RESUMEN
Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.
Asunto(s)
Glioblastoma/terapia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/virología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Flufenazina/farmacología , Glioblastoma/tratamiento farmacológico , Glioblastoma/virología , Células HCT116 , Humanos , Indoles/farmacología , Virus Oncolíticos/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Replication-conditional herpes simplex virus (HSV)-based vectors have great potential in the treatment of various types of cancers including brain tumors. HSV mutants lacking the U(L)39 gene and both copies of the gamma(1)34.5 gene (e.g. MGH1, G207) have been demonstrated to possess oncolytic effects as well as potent anticancer vaccination effects without compromising safety. Such mutants thus provide optimal templates to produce novel oncolytic HSV vectors for cancer gene therapy applications. In order to accomplish quick and efficient construction of oncolytic HSV vectors, a novel BAC-based method designated as 'HSVQuik system' was developed. This system sequentially utilizes two different site-specific recombination systems to introduce virtually any transgene cassettes of interest into the deleted U(L)39 locus (Flp-FRT in Escherichia coli) and to release the vector genome sequence from the procaryotic plasmid backbone (Cre-loxP in Vero cells). Taking advantage of the HSVQuik system, we constructed three oncolytic HSV vectors that express mouse IL4, CD40 ligand and 6CK, respectively. In vivo therapeutic experiments using two luciferase-labeled syngeneic mouse brain tumor models revealed that expression of these immunomodulators significantly enhanced antitumor efficacy of oncolytic HSV. The HSVQuik system, together with luciferase-labeled tumor models, should expedite the process of generating and evaluating oncolytic HSV vectors for cancer gene therapy applications.
Asunto(s)
Vacunas contra el Cáncer/genética , Ingeniería Genética/métodos , Neoplasias/terapia , Viroterapia Oncolítica , Simplexvirus/genética , Animales , Ligando de CD40/genética , Vacunas contra el Cáncer/administración & dosificación , Línea Celular Tumoral , Quimiocina CCL21 , Quimiocinas CC/genética , Chlorocebus aethiops , Femenino , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Interleucina-4/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Animales , Trasplante de Neoplasias , Neoplasias/genética , Virus Oncolíticos/genética , Plásmidos , Células Vero , Replicación ViralRESUMEN
Oncolytic viruses (OVs) are being used as anticancer agents in preclinical and clinical trials. Propagation of OVs inside infected tumors is critical to their efficacy and is mediated by the productive generation of progeny OVs within infected tumor cells. In turn, this progeny can spread the infection to other tumor cells in successive rounds of oncolysis. Previously, we had found that, in rats, cyclophosphamide (CPA) pretreatment increased infection of brain tumors by an intra-arterially administered herpes simplex virus type 1 OV, because it inhibited activation of complement responses, mediated by innate IgM. We also have previously shown that other pharmacologic inhibitors of complement, such as cobra venom factor (CVF), allowed for increased infection. However, in these studies, further inhibition of complement responses by CVF did not result in additional infection of brain tumor cells or in propagation of OV to surrounding tumor cells. In this study, we sought to determine if CPA did lead to increased infection/propagation from initially infected tumor cells. Unlike our results with CVF, we find that CPA administration does result in a time-dependent increase in infection of tumor cells, suggestive of increased propagation, in both syngeneic and athymic models of brain tumors. This increase was due to increased survival of OV within infected tumors and brain surrounding tumors. CPA's effect was not due to a direct enhancement of viral replication in tumor cells, rather was associated with its immunosuppressive effects. RT-PCR analysis revealed that CPA administration resulted in impaired mRNA production by peripheral blood mononuclear cells (PBMCs) of several cytokines (interferons alpha/beta, interferon gamma, TNFalpha, IL-15, and IL-18) with anti-HSV function. These findings suggest that the CPA-mediated facilitation of OV intraneoplastic propagation is associated with a general decrease of antiviral cytokines mRNAs in PBMCs. These findings not only suggest a potential benefit for the addition of transient immunosuppression in clinical applications of oncolytic HSV therapy, but also suggest that innate immunomodulatory pathways may be amenable to manipulation, in order to increase OV propagation and survival within infected tumors.
Asunto(s)
Proteínas Inactivadoras de Complemento/uso terapéutico , Ciclofosfamida/uso terapéutico , Citocinas/metabolismo , Inmunoterapia/métodos , ARN Mensajero/metabolismo , Simplexvirus/fisiología , Animales , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virología , Citocinas/genética , Expresión Génica , Glioma/terapia , Glioma/virología , Modelos Animales , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/efectos de los fármacosRESUMEN
The owl monkey (Aotus trivirgatus) has served as the standard non-human primate model of herpes simplex virus-1 (HSV-1) infection because it is highly susceptible to HSV-1 encephalitis. Owl monkeys, however, are expensive, difficult to obtain, and difficult to maintain in captivity, thus greatly hampering the efficiency of preclinical gene therapy trials for brain tumors using HSV-1-based vectors. We have therefore compared the susceptibility of the common marmoset (Callithrix jacchus) with the owl monkey in a model of intracerebral inoculation of wildtype HSV-1 F-strain at increasing titers. The common marmosets consistently succumbed earlier to viral encephalitis than the owl monkeys. The histological evaluation of the common marmoset revealed extensive HSV-1 infection with a concomitant yet less marked inflammatory response compared to the owl monkeys. PCR for HSV-1 demonstrated a similar extra-CNS shedding route in both experimental models. Our findings show that the common marmoset is at least as susceptible to intracerebral HSV-infection as the owl monkey and that it can therefore serve as a valid and reliable experimental model for the important preclinical safety tests of HSV-based therapeutic viral vector constructs in the brain.
Asunto(s)
Callithrix/virología , Modelos Animales de Enfermedad , Encefalitis por Herpes Simple/patología , Vectores Genéticos/toxicidad , Herpesvirus Humano 1/patogenicidad , Animales , Encéfalo/virología , Susceptibilidad a Enfermedades , Encefalitis por Herpes Simple/etiología , Femenino , Herpesvirus Humano 1/genética , Riñón/virología , Hígado/virología , Masculino , Reacción en Cadena de la Polimerasa/métodos , Tasa de SupervivenciaRESUMEN
Several clinical trials have or are being performed testing the safety and efficacy of different strains of oncolytic viruses (OV) for malignant cancers. OVs represent either naturally occurring or genetically engineered strains of viruses that exhibit relatively selective replication in tumor cells. Several types of OV have been derived from herpes simplex virus 1 (HSV1). Tumor oncolysis depends on the processes of initial OV infection of tumor, followed by subsequent propagation of OV within the tumor itself. The role of the immune responses in these processes has not been extensively studied. On the contrary, effects of the immune response on the processes of wild-type HSV1 infection and propagation in the central nervous system have been studied and described in detail. The first line of defense against a wild-type HSV1 infection in both naive and immunized individuals is provided by innate humoral (complement, cytokines, chemokines) and cellular (macrophages, neutrophils, NK cells, gammadelta T cells, and interferon-producing cells) responses. These orchestrate the lysis of virions and virus-infected cells as well as provide a link to effective adaptive immunity. The role of innate defenses in curtailing the oncolytic effect of genetically engineered HSV has only recently been studied, but several of the same host responses appear to be operative in limiting anticancer effects by the replicating virus. The importance of this knowledge lies in finding avenues to modulate such initial innate responses, in order to allow for increased oncolysis of tumors while minimizing host toxicity.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Neoplasias/terapia , Virus/inmunología , Animales , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Proteínas Inactivadoras de Complemento/uso terapéutico , Proteínas del Sistema Complemento/inmunología , Ciclofosfamida/uso terapéutico , Herpes Simple/complicaciones , Humanos , Inmunosupresores/uso terapéutico , Neoplasias/inmunología , Neoplasias/virología , Replicación ViralAsunto(s)
Arritmias Cardíacas/genética , Defectos del Tabique Interatrial/genética , Proteínas de Homeodominio/genética , Factores de Transcripción , Proteínas de Xenopus , Potenciales de Acción , Animales , Arritmias Cardíacas/fisiopatología , Modelos Animales de Enfermedad , Electrofisiología , Defectos del Tabique Interatrial/fisiopatología , Heterocigoto , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/fisiología , Humanos , Técnicas In Vitro , Ratones , Ratones Mutantes , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologíaRESUMEN
BACKGROUND: Homozygous mutant mice expressing a truncated form of myosin-binding protein C (MyBP-C(t/t)) develop severe dilated cardiomyopathy, whereas the heterozygous mutation (MyBP-C(t/+)) causes mild hypertrophic cardiomyopathy. Adult male MyBP-C(t/t) and MyBP-C(t/+) mice were evaluated for arrhythmia vulnerability with an in vivo electrophysiology study. METHODS AND RESULTS: Surface ECGs were obtained for heart rate, rhythm, and conduction intervals. Atrial, atrioventricular, and ventricular conduction parameters and refractoriness were assessed in 22 MyBP-C(t/t), 10 MyBP-C(t/+), and 17 wild-type MyBP-C(+/+) mice with endocardial pacing and intracardiac electrogram recording. Arrhythmia induction was attempted with standardized programmed stimulation at baseline and with isoproterenol. Heart rate variability and ambient arrhythmia activity were assessed with telemetric ECG monitors. Quantitative histological characterization was performed on serial sections of excised hearts. MyBP-C(t/t) and MyBP-C(t/+) mice have normal ECG intervals and sinus node, atrial, and ventricular conduction and refractoriness. Ventricular tachycardia was reproducibly inducible in 14 of 22 MyBP-C(t/t) mice (64%) during programmed stimulation, compared with 2 of 10 MyBP-C(t/+) mice (20%) and 0 of 17 wild-type controls (P<0.001). Ventricular ectopy was present only in MyBP-C(t/t) mice during ambulatory ECG recordings. There were no differences in heart rate variability parameters. Interstitial fibrosis correlated with genotype but did not predict arrhythmia susceptibility within the MyBP-C(t/t) group. CONCLUSIONS: MyBP-C(t/t) mice, despite prominent histopathology and ventricular dysfunction, exhibit normal conduction and refractoriness, yet are vulnerable to ventricular arrhythmias. Somatic influences between genetically identical mutant mice most likely account for variability in arrhythmia susceptibility. A sarcomeric protein gene mutation leads to a dilated cardiomyopathy and ventricular arrhythmia vulnerability phenotype.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Cardiomiopatías/fisiopatología , Proteínas Portadoras/genética , Ventrículos Cardíacos/fisiopatología , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/genética , Cardiomiopatías/complicaciones , Cardiomiopatías/genética , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Electrocardiografía , Técnicas Electrofisiológicas Cardíacas , Predisposición Genética a la Enfermedad , Sistema de Conducción Cardíaco/fisiopatología , Frecuencia Cardíaca , Heterocigoto , Homocigoto , Masculino , Ratones , Ratones Mutantes , Mutación , Miocardio/patología , Fenotipo , Eliminación de SecuenciaRESUMEN
A DNA nonbinding mutant of the NK2 class homeoprotein Nkx2.5 dominantly inhibits cardiogenesis in Xenopus embryos, causing a small heart to develop or blocking heart formation entirely. Recently, ten heterozygous CSX/NKX2.5 homeoprotein mutations were identified in patients with congenital atrioventricular (AV) conduction defects. All four missense mutations identified in the human homeodomain led to markedly reduced DNA binding. To examine the effect of a DNA binding-impaired mutant of mouse Csx/Nkx2.5 in the embryonic heart, we generated transgenic mice expressing one such allele, I183P, under the beta-myosin heavy chain promoter. Unexpectedly, transgenic mice were born apparently normal, but the accumulation of Csx/Nkx2.5(I183P) mutant protein in the embryo, neonate, and adult myocardium resulted in progressive and profound cardiac conduction defects and heart failure. P-R prolongation observed at 2 weeks of age rapidly progressed into complete AV block as early as 4 weeks of age. Expression of connexins 40 and 43 was dramatically decreased in the transgenic heart, which may contribute to the conduction defects in the transgenic mice. This transgenic mouse model may be useful in the study of the pathogenesis of cardiac dysfunction associated with CSX/NKX2.5 mutations in humans.
Asunto(s)
Nodo Atrioventricular/fisiopatología , Cardiopatías Congénitas/fisiopatología , Insuficiencia Cardíaca/etiología , Proteínas de Homeodominio/metabolismo , Miocardio/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Factores de Edad , Animales , Animales Recién Nacidos , Nodo Atrioventricular/anomalías , Peso Corporal , Conexina 43/metabolismo , Conexinas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Ecocardiografía , Femenino , Corazón/embriología , Corazón/crecimiento & desarrollo , Cardiopatías Congénitas/complicaciones , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Transgénicos , Mutación , Miocardio/patología , Miocardio/ultraestructura , Tamaño de los Órganos , Factores de Transcripción/genética , Proteína alfa-5 de Unión ComunicanteRESUMEN
BACKGROUND: Genetically altered mice will provide important insights into a wide variety of processes in cardiovascular physiology underlying myocardial infarction (MI). Comprehensive and accurate analyses of cardiac function in murine models require implementation of the most appropriate techniques and experimental protocols. OBJECTIVE: In this study we present in vivo, whole-animal techniques and experimental protocols for detailed electrophysiological characterization in a mouse model of myocardial ischemia and infarction. METHODS: FVB mice underwent open-chest surgery for ligation of the left anterior descending coronary artery or sham-operation. By means of echocardiographic imaging, electrocardiography, intracardiac electrophysiology study, and conscious telemetric ECG recording for heart rate variability (HRV) analysis, we evaluated ischemic and post-infarct cardiovascular morphology and function in mice. RESULTS: Coronary artery ligation resulted in antero-apical infarction of the left ventricular wall. MI mice showed decreased cardiac function by echocardiography, infarct-typical pattern on ECG, and increased arrhythmia vulnerability during electrophysiological study. Electrophysiological properties were determined comprehensively, but were not altered significantly as a consequence of MI. Autonomic nervous system function, measured by indices of HRV, did not appear altered in mice during ischemia or infarction. CONCLUSIONS: Cardiac conduction, refractoriness, and heart rate variability appear to remain preserved in a murine model of myocardial ischemia and infarction. Myocardial infarction may increase vulnerability to inducible ventricular tachycardia and atrial fibrillation, similarly to EPS findings in humans. These data may be of value as a reference for comparison with mutant murine models necessitating ischemia or scar to elicit an identifiable phenotype. The limitations of directly extrapolating murine cardiac electrophysiology data to conditions in humans need to be considered.
Asunto(s)
Electrofisiología , Infarto del Miocardio/mortalidad , Animales , Arritmias Cardíacas/etiología , Procedimientos Quirúrgicos Cardiovasculares , Modelos Animales de Enfermedad , Estimulación Eléctrica/métodos , Electrocardiografía , Sistema de Conducción Cardíaco/fisiología , Frecuencia Cardíaca/fisiología , Masculino , Ratones , Modelos Cardiovasculares , Infarto del Miocardio/complicaciones , Infarto del Miocardio/cirugía , Análisis de Supervivencia , Remodelación Ventricular/fisiologíaRESUMEN
BACKGROUND: Atrial tachycardia and fibrillation in humans may be partly consequent to vagal stimulation. Induction of fibrillation in the small heart is considered to be impossible due to lack of a critical mass of > 100-200 mm2. Even with the recent progression of the technology of in vivo and in vitro mouse electrophysiological studies, few reports describe atrial tachycardia or fibrillation in mice. The purpose of this study was to attempt provocation of atrial tachyarrhythmia in mice using transvenous pacing following cholinergic stimulation. METHODS AND RESULTS: In vivo electrophysiology studies were performed in 14 normal mice. A six-lead ECG was recorded from surface limb leads, and an octapolar electrode catheter was inserted via jugular vein cutdown approach for simultaneous atrial and ventricular endocardial recording and pacing. Atrial tachycardia and fibrillation were inducible in one mouse at baseline electrophysiology study and eleven of fourteen mice after carbamyl choline injection. The mean duration of atrial tachycardia was 126 +/- 384 s. The longest episode lasted 35 min and only terminated after atropine injection. Reinduction of atrial tachycardia after administration of atropine was not possible. CONCLUSION: Despite the small mass of the normal mouse atria, sustained atrial tachycardia and fibrillation can be easily and reproducibly inducible with endocardial pacing after cholinergic agonist administration. This finding may contribute to our understanding of the classical theories of arrhythmogenesis and critical substrates necessary for sustaining microreentrant circuits. The techniques of transcatheter parasympathetic agonist-mediated atrial tachycardia induction may be valuable in further murine electrophysiological studies, especially mutant models with potential atrial arrhythmia phenotypes.
Asunto(s)
Fibrilación Atrial/etiología , Taquicardia/etiología , Animales , Fibrilación Atrial/fisiopatología , Sistema Nervioso Autónomo/fisiopatología , Carbacol/farmacología , Estimulación Cardíaca Artificial , Cardiotónicos/farmacología , Agonistas Colinérgicos/farmacología , Modelos Animales de Enfermedad , Electrocardiografía , Femenino , Hemodinámica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Taquicardia/fisiopatologíaRESUMEN
We investigated the expression of c-myc and c-sis/PDGF mRNA and protein products in 20 cases of meningiomas of various grades: 10 benign, 5 atypical and 5 anaplastic meningiomas. All cases of atypical and anaplastic meningiomas were positive for c-myc protein and mRNA by immunohistochemistry and in situ hybridisation, respectively, while all 10 benign meningiomas were negative for c-myc immunostaining, with only one benign tumour positive for c-myc mRNA. Expression of PDGF-BB protein and c-sis mRNA were seen in more than 80% of the meningioma cases and was not restricted to the histological grades of meningiomas. Semiquantitative analysis showed that the frequency of c-myc immunopositive cells positively correlated with Ki-67 proliferative indices. Our findings suggest that c-myc, but not c-sis/PDGF, has some concern to the malignancy of meningiomas.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Meníngeas/patología , Meningioma/patología , Proteínas de Neoplasias/análisis , Fosfoproteínas/análisis , Proteínas Proto-Oncogénicas c-myc/análisis , Proteínas Proto-Oncogénicas c-sis/análisis , Adulto , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Neoplasias Meníngeas/química , Neoplasias Meníngeas/clasificación , Neoplasias Meníngeas/genética , Meningioma/química , Meningioma/clasificación , Meningioma/genética , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-sis/genética , ARN Mensajero/análisisRESUMEN
Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Modelos Animales de Enfermedad , Actinas/genética , Alelos , Animales , Factor Natriurético Atrial/genética , Northern Blotting , Proteínas Portadoras/genética , Ecocardiografía , Electrofisiología , Salud de la Familia , Masculino , Ratones , Mutación , Mutación Missense , Miocardio/química , Miocardio/patología , Empalme del ARN , ARN Mensajero/metabolismo , Sarcómeros/química , Factores de Tiempo , Transgenes , Disfunción Ventricular IzquierdaRESUMEN
INTRODUCTION: Endothelial nitric oxide synthase (eNOS) mediates attenuation of the L-type calcium channel and modulates myocyte contractility. Arrhythmogenic afterdepolarizations are seen in vitro in ouabain-treated isolated myocytes from eNOS-deficient mice. The aim of these studies was to characterize the baseline electrophysiologic (EP) phenotype of eNOS-deficient mice and their potential susceptibility to cardiac conduction abnormalities and inducible arrhythmias. METHODS AND RESULTS: Surface ECG and in vivo intracardiac EP studies were performed in 27 mice lacking the eNOS gene and 21 wild-type littermate control mice. Baseline studies were performed in 10 eNOS-deficient mice and 10 wild-type controls. Subsequently, 17 eNOS-deficient mice and 11 wild-type controls were pretreated with digoxin, and ECG and EP testing were repeated. Data analysis revealed no significant differences in ECG intervals or cardiac conduction parameters, except sinus cycle length was higher in eNOS-deficient mice than wild-type mice (P < 0.01). After digoxin pretreatment, 7 of 17 eNOS-deficient mice had inducible ventricular tachycardia and 2 others had frequent ventricular premature beats, compared with only 3 of 11 wild-type mice with inducible ventricular tachycardia. In addition, 2 digoxin-treated eNOS-deficient mice and 1 wild-type mouse had inducible nonsustained atrial fibrillation. CONCLUSION: Mice with a homozygous targeted disruption of the eNOS gene have slower heart rates but no other distinguishable EP characteristics under basal sedated conditions. Partial inhibition of the Na+/K+ ATPase pump with digoxin administration increases ventricular ectopic activity in eNOS-/- mice, a phenotype analogous to afterdepolarizations seen in vitro in this eNOS-deficient mouse model.
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Técnicas Electrofisiológicas Cardíacas , Endotelio Vascular/enzimología , Óxido Nítrico Sintasa/deficiencia , Animales , Arritmias Cardíacas/enzimología , Arritmias Cardíacas/genética , Atropina/administración & dosificación , Digoxina/farmacología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Electrocardiografía , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Genotipo , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/enzimología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/genética , Masculino , Ratones , Modelos Cardiovasculares , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Variaciones Dependientes del Observador , Parasimpatolíticos/administración & dosificación , Propranolol/administración & dosificación , Simpaticolíticos/administración & dosificaciónRESUMEN
INTRODUCTION: Gap junctions consist of connexin (Cx) proteins that enable electrical coupling of adjacent cells and propagation of action potentials. Cx40 is solely expressed in the atrium and His-Purkinje system. The purpose of this study was to evaluate atrioventricular (AV) conduction in mice with a homozygous deletion of Connexin40 (Cx40(-/-)). METHODS: Surface ECGs, intracardiac electrophysiology (EP) studies, and ambulatory telemetry were performed in Cx40(-/-) mutant mice and wild-type (WT) controls. Atrioventricular (AV) conduction parameters and arrhythmia inducibility were evaluated using programmed stimulation. Analysis of heart rate variability was based on results of ambulatory monitoring. RESULTS: Significant findings included prolonged measures of AV refractoriness and conduction in connexin40-deficient mice, including longer PR, AH, and HV intervals, increased AV refractory periods, and increased AV Wenckebach and 2:1 block cycle lengths. Connexin40-deficient mice also had an increased incidence of inducible ventricular tachycardia, decreased basal heart rates, and increased heart rate variability. CONCLUSION: A homozygous disruption of Cx40 results in prolonged AV conduction parameters due to abnormal electrical coupling in the specialized conduction system, which may also predispose to arrhythmia vulnerability.
Asunto(s)
Nodo Atrioventricular/fisiopatología , Conexinas/deficiencia , Uniones Comunicantes/metabolismo , Taquicardia Ventricular/fisiopatología , Animales , Estimulación Eléctrica , Electrocardiografía , Electrodos , Electrofisiología , Frecuencia Cardíaca , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Mutantes , Modelos Animales , Probabilidad , Valores de Referencia , Estadísticas no Paramétricas , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/etiología , Proteína alfa-5 de Unión ComunicanteRESUMEN
We developed a technology for heart rate (HR) variability (HRV) analysis in the mouse for characterization of HR dynamics, modulated by vagal and sympathetic activity. The mouse is the principal animal model for studying biological processes. Mouse strains are now available harboring gene mutations providing fundamental insights into molecular mechanisms underlying cardiac electrical diseases. Future progress depends on enhanced understanding of these fundamental mechanisms and the implementation of methods for the functional analysis of mouse cardiovascular physiology. By telemetric techniques, standard time and frequency-domain measures of HRV were computed with and without autonomic blockade, and baroreflex sensitivity testing was performed. HR modulation in the high-frequency component is predominantly mediated by the parasympathetic nervous system, whereas the low-frequency component is under the influence of both the parasympathetic and sympathetic systems. The presented technology and protocol allow for assessment of autonomic regulation of the murine HR. Phenotypic screening for HR regulation in mice will further enhance the value of the mouse as a model of heritable electrophysiological human disease.
Asunto(s)
Electrocardiografía , Frecuencia Cardíaca/genética , Ratones Endogámicos C57BL/genética , Animales , Atropina/farmacología , Sistema Nervioso Autónomo/fisiología , Barorreflejo/fisiología , Electrocardiografía/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Ratones , Fenotipo , Propranolol/farmacología , Especificidad de la Especie , TelemetríaRESUMEN
We examined the distributions of the activation recovery interval (ARI), which is correlated with the local action potential duration (APD), to clarify the origin of the repolarization changes in ASD. The ECGs, QRST isointegral maps and ARI isochronal maps of 21 children with ASD from 3 to 5 years old in age were studied in comparison with 21 age-matched normal children. A conventional and 87 unipolar body surface ECG were simultaneously recorded. The ARIs were determined from the first derivatives of the ECG waveforms. Abnormal ST-T patterns were observed in 11 of 21 ASD, but only in two normal children. The QRST maps of a split positive area pattern were seen in 15 of ASD but none of the normal. In the ARI maps, all the normal children exhibited a short-ARI area on the left and a long-ARI area on the right side of the chest. In 19 of ASD, the ARI distribution revealed a leftward extension of the long-ARI area on the anterior chest, a relative shortening on the right anterior chest, and a localized prolonged ARI on the left anterior chest. The results suggest that right ventricular (RV) volume overload in ASD produces a localized prolongation of the APD on the RV epicardium.
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Electrocardiografía , Defectos del Tabique Interatrial/diagnóstico , Disfunción Ventricular Derecha/diagnóstico , Preescolar , Femenino , Humanos , Masculino , Probabilidad , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Dual AV nodal physiology is characterized by discontinuous conduction from the atrium to His bundle during programmed atrial extrastimulus testing (A2V2 conduction curves), AV nodal echo beats, and induction of AV nodal reentry tachycardia (AVNRT). The purpose of this study was to characterize in vivo murine maturational AV nodal conduction properties and determine the frequency of dual AV nodal physiology and inducible AVNRT. METHODS AND RESULTS: A complete transvenous in vivo electrophysiologic study was performed on 30 immature and 19 mature mice. Assessment of AV nodal conduction included (1) surface ECG and intracardiac atrial and ventricular electrograms; (2) decremental atrial pacing to the point of Wenckebach block and 2:1 conduction; and (3) programmed premature atrial extrastimuli to determine AV effective refractory periods (AVERP), construct A2V2 conduction curves, and attempt arrhythmia induction. The mean Wenckebach block interval was 73 +/- 12 msec, 2:1 block pacing cycle length was 61 +/- 11 msec, and mean AVERP100 was 54 +/- 11 msec. The frequency of dual AV nodal physiology increased with chronologic age, with discontinuous A2V2 conduction curves or AV nodal echo beats in 27% of young mice < 8 weeks and 58% in adult mice (P = 0.03). CONCLUSION: These data suggest that mice, similar to humans, have maturation of AV nodal physiology, but they do not have inducible AVNRT. Characterization of murine electrophysiology may be of value in studying genetically modified animals with AV conduction abnormalities. Furthermore, extrapolation to humans may help explain the relative rarity of AVNRT in the younger pediatric population.
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Envejecimiento/fisiología , Nodo Atrioventricular/fisiología , Animales , Fascículo Atrioventricular/fisiopatología , Bloqueo de Rama/fisiopatología , Bloqueo de Rama/terapia , Cateterismo Cardíaco , Estimulación Cardíaca Artificial , Electrocardiografía , Femenino , Frecuencia Cardíaca/fisiología , Masculino , Ratones , Ratones Endogámicos , Caracteres Sexuales , Taquicardia por Reentrada en el Nodo Atrioventricular/fisiopatología , Taquicardia por Reentrada en el Nodo Atrioventricular/terapiaRESUMEN
Intravascular routes of administration can provide a means to target gene- and virus-based therapies to multiple tumor foci located within an organ, such as the brain. However, we demonstrate here that rodent plasma inhibits cell transduction by replication-conditional (oncolytic) herpes simplex viruses (HSV), replication-defective HSV, and adenovirus vectors. In vitro depletion of complement with mild heat treatment or in vivo depletion by treatment of athymic rats with cobra venom factor (CVF) partially reverses this effect. Without CVF, inhibition of cell infection by HSV is observed at plasma dilution as high as 1:32, while plasma from CVF-treated animals displays anti-HSV activity at lower dilutions (1:8). When applied to the therapy of intracerebral brain tumors, in vivo complement depletion facilitates the initial infection (assayed at the 2-day time point) by an intra-arterial replication-conditional HSV of tumor cells, located within three separate and distinct human glioma masses. However, at the 4-day time point, no propagation of HSV from initially infected tumor cells could be observed. Previously, we have shown that the immunosuppressive agent, cyclophosphamide (CPA), facilitates the in vivo propagation of an oncolytic HSV, delivered intravascularly, within infected multiple intracerebral masses, by inhibition of both innate and elicited anti-HSV neutralizing antibody response (K. Ikeda et al., Nat. Med. 5:881-889, 1999). In this study, we thus show that the addition of CPA to the CVF treatment results in a significant increase in viral propagation within infected tumors, measured at the 4-day time period. The concerted action of CVF and CPA significantly increases the life span of athymic rodents harboring three separate and large glioma xenografts after treatment with intravascular, oncolytic HSV. Southern analysis of viral genomes analyzed by PCR reveals the presence of the oncolytic virus in the brains, livers, spleens, kidneys, and intestine of treated animals, although none of these tissues displays evidence of HSV-mediated gene expression. In light of clinical trials of oncolytic HSV for malignant brain tumors, these findings suggest that antitumor efficacy may be limited by the host innate and elicited humoral responses.
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Neoplasias Encefálicas/virología , Proteínas del Sistema Complemento/fisiología , Glioma/virología , Simplexvirus/fisiología , Adenoviridae/genética , Animales , Encéfalo/patología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/terapia , Proteínas Inactivadoras de Complemento/farmacología , Ciclofosfamida/farmacología , Venenos Elapídicos/farmacología , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Glioma/mortalidad , Glioma/terapia , Herpes Simple/virología , Humanos , Inmunosupresores/farmacología , Mutación , Ratas , Ratas Desnudas , Simplexvirus/genética , Activación Viral , Replicación ViralRESUMEN
INTRODUCTION: QRS morphology during narrow QRS supraventricular tachycardia in patients without ventricular preexcitation generally is considered the same as that seen during sinus rhythm. This study presents a new ECG observation that the QRS amplitude increased significantly in leads V2 through V5 during tachycardia. METHODS AND RESULTS: Using the same ECG machine and the same electrode patches applied to the same electrode positions, 12-lead ECGs during sinus rhythm and narrow QRS tachycardia were analyzed comparatively in 23 patients without ventricular preexcitation. Precordial QRS amplitudes were measured as the vertical distance from the peak of the R to the nadir of the S wave. The amplitudes also were measured during atrial rapid pacing and extrastimulation. Furthermore, ventricular excitation during sinus rhythm and tachycardia was studied using body surface mapping. Body surface distributions of QRS potentials and ventricular activation time (VAT) were displayed as maps. Gross area of QRS (AQRS, equivalent to the QRS amplitude) was compared during sinus rhythm versus tachycardia. During tachycardia, QRS amplitude significantly increased in leads V2 through V5, without any noticeable change in the transitional zone or QRS wave duration. Increase of QRS amplitude also was noted during atrial rapid pacing and extrastimulation. Gross AQRS values during tachycardia significantly increased in the left parasternal area, whereas QRS isopotential and VAT isochronal maps were similar during sinus rhythm and tachycardia, suggesting a minimal role of conduction delay in the increase of QRS amplitude. CONCLUSION: QRS wave amplitude significantly increased in leads V2 through V5 during narrow QRS tachycardia compared with QRS waves in sinus rhythm. Increase of QRS amplitude seemed unlikely due to a conduction delay within the ventricular myocardium.
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Electrocardiografía , Taquicardia/fisiopatología , Adolescente , Adulto , Función Atrial , Mapeo del Potencial de Superficie Corporal , Estimulación Cardíaca Artificial , Ecocardiografía , Femenino , Frecuencia Cardíaca , Humanos , Masculino , Persona de Mediana Edad , Taquicardia/diagnóstico por imagen , Factores de Tiempo , Función VentricularRESUMEN
The occurrence of multiple tumors in an organ heralds a rapidly fatal course. Although intravascular administration may deliver oncolytic viruses/vectors to each of these tumors, its efficiency is impeded by an antiviral activity present in complement-depleted plasma of rodents and humans. Here, this activity was shown to interact with complement in a calcium-dependent fashion, and antibody neutralization studies indicated preimmune IgM has a contributing role. Short-term exposure to cyclophosphamide (CPA) partially suppressed this activity in rodents and humans. At longer time points, cyclophosphamide also abrogated neutralizing antibody responses. Cyclophosphamide treatment of rats with large single or multiple intracerebral tumors substantially increased viral survival and propagation, leading to neoplastic regression.