RESUMO
The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. The SERCA-LVAD trial was one of a program of AAV1/SERCA2a cardiac gene therapy trials including CUPID1, CUPID 2 and AGENT trials. Enroled subjects were randomised to receive a single intracoronary infusion of 1 × 1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients at 3 years follow up, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort which may help guide future trial design.
Assuntos
Insuficiência Cardíaca , Coração Auxiliar , Adulto , Estudos de Viabilidade , Terapia Genética , Vetores Genéticos/genética , Insuficiência Cardíaca/terapia , Humanos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Gene therapy based on viral vectors offers great potential for the study and the treatment of cardiac diseases. Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac tissue from failing (dilated cardiomyopathy) hearts was also used to validate the model in a more translational setting. LMS were cultured at physiological sarcomere length for 72-h under electrical stimulation. Two recombinant AAV serotypes (AAV6 and AAV9) at different multiplicity of infection (MOI) expressing enhanced green fluorescent protein (eGFP) were added to the surface of rat LMS. AAV6 at 20,000 MOI proved to be the most suitable serotype without affecting LMS contractility or kinetics and showing high transduction and penetrability efficiency in rat LMS. This serotype exhibited 40% of transduction efficiency in cardiomyocytes and stromal cells while 20% of the endothelial cells were transduced. With great translational relevance, this protocol introduces the use of LMS as a model for nucleic acid-based therapies, allowing the acceleration of preclinical studies for cardiac diseases.
RESUMO
A special in vitro model maintained with ultrathin cardiac slices with a preserved architecture, multi-cellularity, and physiology of the heart tissue was used. In our experiments, we performed label-free quantitative SWATH-MS proteomic analysis of the adult myocardial slices in vitro after biomimetic electromechanical stimulation. Rat myocardial slices were stretched to sarcomere lengths (SL) within the physiological range of 1.8-2.2 µm. Electromechanically stimulated slices were compared with slices cultured without electromechanical stimulation (unloaded and nonstimulated-TW) on a liquid-air interface and with fresh myocardial slices (0 h-C). Quantitative (relative) proteomic analyses were performed using a label-free SWATH-MS technique on a high-resolution microLC-MS/MS TripleTOF 5600+ system (SCIEX). The acquired MS/MS spectra from the DDA LC-MS/MS analyses of the rat heart samples were searched against the UniProt Rattus norvegicus database (version of 15.05.2018) using the Paragon algorithm incorporated into ProteinPilot 4.5 (SCIEX) software. The highest number of differential proteins was observed in the TW group-121 when compared to the C group. In the 1.8 and 2.2 groups, 79 and 52 proteins present at a significantly different concentration from the control samples were found, respectively. A substantial fraction of these proteins were common for two or more comparisons, resulting in a list of 169 significant proteins for at least one of the comparisons. This study found the most prominent changes in the proteomic pattern related to mitochondrial respiration, energy metabolism, and muscle contraction in the slices that were stretched and fresh myocardial slices cultured without electromechanical stimulation.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Animais , Biomimética , Cromatografia Líquida , Miocárdio , Proteômica/métodos , Ratos , Espectrometria de Massas em Tandem/métodosRESUMO
We recently demonstrated that patterned Parylene C films could be effectively used as a mask for directly copolymerizing proteins on polyacrylamide hydrogel (PAm). In this work, we have proved the applicability of this technique for studying the effect such platforms render on neonatal rat ventricular myocytes (NRVMs). Firstly, we have characterised topographically and mechanically the scaffolds in liquid at the nano-scale level. We thus establish that such platforms have physical properties that closely mimics the in vivo extracellular environment of cells. We have then studied the cell morphology and physiology by comparing cultures on flat uniformly-covered and collagen-patterned scaffolds. We show that micro-patterns promote the elongation of cells along the principal axis of the ridges coated with collagen. In several cases, cells also tend to create bridges across the grooves. We have finally studied cell contraction, monitoring Ca2+ cycling at a certain stimulation. Cells seeded on patterned scaffolds present significant responses in comparison to the isotropic ones.
Assuntos
Resinas Acrílicas , Ventrículos do Coração/citologia , Hidrogéis , Miócitos Cardíacos/citologia , Animais , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Módulo de Elasticidade , Matriz Extracelular , Humanos , Multimerização Proteica , RatosRESUMO
Conjugated polymers have been proposed as promising materials for scaffolds in tissue engineering applications. The restricted processability and biodegradability of conjugated polymers limit their use for biomedical applications however. Here we synthesised a block-co-polymer of aniline tetramer and PCL (AT-PCL), and processed it into fibrous non-woven scaffolds by electrospinning. We showed that fibronectin (Fn) adhesion was dependant on the AT-PCL oxidative state, with a reduced Fn unfolding length on doped membranes. Furthermore, we demonstrated the cytocompatibility and potential of these membranes to support the growth and osteogenic differentiation of MC3T3-E1 over 21 days.
RESUMO
BACKGROUND: Left ventricular assist device (LVAD) treatment is known to lead to structural and functional cellular modifications in the heart. The relevance of these changes for clinical recovery is unknown. METHODS AND RESULTS: We compared properties of cardiomyocytes obtained from tissue taken at explantation of the LVAD in patients with clinical recovery with those obtained from hearts of patients who did not show clinical recovery, thus requiring transplantation. Compared with myocytes taken at implantation, both the recovery and nonrecovery groups showed approximately 50% reduction in cell capacitance, an index of cell size. However, action potential duration shortened, L-type Ca2+ current fast inactivation was more rapid, and sarcoplasmic reticulum Ca2+ content was increased in the recovery compared with the nonrecovery group. CONCLUSIONS: These results show that specific changes in excitation-contraction coupling, and not regression of cellular hypertrophy, are specifically associated with clinical recovery after LVAD and further identify sarcoplasmic reticulum Ca2+ handling as a key functional determinant in patients with heart failure.
Assuntos
Sinalização do Cálcio , Cálcio/análise , Insuficiência Cardíaca/terapia , Coração Auxiliar , Miócitos Cardíacos/química , Retículo Sarcoplasmático/ultraestrutura , Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Fármacos Cardiovasculares/uso terapêutico , Tamanho Celular , Terapia Combinada , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Ventrículos do Coração/patologia , Humanos , Transporte de Íons/efeitos dos fármacos , Miócitos Cardíacos/patologia , Indução de Remissão , Retículo Sarcoplasmático/química , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/terapiaRESUMO
BACKGROUND: Myocytes from failing hearts produce slower and smaller Ca(2+) transients associated with reduction in expression of sarcoplasmic reticulum (SR) Ca(2+) ATPase and an overexpression of Na(+)/Ca(2+) exchanger. Since the physiological role of both these proteins is competing for, and removing, Ca(2+) from the cytoplasm, overexpression of the exchanger may compensate for less effective SR Ca(2+) uptake. This study demonstrates this compensatory effect and provides a quantitative description of the results. METHODS: Ventricular myocytes from transgenic mice overexpressing the Na(+)/Ca(2+) exchanger (TR) and nontransgenic littermates (NON) were used. Cell shortening, cytoplasmic [Ca] (using indo-1 AM) and electrophysiological parameters were monitored. RESULTS: TR myocytes displayed faster Ca(2+) transients and twitches compared with NON myocytes. Superfusion with thapsigargin prolonged the time-course of Ca(2+) transients of TR myocytes until these were equal to the ones measured in NON myocytes. The amount of SR Ca(2+)-ATPase (SERCA) inhibition needed to obtain such transients was calculated as a function of V(max) for the Ca(2+) flux via SERCA and found to be 28%. In TR myocytes V(max) for the Ca(2+) flux via Na(+)/Ca(2+)exchange was 240% of NON myocytes. When Ca(2+) transients in TR myocytes were slowed by thapsigargin to similar values to the ones recorded in NON myocytes, SR Ca(2+) content was also correspondingly reduced. CONCLUSIONS: The results suggest that in pathophysiological conditions where there is a reduction in SERCA function, overexpression of Na(+)/Ca(2+) exchanger can compensate and allow normal Ca(2+) homeostasis to be maintained. In mouse ventricular myocytes a 2.4-fold increase in Na(+)/Ca(2+) exchange activity compensates for a reduction in SERCA function by 28% so maintaining the duration of the Ca(2+) transient.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Miocárdio/metabolismo , Retículo Sarcoplasmático/enzimologia , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Técnicas de Cultura de Células , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Miocárdio/enzimologia , Tapsigargina/farmacologiaRESUMO
Overexpression of the sarcoplasmic reticulum Ca ATPase (SERCA2a) produces positive inotropism and it has been proposed as a promising strategy to counteract defective excitation-contraction coupling in the failing heart. However, the effects of overexpressing SERCA2a on action potential duration (APD), which can affect diastolic parameters in the heart, is unknown. We, therefore, investigated the relationship between SERCA2a overexpression and APD in adult rabbit ventricular myocytes which were cultured for 48 h. Overexpression of SERCA2a was achieved by infection with an adenovirus carrying both SERCA2a and GFP independently driven by CMV promoters, Ad.SERCA2a. Myocytes infected with Ad.GFP only and/or non-infected myocytes were used as controls. Electrophysiological measurements were taken using switch clamping with 15-25 M Omega resistance microelectrodes. In Ad.SERCA2a infected myocytes, APD was significantly reduced compared with both groups of control cells at 0.5 Hz (APD50 (ms) non-infected: 481+/-98, n=12; Ad.GFP: 464+/-85, n=11; Ad.SERCA2a: 285+/-69, n=13 (mean+/-S.E.M.) and at 1 Hz (APD50 (ms) non-infected: 375+/-64, n=22; Ad.GFP: 363+/-47, n=18; Ad.SERCA2a: 231+/-54, n=24). Using AP voltage-clamping, we recorded a 0.2 mM Cd-sensitive current which can be ascribed to Ca current flowing during the AP. The integral of this current was reduced in Ad.SERCA2a myocytes compared with control (non-infected charge (pC): 27.5+/-4.2, n=8; Ad.SERCA2a: 15.5+/-4.1, n=11; P<0.01). Using AP clamping during the loading protocol, to take into account changes in APD, SR Ca content (assessed by integrating a 20 mM caffeine-induced inward current) was significantly larger in Ad.SERCA2a compared with both controls (SR Ca content (microM/l non-mitochondrial volume): non-infected: 25.5+/-7, n=8; Ad.GFP: 25.7+/-11, n=6; Ad.SERCA2a: 80.5+/-19, n=8). In conclusion, this study shows that SR Ca content is increased despite decreased Ca entry after overexpression of SERCA2a, and this can lead to positive inotropism. This effect coupled with shorter APD may be a useful therapeutic modality in heart failure.
Assuntos
Potenciais de Ação/fisiologia , ATPases Transportadoras de Cálcio/metabolismo , Miócitos Cardíacos/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Cádmio/metabolismo , Canais de Cálcio Tipo L/metabolismo , ATPases Transportadoras de Cálcio/genética , Células Cultivadas , Masculino , Contração Muscular/fisiologia , Técnicas de Patch-Clamp , Coelhos , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , TransfecçãoRESUMO
An increased phospholamban (PLB)-to-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) ratio has been suggested to contribute to the slowing of relaxation in failing human ventricle. We have used an adenoviral vector carrying the sequence for PLB to increase this ratio in isolated adult rat ventricular myocytes, and we have examined the functional consequences. With use of adenoviral vectors, the PLB content of adult rat myocytes was increased 2.73-fold, with SERCA2a levels unchanged. Maximum contraction amplitude of PLB-overexpressing myocytes was decreased to 6.9 +/- 0.3% shortening compared with 11.2 +/- 0.8% for 24-h controls (Con; P < 0.001, 5 preparations, 103 myocytes). Maximum rates of shortening and relengthening were also significantly decreased. Ca(2+) transient amplitudes were slightly depressed, and time to 50% decay of the transients was significantly increased: 237 +/- 18 (n = 14 myocytes) and 432 +/- 32 ms in Con and PLB (n = 15) myocytes, respectively (P < 0.001). The amount of Ca(2+) in the sarcoplasmic reticulum stores was reduced by 21% (P < 0.05). Relaxation was significantly slower in PLB than in Con myocytes when the Na(+)/Ca(2+) exchanger was blocked but not when sarcoplasmic reticulum Ca(2+) uptake was inhibited. Adenovirus infection with Ad.RSV.PLB was therefore able to produce functional changes in adult cardiac myocytes within 24 h, consistent with overexpression of PLB and similar to those seen in failing human heart.
Assuntos
Adenoviridae/genética , Proteínas de Ligação ao Cálcio/genética , Função Ventricular , Animais , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , DNA Recombinante , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Contração Miocárdica/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Tapsigargina/farmacologia , Transfecção , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
We demonstrate a simple, accurate and versatile method to manipulate Parylene C, a material widely known for its high biocompatibility, and transform it to a substrate that can effectively control the cellular microenvironment and consequently affect the morphology and function of the cells in vitro. The Parylene C scaffolds are fabricated by selectively increasing the material's surface water affinity through lithography and oxygen plasma treatment, providing free bonds for attachment of hydrophilic biomolecules. The micro-engineered constructs were tested as culture scaffolds for rat ventricular fibroblasts and neonatal myocytes (NRVM), toward modeling the unique anisotropic architecture of native cardiac tissue. The scaffolds induced the patterning of extracellular matrix compounds and therefore of the cells, which demonstrated substantial alignment compared to typical unstructured cultures. Ca(2+) cycling properties of the NRVM measured at rates of stimulation 0.5-2 Hz were significantly modified with a shorter time to peak and time to 90% decay, and a larger fluorescence amplitude (p < 0.001). The proposed technique is compatible with standard cell culturing protocols and exhibits long-term pattern durability. Moreover, it allows the integration of monitoring modalities into the micro-engineered substrates for a comprehensive interrogation of physiological parameters.
Assuntos
Técnicas de Cultura de Células/instrumentação , Polímeros/química , Biologia Sintética/instrumentação , Alicerces Teciduais/química , Xilenos/química , Animais , Células Cultivadas , Interações Hidrofóbicas e Hidrofílicas , Miócitos Cardíacos/citologia , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/instrumentaçãoRESUMO
The sodium-calcium exchanger (NCX) is an electrogenic transporter that is widely expressed in different tissues. In the heart, the NCX plays important roles in calcium ion homeostasis, excitation-contraction coupling and the electrophysiological properties of cardiac myocytes. Precise determination of the roles of the NCX has somewhat been hampered by a lack of selective small molecule inhibitors. In this issue of the BJP, Jost and colleagues present data on a new NCX inhibitor, ORM-10103, which has submicromolar EC50 values against cardiac forward and reverse exchange activity. The compound exhibits improved selectivity over existing small molecule NCX inhibitors and, in particular, appears to be without effect on L-type calcium channels at high concentrations. ORM-10103 could therefore have significant value for studies of the (patho)physiological roles of the NCX in the heart. Further pharmacological studies are required to investigate the actions of ORM-10103 on cardiac cells and tissues and to determine its effects on non-cardiac NCX isoforms.
Assuntos
Antiarrítmicos/farmacologia , Benzopiranos/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Piridinas/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Animais , Feminino , MasculinoAssuntos
Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Citoplasma/metabolismo , Modelos Animais de Doenças , Humanos , Contração Miocárdica , Fosforilação , Ligação Proteica , RatosRESUMO
Clenbuterol, a compound classified as a beta2-adrenoceptor (AR) agonist, has been employed in combination with left ventricular assist devices (LVADs) to treat patients with severe heart failure. Previous studies have shown that chronic administration of clenbuterol affects cardiac excitation-contraction coupling. However, the acute effects of clenbuterol and the signaling pathway involved remain undefined. We investigated the acute effects of clenbuterol on isolated ventricular myocyte sarcomere shortening, Ca2+ transients, and L-type Ca2+ current and compared these effects to two other clinically used beta2-AR agonists: fenoterol and salbutamol. Clenbuterol (30 microM) produced a negative inotropic response, whereas fenoterol showed a positive inotropic response. Salbutamol had no significant effects. Clenbuterol reduced Ca2+ transient amplitude and L-type Ca2+ current. Selective beta1-AR blockade did not affect the action of clenbuterol on sarcomere shortening but significantly reduced contractility in the presence of fenoterol and salbutamol (P < 0.05). Incubation with 2 microg/ml pertussis toxin significantly reduced the negative inotropic effects of 30 microM clenbuterol. In addition, overexpression of inhibitory G protein (Gi) by adenoviral transfection induced a stronger clenbuterol-mediated negative inotropic effect, suggesting the involvement of the Gi protein. We conclude that clenbuterol does not increase and, at high concentrations, significantly depresses contractility of isolated ventricular myocytes, an effect not seen with fenoterol or salbutamol. In its negative inotropism, clenbuterol predominantly acts through Gi, and the consequent downstream signaling pathways activation may explain the beneficial effects observed during chronic administration of clenbuterol in patients treated with LVADs.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Clembuterol/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Animais , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fenoterol/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Homeostase , Antagonistas Muscarínicos/farmacologia , Miócitos Cardíacos/metabolismo , Ratos , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , TransfecçãoRESUMO
The direct influence of Na+-K+ pump activity on the ability of the Na+-Ca2+ exchanger to remove Ca2+ was investigated in isolated adult rabbit ventricular myocytes. Cell shortening was measured using an edge-detection system. Cytoplasmic [Ca2+] was monitored using the fluorescent indicator indo-1. Electrophysiological parameters were recorded using high-resistance microelectrodes. The Na+-K+ pump was rapidly inhibited by removal of extracellular K+ and measurements were taken almost immediately to minimise effects on other cellular compartments. Activity of the Na+-Ca2+ exchanger was monitored during release of Ca2+ from the sarcoplasmic reticulum (SR) elicited by rapid application of 15 mM caffeine. When Na+-K+ pump activity was affected by K+ removal, cell relaxation and indo-1 fluorescence decline were slowed by approximately 40 %. The charge calculated by integrating the caffeine-induced transient inward current was unchanged, suggesting that there was no difference in the SR Ca2+ content in the two conditions. However Ca2+ flux via the Na+-Ca2+ exchanger was slower when the Na+-K+ pump was inhibited. Similar experiments were performed by inhibiting the Na+-K+ pump using 0.5 mM strophanthidin. In this condition similar results to the ones observed by K+ removal were obtained, suggesting a specific role of the Na+-K+ pump in the phenomenon observed. This study suggests that the activity of the Na+-K+ pump influences Na+-Ca2+ exchanger function in the absence of changes in SR Ca2+ content. This can be explained by a slower removal of Na+ from the subsarcolemmal space. The source of the increase in subsarcolemmal [Na+] requires further investigation. However, calculations derived from modelling suggest that the Na+-Ca2+ exchanger itself could be involved.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/citologia , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Cafeína/farmacologia , Corantes Fluorescentes/farmacologia , Ventrículos do Coração/citologia , Técnicas In Vitro , Indóis/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Potássio/farmacocinética , Coelhos , Retículo Sarcoplasmático/metabolismo , Sódio/metabolismoRESUMO
We investigated the effect of intracellular acidosis (imposed by NH4Cl prepulses) on the relaxation and decline in intracellular Ca2+ (using indo 1 fluorescence) of isolated cardiac myocytes from the guinea pig. Acidosis produced a decrease in contraction and a prolongation of the fluorescence transient. The rate of decline in fluorescence after a rapid-cooling contracture was slower in acidosis compared with control. The decline in fluorescence after a rapid-cooling contracture in the presence of 10 mM caffeine was greatly slowed during acidosis, suggesting that Na+/Ca2+ exchange is affected. We recorded indo 1 fluorescence and the transient inward current in voltage-clamped cells on rapid application of 10 mM caffeine under control conditions and in acidosis. The amplitude of the transient increase in fluorescence was reduced in acidosis and the decline in fluorescence slowed. The current showed no difference in amplitude in acidosis, but the time to 50% recovery was increased by 57%. When amiloride or ethylisopropylamiloride was present, no differences in the current were found between control and acidosis, and the times to 50% recovery were similar. We conclude that intracellular acidosis slows Ca2+ efflux via Na+/Ca2+ exchange because of an increase in intracellular Na+ due to enhanced Na+/H+ exchange activity.
Assuntos
Acidose/metabolismo , Acidose/fisiopatologia , Proteínas de Transporte/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Acidose/induzido quimicamente , Cloreto de Amônio/farmacologia , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Temperatura Baixa , Eletrofisiologia , Cobaias , Coração/efeitos dos fármacos , Coração/fisiopatologia , Indóis , Contração Miocárdica/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio , Fatores de TempoRESUMO
This study investigates the contribution of Ca2+ entry via sarcolemmal (SL) Ca2+ channels to the Ca2+ transient and its relationship with sarcoplasmic reticulum (SR) Ca2+ content during steady-state contraction in guinea pig and rat ventricular myocytes. The action potential clamp technique was used to obtain physiologically relevant changes in membrane potential. A method is shown that allows calculation of Ca2+ entry through the SL Ca2+ channels by measuring Cd(2+)-sensitive current during the whole cardiac cycle. SR Ca2+ content was calculated from caffeine-induced transient inward current. In guinea pig cardiac myocytes stimulated at 0.5 Hz and 0.2 Hz, Ca2+ entry through SL Ca2+ channels during a cardiac cycle was approximately 30% and approximately 50%, respectively, of the SR Ca2+ content. In rat myocytes Ca2+ entry via SL Ca2+ channels at 0.5 Hz was approximately 3.5% of the SR Ca2+ content. In the presence of 500 nM thapsigargin Ca2+ entry via SL Ca2+ channels in guinea pig cardiac cells was 39% greater than in controls, suggesting a larger contribution of this mechanism to the Ca2+ transient when the SR is depleted of Ca2+. These results provide quantitative support to the understanding of the relationship between Ca2+ entry and the SR Ca2+ content and may help to explain differences in the Ca2+ handling observed in different species.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Coração/fisiologia , Retículo Sarcoplasmático/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cádmio/farmacologia , Cafeína/farmacologia , Células Cultivadas , Condutividade Elétrica , Cobaias , Coração/efeitos dos fármacos , Ventrículos do Coração , Cinética , Miocárdio/metabolismo , Ácido Niflúmico/farmacologia , Técnicas de Patch-Clamp , Ratos , Retículo Sarcoplasmático/fisiologia , Tapsigargina/farmacologia , Fatores de TempoRESUMO
1. The aim of this study was to investigate the effects of 20 mM extracellular lactate on Ca2+ regulation mechanisms in enzymatically isolated single guinea-pig cardiac myocytes. 2. The activities of the Ca2+ regulation mechanisms during application of lactate were studied using rapid cooling contractures (RCCs) and fast application of caffeine. Cytoplasmic Ca2+ was monitored using the fluorescent indicator indo-1. 3. After application of 20 mM lactate for 5 min, the diastolic level of Ca2+ was increased. The change in cytoplasmic Ca2+ elicited by stimulation (Ca2+ transient) was also changed. With lactate, the amplitude of the Ca2+ transient was smaller, and its time course was slower compared with control. 4. The recovery of cytoplasmic Ca2+ during rewarming after rapid cooling in lactate was slower than under control conditions. When the rewarming was performed either in Na(+)- and Ca(2+)-free solution or in the presence of 10 mM caffeine, the rate of recovery of cytoplasmic Ca2+ in lactate was slower than under control conditions, suggesting that the activity of both SR Ca2+ uptake and Na(+)-Ca2+ exchange is affected by lactate. 5. Cytoplasmic Ca2+ recovery during application of 10 mM caffeine in lactate was slower than in the control. The rate of recovery of the caffeine-induced transient inward current was also slower supporting the hypothesis of a slower Ca2+ extrusion brought about by Na(+)-Ca2+ exchange. 6. The relative contribution of the Ca2+ extrusion mechanisms in the presence of lactate was investigated using paired RCCs. In lactate, a second RCC (RCC2) induced immediately after recovery from the first (RCC1) was greatly reduced compared with the control. RCC2/RCC1 x 100 in lactate was 39% and RCC2/RCC1 x 100 in control conditions was 60%, suggesting that the net sarcoplasmic reticulum Ca2+ uptake is smaller in the presence of lactate. 7. When Na(+)-free Ca2+ solution was used during the paired RCCs and rewarming, RCC2/RCC1 x 100 was increased to 96 and 95% in lactate and control conditions, respectively, implying that Ca2+ efflux from the cell can be maintained by the Na(+)-Ca2+ exchanger and that other Ca2+ removal mechanisms (mitochondria and sarcolemmal Ca(2+)-ATPase) remain largely unchanged in the presence of lactate.
Assuntos
Cálcio/metabolismo , Ácido Láctico/farmacologia , Contração Miocárdica/efeitos dos fármacos , Animais , Cafeína/farmacologia , Temperatura Baixa , Eletrofisiologia , Corantes Fluorescentes , Cobaias , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Indóis , Masculino , Miocárdio/citologia , Miocárdio/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Sódio/metabolismo , Sódio/fisiologiaRESUMO
The effects of rest on a Ca(2+)-depleted sarcoplasmic reticulum (SR) in guinea pig ventricular myocytes were investigated. Cell shortening was measured using a video edge-detection system, and cytoplasmic Ca2+ was monitored using the fluorescent indicator indo-1. Rapid cooling and rewarming in the presence of 10 mM caffeine were used to deplete the SR of Ca2+. The resting cell was then superfused for variable time intervals with a normal Tyrode solution containing 2 mM Ca2+. Another rapid cooling in caffeine was performed to assess the SR Ca2+ load at the end of rest. Rapid cooling after 1- and 2-min rest elicited an increase of indo-1 fluorescence of 51.9 +/- 7.7 (n = 17) and 72.7 +/- 6.7% of control (n = 9), respectively. This increase was not detectable when Ca2+ was absent from the superfusing solution. In contrast, the increase was larger when external Ca2+ was elevated to 4 mM. Nickel (5 mM) and nifedipine (20 microM) added to the superfusing solution during the rest interval did not alter the increase in indo-1 fluorescence. We conclude that Ca2+ is reaccumulated by a depleted SR during rest. Although this Ca2+ seems to originate from the extracellular space, its route from there to the SR is unclear.
Assuntos
Cálcio/deficiência , Cálcio/metabolismo , Miocárdio/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Cafeína/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Temperatura Baixa , Cobaias , Ventrículos do Coração , Indóis , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Níquel/farmacologia , Nifedipino/farmacologia , Concentração Osmolar , Descanso , Temperatura , Fatores de TempoRESUMO
We have estimated sarcoplasmic reticulum calcium content using rapid application of caffeine on voltage clamped, isolated guinea-pig ventricular myocytes. Caffeine induces the release of calcium from the sarcoplasmic reticulum and this calcium is extruded from the cells by the sarcolemmal Na/Ca exchange. Integrating the inward Na/Ca exchange current thus allows estimations of sarcoplasmic reticulum calcium content. Ventricular myocytes were stimulated to reach new steady-states by action potential voltage clamps of varying duration. Once contractile steady-state had been reached caffeine was rapidly applied in place of the next action potential and sarcoplasmic reticulum calcium content measured. Prolonging the action potential duration increased sarcoplasmic reticulum calcium content and vice-versa. This calcium loading may underlie the positive inotropic effect of increased action potential duration.