excitation-contraction coupling Dotaz Zobrazit nápovědu
The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.
- Klíčová slova
- Atomic force microscopy, Calcium imaging, Cardiac muscle mechanics, Cardiomyocyte, Electrophysiology, In vitro models,
- MeSH
- analýza dat MeSH
- elektrofyziologické jevy * MeSH
- kardiomyocyty cytologie fyziologie MeSH
- mechanické jevy * MeSH
- mikroskopie atomárních sil * přístrojové vybavení metody MeSH
- molekulární zobrazování MeSH
- spřažení excitace a kontrakce * MeSH
- vápníková signalizace MeSH
- Publikační typ
- časopisecké články MeSH
Vascular smooth muscle cells (VSMC) display considerable phenotype plasticity which can be studied in vivo on vascular remodeling which occurs during acute or chronic vascular injury. In differentiated cells, which represent contractile phenotype, there are characteristic rapid transient changes of intracellular Ca(2+) concentration ([Ca(2+)]i), while the resting cytosolic [Ca(2+)]i concentration is low. It is mainly caused by two components of the Ca(2+) signaling pathways: Ca(2+) entry via L-type voltage-dependent Ca(2+) channels and dynamic involvement of intracellular stores. Proliferative VSMC phenotype is characterized by long-lasting [Ca(2+)]i oscillations accompanied by sustained elevation of basal [Ca(2+)]i. During the switch from contractile to proliferative phenotype there is a general transition from voltage-dependent Ca(2+) entry to voltage-independent Ca(2+) entry into the cell. These changes are due to the altered gene expression which is dependent on specific transcription factors activated by various stimuli. It is an open question whether abnormal VSMC phenotype reported in rats with genetic hypertension (such as spontaneously hypertensive rats) might be partially caused by a shift from contractile to proliferative VSMC phenotype.
- MeSH
- cévy metabolismus patologie MeSH
- genetická transkripce fyziologie MeSH
- hypertenze metabolismus patologie MeSH
- lidé MeSH
- remodelace cév fyziologie MeSH
- signální transdukce fyziologie MeSH
- spřažení excitace a kontrakce fyziologie MeSH
- svaly hladké cévní metabolismus patologie MeSH
- vápníková signalizace fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Recent studies have shown that left ventricle (LV) exhibits considerable transmural differences in active mechanical properties induced by transmural differences in electrical activity, excitation-contraction coupling, and contractile properties of individual myocytes. It was shown that the time between electrical and mechanical activation of myocytes (electromechanical delay: EMD) decreases from subendocardium to subepicardium and, on the contrary, the myocyte shortening velocity (MSV) increases in the same direction. To investigate the physiological importance of this inhomogeneity, we developed a new finite element model of LV incorporating the observed transmural gradients in EMD and MSV. Comparative simulations with the model showed that when EMD or MSV or both were set constant across the LV wall, the LV contractility during isovolumic contraction (IVC) decreased significantly ((dp/dt)max was reduced by 2 to 38% and IVC was prolonged by 18 to 73%). This was accompanied by an increase of transmural differences in wall stress. These results suggest that the transmural differences in EMD and MSV play an important role in physiological contractility of LV by synchronising the contraction of individual layers of ventricular wall during the systole. Reduction or enhancement of these differences may therefore impair the function of LV and contribute to heart failure.
- MeSH
- kontrakce myokardu fyziologie MeSH
- lidé MeSH
- modely kardiovaskulární * MeSH
- počítačová simulace * MeSH
- srdce - funkce komor fyziologie MeSH
- srdeční komory * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- draslík farmakologie MeSH
- korýši MeSH
- membránové potenciály účinky léků MeSH
- myofibrily účinky léků MeSH
- svalová kontrakce účinky léků MeSH
- techniky in vitro MeSH
- vápník farmakologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- draslík MeSH
- vápník MeSH
Action potentials (APs) and the force of isometric contractions of the right ventricular papillary muscles were measured in adult and newborn guinea-pigs. The measurements were carried out in the steady state with the rate of stimulation of 0.1, 1, and 2 Hz, and further some measurements were done in which Sr2+ was substituted for Ca2. The duration of APs of the newborn animals without pharmacological treatment was significantly shorter in comparison with that of the adults at all the used stimulation frequencies. An analogous sensitivity was found in the contractile force to increased stimulation frequency and when the steady state stimulation was discontinued by the insertion of interpolated extrasystoles in papillary muscles of adult or newborn animals. The biphasic contractions of papillary muscles were evoked in both groups of animals by the incomplete substitution of Sr2+ by Ca2+ in the presence of isoprenaline. The early component of the biphasic contractions had a faster course as compared to the late component and disappeared in the presence of caffeine in both groups of animals. Our results suggest that the heart cells of newborn guinea-pigs probably possess the sarcoplasmic reticulum (SR), whose function does not differ in quality from that of the adult guinea-pigs. The postnatal prolongation of APs is therefore not probably the result of postnatal development changes of the functions of SR, but could be related to changes in the relations between the surface and volume of the heart cell during its growth.
- MeSH
- akční potenciály MeSH
- elektrofyziologie MeSH
- isoprenalin farmakologie MeSH
- kofein farmakologie MeSH
- kontrakce myokardu * účinky léků MeSH
- morčata MeSH
- novorozená zvířata MeSH
- papilární svaly účinky léků fyziologie MeSH
- stárnutí fyziologie MeSH
- stroncium farmakologie MeSH
- vápník farmakologie MeSH
- zvířata MeSH
- Check Tag
- morčata MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- isoprenalin MeSH
- kofein MeSH
- stroncium MeSH
- vápník MeSH
T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.
The aim of the present work was to check changes in functional characteristics of isolated muscle cells, operating on the calcium electrogenesis principle, while kept in culture media for several days. Skeletal muscle cells of the crayfish Astacus fluviatilis were used to study potassium/caffeine contractures and single/tetanic contractions; simultaneous electrical and mechanical responses were recorded by the microelectrode technique, and kinetics of calcium ionic currents was studied under vaseline-gap voltage clamp. In cultured fibers, active membrane responses and calcium current kinetics remained unchanged, or slightly increased, whereas contractile responses were substantially reduced. A gradual excitation-contraction decoupling was observed. The fiber maintained the ability to respond to direct activation (by caffeine) of the contractile apparatus. Subthreshold caffeine concentrations (0.2-0.5 mmol/l) and adrenaline (6.0(-6), 6.10(-5) mol/l) enhanced the inhibited (due to the culturing) single contractile responses.
- MeSH
- akční potenciály účinky léků MeSH
- draslík farmakologie MeSH
- kofein farmakologie MeSH
- kultivační techniky MeSH
- membránové potenciály účinky léků MeSH
- severní raci MeSH
- svalová kontrakce účinky léků fyziologie MeSH
- vápník fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Názvy látek
- draslík MeSH
- kofein MeSH
- vápník MeSH
The transverse-axial tubular system (tubular system) of cardiomyocytes plays a key role in excitation-contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed new approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separate capacitances of the tubular and surface membranes. Based on the analysis of the time course of the capacitive current, computational relations were derived to quantify the elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. Experiments on rat ventricular cardiomyocytes (n = 20) resulted in the surface and tubular capacitance values implying the fraction of tubular capacitance/area of 0.327 ± 0.018. We conclude that the newly proposed method provides results comparable to the data obtained by the currently used detubulation method and, in addition, by being reversible, allows repeated evaluation of surface and tubular membrane parameters on the same cell.
- Klíčová slova
- Cardiomyocyte, Novel method, Sucrose, Tubular membrane capacitance, Tubular system,
- MeSH
- elektrická vodivost MeSH
- kardiomyocyty * MeSH
- krysa rodu Rattus MeSH
- osmotický tlak MeSH
- sacharosa MeSH
- spřažení excitace a kontrakce * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- sacharosa MeSH
- MeSH
- kontrakce myokardu * MeSH
- lidé MeSH
- myokard metabolismus MeSH
- srdce fyziologie MeSH
- svalová kontrakce účinky léků MeSH
- svaly metabolismus fyziologie MeSH
- vápník metabolismus fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- vápník MeSH
Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by the lack of functional dystrophin. DMD is associated with progressive dilated cardiomyopathy, eventually leading to heart failure as the main cause of death in DMD patients. Although several molecular mechanisms leading to the DMD cardiomyocyte (DMD-CM) death were described, mostly in mouse model, no suitable human CM model was until recently available together with proper clarification of the DMD-CM phenotype and delay in cardiac symptoms manifestation. We obtained several independent dystrophin-deficient human pluripotent stem cell (hPSC) lines from DMD patients and CRISPR/Cas9-generated DMD gene mutation. We differentiated DMD-hPSC into cardiac cells (CC) creating a human DMD-CC disease model. We observed that mutation-carrying cells were less prone to differentiate into CCs. DMD-CCs demonstrated an enhanced cell death rate in time. Furthermore, ion channel expression was altered in terms of potassium (Kir2.1 overexpression) and calcium handling (dihydropyridine receptor overexpression). DMD-CCs exhibited increased time of calcium transient rising compared to aged-matched control, suggesting mishandling of calcium release. We observed mechanical impairment (hypocontractility), bradycardia, increased heart rate variability, and blunted β-adrenergic response connected with remodeling of β-adrenergic receptors expression in DMD-CCs. Overall, these results indicated that our DMD-CC models are functionally affected by dystrophin-deficiency associated and recapitulate functional defects and cardiac wasting observed in the disease. It offers an accurate tool to study human cardiomyopathy progression and test therapies in vitro.
- Klíčová slova
- DMD, adrenergic response, cardiomyocyte death, cardiomyocytes, duchenne muscular dystrophy, excitation-contraction coupling, human pluripotent stem cells, intracellular calcium,
- Publikační typ
- časopisecké články MeSH
