RESUMEN
Background and Objective. This study addresses the Force-Frequency relationship, a fundamental characteristic of cardiac muscle influenced byß1-adrenergic stimulation. This relationship reveals that heart rate (HR) changes at the sinoatrial node lead to alterations in ventricular cell contractility, increasing the force and decreasing relaxation time for higher beat rates. Traditional models lacking this relationship offer an incomplete physiological depiction, impacting the interpretation of in silico experiment results. To improve this, we propose a new mathematical model for ventricular myocytes, named 'Feed Forward Modeling' (FFM).Methods. FFM adjusts model parameters like channel conductance and Ca2+pump affinity according to stimulation frequency, in contrast to fixed parameter values. An empirical sigmoid curve guided the adaptation of each parameter, integrated into a rabbit ventricular cell electromechanical model. Model validation was achieved by comparing simulated data with experimental current-voltage (I-V) curves for L-type Calcium and slow Potassium currents.Results. FFM-enhanced simulations align more closely with physiological behaviors, accurately reflecting inotropic and lusitropic responses. For instance, action potential duration at 90% repolarization (APD90) decreased from 206 ms at 1 Hz to 173 ms at 4 Hz using FFM, contrary to the conventional model, where APD90 increased, limiting high-frequency heartbeats. Peak force also showed an increase with FFM, from 8.5 mN mm-2at 1 Hz to 11.9 mN mm-2at 4 Hz, while it barely changed without FFM. Relaxation time at 50% of maximum force (t50) similarly improved, dropping from 114 ms at 1 Hz to 75.9 ms at 4 Hz with FFM, a change not observed without the model.Conclusion. The FFM approach offers computational efficiency, bypassing the need to model all beta-adrenergic pathways, thus facilitating large-scale simulations. The study recommends that frequency change experiments include fractional dosing of isoproterenol to better replicate heart conditionsin vivo.
Asunto(s)
Potenciales de Acción , Simulación por Computador , Ventrículos Cardíacos , Contracción Miocárdica , Miocitos Cardíacos , Conejos , Animales , Miocitos Cardíacos/fisiología , Contracción Miocárdica/fisiología , Modelos Cardiovasculares , Frecuencia Cardíaca/fisiología , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Nodo Sinoatrial/fisiología , Modelos TeóricosRESUMEN
In skeletal muscle, the Ca2+ release flux elicited by a voltage clamp pulse rises to an early peak that inactivates rapidly to a much lower steady level. Using a double pulse protocol the fast inactivation follows an arithmetic rule: if the conditioning depolarization is less than or equal to the test depolarization, then decay (peak minus steady level) in the conditioning release is approximately equal to suppression (unconditioned minus conditioned peak) of the test release. This is due to quantal activation by voltage, analogous to the quantal activation of IP3 receptor channels. Two mechanisms are possible. One is the existence of subsets of channels with different sensitivities to voltage. The other is that the clusters of Ca2+-gated Ryanodine Receptor (RyR) ß in the parajunctional terminal cisternae might constitute the quantal units. These Ca2+-gated channels are activated by the release of Ca2+ through the voltage-gated RyR α channels. If the RyR ß were at the basis of quantal release, it should be modified by strong inhibition of the primary voltage-gated release. This was attained in two ways, by sarcoplasmic reticulum (SR) Ca2+ depletion and by voltage-dependent inactivation. Both procedures reduced global Ca2+ release flux, but SR Ca2+ depletion reduced the single RyR current as well. The effect of both interventions on the quantal properties of Ca2+ release in frog skeletal muscle fibers were studied under voltage clamp. The quantal properties of release were preserved regardless of the inhibitory maneuver applied. These findings put a limit on the role of the Ca2+-activated component of release in generating quantal activation.
Asunto(s)
Músculo Esquelético , Retículo Sarcoplasmático , Retículo Sarcoplasmático/metabolismo , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/farmacología , Señalización del Calcio , Calcio/metabolismoRESUMEN
In skeletal muscle (SM), inward Ca2+-currents have no apparent role in excitation-contraction coupling (e-c coupling), however the Ca2+-channel blocker can affect twitch and tetanic muscle in mammalian SM. Experiments were conducted to study how diltiazem (DLZ) facilitates e-c coupling and inhibits contraction. 1) In complete Extensor Digitorum Longus (EDL) muscle and single intact fibres, 0.03 mM DLZ causes twitch potentiation and decreases force during tetanic activity, with increased fatigue. 2) In split open fibres isolated from EDL fibres, DLZ inhibits sarcoplasmic reticulum (SR) Ca2+-loading in a dose-dependent manner and has a potentiating effect on caffeine-induced SR Ca2+-release. 3) In isolated light SR (LSR) vesicles, SERCA1 hydrolytic activity is not affected by DLZ up to 0.2 mM. However, ATP-dependent Ca2+-uptake was inhibited in a dose-dependent manner at a concentration where e-c coupling is changed. 4) The passive Ca2+-efflux from LSR was reduced by half with 0.03 mM diltiazem, indicating that SR leaking does not account for the decreased Ca2+-uptake. 5) The denaturation profile of the SERCA Ca2+-binding domain has lower thermal stability in the presence of DLZ in a concentration-dependent manner, having no effect on the nucleotide-binding domain. We conclude that the effect of DLZ on SM is exerted by crossing the sarcolemma and interacting directly with the SERCA Ca2+-binding domain, affecting SR Ca2+-loading during relaxation, which has a consequence on SM contractility. Diltiazem effect on SM could be utilized as a tool to understand SM e-c coupling and muscle fatigue.
Asunto(s)
Diltiazem , Músculo Esquelético , Animales , Diltiazem/farmacología , Retículo Sarcoplasmático , Fatiga Muscular , Cafeína/farmacología , Mamíferos , Contracción Muscular , Calcio/farmacologíaRESUMEN
Fipronil is widely used as a broad-spectrum insecticide in agriculture, urban environments, and veterinary medicine. Fipronil can enter aquatic ecosystems and spread to sediment and organic matter, representing a risk to non-target species. This study aimed to evaluate the effects of short-term (96 h) exposure to a low and realistic concentration of sediment-associated fipronil (4.2 µg.kg-1 of Regent® 800 WG) on myocardial contractility of armored catfish Hypostomus regain, a benthic fish species. Fipronil exposure induced increased inotropism and acceleration of contractile kinetics, although no alterations in the relative ventricular mass were observed. This better cardiac function was associated with an elevated expression and/or function of the Na+/Ca2+ exchanger and its marked contribution to contraction and relaxation, probably due to a stress-induced adrenergic stimulation. Ventricle strips of exposed fish also exhibited a faster relaxation and a higher cardiac pumping capacity, indicating that armored catfish were able to perform cardiac adjustments to face the exposure. However, a high energetic cost to maintain an increased cardiac performance can make fish more susceptible to other stressors, impairing developmental processes and/or survival. These findings highlight the need for regulations of emerging contaminants, such as fipronil, to ensure adequate protection of the aquatic system.
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Bagres , Animales , Ecosistema , Corazón , Pirazoles/toxicidadRESUMEN
The excitation-contraction coupling (ECC) in skeletal muscle refers to the Ca2+-mediated link between the membrane excitation and the mechanical contraction. The initiation and propagation of an action potential through the membranous system of the sarcolemma and the tubular network lead to the activation of the Ca2+-release units (CRU): tightly coupled dihydropyridine and ryanodine (RyR) receptors. The RyR gating allows a rapid, massive, and highly regulated release of Ca2+ from the sarcoplasmic reticulum (SR). The release from triadic places generates a sarcomeric gradient of Ca2+ concentrations ([Ca2+]) depending on the distance of a subcellular region from the CRU. Upon release, the diffusing Ca2+ has multiple fates: binds to troponin C thus activating the contractile machinery, binds to classical sarcoplasmic Ca2+ buffers such as parvalbumin, adenosine triphosphate and, experimentally, fluorescent dyes, enters the mitochondria and the SR, or is recycled through the Na+/Ca2+ exchanger and store-operated Ca2+ entry (SOCE) mechanisms. To commemorate the 7th decade after being coined, we comprehensively and critically reviewed "old", historical landmarks and well-established concepts, and blended them with recent advances to have a complete, quantitative-focused landscape of the ECC. We discuss the: 1) elucidation of the CRU structures at near-atomic resolution and its implications for functional coupling; 2) reliable quantification of peak sarcoplasmic [Ca2+] using fast, low affinity Ca2+ dyes and the relative contributions of the Ca2+-binding mechanisms to the whole concert of Ca2+ fluxes inside the fibre; 3) articulation of this novel quantitative information with the unveiled structural details of the molecular machinery involved in mitochondrial Ca2+ handing to understand how and how much Ca2+ enters the mitochondria; 4) presence of the SOCE machinery and its different modes of activation, which awaits understanding of its magnitude and relevance in situ; 5) pharmacology of the ECC, and 6) emerging topics such as the use and potential applications of super-resolution and induced pluripotent stem cells (iPSC) in ECC. Blending the old with the new works better!
RESUMEN
In many reptiles, digestion has been associated with the selection of higher body temperatures, the so-called post-prandial thermophilic response. This study aimed to investigate the excitation-contraction (E-C) coupling in postprandial broad-snouted caimans (Caiman latirostris) in response to acute warming within a preferred body temperature range of crocodiles. Isometric preparations subjected to a temperature transition from 25°C to 30°C were used to investigate myocardial contractility of postprandial caimans, that is, 48 h after the animals ingested a rodent meal corresponding to 15% of body mass. The caiman heart exhibits a negative force-frequency relationship that is independent of the temperature. At 25°C, cardiac muscle was able to maintain a constant force up to 36 bpm, above which it decreased significantly, reaching minimum values at the highest frequency of 84 bpm. Moreover, E-C coupling is predominantly dependent on transsarcolemmal Ca2+ transport denoted by the lack of significant ryanodine effects on force generation. On the contrary, ventricular strips at 30°C were able to sustain the cardiac contractility at higher pacing frequencies (from 12 to 144 bpm) due to an important role of Na+ /Ca2+ exchanger in Ca2+ cycling, as indicated by the decay of the post-rest contraction, and a significant contribution of the sarcoplasmic reticulum above 72 bpm. Our results demonstrated that the myocardium of postprandial caimans exhibits a significant degree of thermal plasticity of E-C coupling during acute warming. Therefore, myocardial contractility can be maximized when postprandial broad-snouted caimans select higher body temperatures (preferred temperature zone) following feeding.
Asunto(s)
Caimanes y Cocodrilos/fisiología , Digestión/fisiología , Tracto Gastrointestinal/fisiología , Contracción Miocárdica/fisiología , Temperatura , Animales , Femenino , Corazón/fisiología , MasculinoRESUMEN
AIMS: Amiodarone (AMIO) is currently used in medical practice to reverse ventricular tachycardia. Here we determine the effects of AMIO in the electromechanical properties of isolated left ventricle myocyte (LVM) from mice and guinea pig and in a cellular model of Long QT Syndrome Type 3 (LQTS-3) using anemone neurotoxin 2 (ATX II), which induces increase of late sodium current in LVM. MAIN METHODS AND KEY FINDINGS: Using patch-clamp technique, fluorescence imaging to detect cellular Ca2+ transient and sarcomere detection systems we evaluate the effect of AMIO in healthy LVM. AMIO produced a significant reduction in the percentage of sarcomere shortening (0.1, 1 and 10 µM) in a range of pacing frequencies, however, without significant attenuation of Ca2+ transient. Also, 10 µM of AMIO caused the opposite effect on action potential repolarization of mouse and guinea pig LVM. When LVM from mouse and guinea pig were paced in a range of pacing frequencies and exposed to ATX (10 nM), AMIO (10 µM) was only able to abrogate electromechanical arrhythmias in LVM from guinea pig at lower pacing frequency. SIGNIFICANCE: AMIO has negative inotropic effect with opposite effect on action potential waveform in mouse and guinea pig LVM. Furthermore, the antiarrhythmic action of AMIO in LQTS-3 is species and frequency-dependent, which indicates that AMIO may be beneficial for some types of arrhythmias related to late sodium current.
Asunto(s)
Amiodarona/farmacología , Antiarrítmicos/farmacología , Trastorno del Sistema de Conducción Cardíaco/tratamiento farmacológico , Síndrome de QT Prolongado/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Amiodarona/administración & dosificación , Animales , Antiarrítmicos/administración & dosificación , Trastorno del Sistema de Conducción Cardíaco/fisiopatología , Relación Dosis-Respuesta a Droga , Cobayas , Ventrículos Cardíacos/citología , Síndrome de QT Prolongado/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Especificidad de la EspecieRESUMEN
The skeletal muscle and myocardial cells present highly specialized structures; for example, the close interaction between the sarcoplasmic reticulum (SR) and mitochondria-responsible for excitation-metabolism coupling-and the junction that connects the SR with T-tubules, critical for excitation-contraction (EC) coupling. The mechanisms that underlie EC coupling in these two cell types, however, are fundamentally distinct. They involve the differential expression of Ca2+ channel subtypes: CaV1.1 and RyR1 (skeletal), vs. CaV1.2 and RyR2 (cardiac). The CaV channels transform action potentials into elevations of cytosolic Ca2+, by activating RyRs and thus promoting SR Ca2+ release. The high levels of Ca2+, in turn, stimulate not only the contractile machinery but also the generation of mitochondrial reactive oxygen species (ROS). This forward signaling is reciprocally regulated by the following feedback mechanisms: Ca2+-dependent inactivation (of Ca2+ channels), the recruitment of Na+/Ca2+ exchanger activity, and oxidative changes in ion channels and transporters. Here, we summarize both well-established concepts and recent advances that have contributed to a better understanding of the molecular mechanisms involved in this bidirectional signaling.
Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/fisiología , Citosol/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Humanos , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcolema/fisiología , Retículo Sarcoplasmático/fisiología , Transducción de SeñalRESUMEN
Pomolic acid (PA) isolated from Licania pittieri has hypotensive effects in rats, inhibits human platelet aggregation and elicits endothelium-dependent relaxation in rat aortic rings. The present study was designed to investigate the effects of PA on cardiomyocytes. Trabeculae and enzymatically isolated cardiomyocytes from rats were used to evaluate the concentration-dependent effects of PA on cardiac muscle tension and excitation-contraction coupling (ECC) by recording Ca2+ transients reported with Fluo-3 and Fura-2, as well as L-type Ca2+ currents (LTCC). PA reduced the contractile force in rat cardiac trabeculae with an EC50 =â¯14.3⯱â¯2.4⯵M. PA also reduced the amplitude of Ca2+ transients in a concentration-dependent manner, with an EC50 =â¯10.5⯱â¯1.3⯵M, without reducing sarcoplasmic reticulum (SR) Ca2+ loading. PA decreased the half width of the Ca2+ transient by 31.7⯱â¯3.3% and increased the decay time and decay time constant (τ) by 7.6⯱â¯2.7% and 75.6⯱â¯3.7%, respectively, which was associated with increased phospholamban (PLN) phosphorylation. PA also reversibly reduced the macroscopic LTCC in the cardiomyocyte membrane, but did not demonstrate any effects on skeletal muscle ECC. In conclusion, PA reduces LTCC, Ca2+ transients and cardiomyocyte force, which along with its vasorelaxant effects explain its hypotensive properties. Increased PLN phosphorylation protected the SR from Ca2+ depletion. Considering the effects of PA on platelet aggregation and the cardiovascular system, we propose it as a new potential, multitarget cardiovascular agent with a demonstrated safety profile.
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Acoplamiento Excitación-Contracción/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ácido Oleanólico/análogos & derivados , Animales , Canales de Calcio Tipo L/metabolismo , Masculino , Miocitos Cardíacos/citología , NG-Nitroarginina Metil Éster/farmacología , Ácido Oleanólico/farmacología , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
[Ca2+] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca2+ release of extrinsic Ca2+ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca2+ buffer is added to the myoplasm, part of the released Ca2+ binds to it, reducing the Ca2+ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca2+ released. Monitoring release by measuring [Ca2+] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca2+] in the SR ([Ca2+]SR) continuously from a starting value close to 400 µM reaching the range of 100 µM in about half an hour. The effect of reducing resting [Ca2+]SR on the Ca2+ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca2+] ([Ca2+]myo) transients were simultaneously recorded with Rhod2. The Ca2+ release flux was calculated from [Ca2+]myo and divided by [Ca2+]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005 ms-1 at resting [Ca2+]SR = 372 ± 5 µM to 0.021 ± 0.004 ms-1 at resting [Ca2+]SR = 120 ± 16 µM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003 ms-1, at the higher and lower [Ca2+]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca2+]SR (Δ[Ca2+]SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca2+]SR = 30.2 ± 3.5 µM from resting [Ca2+]SR = 88.8 ± 13.6 µM, n = 5) than in 40 mM EGTA (Δ[Ca2+]SR = 72.2 ± 10.4 µM from resting [Ca2+]SR = 98.2 ± 15.6 µM, n = 4) suggesting that a Ca2+ activated component of release was suppressed by BAPTA.
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Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Fibras Musculares Esqueléticas/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , RanidaeRESUMEN
Muscle fibres, isolated from frog tibialis anterior and mouse flexor digitorum brevis (FDB) were loaded with the fast dye MagFluo-4 to study the effects of potentiators caffeine, nitrate, Zn2+ and perchlorate on Ca2+ transients elicited by single action potentials. Overall, the potentiators doubled the transients amplitude and prolonged by about 1.5-fold their decay time. In contrast, as shown here for the first time, nitrate and Zn2+, but not caffeine, activated a late, secondary component of the transient rising phase of frog but not mouse, fibres. The rise time was increased from 1.9 ms in normal solution (NR) to 3.3 ms (nitrate) and 4.4 ms (Zn2+). In NR, a single exponential, fitted the rising phase of calcium transients of frog (τ1 = 0.47 ms) and mouse (τ1 = 0.28 ms). In nitrate and Zn2+ only frog transients showed a secondary exponential component, τ2 = 0.72 ms (nitrate) and 0.94 ms, (Zn2+). We suggest that nitrate and Zn2+ activate a late slower component of the ΔF/F signals of frog but not of mouse fibres, possibly promoting Ca2+ induced Ca2+ release at level of the RyR3, that in frog muscle fibres are localized in the para-junctional region of the triads and are absent in mouse FDB muscle fibres.
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Potenciales de Acción/fisiología , Calcio/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Animales , Anuros , RatonesRESUMEN
Iron intoxication is related to reactive oxygen species (ROS) production and organic damage including the cardiovascular system, and is a leading cause of poisoning deaths in children. In this study we examined whether a range of ferrous iron (Fe(2+)) concentrations can interfere differently on the myocardial mechanics, investigating the ROS-mediated effects. Developed force of isolated rat papillary muscles was depressed with a concentration- and time-dependency by Fe(2+) 100-1000µM. The contractile response to Ca(2+) was reduced, but it was partially reversed by co-incubation with catalase and DMSO, but not TEMPOL. In agreement, in situ detection of OH was increased by Fe(2+) whereas O2(-) was unchanged. The myosin-ATPase activity was significantly decreased. Contractions dependent on the sarcolemal Ca(2+) influx were impaired only by Fe(2+) 1000µM, and antioxidants had no effect. In skinned fibers, Fe(2+) reduced the pCa-force relationship, and pCa50 was right-shifted by 0.55. In conclusion, iron overload can acutely impair myocardial contractility by reducing myosin-ATPase activity and myofibrillar Ca(2+) sensitivity. These effects are mediated by local production of OH and H2O2. Nevertheless, in a such high concentration as 1000µM, Fe(2+) appears to depress force also by reducing Ca(2+) influx, probably due to a competition at Ca(2+) channels.
Asunto(s)
Compuestos Ferrosos/farmacología , Contracción Miocárdica/efectos de los fármacos , Músculos Papilares/efectos de los fármacos , Animales , Calcio/metabolismo , Técnicas In Vitro , Sobrecarga de Hierro/metabolismo , Sobrecarga de Hierro/fisiopatología , Contracción Isométrica/efectos de los fármacos , Masculino , Miosinas/metabolismo , Músculos Papilares/metabolismo , Músculos Papilares/fisiología , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/µF meanwhile the Ca2+ release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 µM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.
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Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Músculo Esquelético/metabolismo , Técnicas de Placa-Clamp/métodos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , AnurosRESUMEN
In cardiac muscle cells both T-and L-type Ca(2+) channels (TTCCs and LTCCs, respectively) are expressed, and the latter are relevant to a process known as excitation-contraction coupling (ECC). Evidence obtained from docking studies suggests that isoindolines derived from α-amino acids bind to the LTCC CaV1.2. In the present study, we investigated whether methyl (S)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate (MDIMP), which is derived from L-leucine, modulates both Ca(2+) channels and ECC. To this end, mechanical properties, as well as Ca(2+) transients and currents, were all investigated in isolated cardiac myocytes. The effects of MDIMP on CaV1.2 (transiently expressed in 293T/17 cells) were also studied. In this system, evidence was found for an inhibitory action that develops and recovers in min, with an IC50 of 450µM. With respect to myocytes: atrial-TTCCs, atrial-LTCCs, and ventricular-LTCCs were also inhibited, in that order of potency. Accordingly, Ca(2+) transients, contractions, and window currents of LTCCs were all reduced more strongly in atrial cells. Interestingly, while the modulation of LTCCs was state-independent in these cells, it was state-dependent, and dual, on the ventricular ones. Furthermore, practically all of the ventricular LTCCs were closed at resting membrane potentials. This could explain their resistance to MDIMP, as they were affected in only open or inactivated states. All these features in turn explain the preferential down-regulation of the atrial ECC. Thus, our results support the view that isoindolines bind to Ca(2+) channels, improve our knowledge of the corresponding structure-function relationship, and may be relevant for conditions where decreased atrial activity is desired.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Isoindoles/farmacología , Ácidos Pentanoicos/farmacología , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Células HEK293 , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Humanos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos , Factores de TiempoRESUMEN
AIMS: This study investigated the mechanisms underlying the vascular effects of terpinen-4-ol in isolated rat aortic ring preparations. MAIN METHODS: The thoracic aortae of healthy rats were submitted to isometric tension recording. Membrane resting potential and input membrane resistance were measured by conventional microelectrode technique. KEY FINDINGS: Terpinen-4-ol reversibly relaxed endothelium-containing preparations pre-contracted with high K(+) and phenylephrine with IC50 values of 421.43 µM and 802.50 µM, respectively. These effects were significantly reduced by vascular endothelium removal. In Ca(2+)-free and high K(+) (80 mM) medium, the contractions produced by Ba(2+) were reduced by terpinen-4-ol (100-1000 µM) in a concentration-dependent manner. In aortic rings maintained under Ca(2+)-free conditions, terpinen-4-ol significantly reduced the contractions induced by either phenylephrine (1 µM) or phorbol 12,13-dibutyrate (1 µM). Terpinen-4-ol (10-1000 µM) also relaxed the contractions evoked by BAYK-8644 (3 µM) with an IC50 of 454.23 µM. Neither membrane resting potential nor input resistance of smooth muscle cells was altered by terpinen-4-ol exposure. SIGNIFICANCE: The present results suggest that terpinen-4-ol induced vascular smooth muscle relaxation that was preferentially due to the inhibition of electromechanical pathways related to calcium influx through voltage-operated calcium channels.
Asunto(s)
Aorta/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Terpenos/farmacología , Vasodilatadores/farmacología , Animales , Aorta/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Endotelio Vascular/efectos de los fármacos , Femenino , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Wistar , Vasodilatación/efectos de los fármacosRESUMEN
Methyl cinnamate (MC) is a safe flavoring agent useful to food industry. Although chemically analog to tyrosine kinase inhibitors, there is little information regarding its biological actions. Here, we aimed at assessing the MC effects on gastrointestinal contractility and the putative involvement of tyrosine kinase in the mediation of these effects. Isometric contractions were recorded in rat isolated strips from stomach, duodenum and colon segments. In gastric strips, MC (3-3000 µM) showed antispasmodic effects against carbachol-induced contractions, which remained unchanged by either l-NAME or tetraethylammonium pretreatment and occurred with potency similar to that obtained against contractions evoked by potassium or U-46619. In colon strips, MC was four times more potent than in gastric ones. MC and the positive control genistein inhibited phasic contractions induced by acetylcholine in Ca2+-free medium, an effect fully prevented by sodium orthovanadate. Both MC and genistein decreased the spontaneous contractions of duodenal strips and shortened the time necessary for gastric fundic tissues to reach 50% of maximal relaxation. In freshly isolated colon myocytes, MC decreased the basal levels of cytoplasmic Ca2+, but not the potassium-elicited cytoplasmic Ca2+ elevation. Colon strips obtained from rats subjected to intracolonic acetic acid instillation showed reduced contractility to potassium, which was partially recovered in MC-treated rats. Inhibitory effect of nifedipine against cholinergic contractions, blunted in acetic acid-induced colitis, was also recovered in MC-treated rats. In conclusion, MC inhibited the gastrointestinal contractility with a probable involvement of tyrosine kinase pathways. In vivo, it was effective to prevent the deleterious effects of colitis resulting from acetic acid injury.
Asunto(s)
Cinamatos/farmacología , Colon/efectos de los fármacos , Duodeno/efectos de los fármacos , Aromatizantes/farmacología , Parasimpatolíticos/farmacología , Estómago/efectos de los fármacos , Ácido Acético , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Carbacol , Cinamatos/uso terapéutico , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/fisiopatología , Colon/fisiología , Duodeno/fisiología , Aromatizantes/uso terapéutico , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Nifedipino/farmacología , Parasimpatolíticos/uso terapéutico , Cloruro de Potasio/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/fisiología , Ratas Wistar , Estómago/fisiologíaRESUMEN
First coined by Alexander Sandow in 1952, the term excitation-contraction coupling (ECC) describes the rapid communication between electrical events occurring in the plasma membrane of skeletal muscle fibres and Ca2+ release from the SR, which leads to contraction. The sequence of events in twitch skeletal muscle involves: (1) initiation and propagation of an action potential along the plasma membrane, (2) spread of the potential throughout the transverse tubule system (T-tubule system), (3) dihydropyridine receptors (DHPR)-mediated detection of changes in membrane potential, (4) allosteric interaction between DHPR and sarcoplasmic reticulum (SR) ryanodine receptors (RyR), (5) release of Ca2+ from the SR and transient increase of Ca2+ concentration in the myoplasm, (6) activation of the myoplasmic Ca2+ buffering system and the contractile apparatus, followed by (7) Ca2+ disappearance from the myoplasm mediated mainly by its reuptake by the SR through the SR Ca2+ adenosine triphosphatase (SERCA), and under several conditions movement to the mitochondria and extrusion by the Na+/Ca2+ exchanger (NCX). In this text, we review the basics of ECC in skeletal muscle and the techniques used to study it. Moreover, we highlight some recent advances and point out gaps in knowledge on particular issues related to ECC such as (1) DHPR-RyR molecular interaction, (2) differences regarding fibre types, (3) its alteration during muscle fatigue, (4) the role of mitochondria and store-operated Ca2+ entry in the general ECC sequence, (5) contractile potentiators, and (6) Ca2+ sparks.
RESUMEN
We tested the effects of low-intensity endurance training (LIET) on the structural and mechanical properties of right (RV) and left ventricular (LV) myocytes. Male Wistar rats (4 mo old) were randomly divided into control (C, n = 7) and trained (T, n = 7, treadmill running at 50-60% of maximal running speed for 8 wk) groups. Isolated ventricular myocyte dimensions, contractility, Ca(2+) transients {intracellular Ca(2+) concentration ([Ca(2+)]i)}, and ventricular [Ca(2+)]i regulatory proteins were measured. LIET augmented cell length (C, 152.5 ± 2.0 µm vs. T, 162.2 ± 2.1 µm; P < 0.05) and volume (C, 5,162 ± 131 µm(3) vs. T, 5,506 ± 132 µm(3); P < 0.05) in the LV but not in the RV. LIET increased cell shortening (C, 7.5 ± 0.3% vs. T, 8.6 ± 0.3%; P < 0.05), the [Ca(2+)]i transient amplitude (C, 2.49 ± 0.06 F/F0 vs. T, 2.82 ± 0.06 F/F0; P < 0.05), the expression of sarcoplasmic reticulum Ca(2+)-ATPase 2a (C, 1.07 ± 0.13 vs. T, 1.59 ± 0.12; P < 0.05), and the levels of phosphorylated phospholamban at serine 16 (C, 0.99 ± 0.11 vs. T, 1.34 ± 0.10; P < 0.05), and reduced the total phospholamban-to-sarcoplasmic reticulum Ca(2+)-ATPase 2a ratio (C, 1.19 ± 0.15 vs. T, 0.40 ± 0.16; P < 0.05) in the LV without changing such parameters in the RV. In conclusion, LIET affected the structure and improved the mechanical properties of LV but not of RV myocytes in rats, helping to characterize the functional and morphological changes that accompany the endurance training-induced cardiac remodeling.
Asunto(s)
Miocitos Cardíacos/fisiología , Condicionamiento Físico Animal/fisiología , Resistencia Física/fisiología , Animales , Western Blotting , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Separación Celular , Ventrículos Cardíacos/citología , Técnicas In Vitro , Masculino , Contracción Muscular/fisiología , Contracción Miocárdica/fisiología , Miocitos Cardíacos/ultraestructura , Ratas , Ratas Wistar , Carrera , Función Ventricular Derecha/fisiologíaRESUMEN
This study analyzed the physiological role of the cardiac sarcoplasmic reticulum (SR) of two neotropical teleosts, the jeju, Hoplerythrinus unitaeniatus (Erythrinidae), and the acara, Geophagus brasiliensis (Cichlidae). While the in vivo heart frequency (fH - bpm) of acara (79.6 ± 6.6) was higher than that of the jeju (50.3 ± 2.7), the opposite was observed for the ventricular inotropism (Fc - mN/mm²) at 12 bpm (acara = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67). A 5 min diastolic pause resulted in a strong potentiation of Fc (≅ 90 percent) of strips from jeju, which was completely abolished by ryanodine. Ryanodine also resulted in a ≅ 20 percent decrease in the Fc developed by strips from jeju at both subphysiological (12 bpm) and physiological (in vivo) frequencies. However, this effect of ryanodine reducing the Fc from jeju was completely compensated by adrenaline increments (10-9 and 10-6 M). In contrast, strips from acara were irresponsive to ryanodine, irrespective of the stimulation frequency, and increases in adrenaline concentration (to 10-9 and 10-6 M) further increased Fc. These results reinforce the hypothesis of the functionality of the SR as a common trait in neotropical ostariophysian (as jeju), while in acanthopterygians (as acara) it seems to be functional mainly in 'athletic' species.(AU)
O presente estudo analisou o papel fisiológico desempenhado pelo retículo sarcoplasmático (RS) de duas espécies de teleósteos neotropicais, o jeju, Hoplerythrinus unitaeniatus (Erythrinidae), e o acará, Geophagus brasiliensis (Cichlidae). Enquanto a frequência cardíaca registrada in vivo (fH - bpm) para o acará (79.6 ± 6.6) foi superior àquela observada para o jeju (50.3 ± 2.7), resposta inversa foi verificada para o inotropismo ventricular (Fc - mN/mm²) na frequência de estimulação de 12 bpm (acará = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67). Uma pausa diastólica de 5 min resultou em uma expressiva potenciação da Fc (≅ 90 por cento) das tiras de jeju, a qual foi completamente abolida pela rianodina. A rianodina também resultou em um decréscimo de ≅ 20 por cento na Fc desenvolvida pelas tiras de jeju tanto a frequências sub-fisiológicas (12 bpm) quanto fisiológicas (in vivo). No entanto, o decréscimo da Fc promovido pela rianodina foi completamente compensado pela adição de adrenalina (10-9 e 10-6 M). Em contraste, as tiras de acará foram irresponsivas à rianodina, independentemente da frequência de estimulação utilizada, fazendo com que a adição de adrenalina (10-9 e 10-6 M) resultasse em incrementos ainda maiores da Fc. Esses resultados reforçam a hipótese de que a funcionalidade do RS seja uma característica comum aos ostariofíseos neotropicais (como o jeju), enquanto nos acantopterígios (como o acará) esta organela parece ser funcional principalmente em espécies ativas.(AU)