RESUMEN
Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.
Asunto(s)
Plaquetas/fisiología , Sistemas Microelectromecánicos/métodos , Pruebas de Función Plaquetaria/métodos , Algoritmos , Técnicas Biosensibles , Plaquetas/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Sistemas Microelectromecánicos/instrumentación , Modelos Teóricos , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Pruebas de Función Plaquetaria/instrumentaciónRESUMEN
Dopamine receptors have been claimed not to directly increase contractility in the human heart. Therefore, we performed contraction experiments in isolated electrically driven human atrial preparations (HAP). For comparison, we performed contraction experiments with left atrial preparations of transgenic mice which harbor a cardiac overexpression of human D1-dopamine receptors (D1-TG). In D1-TG, first we noted that dopamine (10 nM-10 µM cumulatively applied) in the presence of propranolol exerted a concentration- and time-dependent positive inotropic effect in D1-TG. In a similar fashion, dopamine increased force of contraction in the presence of 0.4 µM propranolol in HAP and these effects were amplified by pre-treatment with inhibitor of phosphodiesterase III (1 µM) cilostamide. Moreover, contractile effects of dopamine in the presence of propranolol 0.4 µM in HAP were antagonized by odapipam, haloperidol, or raclopride. Ten micromolars of fenoldopam in the presence of cilostamide increased force of contraction in HAP and this effect was antagonized by SCH 23390. We conclude that stimulation of human D1-dopamine receptors can increase force of contraction in the HAP.
RESUMEN
Dopamine can exert effects in the mammalian heart via five different dopamine receptors. There is controversy whether dopamine receptors increase contractility in the human heart. Therefore, we have generated mice that overexpress the human D1-dopamine receptor in the heart (D1-TG) and hypothesized that dopamine increases force of contraction and beating rate compared to wild-type mice (WT). In D1-TG hearts, we ascertained the presence of D1-dopamine receptors by autoradiography using [3H]SKF 38393. The mRNA for human D1-dopamine receptors was present in D1-TG hearts and absent in WT. We detected by in-situ-hybridization mRNA for D1-dopamine receptors in atrial and ventricular D1-TG cardiomyocytes compared to WT but also in human atrial preparations. We noted that in the presence of 10 µM propranolol (to antagonize ß-adrenoceptors), dopamine alone and the D1- and D5-dopamine receptor agonist SKF 38393 (0.1-10 µM cumulatively applied) exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in left or right atrial preparations from D1-TG. The positive inotropic effects of SKF 38393 in left atrial preparations from D1-TG led to an increased rate of relaxation and accompanied by and probably caused by an augmented phosphorylation state of the inhibitory subunit of troponin. In the presence of 0.4 µM propranolol, 1 µM dopamine could increase left ventricular force of contraction in isolated perfused hearts from D1-TG. In this model, we have demonstrated a positive inotropic and chronotropic effect of dopamine. Thus, in principle, the human D1-dopamine receptor can couple to contractility in the mammalian heart.
Asunto(s)
Ratones Transgénicos , Contracción Miocárdica , Receptores de Dopamina D1 , Animales , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/genética , Humanos , Contracción Miocárdica/efectos de los fármacos , Masculino , Dopamina/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Agonistas de Dopamina/farmacología , Miocardio/metabolismo , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Atrios Cardíacos/metabolismo , Atrios Cardíacos/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/fisiología , Ratones Endogámicos C57BL , Frecuencia Cardíaca/efectos de los fármacosRESUMEN
Heart failure is a syndrome that can result from impaired heart muscle contractions like in dilative cardiomyopathy but also from hypertrophic obstructive cardiomyopathy (HOCOM). A pharmacological therapy might lie in Ca2+-sensitizing or Ca2+-desensitizing drugs, respectively. Such drugs are thought to be omecamtiv mecarbil (OME) and mavacamten (MYK-461), respectively. Their function in contracting human muscle is not fully understood and was the focus of the present study. OME from 1 nM to 10 µM cumulatively applied failed to raise force of contraction in human right atrial preparations strips (HAP) or mouse left atrial preparations (LA). However, OME prolonged time to peak tension and time of relaxation in HAP and LA but did not alter the beating rate in right atrial preparations from mice (RA). In contrast, MYK-461 (10 nM to 10 µM) reduced concentration- and time-dependently force of contraction in HAP and LA. MYK-461 (10 µM) did not affect the beating rate in RA. In summary, the present data failed to detect an increase in force of contraction for OME, in human and mouse atrium. In contrast, a Ca2+ desensitizer studied for comparison was able to reduce force of contraction in HAP and LA. We conclude that putative beneficial effects of OME in dilated cardiomyopathy cannot be explained by positive inotropic effects in the HAP, whereas beneficial functional effects of MYK-461 in HOCOM can be explained by negative inotropic effects in HAP.
Asunto(s)
Fibrilación Atrial , Humanos , Ratones , Animales , Atrios Cardíacos , Bencilaminas/farmacología , Contracción MiocárdicaRESUMEN
Human heart (patho)physiology is now widely studied using human pluripotent stem cells, but the immaturity of derivative cardiomyocytes has largely limited disease modeling to conditions associated with mutations in cardiac ion channel genes. Recent advances in tissue engineering and organoids have, however, created new opportunities to study diseases beyond "channelopathies." These synthetic cardiac structures allow quantitative measurement of contraction, force, and other biophysical parameters in three-dimensional configurations, in which the cardiomyocytes in addition become more mature. Multiple cardiac-relevant cell types are also often combined to form organized cardiac tissue mimetic constructs, where cell-cell, cell-extracellular matrix, and paracrine interactions can be mimicked. In this review, we provide an overview of some of the most promising technologies being implemented specifically in personalized heart-on-a-chip models and explore their applications, drawbacks, and potential for future development.
Asunto(s)
Corazón/fisiología , Miocardio/metabolismo , Ingeniería de Tejidos/métodos , Ingeniería de Tejidos/tendencias , Técnicas de Cultivo de Célula , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Dispositivos Laboratorio en un Chip , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
While interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-ß was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.
Asunto(s)
Dispositivos Laboratorio en un Chip , Preparaciones Farmacéuticas , Células Cultivadas , Fibroblastos/patología , Fibrosis , Humanos , Miocardio/patología , Miocitos Cardíacos/patologíaRESUMEN
Functional cardiac tissue engineering holds promise as a candidate therapy for myocardial infarction and heart failure. Generation of "strong-contracting and fast-conducting" cardiac tissue patches capable of electromechanical coupling with host myocardium could allow efficient improvement of heart function without increased arrhythmogenic risks. Towards that goal, we engineered highly functional 1â¯cmâ¯×â¯1â¯cm cardiac tissue patches made of neonatal rat ventricular cells which after 2 weeks of culture exhibited force of contraction of 18.0⯱â¯1.4â¯mN, conduction velocity (CV) of 32.3⯱â¯1.8â¯cm/s, and sustained chronic activation when paced at rates as high as 8.7⯱â¯0.8â¯Hz. Patches transduced with genetically-encoded calcium indicator (GCaMP6) were implanted onto adult rat ventricles and after 4-6 weeks assessed for action potential conduction and electrical integration by two-camera optical mapping of GCaMP6-reported Ca2+ transients in the patch and RH237-reported action potentials in the recipient heart. Of the 13 implanted patches, 11 (85%) engrafted, maintained structural integrity, and conducted action potentials with average CVs and Ca2+ transient durations comparable to those before implantation. Despite preserved graft electrical properties, no anterograde or retrograde conduction could be induced between the patch and host cardiomyocytes, indicating lack of electrical integration. Electrical properties of the underlying myocardium were not changed by the engrafted patch. From immunostaining analyses, implanted patches were highly vascularized and expressed abundant electromechanical junctions, but remained separated from the epicardium by a non-myocyte layer. In summary, our studies demonstrate generation of highly functional cardiac tissue patches that can robustly engraft on the epicardial surface, vascularize, and maintain electrical function, but do not couple with host tissue. The lack of graft-host electrical integration is therefore a critical obstacle to development of efficient tissue engineering therapies for heart repair.
Asunto(s)
Miocardio/citología , Ingeniería de Tejidos/métodos , Animales , Animales Recién Nacidos , Miocitos Cardíacos/citología , Pericardio/citología , Ratas , Ratas Desnudas , Andamios del Tejido/químicaRESUMEN
A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. We investigated the role of biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM). Following tissue consolidation, EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum and enhanced T-tubulation. We conclude that electro-mechanical stimulation at a physiological frequency supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research.