RESUMO
Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.
Assuntos
Planárias , Animais , Cálcio/metabolismo , Ciclo Celular , DNA/metabolismo , Estimulação Elétrica , Planárias/genética , Planárias/metabolismo , Radiação IonizanteRESUMO
RATIONALE: Assessing the underlying ionic currents during a triggered action potential (AP) in intact perfused hearts offers the opportunity to link molecular mechanisms with pathophysiological problems in cardiovascular research. The developed loose patch photolysis technique can provide striking new insights into cardiac function at the whole heart level during health and disease. OBJECTIVE: To measure transmembrane ionic currents during an AP to determine how and when surface Ca(2+) influx that triggers Ca(2+)-induced Ca(2+) release occurs and how Ca(2+)-activated conductances can contribute to the genesis of AP phase 2. METHODS AND RESULTS: Loose patch photolysis allows the measurement of transmembrane ionic currents in intact hearts. During a triggered AP, a voltage-dependent Ca(2+) conductance was fractionally activated (dis-inhibited) by rapidly photo-degrading nifedipine, the Ca(2+) channel blocker. The ionic currents during a mouse ventricular AP showed a fast early component and a slower late component. Pharmacological studies established that the molecular basis underlying the early component was driven by an influx of Ca(2+) through the L-type channel, CaV 1.2. The late component was identified as an Na(+)-Ca(2+) exchanger current mediated by Ca(2+) released from the sarcoplasmic reticulum. CONCLUSIONS: The novel loose patch photolysis technique allowed the dissection of transmembrane ionic currents in the intact heart. We were able to determine that during an AP, L-type Ca(2+) current contributes to phase 1, whereas Na(+)-Ca(2+) exchanger contributes to phase 2. In addition, loose patch photolysis revealed that the influx of Ca(2+) through L-type Ca(2+) channels terminates because of voltage-dependent deactivation and not by Ca(2+)-dependent inactivation, as commonly believed.
Assuntos
Potenciais de Ação , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Ventrículos do Coração/metabolismo , Microscopia de Fluorescência/métodos , Fotólise , Potenciais de Ação/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Acoplamento Excitação-Contração , Ventrículos do Coração/efeitos dos fármacos , Preparação de Coração Isolado , Cinética , Masculino , Camundongos Endogâmicos BALB C , Nifedipino/química , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoAssuntos
Sinalização do Cálcio , Acoplamento Excitação-Contração , Miócitos Cardíacos/metabolismo , Estresse Mecânico , Potenciais de Ação , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Hidrogéis/química , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp/métodos , CoelhosRESUMO
Abnormal intracellular Ca(2+) cycling plays a key role in cardiac dysfunction, particularly during the setting of ischemia/reperfusion (I/R). During ischemia, there is an increase in cytosolic and sarcoplasmic reticulum (SR) Ca(2+). At the onset of reperfusion, there is a transient and abrupt increase in cytosolic Ca(2++), which occurs timely associated with reperfusion arrhythmias. However, little is known about the subcellular dynamics of Ca(2+) increase during I/R, and a possible role of the SR as a mechanism underlying this increase has been previously overlooked. The aim of the present work is to test two main hypotheses: (1) An increase diastolic Ca(2+) sparks frequency (cspf) constitutes a mayor substrate for the ischemia-induced diastolic Ca(2+) increase; (2) an increase in cytosolic Ca(2+) pro-arrhythmogenic events (Ca(2+) waves), mediates the abrupt diastolic Ca(2+) rise at the onset of reperfusion. We used confocal microscopy on mouse intact hearts loaded with Fluo-4. Hearts were submitted to global I/R (12/30 min) to assess epicardial Ca(2+) sparks in the whole heart. Intact heart sparks were faster than in isolated myocytes whereas cspf was not different. During ischemia, cspf significantly increased relative to preischemia (2.07±0.33 vs. 1.13±0.20 sp/s/100 µm, n=29/34, 7 hearts). Reperfusion significantly changed Ca(2+) sparks kinetics, by prolonging Ca(2+) sparks rise time and decreased cspf. However, it significantly increased Ca(2+) wave frequency relative to ischemia (0.71±0.14 vs. 0.38±0.06 w/s/100 µm, n=32/33, 7 hearts). The results show for the first time the assessment of intact perfused heart Ca(2+) sparks and provides direct evidence of increased Ca(2+) sparks in ischemia that transform into Ca(2+) waves during reperfusion. These waves may constitute a main trigger for reperfusion arrhythmias.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Coração/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Separação Celular , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Técnicas In Vitro , Cinética , Masculino , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Perfusão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismoRESUMO
Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such as Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation but also in cell death, transcriptional activation of hypertrophy, inflammation, and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation, and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Acoplamento Excitação-Contração , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Humanos , Miócitos Cardíacos/fisiologiaRESUMO
Calsequestrin (Casq2) is a low affinity Ca(2+)-binding protein located in sarcoplasmic reticulum (SR) of cardiac myocytes. Casq2 acts as a Ca(2+) buffer regulating free Ca(2+) concentration in the SR lumen and plays a significant role in the regulation of Ca(2+) release from this intracellular organelle. In addition, there is experimental evidence supporting the hypothesis that Casq2 also modulates the activity of the cardiac Ca(2+) release channels, ryanodine receptors (RyR2). In this study, Casq2 knockout mice (Casq2-/-) were used as a model to evaluate the effects of the Casq2 on the cytosolic and intra-SR Ca(2+) dynamics, and the electrical activity in the ventricular epicardial layer of intact beating hearts. Casq2-/- mice have accelerated intra-SR Ca(2+) refilling kinetics (76 ± 22 vs. 136.5 ± 15 ms) and a reduced refractoriness of Ca(2+) release (182 ± 32 ms Casq2+/+ and 111 ± 22 ms Casq2-/- ). In addition, mice display reduced Ca(2+) alternans (67% decline in the amplitude of Ca(2+) alternans at 7 Hz, 21oC) and less T-wave alternans at the electrocardiographic level. The results presented in this paper support the idea of Casq2 acting both as a buffer and a direct regulator of the Ca(2+) release process. Finally, we propose that alterations in Ca(2+) release refractoriness shown here could explain the relationship between Casq2 function and an increase in the risk for ventricular arrhythmias.
Assuntos
Cálcio/metabolismo , Calsequestrina/genética , Miocárdio/metabolismo , Período Refratário Eletrofisiológico , Animais , Sinalização do Cálcio , Citosol/metabolismo , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Contração Miocárdica/genética , Pericárdio/metabolismo , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismoRESUMO
Inositol 1,4,5-trisphosphate (InsP(3)R)-mediated Ca(2+) signaling is a major pathway regulating multiple cellular functions in excitable and non-excitable cells. Although InsP(3)-mediated Ca(2+) signaling has been extensively described, its influence on ventricular myocardium activity has not been addressed in contracting hearts at the whole-organ level. In this work, InsP(3)-sensitive intracellular Ca(2+) signals were studied in intact hearts using laser scanning confocal microscopy and pulsed local-field fluorescence microscopy. Intracellular [InsP(3)] was rapidly increased by UV flash photolysis of membrane-permeant caged InsP(3). Our results indicate that the basal [Ca(2+)] increased after the flash photolysis of caged InsP(3) without affecting the action potential (AP)-induced Ca(2+) transients. The amplitude of the basal [Ca(2+)] elevation depended on the intracellular [InsP(3)] reached after the UV flash. Pretreatment with ryanodine failed to abolish the InsP(3)-induced Ca(2+) release (IICR), indicating that this response was not mediated by ryanodine receptors (RyR). Thapsigargin prevented Ca(2+) release from both RyR- and InsP(3)R-containing Ca(2+) stores, suggesting that these pools have similar Ca(2+) reuptake mechanisms. These results were reproduced in acutely isolated cells where photorelease of InsP(3) was able to induce changes in endothelial cells but not in AP-induced transients from cardiomyocytes. Taken together, these results suggest that IICR does not directly regulate cardiac excitation-contraction coupling. To our knowledge, this is the first demonstration of IICR in intact hearts. Consequently, our work provides a reference framework of the spatiotemporal attributes of the IICR under physiological conditions.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Inositol 1,4,5-Trifosfato/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Camundongos , Miócitos Cardíacos/metabolismo , Ligação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca(2+) release underlying Ca(2+) sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818-821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where â¼25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In â¼10% of the cases, all RyRs (2-10 channels; most frequently 3-4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg(2+) and high (50 mM) luminal Ca(2+) (Ca(lum)) or Sr(2+) solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within â¼300 µs. Coupled RyR1 were heterogeneous, where highly active RyR1 ("drivers") seemed open during the entire coupled event (P(o) = 1), while other RyR1s ("followers") displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca(2+) (Ca(cyt)) or caffeine, whereas followers flicker frequency was Ca(cyt) independent and more sensitive to inhibition by cytosolic Mg(2+). Coupled events were insensitive to varying lumen-to-cytosol Ca(2+) fluxes from â¼1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca(2+)-induced Ca(2+) release. However, coupling requires specific Ca(lum) sites, as it was lost when Ca(lum) was replaced by luminal Ba(2+) or Mg(2+). In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg(2+), Ca(cyt), and Ca(lum,) which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca(2+) sparks.
Assuntos
Trifosfato de Adenosina/fisiologia , Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Magnésio/fisiologia , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/química , Animais , Cálcio/química , Magnésio/química , Músculo Esquelético/fisiologia , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologiaRESUMO
The ventricular action potential (AP) is characterized by a fast depolarizing phase followed by a repolarization that displays a second upstroke known as phase 2. This phase is generally not present in mouse ventricular myocytes. Thus we performed colocalized electrophysiological and optical recordings of APs in Langendorff-perfused mouse hearts founding a noticeable phase 2. Ryanodine as well as nifedipine reduced phase 2. Our hypothesis is that a depolarizing current activated by Ca(2+) released from the sarcoplasmic reticulum (SR) rather than the "electrogenicity" of the L-type Ca(2+) current is crucial in the generation of mouse ventricular phase 2. When Na(+) was partially replaced by Li(+) in the extracellular perfusate or the organ was cooled down, phase 2 was reduced. These results suggest that the Na(+)/Ca(2+) exchanger functioning in the forward mode is driving the depolarizing current that defines phase 2. Phase 2 appears to be an intrinsic characteristic of single isolated myocytes and not an emergent property of the tissue. As in whole heart experiments, ventricular myocytes impaled with microelectrodes displayed a large phase 2 that significantly increases when temperature was raised from 22 to 37°C. We conclude that mouse ventricular APs display a phase 2; however, changes in Ca(2+) dynamics and thermodynamic parameters also diminish phase 2, mostly by impairing the Na(+)/Ca(2+) exchanger. In summary, these results provide important insights about the role of Ca(2+) release in AP ventricular repolarization under physiological and pathological conditions.
Assuntos
Potenciais de Ação/fisiologia , Miócitos Cardíacos/fisiologia , Função Ventricular/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Lítio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Nifedipino/farmacologia , Rianodina/farmacologia , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/fisiologia , Função Ventricular/efeitos dos fármacosRESUMO
Autonomic regulation plays a central role in cardiac contractility and excitability in numerous vertebrate species. However, the role of autonomic regulation is less understood in fish physiology. Here, we used Goldfish as a model to explore the role of autonomic regulation. A transmural electrocardiogram recording showed perfusion of the Goldfish heart with isoproterenol increased the spontaneous heart rate, while perfusion with carbamylcholine decreased the spontaneous heart rate. Cardiac action potentials obtained via sharp microelectrodes exhibited the same modifications of the spontaneous heart rate in response to isoproterenol and carbamylcholine. Interestingly, the duration of the cardiac action potentials lengthened in the presence of both isoproterenol and carbamylcholine. To evaluate cardiac contractility, the Goldfish heart was perfused with the Ca2+ indicator Rhod-2 and ventricular epicardial Ca2+ transients were measured using Pulsed Local Field Fluorescence Microscopy. Following isoproterenol perfusion, the amplitude of the Ca2+ transient significantly increased, the half duration of the Ca2+ transient shortened, and there was an observable increase in the velocity of the rise time and fall time of the Ca2+ transient, all of which are compatible with the shortening of the action potential induced by isoproterenol perfusion. On the other hand, carbamylcholine perfusion significantly reduced the amplitude of the Ca2+ transient and increased the half duration of the Ca2+ transient. These results are interesting because the effect of carbamylcholine is opposite to what happens in classically used models, such as mouse hearts, and the autonomic regulation of the Goldfish heart is strikingly similar to what has been observed in larger mammalian models resembling humans.
RESUMO
Whether intracellular Ca(2+) regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca(2+) regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca(2+) buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca(2+). Prior to introduction of the caged Ca(2+) buffer, spontaneous local Ca(2+) releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r²=0.9). The buffer markedly prolonged the decay time (T50) and moderately reduced the amplitude of the AP-induced Ca(2+) transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca(2+) was acutely released from the caged compound by flash photolysis, intracellular Ca(2+) dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca(2+) dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca(2+) regulates normal SANC automaticity on a beat-to-beat basis.
Assuntos
Relógios Biológicos/fisiologia , Cálcio/metabolismo , Nó Sinoatrial/fisiologia , Potenciais de Ação , Animais , Acoplamento Excitação-Contração , Coelhos , Nó Sinoatrial/metabolismoAssuntos
Sinalização do Cálcio , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Período Refratário Eletrofisiológico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Fibrilação Ventricular/metabolismo , Imagens com Corantes Sensíveis à Voltagem , AnimaisRESUMO
Background: The use of direct current electric stimulation (DCS) is an effective strategy to treat disease and enhance body functionality. Thus, treatment with DCS is an attractive biomedical alternative, but the molecular underpinnings remain mostly unknown. The lack of experimental models to dissect the effects of DCS from molecular to organismal levels is an important caveat. Here, we introduce the planarian flatworm Schmidtea mediterranea as a tractable organism for in vivo studies of DCS. We developed an experimental method that facilitates the application of direct current electrical stimulation to the whole planarian body (pDCS). Materials and Methods: Planarian immobilization was achieved by combining treatment with anesthesia, agar embedding, and low temperature via a dedicated thermoelectric cooling unit. Electric currents for pDCS were delivered using pulled glass microelectrodes. The electric potential was supplied through a constant voltage power supply. pDCS was administered up to six hours, and behavioral and molecular effects were measured by using video recordings, immunohistochemistry, and gene expression analysis. Results: The behavioral immobilization effects are reversible, and pDCS resulted in a redistribution of mitotic cells along the mediolateral axis of the planarian body. The pDCS effects were dependent on the polarity of the electric field, which led to either increase in reductions in mitotic densities associated with the time of pDCS. The changes in mitotic cells were consistent with apparent redistribution in gene expression of the stem cell marker smedwi-1. Conclusion: The immobilization technique presented in this work facilitates studies aimed at dissecting the effects of exogenous electric stimulation in the adult body. Treatment with DCS can be administered for varying times, and the consequences evaluated at different levels, including animal behavior, cellular and transcriptional changes. Indeed, treatment with pDCS can alter cellular and transcriptional parameters depending on the polarity of the electric field and duration of the exposure.
RESUMO
Ca2+ alternans (Ca-Alts) are alternating beat-to-beat changes in the amplitude of Ca2+ transients that frequently occur during tachycardia, ischemia, or hypothermia that can lead to sudden cardiac death. Ca-Alts appear to result from a variation in the amount of Ca2+ released from the sarcoplasmic reticulum (SR) between two consecutive heartbeats. This variable Ca2+ release has been attributed to the alternation of the action potential duration, delay in the recovery from inactivation of RYR Ca2+ release channel (RYR2), or an incomplete Ca2+ refilling of the SR. In all three cases, the RYR2 mobilizes less Ca2+ from the SR in an alternating manner, thereby generating an alternating profile of the Ca2+ transients. We used a new experimental approach, fluorescence local field optical mapping (FLOM), to record at the epicardial layer of an intact heart with subcellular resolution. In conjunction with a local cold finger, a series of images were recorded within an area where the local cooling induced a temperature gradient. Ca-Alts were larger in colder regions and occurred without changes in action potential duration. Analysis of the change in the enthalpy and Q10 of several kinetic processes defining intracellular Ca2+ dynamics indicated that the effects of temperature change on the relaxation of intracellular Ca2+ transients involved both passive and active mechanisms. The steep temperature dependency of Ca-Alts during tachycardia suggests Ca-Alts are generated by insufficient SERCA-mediated Ca2+ uptake into the SR. We found that Ca-Alts are heavily dependent on intra-SR Ca2+ and can be promoted through partial pharmacologic inhibition of SERCA2a. Finally, the FLOM experimental approach has the potential to help us understand how arrhythmogenesis correlates with the spatial distribution of metabolically impaired myocytes along the myocardium.
Assuntos
Cálcio , Miócitos Cardíacos , Cálcio/metabolismo , Sinalização do Cálcio , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Ca(+)-induced Ca(2+) release tightly controls the function of ventricular cardiac myocytes under normal and pathological conditions. Two major factors contributing to the regulation of Ca(2+) release are the cytosolic free Ca(2+) concentration and sarcoplasmic reticulum (SR) Ca(2+) content. We hypothesized that the amount of Ca(2+) released from the SR during each heart beat strongly defines the refractoriness of Ca(2+) release. To test this hypothesis, EGTA AM, a high-affinity, slow-association rate Ca(2+) chelator, was used as a tool to modify luminal SR Ca(2+) content. An analysis of the cytosolic and luminal SR Ca(2+) dynamics recorded from the epicardial layer of intact mouse hearts indicated that the presence of EGTA reduced the diastolic SR free Ca(2+) concentration and fraction of SR Ca(2+) depletion during each beat. In addition, this maneuver shortened the refractory period and accelerated the restitution of Ca(2+) release. As a consequence of the accelerated restitution, the frequency dependence of Ca(2+) alternans was significantly shifted toward higher heart rates, suggesting a role of luminal SR Ca(2+) in the genesis of this highly arrhythmogenic phenomenon. Thus, intra-SR Ca(2+) dynamics set the refractoriness and frequency dependence of Ca(2+) transients in subepicardial ventricular myocytes.
Assuntos
Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Pericárdio/metabolismo , Animais , Soluções Tampão , Citosol/metabolismo , Ácido Egtázico/farmacologia , Masculino , Camundongos , Modelos Animais , Modelos Teóricos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Pericárdio/citologia , Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
Cardiac physiology of fish models is an emerging field given the ease of genome editing and the development of transgenic models. Several studies have described the cardiac properties of zebrafish (Denio rerio). The goldfish (Carassius auratus) belongs to the same family as the zebrafish and has emerged as an alternative model with which to study cardiac function. Here, we propose to acutely study electrophysiological and systolic Ca2+ signaling in intact goldfish hearts. We assessed the Ca2+ dynamics and the electrophysiological cardiac function of goldfish, zebrafish, and mice models, using pulsed local field fluorescence microscopy, intracellular microelectrodes, and flash photolysis in perfused hearts. We observed goldfish ventricular action potentials (APs) and Ca2+ transients to be significantly longer when compared to the zebrafish. The action potential half duration at 50% (APD50) of goldfish was 370.38 ± 8.8 ms long, and in the zebrafish they were observed to be only 83.9 ± 9.4 ms. Additionally, the half duration of the Ca2+ transients was also longer for goldfish (402.1 ± 4.4 ms) compared to the zebrafish (99.1 ± 2.7 ms). Also, blocking of the L-type Ca2+ channels with nifedipine revealed this current has a major role in defining the amplitude and the duration of goldfish Ca2+ transients. Interestingly, nifedipine flash photolysis experiments in the intact heart identified whether or not the decrease in the amplitude of Ca2+ transients was due to shorter APs. Moreover, an increase in temperature and heart rate had a strong shortening effect on the AP and Ca2+ transients of goldfish hearts. Furthermore, ryanodine (Ry) and thapsigargin (Tg) significantly reduced the amplitude of the Ca2+ transients, induced a prolongation in the APs, and altogether exhibited the degree to which the Ca2+ release from the sarcoplasmic reticulum contributed to the Ca2+ transients. We conclude that the electrophysiological properties and Ca2+ signaling in intact goldfish hearts strongly resembles the endocardial layer of larger mammals.
RESUMO
The relationship between cardiac excitability and contractility depends on when Ca2+ influx occurs during the ventricular action potential (AP). In mammals, it is accepted that Ca2+ influx through the L-type Ca2+ channels occurs during AP phase 2. However, in murine models, experimental evidence shows Ca2+ influx takes place during phase 1. Interestingly, Ca2+ influx that activates contraction is highly regulated by the autonomic nervous system. Indeed, autonomic regulation exerts multiple effects on Ca2+ handling and cardiac electrophysiology. In this paper, we explore autonomic regulation in endocardial and epicardial layers of intact beating mice hearts to evaluate their role on cardiac excitability and contractility. We hypothesize that in mouse cardiac ventricles the influx of Ca2+ that triggers excitation-contraction coupling (ECC) does not occur during phase 2. Using pulsed local field fluorescence microscopy and loose patch photolysis, we show sympathetic stimulation by isoproterenol increased the amplitude of Ca2+ transients in both layers. This increase in contractility was driven by an increase in amplitude and duration of the L-type Ca2+ current during phase 1. Interestingly, the ß-adrenergic increase of Ca2+ influx slowed the repolarization of phase 1, suggesting a competition between Ca2+ and K+ currents during this phase. In addition, cAMP activated L-type Ca2+ currents before SR Ca2+ release activated the Na+-Ca2+ exchanger currents, indicating Cav1.2 channels are the initial target of PKA phosphorylation. In contrast, parasympathetic stimulation by carbachol did not have a substantial effect on amplitude and kinetics of endocardial and epicardial Ca2+ transients. However, carbachol transiently decreased the duration of the AP late phase 2 repolarization. The carbachol-induced shortening of phase 2 did not have a considerable effect on ventricular pressure and systolic Ca2+ dynamics. Interestingly, blockade of muscarinic receptors by atropine prolonged the duration of phase 2 indicating that, in isolated hearts, there is an intrinsic release of acetylcholine. In addition, the acceleration of repolarization induced by carbachol was blocked by the acetylcholine-mediated K+ current inhibition. Our results reveal the transmural ramifications of autonomic regulation in intact mice hearts and support our hypothesis that Ca2+ influx that triggers ECC occurs in AP phase 1 and not in phase 2.
RESUMO
In the heart, Ca2+ influx through L-type Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum. In most mammals, this influx occurs during the ventricular action potential (AP) plateau phase 2. However, in murine models, the influx through L-type Ca2+ channels happens in early repolarizing phase 1. The aim of this work is to assess if changes in the open probability of 4-aminopyridine (4-AP)-sensitive Kv channels defining the outward K+ current during phase 1 can modulate Ca2+ currents, Ca2+ transients, and systolic pressure during the cardiac cycle in intact perfused beating hearts. Pulsed local-field fluorescence microscopy and loose-patch photolysis were used to test the hypothesis that a decrease in a transient K+ current (Ito) will enhance Ca2+ influx and promote a larger Ca2+ transient. Simultaneous recordings of Ca2+ transients and APs by pulsed local-field fluorescence microscopy and loose-patch photolysis showed that a reduction in the phase 1 repolarization rate increases the amplitude of Ca2+ transients due to an increase in Ca2+ influx through L-type Ca2+ channels. Moreover, 4-AP induced an increase in the time required for AP to reach 30% repolarization, and the amplitude of Ca2+ transients was larger in epicardium than endocardium. On the other hand, the activation of Ito with NS5806 resulted in a reduction of Ca2+ current amplitude that led to a reduction of the amplitude of Ca2+ transients. Finally, the 4-AP effect on AP phase 1 was significantly smaller when the L-type Ca2+ current was partially blocked with nifedipine, indicating that the phase 1 rate of repolarization is defined by the competition between an outward K+ current and an inward Ca2+ current.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Contração Miocárdica/fisiologia , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Eletrofisiologia/métodos , Ventrículos do Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contração Miocárdica/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
OBJECTIVE: To investigate the importance of the phosphorylation of Ser16 and Thr17 sites of phospholamban (PLN) on intracellular Ca2+ (Cai2+) handling and contractile recovery of the stunned myocardium. METHODS: Cai2+ (Rhod-2, pulsed local-field fluorescence microscopy) and contractility (isovolumic left ventricular developed pressure, LVDP) were simultaneously measured in Langendorff perfused hearts from transgenic mice expressing either intact PLN (PLN-WT) or PLN with both phosphorylation sites mutated to Ala (PLN-DM), subjected to 12 min of global ischemia followed by a reperfusion period of 30 min. RESULTS: Pre-ischemic values of Cai2+ and LVDP were similar in both groups. In PLN-WT, a transient increase in Thr17 phosphorylation at early reperfusion preceded a recovery of Ca2+ transient amplitude, virtually completed by the end of reperfusion. LVDP at 30 min reperfusion was 67.9+/-7.6% of pre-ischemic values, n=14. In contrast, in PLN-DM, there was a poor recovery of Cai2+ transient amplitude and LVDP was significantly lower (28.3+/-6.7%, n=11, 30 min reperfusion) than in PLN-WT hearts. Although myofilament Ca2+ responsiveness and troponin I (TnI) degradation did not differ between groups, the episodes of mechanical alternans, typical of Cai2+ overload, were significantly prolonged in PLN-DM vs. PLN-WT hearts. CONCLUSIONS: PLN phosphorylation appears to be crucial for the mechanical and Cai2+ recovery during stunning and protective against the mechanical abnormalities typical of Cai2+ overload. The importance of PLN phosphorylation would primarily reside in the Thr17 residue, which is phosphorylated during the critical early phase of reperfusion. Our results emphasize that, although ablation of PLN phosphorylation does not affect basal contractility, it does alter Ca2+ handling and mechanical performance under stress situations.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Líquido Intracelular/metabolismo , Miocárdio/metabolismo , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Camundongos , Camundongos Transgênicos , Mutação , Contração Miocárdica , Perfusão , FosforilaçãoRESUMO
In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).