RESUMEN
Decline in cardiac high-energy phosphate metabolism [phosphocreatine-to-ATP (PCr/ATP) ratio] and whole body metabolism increase the risk of heart failure and metabolic diseases. The aim of the present study was to assess the relationship between PCr/ATP ratio and measures of body metabolic function. A total of 35 healthy women (56+/-14.0 years of age) underwent cardiac 31P magnetic resonance spectroscopy to assess PCr/ATP ratio - an index of cardiac high-energy phosphate metabolism. Fasting and 2-hour glucose levels were assessed using oral glucose tolerance test. Indirect calorimetry was performed to determine oxygen consumption and resting metabolic rate. There were no significant relationships between PCr/ATP ratio and resting metabolic rate (r=-0.09, p=0.62), oxygen consumption (r=-0.11, p=0.54), fasting glucose levels (r=-0.31, p=0.07), and 2-hour plasma glucose (r=-0.10, p=0.58). Adjusted analysis for covariates including age, body mass index, fat mass, and physical activity, had no significant influence on the relationship between PCr/ATP ratio and body metabolism. In conclusion, the lack of relationship between cardiac PCr/ATP ratio, glucose control and metabolic rate may suggest that overall metabolic function does not influence cardiac high-energy phosphate metabolism.
Asunto(s)
Adenosina Trifosfato/metabolismo , Metabolismo/fisiología , Miocardio/metabolismo , Fosfocreatina/metabolismo , Adiposidad , Adulto , Anciano , Envejecimiento , Glucemia/análisis , Índice de Masa Corporal , Ejercicio Físico , Femenino , Humanos , Persona de Mediana Edad , Consumo de OxígenoRESUMEN
AIMS: The aims of this study were to determine the diagnostic yield of a dedicated heart failure diagnosis clinic and the impact of using different guideline recommended N-terminal pro B-type natriuretic peptide (NT-proBNP) referral thresholds on diagnosis and referral patterns. METHODS AND RESULTS: Patients referred by primary care between September 2011 and May 2013 were included in the analysis. Data collected included baseline characteristics, NT-proBNP levels, echocardiographic and clinical findings, final diagnosis and outcome. The impact of using Newcastle (locally modified age-adjusted NT-proBNP thresholds), National Institute for Health and Care Excellence (NICE) and European Society of Cardiology (ESC) NT-proBNP thresholds on diagnosis and referrals was determined by applying the different guidelines to this population. A total of 208 patients were referred; median age 77.5 years and 62.5% were women. Thirty-four patients (16.3%) had systolic heart failure, 50 patients (24.0%) had heart failure with preserved ejection fraction. One hundred and six patients (51.0%) did not have heart failure. Using NICE guidelines (NT-proBNP ≥ 400 ng/l) instead of the Newcastle age-adjusted NT-proBNP referral thresholds results in 59 fewer referrals, but eight heart failure diagnoses were missed. Using the ESC cut-off of NT-proBNP ≥ 125 ng/l would result in 88 additional referrals; one diagnosis of heart failure would be missed. Over a mean follow-up of 16.8 ± 6 months there were 21 deaths and 47 hospital admissions. CONCLUSION: The Newcastle age-adjusted thresholds led to more referrals in comparison to NICE guidelines but are more sensitive in diagnosing heart failure. Using ESC recommended thresholds results in a similar diagnostic yield to our age-adjusted thresholds, but has the potential to significantly increase the referrals in patients ≥ 75 years, which may result in a lower diagnostic yield.
Asunto(s)
Insuficiencia Cardíaca/sangre , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Errores Diagnósticos/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Valores de Referencia , Derivación y Consulta/estadística & datos numéricosRESUMEN
BACKGROUND: Bioreactance is a novel noninvasive method for cardiac output measurement that involves analysis of blood flow-dependent changes in phase shifts of electrical currents applied across the thorax. The present study evaluated the test-retest reliability of bioreactance for assessing haemodynamic variables at rest and during exercise. METHODS: 22 healthy subjects (26 (4) yrs) performed an incremental cycle ergometer exercise protocol relative to their individual power output at maximal O2 consumption (Wmax) on two separate occasions (trials 1 and 2). Participants cycled for five 3 min stages at 20, 40, 60, 80 and 90% Wmax. Haemodynamic and cardiorespiratory variables were assessed at rest and continuously during the exercise protocol. RESULTS: Cardiac output was not significantly different between trials at rest (P=0.948), or between trials at any stage of the exercise protocol (all P>0.30). There was a strong relationship between cardiac output estimates between the trials (ICC=0.95, P<0.001) and oxygen consumption (ICC=0.99, P<0.001). Stroke volume was also not significantly different between trials at rest (P=0.989) or during exercise (all P>0.15), and strong relationships between trials were found (ICC=0.83, P<0.001). CONCLUSIONS: The bioreactance method demonstrates good test-retest reliability for estimating cardiac output at rest and during different stages of graded exercise testing including maximal exertion.
Asunto(s)
Gasto Cardíaco/fisiología , Prueba de Esfuerzo/métodos , Ejercicio Físico/fisiología , Monitoreo Fisiológico/métodos , Descanso/fisiología , Adulto , Femenino , Humanos , Masculino , Consumo de Oxígeno/fisiología , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVES: Impaired cardiac function has been confirmed in patients with chronic fatigue syndrome (CFS). Magnetic resonance cardiac tagging is a novel technique that assesses myocardial wall function in vivo. We hypothesized that patients with CFS may have impaired development and release of myocardial torsion and strain. METHODS: Cardiac morphology and function were assessed using magnetic resonance imaging and cardiac tagging methodology in 12 CFS patients (Fukuda) and 10 matched controls. RESULTS: Compared to controls, the CFS group had substantially reduced left ventricular mass (reduced by 23%), end-diastolic volume (30%), stroke volume (29%) and cardiac output (25%). Residual torsion at 150% of the end-systolic time was found to be significantly higher in the patients with CFS (5.3 ± 1.6°) compared to the control group (1.7 ± 0.7°, P = 0.0001). End-diastolic volume index correlated negatively with both torsion-to-endocardial-strain ratio (TSR) (r = -0.65, P = 0.02) and the residual torsion at 150% end-systolic time (r = -0.76, P = 0.004), so decreased end-diastolic volume is associated with raised TSR and torsion persisting longer into diastole. Reduced end-diastolic volume index also correlated significantly with increased radial thickening (r = -0.65, P = 0.03) and impaired diastolic function represented by the ratio of early to late ventricular filling velocity (E/A ratio, r = 0.71, P = 0.009) and early filling percentage (r = 0.73, P = 0.008). CONCLUSION: Patients with CFS have markedly reduced cardiac mass and blood pool volumes, particularly end-diastolic volume: this results in significant impairments in stroke volume and cardiac output compared to controls. The CFS group appeared to have a delay in the release of torsion.
Asunto(s)
Gasto Cardíaco/fisiología , Síndrome de Fatiga Crónica/diagnóstico , Imagen por Resonancia Cinemagnética/métodos , Disfunción Ventricular/diagnóstico , Función Ventricular/fisiología , Adulto , Estudios de Casos y Controles , Síndrome de Fatiga Crónica/fisiopatología , Femenino , Humanos , Imagen por Resonancia Cinemagnética/normas , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estadística como Asunto , Torsión Mecánica , Disfunción Ventricular/fisiopatologíaRESUMEN
AIMS: To determine which factors predict outcomes in a group of patients with advanced heart failure, and in particular if NT-proBNP provides additional clinical and prognostic information to other haemodynamic and biochemical data. METHODS AND RESULTS: Ninety-one patients were studied who were being evaluated for heart transplantation, with 166 assessments. The patients had advanced heart failure as determined by median cardiac index of 2.0 l/min/m(2), left ventricular end-diastolic diameter of 7.0 mm and levels of NT-proBNP of 2473 pg/ml. Median follow-up time was 359 days. Clinicians were blinded to NT-proBNP levels. NT-proBNP significantly correlated with cardiac index (R = -0.44, p < 0.001), right atrial pressure (R = 0.40, p < 0.001), pulmonary arterial wedge pressure (R = 0.38, p < 0.001) and albumin (R = -0.52, p < 0.001), and total bilirubin with right atrial pressure (R = 0.59, p < 0.001). Cardiac index was the most important independent predictor of outcome (p = 0.0001), although bilirubin (p = 0.001) and NT-proBNP (p < 0.05) were also significant. In patients with a 50% increase in NT-proBNP, 64% had adverse outcomes, whereas those in whom levels were stable, 22% had adverse outcomes (p < 0.05). CONCLUSION: Cardiac index is the primary independent predictor of outcome in advanced heart failure when haemodynamic deterioration is evident. In situations where invasive haemodynamics are not available, total bilirubin (reflecting hepatic congestion) and NT-proBNP (related to haemodynamics) also provide important prognostic information.
Asunto(s)
Insuficiencia Cardíaca/cirugía , Péptido Natriurético Encefálico/metabolismo , Fragmentos de Péptidos/metabolismo , Biomarcadores/metabolismo , Femenino , Insuficiencia Cardíaca/sangre , Trasplante de Corazón , Hemodinámica/efectos de los fármacos , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
AIMS: The aim of this study was to assess the spatial and temporal variability of dominant fibrillation frequencies in a rare case of sustained human ventricular fibrillation (VF). METHODS AND RESULTS: Body surface potential mapping was performed in a patient with sustained VF and who was supported by a biventricular assist device. Dominant frequencies at 54 body sites were calculated from two recordings obtained 38 days apart. Variability of dominant frequencies between recordings and across body sites was quantified. Median dominant frequencies within recordings varied between 6.1 and 7.2 Hz in recording 1 and 5.6 and 6.6 Hz in recording 2, indicating a significant reduction in dominant frequencies between the recordings (P < 0.0001). Dominant frequencies differed across body sites by a mean (range) of 1.7 (0.4-2.8) Hz. CONCLUSION: In this rare case of sustained VF, there was significant spatial and temporal variability of VF dominant frequencies. These findings should be considered in future ECG studies on VF where the spatial variability of dominant frequency might not otherwise have been considered.
Asunto(s)
Mapeo del Potencial de Superficie Corporal/métodos , Electrocardiografía/métodos , Fibrilación Ventricular/diagnóstico , Enfermedad Crónica , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Raras/diagnósticoRESUMEN
AIMS: As oral corticosteroids have a beneficial effect on muscle strength in Duchenne muscular dystrophy, it has been suggested that they may also be a useful treatment in the pathologically related sarcoglycanopathies. The delta-sarcoglycan-deficient mouse (Sgcd-null) is a model for both limb girdle muscular dystrophy 2F (LGMD2F) and dilated cardiomyopathy. METHODS AND RESULTS: To study the effect of oral corticosteroids on cardiac function, we treated 8-week-old Sgcd-null mice with prednisolone (1.5 mg/kg body weight/day orally) for 8 weeks. In vivo cardiac function was assessed by pressure-volume loops using a conductance catheter. We found a well-compensated cardiomyopathy at baseline in Sgcd-null mice with decreased myocardial contractility, increased preload, and decreased afterload, maintaining a high cardiac output. Cardiac haemodynamics, surprisingly, did not improve in prednisolone-treated mice, but instead deteriorated with evidence of ventricular stiffening. On histology, after steroid treatment there was increased myocardial cell damage and increased myocardial fibrosis. CONCLUSION: Prednisolone led to a decompensation of cardiac haemodynamics in Sgcd-null mice and induced additional cardiac damage. On the basis of these findings, although mouse models may not completely replicate the human situation for LGMD2F, we conclude that careful cardiac monitoring is clearly indicated in patients on long-term corticosteroids.
Asunto(s)
Corticoesteroides/farmacología , Cardiomiopatía Dilatada/tratamiento farmacológico , Hemodinámica/efectos de los fármacos , Miocardio/patología , Prednisolona/farmacología , Sarcoglicanos/deficiencia , Función Ventricular Izquierda/efectos de los fármacos , Administración Oral , Corticoesteroides/administración & dosificación , Corticoesteroides/efectos adversos , Agonistas Adrenérgicos beta/administración & dosificación , Animales , Peso Corporal/efectos de los fármacos , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Modelos Animales de Enfermedad , Dobutamina/administración & dosificación , Fibrosis , Infusiones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Prednisolona/administración & dosificación , Prednisolona/efectos adversos , ARN Mensajero/metabolismo , Sarcoglicanos/genética , Volumen Sistólico/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Presión Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: In the most severely affected patients the mortality for congestive heart failure exceeds that of many cancers. While therapies are largely aimed at attenuating neurohumoral responses recent molecular insights reveal other potential targets for therapy. AIMS: To summarise some of the recent developments in the management of heart failure and provide the clinician who treats heart failure with new insights into emerging approaches. METHODS: A literature review was conducted of the recent literature together with personal research data. RESULTS: Large randomised trials will provide a more comprehensive understanding of the interaction of beta-blockers and other heart failure therapies with gene polymorphisms. Cytokines are important in the progression of heart failure, yet therapy aimed at blocking cytokine effects has not been successful. More selective use of anti-cytokine therapy may have beneficial effects. Gene therapy to improve heart failure has not yet reached clinical trials. The molecular genetics of hypertrophic and dilated cardiomyopathy is rapidly improving our understanding so that genetic diagnostics and counselling may soon be performed for patients and families. CONCLUSIONS: The emergence of a molecular based understanding of heart failure will hopefully improve therapy of this common condition.
Asunto(s)
Cardiomiopatía Dilatada/fisiopatología , Insuficiencia Cardíaca/fisiopatología , Animales , Calcio/fisiología , Cardiomiopatía Dilatada/terapia , Citocinas/fisiología , Técnicas de Transferencia de Gen , Terapia Genética , Insuficiencia Cardíaca/terapia , Humanos , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Transgenic mice overexpressing beta-tropomyosin have increased myofilament Ca(2+) sensitivity that we hypothesized would result in altered relationships among pressure and heart rates, intracellular Ca(2+), and myocardial O(2) consumption. In perfused hearts from transgenic mice there was a marked negative force-frequency response between 6 and 10 Hz with a 30 +/- 3% reduction in peak-positive first derivative of pressure development over time (dP/dt) compared with 14 +/- 2% in wild-type mice (P < 0.001). At 8 Hz systolic pressures were normal, though peak systolic intracellular Ca(2+) was significantly reduced in transgenic mice versus wild type (726 +/- 61 vs. 936 +/- 67 nM, P < 0.05) indicating an alteration in the pressure-Ca(2+) relationship. Over a wide range of positive and negative inotropic interventions there were normal developed pressures, though marked elevations in myocardial O(2) consumption (15-54%). Because pressures are normal and intracellular Ca(2+) decreased and myocardial O(2) consumption increased, this suggests that these abnormalities are at least in part compensatory mechanisms to the altered myofilament function.
Asunto(s)
Calcio/metabolismo , Miocardio/metabolismo , Consumo de Oxígeno/fisiología , Tropomiosina/genética , Animales , Presión Sanguínea/fisiología , Señalización del Calcio/fisiología , Cardiotónicos/farmacología , Dobutamina/farmacología , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , PerfusiónRESUMEN
BACKGROUND: Impaired functional capacity during exercise is used to assess need for transplantation in congestive heart failure patients, although impaired capacity is present in several chronic illnesses. The purpose of this study was to test the hypothesis that ventilatory abnormalities during exercise, rather than functional capacity, are specific to congestive heart failure patients. METHODS: We compared exercise-related gas exchange among a group of congestive heart failure patients and a group of patients who had chronic liver disease and normal cardiac function, matched for functional impairment, and a group of normal controls. RESULTS: Patients with congestive heart failure and patients with chronic liver disease experienced marked reduction in peak exercise oxygen consumption compared with normal controls (14.0 +/- 1.4 and 14.2 +/- 3.7 ml/kg/min, respectively, vs 25.8 +/- 5.6 ml/kg/min, p < 0.01). Minute ventilation at peak exercise was significantly higher in congestive heart failure subjects than in chronic liver disease patients (59.3 +/- 16.8 liter/min vs 41.4 +/- 14.2 liter/min, p < 0.05), although carbon dioxide production was similar (1,380 +/- 308 ml vs 1,180 +/- 389 ml, p = not significant), so that the ratio of minute ventilation to carbon dioxide production (ventilatory equivalent for carbon dioxide, an index of ventilatory drive) was significantly elevated in congestive heart failure subjects (43 +/- 9 vs 36 +/- 7, p < 0.05). CONCLUSIONS: Although functional impairment characterizes both congestive heart failure and chronic liver disease, only congestive heart failure patients exhibit exercise-related ventilatory abnormalities. Exercise-related ventilatory abnormalities may be more specific to the underlying pathophysiology of chronic heart failure and should be considered when evaluating patients for heart transplantation.
Asunto(s)
Prueba de Esfuerzo , Insuficiencia Cardíaca/fisiopatología , Consumo de Oxígeno/fisiología , Fenómenos Fisiológicos Respiratorios , Presión Sanguínea/fisiología , Dióxido de Carbono/análisis , Enfermedad Crónica , Frecuencia Cardíaca/fisiología , Humanos , Hepatopatías/fisiopatología , Oxígeno/análisis , Intercambio Gaseoso Pulmonar/fisiologíaRESUMEN
Recently the dual properties of manganese ion (Mn(2+)) as an MRI contrast agent and a calcium analogue to enter excitable cells has been used to mark specific cells in brain and as a potential intracellular cardiac contrast agent. Here the hypothesis that in vivo manganese-enhanced MRI (MEMRI) can detect changes in inotropy in the mouse heart has been tested. T(1)-weighted images were acquired every minute during an experimental time course of 75 min. Varying doses of Mn(2+) (3.3-14.0 nmoles/min/g BW) were infused during control and altered inotropy with dobutamine (positive inotropy due to increased calcium influx) and the calcium channel blocker diltiazem (negative inotropy). Infusion of MnCl(2) led to a significant increase in signal enhancement in mouse heart that saturated above 3.3 +/- 0.1 nmoles/min/g BW Mn(2+) infusion. At the highest Mn(2+) dose infused there was a 41-47% increase in signal intensity with no alteration in cardiac function as measured by MRI-determined ejection fractions. Dobutamine increased both the steady-state level of enhancement and the rate of MRI signal enhancement. Diltiazem decreased both the steady-state level of enhancement and the rate of MRI signal enhancement. These results are consistent with the model that Mn(2+)-induced enhancement of cardiac signal is indicative of the rate of calcium influx into the heart. Thus, the simultaneous measurement of global function and calcium influx using MEMRI may provide a useful method of evaluating in vivo responses to inotropic therapy.
Asunto(s)
Cloruros , Corazón/anatomía & histología , Imagen por Resonancia Magnética/métodos , Compuestos de Manganeso , Contracción Miocárdica , Animales , Diltiazem , Dobutamina , Masculino , RatonesRESUMEN
BACKGROUND: In perfused hearts, high calcium-induced inotropy results in less developed pressure relative to myocardial oxygen consumption compared to the beta-adrenergic agonist dobutamine. Calcium handling is an important determinant of myocardial oxygen consumption. Therefore, we hypothesized that this phenomenon was due to reduced myofilament responsiveness to calcium, related to protein kinase C activation. RESULTS: Developed pressure was significantly higher with dobutamine compared to high perfusate calcium of 3.5 mM (73 +/- 10 vs 63 +/- 10 mmHg, p < 0.05), though peak systolic intracellular calcium was not significantly different, suggesting reduced myofilament responsiveness to intracellular calcium with high perfusate calcium. The ratio of developed pressure to myocardial oxygen consumption, an index of economy of contraction, was significantly increased with dobutamine compared to high perfusate calcium (1.35 +/- 0.15 vs 1.15 +/- 0.15 mmHg/micromoles/min/g dry wt, p < 0.05), suggesting energetic inefficiency with high perfusate calcium. The specific protein kinase C inhibitor, chelerythrine, significantly attenuated the expected increase in developed pressure when increasing perfusate calcium from 2.5 to 3.5 mM (3.5 mM: 64 +/- 8 vs 3.5 mM + chelerythrine: 55 +/- 5 mmHg, p < 0.05), though had no effects on dobutamine, or lower levels of perfusate calcium (1.5 to 2.5 mM). CONCLUSIONS: By measuring intracellular calcium, developed pressures and myocardial oxygen consumption in perfused mouse hearts, these results demonstrate that high perfusate calcium positive inotropy compared to dobutamine results in reduced myofilament responsiveness to intracellular calcium, which is associated with energetic inefficiency and evidence of protein kinase C activation.
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Citoesqueleto de Actina/metabolismo , Calcio/farmacología , Cardiotónicos/farmacología , Dobutamina/farmacología , Miocardio/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Alcaloides , Animales , Benzofenantridinas , Calcio/metabolismo , Metabolismo Energético , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Masculino , Ratones , Miocardio/enzimología , Técnicas de Cultivo de Órganos , Consumo de Oxígeno , Fenantridinas/farmacologíaRESUMEN
BACKGROUND: Activation of the renin-angiotensin and sympathetic nervous systems adversely affect heart failure progression. The ACE deletion allele (ACE D) is associated with increased renin-angiotensin activation; however, its influence on patient outcomes remains uncertain, and the pharmacogenetic interactions with beta-blocker therapy have not been previously evaluated. METHODS AND RESULTS: We prospectively followed 328 patients (age, 56.1+/-11.9 years) with systolic dysfunction (left ventricular ejection fraction, 0.24+/-0.08) to assess the impact of the ACE D allele on transplant-free survival (median follow-up, 21 months). Transplant-free survival was compared by genotype for the whole cohort and separately in patients with (n=120) and those without beta-blocker therapy (n=208) at the time of entry. Transplant-free survival was significantly poorer for patients with the D: allele (1-year percent survival II/ID/DD=94/77/75; 2-year=78/65/60; ordered log-rank test, P:=0.044). In patients not treated with beta-blockers, the adverse impact of ACE D allele was dramatically increased (1-year percent survival II/ID/DD=95/75/67; 2-year=81/61/48; P:=0.005). In contrast, in patients receiving beta-blocker therapy, no influence of ACE genotype on transplant-free survival was evident (1-year percent survival II/ID/DD=91/80/86; 2-year=70/71/77; P:=0.73). CONCLUSIONS: In a cohort of patients with systolic dysfunction, the ACE D allele was associated with a significantly poorer transplant-free survival. This effect was primarily evident in patients not treated with beta-blockers and was not seen in patients receiving therapy. These findings suggest a potential pharmacogenetic interaction between the ACE D/I polymorphism and therapy with beta-blockers in the determination of heart failure survival.
Asunto(s)
Antagonistas Adrenérgicos beta/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/genética , Peptidil-Dipeptidasa A/genética , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Pruebas Genéticas , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Farmacogenética , Polimorfismo Genético/genética , Estudios Prospectivos , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/genética , Eliminación de Secuencia , Resultado del TratamientoRESUMEN
Both theoretical and experimental results are presented for in vivo calibration of the dissociation constant K(Ca)(d)of the calcium-sensitive fluorescent dye Rhod(2)in the perfused mouse heart, using manganese quenching of fluorescence transients. An analytical model is derived, based on the biochemical equilibrium of manganese competition with calcium for Rhod(2)binding. Expressing the differential of the changes between systole and diastole in fluorescence transient (delta Delta F(sys-dia)). delta DeltaF(sys-dia)in a beating heart as a function of the perfusate manganese concentration [Mn(2+)](p)allows correlation of the measured differential transient changes delta Delta F(sys-dia)with the calcium dissociation constant K(Ca)(d)of Rhod(2)and the calcium concentration in the heart. Numerical modeling indicates that the K(Ca)(d)predominantly affects the asymptotic slope of the delta Delta F(sys-dia)versus [Mn(2+)](p)curve at certain manganese concentrations, which suggests that the K(Ca)(d)can be inversely calculated by partially fitting the delta Delta F(sys-dia)distribution as a function of the perfusate manganese concentration. The feasibility of this approach is confirmed by quenching of calcium transients by manganese infusion into isolated perfused beating mouse hearts. The resulting calculated dissociation constant K(Ca)(d)of Rhod(2)is 720nM. Using the same approach, we are able to also estimate intracellular calcium concentrations of 700nM at peak systole and 300nM in diastole. This is in good agreement with values obtained by calibration of fluorescence values with a calcium saturation tetanization procedure in the same perfused mouse heart model.
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Calcio/análisis , Calcio/metabolismo , Colorantes Fluorescentes/análisis , Modelos Químicos , Miocardio/metabolismo , Animales , Unión Competitiva , Calcio/química , Calibración , Colorantes Fluorescentes/química , Fluorometría , Compuestos Heterocíclicos con 3 Anillos , Técnicas In Vitro , Cinética , Manganeso/análisis , Manganeso/química , Manganeso/metabolismo , Ratones , Miocardio/química , PerfusiónRESUMEN
We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye rhod-2. In Langendorff perfused isolated mouse hearts, rhod-2 is bolused through the perfusate, resulting in a 6.2+/-1.9-fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of 33+/-9%. Quantification of the relative amount of rhod-2 in the heart was done by taking the ratio of absorbance at 524 nm (rhod-2 sensitive) to 589 nm (rhod-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 microM). Electron microscopy was used to determine the subcellular localization of rhod-2, by fixing rhod-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain rhod-2. These images demonstrated prominent cytosolic rhod-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of rhod-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 microM), which increases endothelial cell calcium, had no effects on mean fluorescence (N=4, p=NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686+/-237 nM at peak systole, and 360+/-101 nM in diastole, and with high perfusate calcium (3.5 mM) were 1199+/-215 and 544+/-53 nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice.
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Calcio/metabolismo , Colorantes Fluorescentes , Membranas Intracelulares/metabolismo , Miocardio/metabolismo , Animales , Calcio/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Compuestos Heterocíclicos con 3 Anillos , Técnicas In Vitro , Masculino , Ratones , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Contracción Miocárdica/fisiología , Perfusión , Fracciones Subcelulares/metabolismo , Distribución TisularRESUMEN
Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.
Asunto(s)
Calcio/metabolismo , Miocardio/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Citosol/metabolismo , Estimulación Eléctrica , Colorantes Fluorescentes , Compuestos Heterocíclicos con 3 Anillos , Técnicas In Vitro , Manganeso/farmacología , Ratones , Modelos Cardiovasculares , Oxígeno/metabolismo , Perfusión , Fotones , Dispersión de Radiación , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts (P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium (P < 0.01) and diastolic calcium (P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12-27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 +/- 18 vs. 69 +/- 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.