RESUMO
Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.
Assuntos
Proteínas de Transporte , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Proteínas de Transporte/metabolismo , Ligação Proteica/genética , Sarcômeros/metabolismo , Miosinas/genética , Miosinas/metabolismo , Miocárdio/metabolismoRESUMO
BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.
Assuntos
Dinamina II , Miócitos Cardíacos , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dinamina II/genética , Dinamina II/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.
Assuntos
Arritmias Cardíacas , Cálcio , Doença do Sistema de Condução Cardíaco , Proteínas do Citoesqueleto , Animais , Humanos , Camundongos , Arritmias Cardíacas/genética , Cálcio/metabolismo , Doença do Sistema de Condução Cardíaco/genética , Contactinas/metabolismo , Proteínas do Citoesqueleto/genética , Átrios do Coração/metabolismo , Miosinas/metabolismo , Ramos Subendocárdicos , TaquicardiaRESUMO
BACKGROUND: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K+ current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. METHODS: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. RESULTS: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKACh by a mechanism involving frequency-dependent activation of PKCε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. CONCLUSIONS: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.
Assuntos
Fibrilação Atrial , Remodelamento Atrial , Regulação Enzimológica da Expressão Gênica , Terapia Genética , NADPH Oxidase 2 , RNA Interferente Pequeno , Animais , Fibrilação Atrial/enzimologia , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/terapia , Cães , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , NADPH Oxidase 2/biossíntese , NADPH Oxidase 2/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The goal of this work was to investigate the role of t-tubule (TT) remodeling in abnormal Ca2+ cycling in ventricular myocytes of failing dog hearts. Heart failure (HF) was induced using rapid right ventricular pacing. Extensive changes in echocardiographic parameters, including left and right ventricular dilation and systolic dysfunction, diastolic dysfunction, elevated left ventricular filling pressures, and abnormal cardiac mechanics, indicated that severe HF developed. TT loss was extensive when measured as the density of total cell volume, derived from three-dimensional confocal image analysis, and significantly increased the distances in the cell interior to closest cell membrane. Changes in Ca2+ transients indicated increases in heterogeneity of Ca2+ release along the cell length. When critical properties of Ca2+ release variability were plotted as a function of TT organization, there was a complex, nonlinear relationship between impaired calcium release and decreasing TT organization below a certain threshold of TT organization leading to increased sensitivity in Ca2+ release below a TT density threshold of 1.5%. The loss of TTs was also associated with a greater incidence of triggered Ca2+ waves during rapid pacing. Finally, virtually all of these observations were replicated by acute detubulation by formamide treatment, indicating an important role of TT remodeling in impaired Ca2+ cycling. We conclude that TT remodeling itself is a major contributor to abnormal Ca2+ cycling in HF, reducing myocardial performance. The loss of TTs is also responsible for a greater incidence of triggered Ca2+ waves that may play a role in ventricular arrhythmias arising in HF.NEW & NOTEWORTHY Three-dimensional analysis of t-tubule density showed t-tubule disruption throughout the whole myocyte in failing dog ventricle. A double-linear relationship between Ca2+ release and t-tubule density displays a steeper slope at t-tubule densities below a threshold value (â¼1.5%) above which there is little effect on Ca2+ release (T-tubule reserve). T-tubule loss increases incidence of triggered Ca2+ waves. Chemically induced t-tubule disruption suggests that t-tubule loss alone is a critical component of abnormal Ca2+ cycling in heart failure.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Estimulação Cardíaca Artificial , Modelos Animais de Doenças , Cães , Feminino , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Miócitos Cardíacos/patologia , Função Ventricular Esquerda , Função Ventricular Direita , Pressão VentricularRESUMO
Excitation-contraction coupling in atrial cells is mediated by calcium (Ca) signaling between L-type Ca channels and Ryanodine receptors that occurs mainly at the cell boundary. This unique architecture dictates essential aspects of Ca signaling under both normal and diseased conditions. In this study we apply laser scanning confocal microscopy, along with an experimentally based computational model, to understand the Ca cycling dynamics of an atrial cell subjected to rapid pacing. Our main finding is that when an atrial cell is paced under Ca overload conditions, Ca waves can then nucleate on the cell boundary and propagate to the cell interior. These propagating Ca waves are referred to as "triggered waves" because they are initiated by L-type Ca channel openings during the action potential. These excitations are distinct from spontaneous Ca waves originating from random fluctuations of Ryanodine receptor channels, and which occur after much longer waiting times. Furthermore, we argue that the onset of these triggered waves is a highly nonlinear function of the sarcoplasmic reticulum Ca load. This strong nonlinearity leads to aperiodic response of Ca at rapid pacing rates that is caused by the complex interplay between paced Ca release and triggered waves. We argue further that this feature of atrial cells leads to dynamic instabilities that may underlie atrial arrhythmias. These studies will serve as a starting point to explore the nonlinear dynamics of atrial cells and will yield insights into the trigger and maintenance of atrial fibrillation.
Assuntos
Sinalização do Cálcio , Átrios do Coração/citologia , Miócitos Cardíacos/citologia , Animais , Fibrilação Atrial/patologia , Sinalização do Cálcio/efeitos dos fármacos , Cães , Isoproterenol/farmacologia , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Dinâmica não LinearRESUMO
Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiomyocytes remains poorly understood. This is exemplified by the elusive role of the transcription factor, Foxc1, during cardiac development. The only known Foxc1 target during heart development is Tbx1. Because Foxc1 null mice contain heart mutations that are far more severe than Tbx1 null mice, it is likely that Foxc1 has additional regulatory roles during heart development. The goal of our study was to test whether Foxc1 is critical for ESC differentiation into functional cardiomyocytes through proper regulation of specific downstream gene networks. Converging evidence from Foxc1 deficient and overexpression ESC models reveals a close relationship between Foxc1 levels and early cardiomyogenic factors Isl1, Mef2c, and Nkx2.5 and also the production of functional cardiomyocytes. We show Foxc1 regulates early cardiomyogenesis during a specific window of differentiation, D4-D6. Through whole transcriptome RNA-sequencing analysis, we report pathways regulated by Foxc1 involved in cardiac function including actin cytoskeleton, cell adhesion, tight and gap junctions, and calcium signaling. Our data indicate a novel Foxc1 direct gene target, Myh7, which encodes the predominant myosin heavy chain isoform, MHCß, expressed during cardiac development. These data lead us to conclude that Foxc1 regulates both early cardiomyogenesis and the functional properties of ESC-derived cardiomyocytes. Our findings shed light on the molecular circuitry governing cardiomyogenesis that may lead to the development of better translational strategies for the use of pluripotent stem cells in regenerative medicine towards repairing damaged myocardium. Stem Cells 2016;34:1487-1500.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organogênese , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Doxiciclina/farmacologia , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Fatores de Transcrição Forkhead/deficiência , Proteína Homeobox Nkx-2.5/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Organogênese/efeitos dos fármacos , Organogênese/genética , Análise de Sequência de RNA , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
RATIONALE: Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established. OBJECTIVE: The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. METHODS AND RESULTS: Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. CONCLUSIONS: ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Heme/biossíntese , Mitocôndrias Cardíacas/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Células Cultivadas , Heme/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
Although the development of abnormal myocardial mechanics represents a key step during the transition from hypertension to overt heart failure (HF), the underlying ultrastructural and cellular basis of abnormal myocardial mechanics remains unclear. We therefore investigated how changes in transverse (T)-tubule organization and the resulting altered intracellular Ca(2+) cycling in large cell populations underlie the development of abnormal myocardial mechanics in a model of chronic hypertension. Hearts from spontaneously hypertensive rats (SHRs; n = 72) were studied at different ages and stages of hypertensive heart disease and early HF and were compared with age-matched control (Wistar-Kyoto) rats (n = 34). Echocardiography, including tissue Doppler and speckle-tracking analysis, was performed just before euthanization, after which T-tubule organization and Ca(2+) transients were studied using confocal microscopy. In SHRs, abnormalities in myocardial mechanics occurred early in response to hypertension, before the development of overt systolic dysfunction and HF. Reduced longitudinal, circumferential, and radial strain as well as reduced tissue Doppler early diastolic tissue velocities occurred in concert with T-tubule disorganization and impaired Ca(2+) cycling, all of which preceded the development of cardiac fibrosis. The time to peak of intracellular Ca(2+) transients was slowed due to T-tubule disruption, providing a link between declining cell ultrastructure and abnormal myocardial mechanics. In conclusion, subclinical abnormalities in myocardial mechanics occur early in response to hypertension and coincide with the development of T-tubule disorganization and impaired intracellular Ca(2+) cycling. These changes occur before the development of significant cardiac fibrosis and precede the development of overt cardiac dysfunction and HF.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Hipertensão/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/ultraestrutura , Sarcolema/ultraestrutura , Animais , Pressão Sanguínea , Cálcio/metabolismo , Sinalização do Cálcio , Fibrose/fisiopatologia , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/patologia , Frequência Cardíaca , Hipertensão/diagnóstico por imagem , Hipertensão/patologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , UltrassonografiaRESUMO
Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.
Assuntos
Proliferação de Células , Mitocôndrias Cardíacas , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Camundongos Knockout , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Glucose/metabolismoRESUMO
The treatment of heart failure (HF) is challenging and morbidity and mortality are high. The goal of this study was to determine if inhibition of the late Na(+) current with ranolazine during early hypertensive heart disease might slow or stop disease progression. Spontaneously hypertensive rats (aged 7 mo) were subjected to echocardiographic study and then fed either control chow (CON) or chow containing 0.5% ranolazine (RAN) for 3 mo. Animals were then restudied, and each heart was removed for measurements of t-tubule organization and Ca(2+) transients using confocal microscopy of the intact heart. RAN halted left ventricular hypertrophy as determined from both echocardiographic and cell dimension (length but not width) measurements. RAN reduced the number of myocytes with t-tubule disruption and the proportion of myocytes with defects in intracellular Ca(2+) cycling. RAN also prevented the slowing of the rate of restitution of Ca(2+) release and the increased vulnerability to rate-induced Ca(2+) alternans. Differences between CON- and RAN-treated animals were not a result of different expression levels of voltage-dependent Ca(2+) channel 1.2, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, ryanodine receptor type 2, Na(+)/Ca(2+) exchanger-1, or voltage-gated Na(+) channel 1.5. Furthermore, myocytes with defective Ca(2+) transients in CON rats showed improved Ca(2+) cycling immediately upon acute exposure to RAN. Increased late Na(+) current likely plays a role in the progression of cardiac hypertrophy, a key pathological step in the development of HF. Early, chronic inhibition of this current slows both hypertrophy and development of ultrastructural and physiological defects associated with the progression to HF.
Assuntos
Acetanilidas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Piperazinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Sódio/metabolismo , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Relação Dose-Resposta a Droga , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Hipertensão/complicações , Hipertensão/diagnóstico por imagem , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Masculino , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ranolazina , Ratos , Ratos Endogâmicos SHR , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Canais de Sódio/metabolismo , Trocador de Sódio e Cálcio/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Fatores de Tempo , UltrassonografiaRESUMO
RATIONALE: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. OBJECTIVE: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. METHODS AND RESULTS: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII(+/)â») mice to I/R by coronary ligation. At baseline, HKII(+/)â» mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor-dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O2 consumption, and contractility. CONCLUSIONS: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.
Assuntos
Hexoquinase/metabolismo , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Morte Celular , Metabolismo Energético/genética , Fibrose , Hexoquinase/genética , Camundongos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Proteínas Musculares/genética , Contração Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Consumo de Oxigênio/genética , Fatores de TempoRESUMO
BACKGROUND: Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. METHODS: Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. RESULTS: Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. CONCLUSIONS: We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.
Assuntos
Arritmias Cardíacas , Cafeína , Feminino , Masculino , Animais , Camundongos , Cafeína/farmacologia , Modelos Animais de Doenças , Arritmias Cardíacas/genética , Predisposição Genética para Doença , Epinefrina , Calmodulina/genéticaRESUMO
Traditional anatomically guided ablation and attempts to perform electrogram-guided atrial fibrillation (AF) ablation (CFAE, DF, and FIRM) have not been shown to be sufficient treatment for persistent AF. Using biatrial high-density electrophysiologic mapping in a canine rapid atrial pacing model of AF, we systematically investigated the relationship of electrogram morphology recurrence (EMR) (Rec% and CLR) with established AF electrogram parameters and tissue characteristics. Rec% correlates with stability of rotational activity and with the spatial distribution of parasympathetic nerve fibers. These results have indicated that EMR may therefore be a viable therapeutic target in persistent AF.
RESUMO
The authors have requested that this preprint be removed from Research Square.
RESUMO
BACKGROUND: Abnormalities in intracellular calcium (Ca) cycling during Ca overload can cause triggered activity because spontaneous calcium release (SCR) activates sufficient Ca-sensitive inward currents to induce delayed afterdepolarizations (DADs). However, little is known about the mechanisms relating SCR and triggered activity on the tissue scale. METHODS AND RESULTS: Laser scanning confocal microscopy was used to measure the spatiotemporal properties of SCR within large myocyte populations in intact rat heart. Computer simulations were used to predict how these properties of SCR determine DAD magnitude. We measured the average and standard deviation of the latency distribution of SCR within a large population of myocytes in intact tissue. We found that as external [Ca] is increased, and with faster pacing rates, the average and SD of the latency distribution decreases substantially. This result demonstrates that the timing of SCR occurs with less variability as the sarcoplasmic reticulum (SR) Ca load is increased, causing more sites to release Ca within each cell. We then applied a mathematical model of subcellular Ca cycling to show that a decrease in SCR variability leads to a higher DAD amplitude and is dictated by the rate of SR Ca refilling following an action potential. CONCLUSIONS: Our results demonstrate that the variability of the timing of SCR in a population of cells in tissue decreases with SR load and is dictated by the time course of the SR Ca content.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Miocárdio/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Masculino , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Tempo de Reação/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Fatores de TempoRESUMO
In cardiac myocytes, calcium (Ca) can be released from the sarcoplasmic reticulum independently of Ca influx from voltage-dependent membrane channels. This efflux of Ca, referred to as spontaneous Ca release (SCR), is due to Ryanodine receptor fluctuations, which can induce spontaneous Ca sparks, which propagate to form Ca waves. This release of Ca can then induce delayed after-depolarizations (DADs), which can lead to arrhythmogenic-triggered activity in the heart. However, despite its importance, to date there is no mathematical model of SCR that accounts for experimentally observed features of subcellular Ca. In this article, we present an experimentally based model of SCR that reproduces the timing distribution of spontaneous Ca sparks and key features of the propagation of Ca waves emanating from these spontaneous sparks. We have coupled this model to an ionic model for the rabbit ventricular action potential to simulate SCR within several thousand cells in cardiac tissue. We implement this model to study the formation of an ectopic beat on a cable of cells that exhibit SCR-induced DADs.
Assuntos
Cálcio/metabolismo , Modelos Teóricos , Miócitos Cardíacos/metabolismo , Potenciais de Ação/fisiologia , Animais , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Modelos Animais , Miócitos Cardíacos/citologia , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fatores de TempoRESUMO
Optical mapping of intact cardiac tissue reveals that, in some cases, intracellular calcium (Ca) release can alternate from one beat to the next in a large-small-large sequence, also referred to as Ca transient (CaT) alternans. CaT alternans can also become spatially phase-mismatched within a single cell, when one part of the cell alternates in a large-small-large sequence, whereas a different part alternates in a small-large-small sequence, a phenomenon known as subcellular discordant alternans. The mechanisms for the formation and spatiotemporal evolution of these phase-mismatched patterns are not known. We used confocal Ca imaging to measure CaT alternans at the sarcomeric level within individual myocytes in the intact rat heart. After a sudden change in cycle length (CL), 2 distinct spatial patterns of CaT alternans emerge. CaTs can form spatially phase-mismatched alternans patterns after the first few beats following the change in CL. The phase mismatch persists for many beats, after which it gradually becomes phase matched via the movement of nodes, which are junctures between phase-mismatched cell regions. In other examples, phase-matched alternans gradually become phase-mismatched, via the formation and movement of nodes. In these examples, we observed large beat-to-beat variations in the cell activation times, despite constant CL pacing. Using computer simulations, we explored the underlying mechanisms for these dynamical phenomena. Our results show how heterogeneity at the sarcomeric level, in conjunction with the dynamics of Ca cycling and membrane voltage, can lead to complex spatiotemporal phenomena within myocytes of the intact heart.
Assuntos
Sinalização do Cálcio , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Potenciais de Ação , Animais , Estimulação Cardíaca Artificial , Simulação por Computador , Técnicas In Vitro , Microscopia Confocal , Modelos Cardiovasculares , Perfusão , Ratos , Ratos Sprague-Dawley , Processamento de Sinais Assistido por Computador , Fatores de TempoRESUMO
BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cß1 (PLCß1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.
RESUMO
Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.