The sarcolemmal Ca2+ efflux pathways, Na+-Ca2+-exchanger (NCX) and Ca2+-ATPase (PMCA), play a crucial role in the regulation of intracellular Ca2+ load and Ca2+ transient in cardiomyocytes. The distribution of these pathways between the t-tubular and surface membrane of ventricular cardiomyocytes varies between species and is not clear in human. Moreover, several studies suggest that this distribution changes during the development and heart diseases. However, the consequences of NCX and PMCA redistribution in human ventricular cardiomyocytes have not yet been elucidated. In this study, we aimed to address this point by using a mathematical model of the human ventricular myocyte incorporating t-tubules, dyadic spaces, and subsarcolemmal spaces. Effects of various combinations of t-tubular fractions of NCX and PMCA were explored, using values between 0.2 and 1 as reported in animal experiments under normal and pathological conditions. Small variations in the action potential duration (≤ 2%), but significant changes in the peak value of cytosolic Ca2+ transient (up to 17%) were observed at stimulation frequencies corresponding to the human heart rate at rest and during activity. The analysis of model results revealed that the changes in Ca2+ transient induced by redistribution of NCX and PMCA were mainly caused by alterations in Ca2+ concentrations in the subsarcolemmal spaces and cytosol during the diastolic phase of the stimulation cycle. The results suggest that redistribution of both transporters between the t-tubular and surface membranes contributes to changes in contractility in human ventricular cardiomyocytes during their development and heart disease and may promote arrhythmogenesis.
- Klíčová slova
- Calcium ATPase, Calcium cycling, Human ventricular cell model, Membrane protein distribution, NCX, PMCA, Sodium‑calcium exchanger, T-tubules,
- MeSH
- akční potenciály MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- kardiomyocyty * metabolismus MeSH
- lidé MeSH
- modely kardiovaskulární MeSH
- pumpa pro výměnu sodíku a vápníku * metabolismus MeSH
- sarkolema * metabolismus MeSH
- srdeční komory * metabolismus MeSH
- vápník * metabolismus MeSH
- vápníková signalizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- pumpa pro výměnu sodíku a vápníku * 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 ratio between Na+-Ca2+ exchange current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes (JNaCa-ratio) estimated from electrophysiological data published to date yields strikingly different values between 1.7 and nearly 40. Possible reasons for such divergence were analysed by Monte Carlo simulations assuming both normal and log-normal distribution of the measured data. The confidence intervals CI95 of the mean JNaCa-ratios computed from the reported data showed an overlap of values between 1 and 3, and between 0.3 and 4.3 in the case of normal and log-normal distribution, respectively. Further analyses revealed that the published high values likely result from a large scatter of data due to transmural differences in JNaCa, dispersion of cell membrane capacitances and variability in incomplete detubulation. Taking into account the asymmetric distribution of the measured data, the reduction of mean current densities after detubulation and the substantially smaller CI95 of lower values of the mean JNaCa-ratio, the values between 1.6 and 3.2 may be considered as the most accurate estimates. This implies that 40 to 60% of Na+-Ca2+ exchanger is located at the t-tubular membrane of adult rat ventricular cardiomyocytes.
- Klíčová slova
- Cardiac myocyte, Log-normal distribution, Membrane current density, Monte Carlo simulation, Na-Ca exchanger, t-tubules,
- MeSH
- kardiomyocyty * metabolismus MeSH
- krysa rodu Rattus MeSH
- pumpa pro výměnu sodíku a vápníku MeSH
- sarkolema metabolismus MeSH
- sodík metabolismus MeSH
- srdeční komory metabolismus MeSH
- vápník * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- pumpa pro výměnu sodíku a vápníku MeSH
- sodík MeSH
- vápník * MeSH
We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca(2+) efflux pathways (SERCA, Na(+)/Ca(2+) exchange, and sarcolemmal Ca(2+) ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca(2+) buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca(2+) in the dyad and bulk cytoplasm, on cellular Ca(2+) cycling. Increasing the dyadic fraction of a particular Ca(2+) efflux pathway increases the amount of Ca(2+) removed by that pathway, with corresponding changes in Ca(2+) efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca(2+) removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca(2+)-dependent inactivation of the L-type Ca(2+) current, resulted from the buffers acting as slow and fast "shuttles," respectively, removing Ca(2+) from the dyadic space. The data suggest that complex changes in dyadic Ca(2+) and cellular Ca(2+) cycling occur as a result of changes in the location of Ca(2+) removal pathways or the presence of exogenous Ca(2+) buffers, although changing the distribution of Ca(2+) efflux pathways has relatively small effects on the systolic Ca(2+) transient.
- MeSH
- biologické modely MeSH
- časové faktory MeSH
- EGTA analogy a deriváty farmakologie MeSH
- gating iontového kanálu účinky léků MeSH
- intracelulární prostor účinky léků metabolismus MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- kompartmentace buňky MeSH
- krysa rodu Rattus MeSH
- počítačová simulace MeSH
- pufry MeSH
- pumpa pro výměnu sodíku a vápníku metabolismus MeSH
- sarkolema účinky léků metabolismus MeSH
- srdeční komory cytologie MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid MeSH Prohlížeč
- EGTA MeSH
- pufry MeSH
- pumpa pro výměnu sodíku a vápníku MeSH
- vápník MeSH
We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca2+]i-transient was explored as a function of critical fractions of ion transporters in t-tubular membrane (not documented for human CVM). Depletion of Ca2+ and accumulation of K+ occurring in extracellular spaces slightly affected the transmembrane Ca2+ flux, but not the action potential duration (APD90). The [Ca2+]i-transient was reduced (by 2%-9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1-2.5 Hz) caused a temporal decrease of [Ca2+] in both extracellular spaces, a reduction of [Ca2+]i-transient (by 15%) and APD90 (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca2+ turnover.
- MeSH
- akční potenciály MeSH
- biologické modely * MeSH
- extracelulární prostor metabolismus MeSH
- iontový transport MeSH
- ionty chemie metabolismus MeSH
- kardiomyocyty cytologie metabolismus MeSH
- lidé MeSH
- sarkolema metabolismus MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ionty MeSH
- vápník MeSH
Membrane fluidity is a widely recognized biophysical variable that provides information about structural organization of the subcellular membranes exhibiting physical characteristics of liquid crystals. The term "fluidity" reflects in this case the tightness in packing of acyl parts of the membrane phospholipid molecules, a feature that may influence considerably the molecular mobility and via that also the sensitivity and reactivity of membrane-bound transporters, receptors and enzyme systems. Data presented in this review are aimed to demonstrate the substantial role of changes in membrane fluidity occurring in the processes associated with endogenous protection observed in cardiac sarcolemma and mitochondria in diverse pathologies, particularly in diabetes and hypertension.
- MeSH
- buněčná membrána metabolismus MeSH
- fluidita membrány fyziologie MeSH
- fosfolipidy metabolismus MeSH
- krysa rodu Rattus MeSH
- membránové lipidy metabolismus MeSH
- myokard metabolismus MeSH
- sarkolema metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- fosfolipidy MeSH
- membránové lipidy MeSH
The adaptation to chronic hypoxia confers long-lasting cardiac protection against acute ischemia-reperfusion injury. Protein kinase C (PKC) appears to play a role in the cardioprotective mechanism but the involvement of individual PKC isoforms remains unclear. The aim of this study was to examine the effects of chronic intermittent hypoxia (CIH; 7,000 m, 8 h/day) and acute administration of PKC-δ inhibitor (rottlerin, 0.3 mg/kg) on the expression and subcellular distribution of PKC-δ and PKC-ε in the left ventricular myocardium of adult male Wistar rats by Western blot and quantitative immunofluorescence microscopy. CIH decreased the total level of PKC-ε in homogenate without affecting the level of phosphorylated PKC-ε (Ser729). In contrast, CIH up-regulated the total level of PKC-δ as well as the level of phosphorylated PKC-δ (Ser643) in homogenate. Rottlerin partially reversed the hypoxia-induced increase in PKC-δ in the mitochondrial fraction. Immunofluorescent staining of ventricular cryo-sections revealed increased co-localization of PKC-δ with mitochondrial and sarcolemmal membranes in CIH hearts that was suppressed by rottlerin. The formation of nitrotyrosine as a marker of oxidative stress was enhanced in CIH myocardium, particularly in mitochondria. The expression of total oxidative phosphorylation complexes was slightly decreased by CIH mainly due to complex II decline. In conclusion, up-regulated PKC-δ in CIH hearts is mainly localized to mitochondrial and sarcolemmal membranes. The inhibitory effects of rottlerin on PKC-δ subcellular redistribution and cardioprotection (as shown previously) support the view that this isoform plays a role in the mechanism of CIH-induced ischemic tolerance.
- MeSH
- chronická nemoc MeSH
- fosforylace MeSH
- hypoxie enzymologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- krysa rodu Rattus MeSH
- mitochondrie metabolismus MeSH
- myokard metabolismus patologie MeSH
- ochranné látky MeSH
- potkani Wistar MeSH
- proteinkinasa C-delta genetika metabolismus fyziologie MeSH
- sarkolema metabolismus MeSH
- tkáňová distribuce MeSH
- upregulace * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- inhibitory proteinkinas MeSH
- ochranné látky MeSH
- proteinkinasa C-delta MeSH
Diabetes is a recognized risk factor of heart disease. The abnormalities related to a decreased heart performance probably arise at cellular and molecular levels already in the asymptomatic phase of diabetes. However, the early alterations initiating a sequence of events that culminates in the clinical signs have not been fully elucidated yet. This review deals with some biophysical methods applied to investigation of left ventricular myocytes in rats with streptozotocin diabetes, as well as our most important findings concerning diabetes-induced cell changes which cannot be captured by other techniques. The observed decrease in sarcolemmal membrane fluidity is causatively associated with increased glycation and glycoxidation. On the other hand, an increase in the mitochondrial membrane fluidity may be attributed to augmented energy transduction through the membranes. We reported for the first time concurrent measurements of membrane potential and dynamics, and respiratory chain activities in rat heart mitochondria, as well as calcium transients in the myocytes from diabetic hearts together with the assessed quantitative relationships among these variables. We were able to detect some significant alterations that may underlie myocyte dysfunction and subsequent remodeling of the heart. We suppose that not all these changes reflect mechanisms leading to pathology; some may represent adaptive and compensatory responses to diabetes.
- MeSH
- biologické modely MeSH
- energetický metabolismus MeSH
- experimentální diabetes mellitus komplikace metabolismus patofyziologie MeSH
- fluidita membrány MeSH
- kardiomyocyty metabolismus MeSH
- krysa rodu Rattus MeSH
- membránové potenciály MeSH
- mitochondriální membrány metabolismus MeSH
- sarkolema metabolismus MeSH
- srdeční komory metabolismus patofyziologie MeSH
- srdeční mitochondrie metabolismus MeSH
- vápníková signalizace MeSH
- velikost buňky MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The transverse (t-) tubules of cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. Many of the key proteins involved in excitation-contraction coupling appear to be located predominantly at the t-tubule membrane. Despite their importance, the fraction of cell membrane within the t-tubules remains unclear: measurement of cell capacitance following detubulation suggests approximately 32%, whereas optical measurements suggest up to approximately 65%. We have, therefore, investigated the factors that may account for this discrepancy. Calculation of the combinations of t-tubule radius, length and density that produce t-tubular membrane fractions of 32% or 56% suggest that the true fraction is at the upper end of this range. Assessment of detubulation using confocal and electron microscopy suggests that incomplete detubulation can account for some, but not all of the difference. High cholesterol, and a consequent decrease in specific capacitance, in the t-tubule membrane, may also cause the t-tubule fraction calculated from the loss of capacitance following detubulation to be underestimated. Correcting for both of these factors results in an estimate that is still lower than that obtained from optical measurements suggesting either that optical methods overestimate the fraction of membrane in the t-tubules, or that other, unknown, factors, reduce the apparent fraction obtained by detubulation. A biophysically realistic computer model of a rat ventricular myocyte, incorporating a t-tubule network, is used to assess the effect of the altered estimates of t-tubular membrane fraction on the calculated distribution of ion flux pathways.
- MeSH
- kardiomyocyty chemie metabolismus MeSH
- krysa rodu Rattus MeSH
- proteiny analýza metabolismus MeSH
- sarkolema chemie metabolismus MeSH
- sarkoplazmatické retikulum chemie metabolismus MeSH
- srdeční komory chemie cytologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- proteiny MeSH
We examined the effect of MCC-134, a novel inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels and activator of sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels, on cardioprotection conferred by adaptation to chronic hypoxia. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 5-6 weeks) and susceptibility of their hearts to ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion on the day after the last hypoxic exposure. MCC-134 was administered intravenously 10 min before ischemia and 5 min before reperfusion in a total dose of 0.3 mg/kg or 3 mg/kg divided into two equal boluses. The infarct size (tetrazolium staining) was reduced from 59.2+/-4.4 % of the area at risk in normoxic controls to 43.2+/-3.3 % in the chronically hypoxic group. Chronic hypoxia decreased the reperfusion arrhythmia score from 2.4+/-0.5 in normoxic animals to 0.7+/-0.5. Both doses of MCC-134 completely abolished the antiarrhythmic protection (score 2.4+/-0.7 and 2.5+/-0.5, respectively) but only the high dose blocked the infarct size-limiting effect of chronic hypoxia (54.2+/-3.7 %). MCC-134 had no effect in the normoxic group. These results support the view that the opening of mitoKATP channels but not sarcKATP channels plays a crucial role in the mechanism by which chronic hypoxia improves cardiac tolerance to ischemia/reperfusion injury.
- MeSH
- ABC transportéry agonisté MeSH
- blokátory kalciových kanálů farmakologie MeSH
- draslíkové kanály dovnitř usměrňující agonisté MeSH
- fyziologická adaptace účinky léků fyziologie MeSH
- hypoxie patofyziologie MeSH
- imidazoly farmakologie MeSH
- infarkt myokardu patologie prevence a kontrola MeSH
- KATP kanály MeSH
- krevní tlak účinky léků MeSH
- krysa rodu Rattus MeSH
- potkani Wistar MeSH
- reperfuzní poškození myokardu patologie prevence a kontrola MeSH
- sarkolema účinky léků metabolismus MeSH
- srdeční frekvence účinky léků MeSH
- srdeční mitochondrie účinky léků MeSH
- techniky in vitro MeSH
- thioamidy farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ABC transportéry MeSH
- blokátory kalciových kanálů MeSH
- draslíkové kanály dovnitř usměrňující MeSH
- imidazoly MeSH
- KATP kanály MeSH
- MCC 134 MeSH Prohlížeč
- thioamidy MeSH
- uK-ATP-1 potassium channel MeSH Prohlížeč
