RESUMO
We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered "undruggable".
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala/métodos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Técnicas Biossensoriais , Proteínas de Ligação ao Cálcio/química , Sobrevivência Celular , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/químicaRESUMO
RATIONALE: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. OBJECTIVE: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. METHODS AND RESULTS: Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). CONCLUSIONS: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.
Assuntos
Simulação por Computador , Acoplamento Excitação-Contração/fisiologia , Células-Tronco Mesenquimais/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Comunicação Parácrina/fisiologia , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Camundongos , RatosRESUMO
Autophagy, macroautophagy and chaperone-mediated autophagy (CMA), are upregulated in pressure overload (PO) hypertrophy. In this study, we targeted this process at its induction using 3 methyladenine and at the lysosomal level using chloroquine and evaluated the effects of these modulations on cardiac function and myocyte ultrastructure. Sprague-Dawley rats weighing 200 g were subjected to ascending aortic banding. After 1 week of PO, animals were randomized to receive 3 methyladenine versus chloroquine, intraperitoneally, for 2 weeks at a dose of 40 and 50 mg/kg/day, respectively. Saline injection was used as control. Chloroquine treatment, in PO, resulted in regression in cardiac hypertrophy but with significant impairments in cardiac relaxation and contractility. Ultrastructurally, chloroquine accentuated mitochondrial fragmentation and cristae destruction with a plethora of autophagosomes containing collapsed mitochondria and lysosomal lamellar bodies. In contrast, 3 methyladenine improved cardiac function and attenuated mitochondrial fragmentation and autophagososme formation. Markers of macroautophagy and CMA were significantly decreased in the chloroquine group; whereas 3 methyladenine treatment significantly attenuated macroautophagy with a compensatory increase in CMA. Furthermore, chloroquine accentuated PO induced oxidative stress through the further decrease in the expression of manganese superoxide dismutase; whereas, 3 MA had a completely opposite effect. Taken together, these data suggest that high-dose chloroquine, in addition to its effect on the autophagy-lysosome pathway, significantly impairs mitochondrial antioxidant buffering capacity and accentuates oxidative stress and mitochondrial dysfunction in PO hypertrophy; highlighting, the cautious administration of this drug in high oxidative stress conditions, such as pathological hypertrophy or heart failure.
RESUMO
RATIONALE: The Calcium Up-Regulation by Percutaneous Administration of Gene Therapy In Cardiac Disease (CUPID 1) study was a phase 1/phase 2 first-in-human clinical gene therapy trial using an adeno-associated virus serotype 1 (AAV1) vector carrying the sarcoplasmic reticulum calcium ATPase gene (AAV1/SERCA2a) in patients with advanced heart failure. The study explored potential benefits of the therapy at 12 months, and results were previously reported. OBJECTIVE: To report long-term (3-year) clinical effects and transgene expression in the patients in CUPID 1. METHODS AND RESULTS: A total of 39 patients with advanced heart failure who were on stable, optimal heart failure therapy were randomized to receive intracoronary infusion of AAV1/SERCA2a in 1 of 3 doses (low-dose, 6×10(11) DNase-resistant particles; mid-dose, 3×10(12) DNase-resistant particles; and high-dose, 1×10(13) DNase-resistant particles) versus placebo. The following recurrent cardiovascular and terminal events were tracked for 3 years in all groups: myocardial infarction, worsening heart failure, heart failure-related hospitalization, ventricular assist device placement, cardiac transplantation, and death. The number of cardiovascular events, including death, was highest in the placebo group, high but delayed in the low- and mid-dose groups, and lowest in the high-dose group. Evidence of long-term transgene presence was also observed in high-dose patients. The risk of prespecified recurrent cardiovascular events was reduced by 82% in the high-dose versus placebo group (P=0.048). No safety concerns were noted during the 3-year follow-up. CONCLUSIONS: After a single intracoronary infusion of AAV1/SERCA2a in patients with advanced heart failure, positive signals of cardiovascular events persist for years.
Assuntos
Terapia Genética , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Adulto , Idoso , Dependovirus/genética , Método Duplo-Cego , Feminino , Vetores Genéticos/genética , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/fisiopatologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Tempo , Transgenes/genética , Resultado do TratamentoRESUMO
Recently, the impact of small ubiquitin-related modifier 1 (SUMO-1) on the regulation and preservation of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2a) function was discovered. The amount of myocardial SUMO-1 is decreased in failing hearts, and its knockdown results in severe heart failure (HF) in mice. In a previous study, we showed that SUMO-1 gene transfer substantially improved cardiac function in a murine model of pressure overload-induced HF. Toward clinical translation, we evaluated in this study the effects of SUMO-1 gene transfer in a swine model of ischemic HF. One month after balloon occlusion of the proximal left anterior descending artery followed by reperfusion, the animals were randomized to receive either SUMO-1 at two doses, SERCA2a, or both by adeno-associated vector type 1 (AAV1) gene transfer via antegrade coronary infusion. Control animals received saline infusions. After gene delivery, there was a significant increase in the maximum rate of pressure rise [dP/dt(max)] that was most pronounced in the group that received both SUMO-1 and SERCA2a. The left ventricular ejection fraction (LVEF) improved after high-dose SUMO-1 with or without SERCA2a gene delivery, whereas there was a decline in LVEF in the animals receiving saline. Furthermore, the dilatation of LV volumes was prevented in the treatment groups. SUMO-1 gene transfer therefore improved cardiac function and stabilized LV volumes in a large-animal model of HF. These results support the critical role of SUMO-1 in SERCA2a function and underline the therapeutic potential of SUMO-1 for HF patients.
Assuntos
Modelos Animais de Doenças , Técnicas de Transferência de Genes , Insuficiência Cardíaca/fisiopatologia , Coração/fisiopatologia , Proteína SUMO-1/genética , Animais , Dependovirus/genética , Insuficiência Cardíaca/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , SuínosRESUMO
AIMS: Impaired myocardial sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) activity is a hallmark of failing hearts, and SERCA2a gene therapy improves cardiac function in animals and patients with heart failure (HF). Deregulation of microRNAs has been demonstrated in HF pathophysiology. We studied the effects of therapeutic AAV9.SERCA2a gene therapy on cardiac miRNome expression and focused on regulation, expression, and function of miR-1 in reverse remodelled failing hearts. METHODS AND RESULTS: We studied a chronic post-myocardial infarction HF model treated with AAV9.SERCA2a gene therapy. Heart failure resulted in a strong deregulation of the cardiac miRNome. miR-1 expression was decreased in failing hearts, but normalized in reverse remodelled hearts after AAV9.SERCA2a gene delivery. Increased Akt activation in cultured cardiomyocytes led to phosphorylation of FoxO3A and subsequent exclusion from the nucleus, resulting in miR-1 gene silencing. In vitro SERCA2a expression also rescued miR-1 in failing cardiomyocytes, whereas SERCA2a inhibition reduced miR-1 levels. In vivo, Akt and FoxO3A were highly phosphorylated in failing hearts, but reversed to normal by AAV9.SERCA2a, leading to cardiac miR-1 restoration. Likewise, enhanced sodium-calcium exchanger 1 (NCX1) expression during HF was normalized by SERCA2a gene therapy. Validation experiments identified NCX1 as a novel functional miR-1 target. CONCLUSION: SERCA2a gene therapy of failing hearts restores miR-1 expression by an Akt/FoxO3A-dependent pathway, which is associated with normalized NCX1 expression and improved cardiac function.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Terapia Genética/métodos , Insuficiência Cardíaca/terapia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Células Cultivadas , Vasos Coronários , Regulação para Baixo , Proteína Forkhead Box O3 , Lactonas/farmacologia , Ligadura , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sesquiterpenos/farmacologia , Transdução de Sinais/fisiologia , Trocador de Sódio e Cálcio/metabolismoRESUMO
BACKGROUND: Adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase was assessed in a randomized, double-blind, placebo-controlled, phase 2 study in patients with advanced heart failure. METHODS AND RESULTS: Thirty-nine patients received intracoronary adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase or placebo. Seven efficacy parameters were assessed in 4 domains: symptoms (New York Heart Association class, Minnesota Living With Heart Failure Questionnaire), functional status (6-minute walk test, peak maximum oxygen consumption), biomarker (N-terminal prohormone brain natriuretic peptide), and left ventricular function/remodeling (left ventricular ejection fraction, left ventricular end-systolic volume), plus clinical outcomes. The primary end point success criteria were prospectively defined as achieving efficacy at 6 months in the group-level (concordant improvement in 7 efficacy parameters and no clinically significant worsening in any parameter), individual-level (total score for predefined clinically meaningful changes in 7 efficacy parameters), or outcome end points (cardiovascular hospitalizations and time to terminal events). Efficacy in 1 analysis had to be associated with at least a positive trend in the other 2 analyses. This combination of requirements resulted in a probability of success by chance alone of 2.7%. The high-dose group versus placebo met the prespecified criteria for success at the group-level, individual-level, and outcome analyses (cardiovascular hospitalizations) at 6 months (confirmed at 12 months) and demonstrated improvement or stabilization in New York Heart Association class, Minnesota Living With Heart Failure Questionnaire, 6-minute walk test, peak maximum oxygen consumption, N-terminal prohormone brain natriuretic peptide levels, and left ventricular end-systolic volume. Significant increases in time to clinical events and decreased frequency of cardiovascular events were observed at 12 months (hazard ratio=0.12; P=0.003), and mean duration of cardiovascular hospitalizations over 12 months was substantially decreased (0.4 versus 4.5 days; P=0.05) on high-dose treatment versus placebo. There were no untoward safety findings. CONCLUSIONS: The Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) study demonstrated safety and suggested benefit of adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase in advanced heart failure, supporting larger confirmatory trials. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT00454818.
Assuntos
Cálcio/metabolismo , Terapia Genética/métodos , Cardiopatias/terapia , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Regulação para Cima/fisiologia , Adenoviridae/genética , Adulto , Idoso , Progressão da Doença , Método Duplo-Cego , Teste de Esforço , Feminino , Terapia Genética/efeitos adversos , Cardiopatias/fisiopatologia , Humanos , Injeções Intra-Arteriais , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio/fisiologia , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Apolipoprotein A-I (ApoA-I)/high-density lipoprotein (HDL)-raising treatments are effective antiatherosclerotic strategies. We have compared the antiatherogenic effects of human ApoA-I (hApoA-I) overexpression by intraportal and intramuscular gene transfer in atherosclerotic ApoE-knockout mice. Atherosclerotic lesions were induced by atherogenic diet. After atherosclerosis induction, a group of animals was killed and served as atherosclerosis baseline-control group. The remaining animals were randomized into the following groups: (1) atherosclerosis-progression-control, (2) intraportal/vector administration, and (3) intramuscular/vector administration. Aortas and hearts were processed for atherosclerotic quantification by en face Sudan IV and Oil Red-O, respectively. Liver and muscle specimens were processed for protein/gene expression analysis. A sustained increase in hApoA-I/HDL plasma levels was observed in both transduced groups. hApoA-I overexpression abolished plaque progression versus progression-control group. hApoA-I overexpression significantly reduced lesion macrophage, feature indicative of plaque stabilization. Scavenger receptor class-B type I (SR-BI), but not ATP-binding cassette, sub-family A (ABCA), member 1 (ABCA-1), was significantly upregulated in treated groups versus progression-controls. The results of this study show a similar effect of hApoA-I/HDL overexpression on plaque progression/stabilization by 2 different routes of administration. Our results showing similar effects using either intramuscular administration and intraportal route of administration may have significant clinical implications, given the reduced medical risk to patient and cost of intramuscular injections.
Assuntos
Aorta/efeitos dos fármacos , Apolipoproteína A-I/genética , Apolipoproteína A-I/uso terapêutico , Apolipoproteínas E/genética , Aterosclerose/tratamento farmacológico , Dependovirus/metabolismo , Fígado/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aorta/patologia , Apolipoproteína A-I/administração & dosagem , Apolipoproteína A-I/sangue , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , HDL-Colesterol/sangue , Dependovirus/genética , Dieta Aterogênica , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Vetores Genéticos , Humanos , Injeções Intramusculares , Injeções Intravenosas , Fígado/anatomia & histologia , Fígado/fisiopatologia , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Receptores Depuradores Classe B/análise , Receptores Depuradores Classe B/genética , Fatores de Tempo , Transdução GenéticaRESUMO
BACKGROUND: Despite the FDA guidelines for studies to be performed to rule out potential cardiac toxicity, many drugs have nevertheless entered the market only to be later withdrawn from the market owing to cardiac toxicity. Cardiac toxicity may result from drugs causing impaired function or death of cardiomyocytes, valvular damage, myocardial ischemia and/or ventricular arrhythmias. Negative cardiovascular events have been implicated in 28% of drug withdrawals in the USA. The significance for patients, regulators and the pharmaceutical industry is immense. OBJECTIVE: We address whether a more rigorous and integrative approach is needed for cardiovascular safety screening of all new drug candidates. Furthermore, we will present a cardionomics approach that looks at several in vitro and in vivo models that can be applied to all drugs independent of category, therapeutic area or class. METHODS: We present examples of drugs demonstrating cardiac toxicity and provide an in-depth review of how calcium homeostasis may be a unifying theme in clinically observed cardiotoxic events. We introduce a cardionomics approach that detects clinical cardiac toxicity early in the drug discovery process, thus, preventing costly late attrition. CONCLUSION: The consequences of a failure to detect potential cardiovascular safety issues before clinical launch can have an enormous cost for the pharmaceutical industry, when major drugs are withdrawn due to lawsuits as well as loss of time and resources. An integrated cardionomics approach may reduce the risk of drug withdrawals as a result of unexpected clinical cardiac safety issues.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/prevenção & controle , Pesquisa Biomédica/métodos , Fármacos Cardiovasculares/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Humanos , Preparações Farmacêuticas/administração & dosagemRESUMO
OBJECTIVES: We investigated the effects of erbB2 inhibition by anti-erbB2 antibody on cardiomyocyte survival and mitochondrial function. BACKGROUND: ErbB2 is an important signal integrator for the epidermal growth factor family of receptor tyrosine kinases. Herceptin, an inhibitory antibody to the erbB2 receptor, is a potent chemotherapeutic but causes cardiac toxicity. METHODS: Primary cultures of neonatal rat ventricular myocytes were exposed to anti-erbB2 antibody (Ab) (7.5 mug/ml) for up to 24 h. Cell viability, mitochondrial function, and apoptosis were measured using multiple complementary techniques. RESULTS: ErbB2 inhibition was associated with a dramatic increase in expression of the pro-apoptotic Bcl-2 family protein Bcl-xS and decreased levels of anti-apoptotic Bcl-xL. There was a time-dependent increase in mitochondrial translocation and oligomerization of bcl-associated protein (BAX), as indicated by 1,6-bismaleimidohexane crosslinking. The BAX oligomerization was associated with cytochrome c release and caspase activation. These alterations induced mitochondrial dysfunction, a loss of mitochondrial membrane potential (psi) (76.9 +/- 2.4 vs. 51.7 +/- 0.1; p < 0.05; n = 4), a 35% decline in adenosine triphosphate levels (p < 0.05), and a loss of redox capacity (0.72 +/- 0.04 vs. 0.64 +/- 0.02; p< 0.01). Restoration of Bcl-xL levels through transactivating regulatory protein-mediated protein transduction prevented the decline in psi mitochondrial reductase activity and cytosolic adenosine triphosphate. CONCLUSIONS: Anti-erbB2 activates the mitochondrial apoptosis pathway through a previously undescribed modulation of Bcl-xL and -xS, causing impairment of mitochondrial function and integrity and disruption of cellular energetics.
Assuntos
Anticorpos Monoclonais/farmacologia , Genes erbB-2/efeitos dos fármacos , Mitocôndrias Cardíacas/fisiologia , Miócitos Cardíacos/citologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais Humanizados , Apoptose/fisiologia , Fracionamento Celular , Células Cultivadas , Reagentes de Ligações Cruzadas , Genes erbB-2/fisiologia , Marcação In Situ das Extremidades Cortadas , Mitocôndrias Cardíacas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Transdução Genética , Trastuzumab , Proteína bcl-XRESUMO
BACKGROUND: Beta blocker treatment has emerged as an effective treatment modality for heart failure. Interestingly, beta-blockers can activate both pro-apoptotic and anti-apoptotic pathways. Nevertheless, the mechanism for improved cardiac function seen with beta-blocker treatment remains largely unknown. Carvedilol is a non-selective beta-blocker with alpha-receptor blockade and antioxidant properties. We therefore studied the impact of the effects of carvedilol in an animal model of end-stage heart failure. RESULTS: To test whether chronic treatment with beta-blockade decreases apoptosis, we treated myopathic turkeys with two dosages of carvedilol, 1 mg/kg (DCM1) and 20 mg/kg (DCM20), for four weeks and compared them to non-treated DCM animals (DCM0) and to control turkeys (CON). Echocardiographic measurements showed that non-treated DCM animals had a significantly lower fractional shortening (FS) when compared to CON (68.73 +/- 1.37 vs. 18.76 +/- 0.59%, p < 0.001). Both doses of carvedilol significantly improved FS (33.83 +/- 10.11 and 27.73 +/- 6.18% vs. 18.76 +/- 0.59% for untreated DCM, p < 0.001). DCM left ventricles were characterized by a higher percentage of apoptotic nuclei when compared to CON (5.64 +/- 0.49 vs. 1.72 +/- 0.12%, respectively p < 0.001). Both doses of carvedilol significantly reduced the number of apoptotic nuclei (2.32 +/- 0.23% and 2.36 +/-6% 1 mg and 20 mg/kg respectively). CONCLUSIONS: Carvedilol improves ventricular function. Furthermore, treatment with carvedilol decreased the incidence of apoptosis in cardiac myocytes from failing hearts at both doses. These data suggest that the inhibition of apoptosis with carvedilol may lead to improvement in ventricular function and may underlie a beneficial effect of beta-blockade independent of heart rate lowering effects.
Assuntos
Apoptose/efeitos dos fármacos , Carbazóis/uso terapêutico , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/prevenção & controle , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Propanolaminas/uso terapêutico , Função Ventricular/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/patologia , Carvedilol , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Furazolidona/efeitos adversos , Insuficiência Cardíaca/induzido quimicamente , Perus , Disfunção Ventricular/induzido quimicamente , Disfunção Ventricular/tratamento farmacológico , Disfunção Ventricular/patologia , Função Ventricular/fisiologiaRESUMO
The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.