-dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length-dependent Ca2+-troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation of the myocardium.
Asunto(s)
Calcio/metabolismo , Miocardio/metabolismo , Proteínas Quinasas/metabolismo , Sarcómeros/metabolismo , Troponina C/metabolismo , Actinas/metabolismo , Animales , Ratones , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Miosinas/metabolismo , Dominios Proteicos/fisiologíaRESUMEN
Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome characterized by a preserved ejection fraction but increased diastolic stiffness and abnormalities of filling. Although the prevalence of HFpEF is high and continues to rise, no effective therapies exist; however, the diabetic drug metformin has been associated with improved diastolic function in diabetic patients. Here we determine the therapeutic potential of metformin for improving diastolic function in a mouse model with HFpEF-like symptoms. We combine transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) supplementation to obtain a mouse model with increased diastolic stiffness and exercise intolerance. Echocardiography and pressure-volume analysis reveal that providing metformin to TAC/DOCA mice improves diastolic function in the left ventricular (LV) chamber. Muscle mechanics show that metformin lowers passive stiffness of the LV wall muscle. Concomitant with this improvement in diastolic function, metformin-treated TAC/DOCA mice also demonstrate preserved exercise capacity. No metformin effects are seen in sham operated mice. Extraction experiments on skinned ventricular muscle strips show that the metformin-induced reduction of passive stiffness in TAC/DOCA mice is due to an increase in titin compliance. Using phospho-site-specific antibodies, we assay the phosphorylation of titin's PEVK and N2B spring elements. Metformin-treated mice have unaltered PEVK phosphorylation but increased phosphorylation of PKA sites in the N2B element, a change which has previously been shown to lower titin's stiffness. Consistent with this result, experiments with a mouse model deficient in the N2B element reveal that the beneficial effect of metformin on LV chamber and muscle stiffness requires the presence of the N2B element. We conclude that metformin offers therapeutic benefit during HFpEF by lowering titin-based passive stiffness.
Asunto(s)
Diástole/efectos de los fármacos , Insuficiencia Cardíaca/tratamiento farmacológico , Metformina/farmacología , Proteínas Quinasas/metabolismo , Animales , Acetato de Desoxicorticosterona/farmacología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Fosforilación/efectos de los fármacos , Volumen Sistólico/efectos de los fármacosRESUMEN
The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin's C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.
Asunto(s)
Conectina/fisiología , Sarcómeros/ultraestructura , Animales , Cardiomiopatía Dilatada/fisiopatología , Conectina/genética , Masculino , Ratones , Contracción Muscular , Eliminación de SecuenciaAsunto(s)
Conectina , Volumen Sistólico , Animales , Diástole , Insuficiencia Cardíaca , Ratones , Motivos de Unión al ARN , Proteínas de Unión al ARNRESUMEN
BACKGROUND: Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction. METHODS: Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene), with dimethyl sulfoxide (DMSO)-injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels. RESULTS: cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. CONCLUSIONS: Inhibition of the RNA binding motif-20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.
Asunto(s)
Diástole/genética , Tolerancia al Ejercicio/genética , Insuficiencia Cardíaca/genética , Proteínas de Unión al ARN/genética , Función Ventricular Izquierda/genética , Animales , Adaptabilidad , Conectina/fisiología , Diástole/fisiología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Hipertrofia Ventricular Izquierda/metabolismo , Ratones , Ratones Transgénicos , Motivos de Unión al ARN/genética , Volumen Sistólico/fisiología , Función Ventricular Izquierda/fisiologíaRESUMEN
Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20 (ΔRRM) ) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20 (ΔRRM) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20 (ΔRRM) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca(2+)-activated, skinned papillary muscle strips from Rbm20 (ΔRRM) and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca(2+)-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20 (ΔRRM) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 µm, length-dependent activation of contraction was eliminated in the Rbm20 (ΔRRM) myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.
RESUMEN
RATIONALE: Patients with heart failure with preserved ejection fraction (HFpEF) experience elevated filling pressures and reduced ventricular compliance. The splicing factor RNA-binding motif 20 (RBM20) regulates the contour length of titin's spring region and thereby determines the passive stiffness of cardiomyocytes. Inhibition of RBM20 leads to super compliant titin isoforms (N2BAsc) that reduce passive stiffness. OBJECTIVE: To determine the therapeutic potential of upregulating compliant titin isoforms in an HFpEF-like state in the mouse. METHODS AND RESULTS: Constitutive and inducible cardiomyocyte-specific RBM20-inhibited mice were produced on a Ttn(ΔIAjxn) background to assess the effect of upregulating compliant titin at the cellular and organ levels. Genetic deletion of the I-band-A-band junction (IAjxn) in titin increases strain on the spring region and causes a HFpEF-like syndrome in the mouse without pharmacological or surgical intervention. The increased strain represents a mechanical analog of deranged post-translational modification of titin that results in increased passive myocardial stiffness in patients with HFpEF. On inhibition of RBM20 in Ttn(ΔIAjxn) mice, compliant titin isoforms were expressed, diastolic function was normalized, exercise performance was improved, and pathological hypertrophy was attenuated. CONCLUSIONS: We report for the first time a benefit from upregulating compliant titin isoforms in a murine model with HFpEF-like symptoms. Constitutive and inducible RBM20 inhibition improves diastolic function resulting in greater tolerance to exercise. No effective therapies exists for treating this pervasive syndrome; therefore, our data on RBM20 inhibition are clinically significant.
Asunto(s)
Empalme Alternativo/fisiología , Presión Sanguínea/fisiología , Conectina/biosíntesis , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Volumen Sistólico/fisiología , Animales , Conectina/genética , Insuficiencia Cardíaca/genética , Masculino , Ratones , Ratones Transgénicos , Miocitos Cardíacos/fisiología , Condicionamiento Físico Animal/fisiología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genéticaRESUMEN
Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05µm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1µm per 0.35µm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0µm where TFL was ~1.05µm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.
Asunto(s)
Conectina/metabolismo , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Sarcómeros/fisiología , Animales , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Miofibrillas , Cadenas Pesadas de Miosina/deficiencia , Cadenas Pesadas de Miosina/genéticaRESUMEN
Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the thick filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (â¼ 40-fold higher) but low, relative to that of the myosin-based thick filament (â¼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for thick filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.
Asunto(s)
Conectina/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Sarcómeros/metabolismo , Secuencia de Aminoácidos , Animales , Fenómenos Biomecánicos , Presión Sanguínea/fisiología , Western Blotting , Células Cultivadas , Conectina/genética , Ecocardiografía , Perfilación de la Expresión Génica , Modelos Lineales , Mecanotransducción Celular , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Miocardio/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcómeros/ultraestructura , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography. METHODS AND RESULTS: Compliant titins were upregulated through deletion of the RNA Recognition Motif of the splicing factor RBM20 (Rbm20(ΔRRM)mice). A genome-wide exon expression analysis and a candidate approach revealed that the phenotype is likely to be dominated by greatly increased lengths of titin's spring elements. At both cardiomyocyte and left ventricular chamber levels, diastolic stiffness was reduced in heterozygous (+/-) Rbm20(ΔRRM)mice with a further reduction in homozygous (-/-) mice at only the intact myocyte level. Fibrosis was present in only -/- Rbm20(ΔRRM) hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20(ΔRRM) mice, at both the cardiomyocyte and left ventricular chamber levels. Exercise tests revealed an increase in exercise capacity in +/- mice. CONCLUSIONS: Titin is not only important in diastolic but also in systolic cardiac function. Upregulating compliant titins reduces diastolic chamber stiffness owing to the increased compliance of myocytes, but it depresses end-systolic elastance; under conditions of exercise, the beneficial effects on diastolic function dominate. Therapeutic manipulation of the RBM20-based splicing system might be able to minimize effects on fibrosis and systolic function while improving the diastolic function in patients with heart failure.