RESUMO
BACKGROUND: Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. METHODS: Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. RESULTS: We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. CONCLUSIONS: Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.
Assuntos
Miócitos Cardíacos , Sarcômeros , Ratos , Camundongos , Animais , Sarcômeros/metabolismo , Conectina/genética , Conectina/metabolismo , Miócitos Cardíacos/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismoRESUMO
Mechanical load is one of the main determinants of cardiac structure and function. Mechanical load is studied in vitro using cardiac preparations together with loading protocols (e.g., auxotonic, isometric). However, such studies are often limited by reductionist models and poorly simulated mechanical load profiles. This hinders the physiological relevance of findings. Living myocardial slices have been used to study load in vitro. Living myocardial slices (LMS) are 300-µm-thick intact organotypic preparations obtained from explanted animal or human hearts. They have preserved cellular populations and the functional, structural, metabolic and molecular profile of the tissue from which they are prepared. Using a three-element Windkessel (3EWK) model we previously showed that LMSs can be cultured while performing cardiac work loops with different preload and afterload. Under such conditions, LMSs remodel as a function of the mechanical load applied to them (physiological load, pressure or volume overload). These studies were conducted in commercially available length actuators that had to be extensively modified for culture experiments. In this paper, we demonstrate the design, development and validation of a novel device, MyoLoop. MyoLoop is a bioreactor that can pace, thermoregulate, acquire and process data, and chronically load LMSs and other cardiac tissues in vitro. In MyoLoop, load is parametrised using a 3EWK model, which can be used to recreate physiological and pathological work loops and the remodelling response to these. We believe MyoLoop is the next frontier in basic cardiovascular research enabling reductionist but physiologically relevant in vitro mechanical studies.
Assuntos
Reatores Biológicos , Coração , Animais , Humanos , Miocárdio , Projetos de PesquisaRESUMO
Chronic obstructive pulmonary disease (COPD) is a clinical entity of increasing significance. COPD involves abnormalities of the airways and, in emphysema, parenchymal pulmonary destruction. Cardiovascular disease has emerged as a significant comorbidity to COPD. Heart failure with preserved ejection fraction (HFpEF) appears to be particularly associated with COPD-emphysema. Traditional treatments have shown limited efficacy in improving COPD-associated HFpEF. This lack of therapeutic efficacy highlights the need to identify potential mechanisms that link COPD-emphysema to HFpEF. Therefore, we aimed to study the delayed cardiac physiological impacts in a rat model with acute exacerbated emphysema. Emphysema was induced by four weekly 4 units elastase (ELA) intratracheal pulmonary instillations and exacerbation by one final additional lipolysaccharide (LPS) instillation in male Wistar rats. At 5 weeks after the ELA and LPS exposure, in vivo and ex vivo pulmonary and cardiac measurements were performed. Experimental exacerbated emphysema resulted in decreased pulmonary function and exercise intolerance. Histological analysis revealed parenchymal pulmonary destruction without signs of inflammation or cardiac fibrosis. In vivo cardiac functional analysis revealed diastolic dysfunction and tachycardia. Ex vivo analysis revealed a cellular cardiomyopathy with decreased myofilament Ca2+ sensitivity, cross-bridge cycling kinetics, and increased adrenergic PKA (protein kinase A)-dependent phosphorylation of troponin-I. Experimental exacerbated emphysema was associated with exercise intolerance that appeared to be secondary to increased ß-adrenergic tone and subsequent cardiac myofilament dysfunction. A ß1-receptor antagonist treatment (bisoprolol) started 24 hours after ELA-LPS instillation prevented in vivo and ex vivo diastolic dysfunction. These results suggest that novel treatment strategies targeted to the cardiac myofilament may be beneficial to combat exacerbated emphysema-associated HFpEF.
Assuntos
Cardiomiopatias , Enfisema , Insuficiência Cardíaca , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Masculino , Ratos , Animais , Insuficiência Cardíaca/complicações , Lipopolissacarídeos , Volume Sistólico/fisiologia , Ratos Wistar , Enfisema Pulmonar/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Cardiomiopatias/complicaçõesRESUMO
The contractility of cardiac muscle is greatly affected by preload via the Frank-Starling mechanism (FSM). It is based on preload-dependent activation of sarcomeres-the elementary contractile units in muscle cells. Recent findings show a natural variability in sarcomere length (SL) in resting cardiomyocytes that, moreover, is altered in an actively contracting myocyte. SL variability may contribute to the FSM, but it remains unresolved whether the change in the SL variability is regulated by activation process per se or simply by changes in cell stretch, i.e., average SL. To separate the roles of activation and SL, we characterized SL variability in isolated, fully relaxed rat ventricular cardiomyocytes (n = 12) subjected to a longitudinal stretch with the carbon fiber (CF) technique. Each cell was tested in three states: without CF attachment (control, no preload), with CF attachment without stretch, and with CF attachment and ~ 10% stretch of initial SL. The cells were imaged by transmitted light microscopy to retrieve and analyze individual SL and SL variability off-line by multiple quantitative measures such as coefficient of variation or median absolute deviation. We found that CF attachment without stretch did not affect the extent of SL variability nor average SL. In stretched myocytes, the averaged SL significantly increased, while the SL variability remained unchanged. This result clearly indicates that the non-uniformity of individual SL is not sensitive to the average SL itself in fully relaxed myocytes. We conclude that SL variability per se does not contribute to the FSM in the heart.
Assuntos
Miócitos Cardíacos , Sarcômeros , Animais , Ratos , Miocárdio , Descanso , Contração Muscular , Fibra de CarbonoRESUMO
Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.
Assuntos
Actinas , Tomografia com Microscopia Eletrônica , Ratos , Animais , Actinas/metabolismo , Miocárdio/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Mamíferos/metabolismoRESUMO
The cardiac isoform of troponin I has a unique N-terminal extension (~ 1-30 amino acids), which contributes to the modulation of cardiac contraction and relaxation. Hearts of various species including humans produce a truncated variant of cardiac troponin I (cTnI-ND) deleting the first ~ 30 amino acids as an adaption in pathophysiological conditions. In this study, we investigated the impact of cTnI-ND chronic expression in transgenic mouse hearts compared to wildtype (WT) controls (biological n = 8 in each group). We aimed to determine the global phosphorylation effects of cTnI-ND on the cardiac proteome, thereby determining the signaling pathways that have an impact on cardiac function. The samples were digested and isobarically labeled and equally mixed for relative quantification via nanoLC-MS/MS. The peptides were then enriched for phospho-peptides and bioinformatic analysis was done with Ingenuity Pathway Analysis (IPA). We found approximately 77% replacement of the endogenous intact cTnI with cTnI-ND in the transgenic mouse hearts with 1674 phospho-proteins and 2971 non-modified proteins. There were 73 significantly altered phospho-proteins; bioinformatic analysis identified the top canonical pathways as associated with integrin, protein kinase A, RhoA, and actin cytoskeleton signaling. Among the 73 phospho-proteins compared to controls cTnI-ND hearts demonstrated a significant decrease in paxillin and YAP1, which are known to play a role in cell mechano-sensing pathways. Our data indicate that cTnI-ND modifications in the sarcomere are sufficient to initiate changes in the phospho-signaling profile that may underly the chronic-adaptive response associated with cTnI cleavage in response to stressors by modifying mechano-sensitive signaling pathways.
Assuntos
Espectrometria de Massas em Tandem , Troponina I , Aminoácidos , Animais , Cálcio/metabolismo , Camundongos , Camundongos Transgênicos , Contração Miocárdica , Miocárdio/metabolismo , Peptídeos , Fosforilação , Transdução de Sinais , Troponina I/química , Troponina I/genética , Troponina I/metabolismoRESUMO
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.
Assuntos
Proteínas de Transporte/genética , Contração Miocárdica/genética , Miocárdio/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Coração/diagnóstico por imagem , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Fosforilação , Sarcômeros/metabolismoRESUMO
Determining transmural mechanical properties in the heart provides a foundation to understand physiological and pathophysiological cardiac mechanics. Although work on mechanical characterisation has begun in isolated cells and permeabilised samples, the mechanical profile of living individual cardiac layers has not been examined. Myocardial slices are 300 µm-thin sections of heart tissue with preserved cellular stoichiometry, extracellular matrix, and structural architecture. This allows for cardiac mechanics assays in the context of an intact in vitro organotypic preparation. In slices obtained from the subendocardium, midmyocardium and subepicardium of rats, a distinct pattern in transmural contractility is found that is different from that observed in other models. Slices from the epicardium and midmyocardium had a higher active tension and passive tension than the endocardium upon stretch. Differences in total myocyte area coverage, and aspect ratio between layers underlined the functional readouts, while no differences were found in total sarcomeric protein and phosphoprotein between layers. Such intrinsic heterogeneity may orchestrate the normal pumping of the heart in the presence of transmural strain and sarcomere length gradients in the in vivo heart.
Assuntos
Miocárdio/metabolismo , Animais , Fenômenos Biomecânicos , Proteínas de Transporte/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Ratos Sprague-Dawley , Sarcômeros/metabolismo , Troponina/metabolismoRESUMO
Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (Tmax) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H2O2) or a reducing agent (DTT) indicated that the effects on Tmax were mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca2+ sensitivity. DOX increased Ca2+ sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca2+ sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser23/Ser24. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca2+ sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function. NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca2+ sensitivity in the presence of estrogen but decreased Ca2+ sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca2+ sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.
Assuntos
Antioxidantes/farmacologia , Doxorrubicina/toxicidade , Estrogênios/farmacologia , Músculos Papilares/metabolismo , Testosterona/farmacologia , Animais , Cálcio/metabolismo , Cardiotoxicidade , Estrogênios/metabolismo , Feminino , Masculino , Contração Miocárdica , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miofibrilas/fisiologia , Estresse Oxidativo , Músculos Papilares/efeitos dos fármacos , Músculos Papilares/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Testosterona/metabolismo , Troponina I/metabolismoRESUMO
The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 µm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.
Assuntos
Conectina/fisiologia , Coração/fisiologia , Animais , Sinalização do Cálcio , Conectina/química , Conectina/genética , Modelos Cardiovasculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miofibrilas/fisiologia , Miosinas/metabolismo , Ratos , Ratos Mutantes , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Estresse Mecânico , Troponina C/genética , Troponina C/metabolismo , Difração de Raios XRESUMO
AIM: Duchenne Muscular Dystrophy (DMD) is associated with progressive depressed left ventricular (LV) function. However, DMD effects on myofilament structure and function are poorly understood. Golden Retriever Muscular Dystrophy (GRMD) is a dog model of DMD recapitulating the human form of DMD. OBJECTIVE: The objective of this study is to evaluate myofilament structure and function alterations in GRMD model with spontaneous cardiac failure. METHODS AND RESULTS: We have employed synchrotron X-rays diffraction to evaluate myofilament lattice spacing at various sarcomere lengths (SL) on permeabilized LV myocardium. We found a negative correlation between SL and lattice spacing in both sub-epicardium (EPI) and sub-endocardium (ENDO) LV layers in control dog hearts. In the ENDO of GRMD hearts this correlation is steeper due to higher lattice spacing at short SL (1.9µm). Furthermore, cross-bridge cycling indexed by the kinetics of tension redevelopment (ktr) was faster in ENDO GRMD myofilaments at short SL. We measured post-translational modifications of key regulatory contractile proteins. S-glutathionylation of cardiac Myosin Binding Protein-C (cMyBP-C) was unchanged and PKA dependent phosphorylation of the cMyBP-C was significantly reduced in GRMD ENDO tissue and more modestly in EPI tissue. CONCLUSIONS: We found a gradient of contractility in control dogs' myocardium that spreads across the LV wall, negatively correlated with myofilament lattice spacing. Chronic stress induced by dystrophin deficiency leads to heart failure that is tightly associated with regional structural changes indexed by increased myofilament lattice spacing, reduced phosphorylation of regulatory proteins and altered myofilament contractile properties in GRMD dogs.
Assuntos
Cardiomiopatias/patologia , Distrofia Muscular de Duchenne/patologia , Miofibrilas/patologia , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Cães , Eletrocardiografia , Espaço Intracelular/metabolismo , Distrofia Muscular de Duchenne/diagnóstico por imagem , Miocárdio/patologia , Miofibrilas/metabolismo , Fosforilação , Sarcômeros/metabolismo , Transdução de Sinais , Troponina/metabolismoRESUMO
The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.
Assuntos
Cardiomiopatia Restritiva , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Sarcômeros , Troponina I , Substituição de Aminoácidos , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Proteína Quinase C/química , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Sarcômeros/química , Sarcômeros/genética , Sarcômeros/metabolismo , Relação Estrutura-Atividade , Troponina I/química , Troponina I/genética , Troponina I/metabolismoRESUMO
The long-lived, hypoxic-tolerant naked mole-rat well-maintains cardiac function over its three-decade-long lifespan and exhibits many cardiac features atypical of similar-sized laboratory rodents. For example, they exhibit low heart rates and resting cardiac contractility, yet have a large cardiac reserve. These traits are considered ecophysiological adaptations to their dank subterranean atmosphere of low oxygen and high carbon dioxide levels and may also contribute to negligible declines in cardiac function during aging. We asked if naked mole-rats had a different myofilament protein signature to that of similar-sized mice that commonly show both high heart rates and high basal cardiac contractility. Adult mouse ventricles predominantly expressed α-myosin heavy chain (97.9 ± 0.4%). In contrast, and more in keeping with humans, ß myosin heavy chain was the dominant isoform (79.0 ± 2.0%) in naked mole-rat ventricles. Naked mole-rat ventricles diverged from those of both humans and mice, as they expressed both cardiac and slow skeletal isoforms of troponin I. This myofilament protein profile is more commonly observed in mice in utero and during cardiomyopathies. There were no species differences in phosphorylation of cardiac myosin binding protein-C or troponin I. Phosphorylation of both ventricular myosin light chain 2 and cardiac troponin T in naked mole-rats was approximately half that observed in mice. Myofilament function was also compared between the two species using permeabilized cardiomyocytes. Together, these data suggest a cardiac myofilament protein signature that may contribute to the naked mole-rat's suite of adaptations to its natural subterranean habitat.
Assuntos
Adaptação Fisiológica/fisiologia , Ventrículos do Coração , Miofibrilas , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos-ToupeiraRESUMO
The benefits of α-mangostin for various tissues have been reported, but its effect on the heart has not been clarified. This study aimed to evaluate the effects of α-mangostin on cardiac function. Using a cardiac sarcoplasmic reticulum (SR) membrane preparation, α-mangostin inhibited SR Ca2+ -ATPase activity in a dose-dependent manner (IC50 of 6.47 ± 0.7 µM). Its suppressive effect was specific to SR Ca2+ -ATPase but not to myofibrillar Ca2+ -ATPase. Using isolated cardiomyocytes, 50 µM of α-mangostin significantly increased the duration of cell relengthening and increased the duration of Ca2+ transient decay, suggesting altered myocyte relaxation. The relaxation effect of α-mangostin was also supported in vivo after intravenous infusion. A significant suppression of both peak pressure and rate of ventricular relaxation (-dP/dt) relative to DMSO infusion was observed. The results from the present study demonstrated that α-mangostin exerts specific inhibitory action on SR Ca2+ -ATPase activity, leading to myocardial relaxation dysfunction.
Assuntos
Diástole/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Xantonas/toxicidade , Animais , Ventrículos do Coração/fisiopatologia , Masculino , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
KEY POINTS: The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes. In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase-1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes. Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea-pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart. The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. ABSTRACT: Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 µm blebbistatin resulted in an â¼3-fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO-1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea-pig model of angiotensin II infusion (400 ng kg(-1) min(-1) ) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n = 4, P < 0.01) during compensated hypertrophy but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01, n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signalling.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Heme Oxigenase-1/fisiologia , Proteínas com Domínio LIM/fisiologia , Proteínas Musculares/fisiologia , Miócitos Cardíacos/fisiologia , Angiotensina II/farmacologia , Animais , Feminino , Cobaias , Heme Oxigenase-1/metabolismo , Histona Desacetilases/fisiologia , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Miocárdio , Ratos Sprague-DawleyRESUMO
A naturally occurring R9C mutation of phospholamban (PLB) triggers cardiomyopathy and premature death by altering regulation of sarco/endoplasmic reticulum calcium-ATPase (SERCA). The goal of this study was to investigate the acute physiological consequences of the R9C-PLB mutation on cardiomyocyte calcium kinetics and contractility. We measured the physiological consequences of R9C-PLB mutation on calcium transients and sarcomere shortening in adult cardiomyocytes. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and ß-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we quantified the effect of R9C on PLB oligomerization and PLB-SERCA binding. FRET measurements in live cells revealed that R9C-PLB exhibited an increased propensity for oligomerization, and this was further increased by oxidative stress. The R9C also decreased PLB binding to SERCA and altered the structure of the PLB-SERCA regulatory complex. The structural change after oxidative modification of R9C-PLB was similar to that observed after PLB phosphorylation. We conclude that R9C mutation of PLB decreases SERCA inhibition by decreasing the amount of the regulatory complex and altering its conformation. This has an acute inotropic/lusitropic effect but yields negative consequences of impaired frequency potentiation and blunted ß-adrenergic responsiveness. We envision a self-reinforcing mechanism beginning with phosphomimetic R9C-PLB oxidation and loss of SERCA inhibition, leading to impaired calcium regulation and heart failure.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cardiomiopatias/genética , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/metabolismo , Células Cultivadas , Cães , Transferência Ressonante de Energia de Fluorescência , Humanos , Dados de Sequência Molecular , Estresse Oxidativo , Mutação Puntual , Multimerização Proteica , CoelhosRESUMO
ß-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 µm and SL = 2.3 µm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that ß-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.
Assuntos
Proteínas de Transporte/fisiologia , Miofibrilas/metabolismo , Troponina I/metabolismo , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Ventrículos do Coração/patologia , Contração Isométrica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células Musculares/citologia , Células Musculares/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Fosforilação , Receptores Adrenérgicos beta/metabolismo , Proteínas Recombinantes/metabolismo , Sarcômeros/metabolismo , Transdução de Sinais , Estresse MecânicoRESUMO
In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR; [Ca]SR) partially determines the amplitude of cytosolic Ca transient that, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best approach available to directly measure [Ca]SR was to use low-affinity Ca indicators (e.g., Fluo-5N). However, this approach presents several limitations, including nonspecific cellular localization, dye extrusion, and species limitation. Recently a new genetically encoded family of Ca indicators has been generated, named Ca-measuring organelle-entrapped protein indicators (CEPIA). Here, we tested the red fluorescence SR-targeted Ca sensor (R-CEPIA1er) as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat ventricular myocytes with an adenovirus expressing the R-CEPIA1er gene displayed prominent localization in the SR and nuclear envelope. Calibration of R-CEPIA1er in myocytes resulted in a Kd of 609 µM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR [Ca]SR dynamics measured with R-CEPIA1er were compared with [Ca]SR measured with Fluo5-N. We found that both the time course of the [Ca]SR depletion and fractional SR Ca release induced by an action potential were similar between these two Ca sensors. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, measurement of [Ca]SR with R-CEPIA1er can be combined with cytosolic [Ca] measurements (with Fluo-4) to obtain more detailed information regarding Ca handling in cardiac myocytes. In conclusion, R-CEPIA1er is a promising tool that can be used to measure [Ca]SR dynamics in myocytes from different animal species.
Assuntos
Técnicas Biossensoriais , Sinalização do Cálcio , Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Proteínas Luminescentes/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Compostos de Anilina/metabolismo , Animais , Células Cultivadas , Corantes Fluorescentes/metabolismo , Ventrículos do Coração/citologia , Cinética , Proteínas Luminescentes/genética , Microscopia Confocal , Coelhos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Xantenos/metabolismo , Proteína Vermelha FluorescenteRESUMO
While ß-adrenergic receptor (ß-AR) stimulation ensures adequate cardiac output during stress, it can also trigger life-threatening cardiac arrhythmias. We have previously shown that proarrhythmic Ca(2+) waves during ß-AR stimulation temporally coincide with augmentation of reactive oxygen species (ROS) production. In this study, we tested the hypothesis that increased energy demand during ß-AR stimulation plays an important role in mitochondrial ROS production and Ca(2+)-wave generation in rabbit ventricular myocytes. We found that ß-AR stimulation with isoproterenol (0.1 µM) decreased the mitochondrial redox potential and the ratio of reduced to oxidated glutathione. As a result, ß-AR stimulation increased mitochondrial ROS production. These metabolic changes induced by isoproterenol were associated with increased sarcoplasmic reticulum (SR) Ca(2+) leak and frequent diastolic Ca(2+) waves. Inhibition of cell contraction with the myosin ATPase inhibitor blebbistatin attenuated oxidative stress as well as spontaneous SR Ca(2+) release events during ß-AR stimulation. Furthermore, we found that oxidative stress induced by ß-AR stimulation caused the formation of disulfide bonds between two ryanodine receptor (RyR) subunits, referred to as intersubunit cross-linking. Preventing RyR cross-linking with N-ethylmaleimide decreased the propensity of Ca(2+) waves induced by ß-AR stimulation. These data suggest that increased energy demand during sustained ß-AR stimulation weakens mitochondrial antioxidant defense, causing ROS release into the cytosol. By inducing RyR intersubunit cross-linking, ROS can increase SR Ca(2+) leak to the critical level that can trigger proarrhythmic Ca(2+) waves.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Técnicas de Cultura de Células , Citosol/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Isoproterenol/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
Mutations in MYBPC3, the gene encoding cardiac myosin binding protein-C (cMyBP-C), account for ~40% of hypertrophic cardiomyopathy (HCM) cases. Most pathological MYBPC3 mutations encode truncated protein products not found in tissue. Reduced protein levels occur in symptomatic heterozygous human HCM carriers, suggesting haploinsufficiency as an underlying mechanism of disease. However, we do not know if reduced cMyBP-C content results from, or initiates the development of HCM. In previous studies, heterozygous (HET) mice with a MYBPC3 C'-terminal truncation mutation and normal cMyBP-C levels show altered contractile function prior to any overt hypertrophy. Therefore, this study aimed to test whether haploinsufficiency occurs, with decreased cMyBP-C content, following cardiac stress and whether the functional impairment in HET MYBPC3 hearts leads to worsened disease progression. To address these questions, transverse aortic constriction (TAC) was performed on three-month-old wild-type (WT) and HET MYBPC3-truncation mutant mice and then characterized at 4 and 12weeks post-surgery. HET-TAC mice showed increased hypertrophy and reduced ejection fraction compared to WT-TAC mice. At 4weeks post-surgery, HET myofilaments showed significantly reduced cMyBP-C content. Functionally, HET-TAC cardiomyocytes showed impaired force generation, higher Ca(2+) sensitivity, and blunted length-dependent increase in force generation. RNA sequencing revealed several differentially regulated genes between HET and WT groups, including regulators of remodeling and hypertrophic response. Collectively, these results demonstrate that haploinsufficiency occurs in HET MYBPC3 mutant carriers following stress, causing, in turn, reduced cMyBP-C content and exacerbating the development of dysfunction at myofilament and whole-heart levels.