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OBJECTIVES: The body composition monitor (BCM) is a bioimpedance spectroscopy device, specifically developed for patients on hemodialysis (HD) to improve ultrafiltration (UF) programming, based on an objective assessment of the degree of overhydration (OH) at the start of HD. However, its acceptance in clinical practice remains limited because of concerns about the accuracy at the individual level. The aim of this study is to examine the performance of the BCM and to identify means of improvement. METHODS: Precision of the OH estimate was assessed by 6 consecutive measurements in 24 patients on HD. Accuracy was examined in 45 patients, by comparing the change in OH (ΔOH) during HD with UF volume. Accuracy was considered acceptable if the volume error in individual patients was ≤0.5 L. RESULTS: The OH estimate had an analytical precision of 1.0 ± 0.4%. The correlation between UF volume and ΔOH was moderate (Slope = 0.66, R2 = 0.44, P < .001) and indicated underestimation of UF volume, in particular for high UF volumes. Accuracy at individual level was highly variable. A volume error >0.5 L occurred in 44% of patients. Accuracy improved over the course of HD, with a decrease in total error range from 2.3 L in the first hour to 1.1 L in the final hour of HD. CONCLUSIONS: The accuracy of BCM volume change estimates is highly variable and below requirements of daily practice. Improvement may be achieved by a switch to an end-of-HD measurement.
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Composição Corporal , Diálise Renal , Humanos , Diálise Renal/métodos , Impedância ElétricaRESUMO
We present a patient at risk of misdiagnosis with multiple myeloma due to pseudohypercalcemia. Examinations showed monoclonal protein, 50% monoclonal plasma cells in bone marrow, and hypercalcemia but no osteolytic bone lesions. Follow-up tests revealed pseudohypercalcemia, with elevated total calcium, but normal ionized calcium: a discrepancy due to calcium binding to monoclonal paraprotein (confirmed by laboratory experiments). Accordingly, the patient was diagnosed with smouldering myeloma. After 900 days, the presence of bone lesions prompted the start of treatment for myeloma. Consequently, monoclonal paraprotein levels declined and pseudohypercalcemia dissolved. Hence, ionized calcium should be measured in monoclonal gammopathies to avoid misdiagnosis.
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There is an ongoing debate on the mechanism underlying the pacemaker activity of sinoatrial node (SAN) cells, focusing on the relative importance of the "membrane clock" and the "Ca(2+) clock" in the generation of the small net membrane current that depolarizes the cell towards the action potential threshold. Specifically, the debate centers around the question whether the membrane clock-driven hyperpolarization-activated current, I f , which is also known as the "funny current" or "pacemaker current," or the Ca(2+) clock-driven sodium-calcium exchange current, I NaCa, is the main contributor to diastolic depolarization. In our contribution to this journal's "Special Issue on Cardiac Electrophysiology," we present a numerical reconstruction of I f and I NaCa in isolated rabbit and human SAN pacemaker cells based on experimental data on action potentials, I f , and intracellular calcium concentration ([Ca(2+)] i ) that we have acquired from these cells. The human SAN pacemaker cells have a smaller I f , a weaker [Ca(2+)] i transient, and a smaller I NaCa than the rabbit cells. However, when compared to the diastolic net membrane current, I NaCa is of similar size in human and rabbit SAN pacemaker cells, whereas I f is smaller in human than in rabbit cells.
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Relógios Biológicos/fisiologia , Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Nó Sinoatrial/fisiologia , Sódio/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Simulação por Computador , Humanos , Ativação do Canal Iônico/fisiologia , Modelos Cardiovasculares , Coelhos , Trocador de Sódio e Cálcio/fisiologia , Especificidade da EspécieRESUMO
Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of â¼200 cp/µL was achieved within â¼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.
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Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca(2+) (Ca(2+)(i)) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca(2+)(i) were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca(2+) current (I(Ca,L)) density, but left densities of sodium current, inward rectifier K(+) current, transient outward K(+) current, and rapid component of the delayed rectifier K(+) current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca(2+)(i), Ca(2+)(i) transient amplitude, and sarcoplasmic reticulum Ca(2+) content. ABPPs did not affect the Na(+)-Ca(2+) exchange current (I(NCX)) in Ca(2+)-buffered conditions. Products secreted from activated human platelets induce changes in I(Ca,L) and Ca(2+)(i), which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis.
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Plaquetas/metabolismo , Miócitos Cardíacos/fisiologia , Ativação Plaquetária/fisiologia , Função Ventricular , Potenciais de Ação/efeitos dos fármacos , Animais , Fatores Biológicos/metabolismo , Fatores Biológicos/farmacologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Tripsina/metabolismoRESUMO
Monitoring of hydration in patients on hemodialysis (HD) by currently available bioelectrical impedance analysis (BIA) methods is hampered by limited accuracy. This may be caused by changes in total body electrical resistance (TBER) that are induced by processes other than ultrafiltration (UF). To identify these sources of error, we examined the impact of UF, diffusion, and postural change (PC), separately. Extracellular TBER (TBERe) was measured by bioimpedance spectroscopy every 30 min in 23 patients on HD, for 2 h during diffusion-only (DO), followed by 2-h UF-only (UFO). The impact of PC from upright to semi-recumbent position was assessed by a 2-h TBERe measurement on the day after HD. TBERe increased by 23.5 ± 12.4 Ω (P < 0.001) during DO and by 40.0 ± 16.2 Ω (P < 0.001) during UFO. PC, evaluated on a separate day, was associated with an increase in TBERe of 27.6 ± 26.0 Ω (P < 0.001). TBERe changes during DO were mainly attributed to PC and to a lesser extent to electrolyte exchange. Extrapolation of the data to a conventional 4-h HD session indicates that about 32% of the total increase in TBERe is not related to UF. In conclusion, a significant part of the increase in TBER during HD is not related to UF but can be attributed to other processes such as the effects of PC and diffusion-related electrolyte exchange. These factors have to be taken into account when TBER-guided UF is considered.NEW & NOTEWORTHY Current BIA methods have limited accuracy in patients on HD. This may be related to the incorrect assumption that all changes in total body electrical resistance (TBER) are caused by changes in body water volumes. The present study indicates that two-thirds of the change in TBER during a conventional 4-h HD session can be attributed to fluid extraction, and that the remaining part is caused by other processes such as postural change and electrolyte exchange. This may cause volume prediction errors when not recognized.
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Diálise Renal , Ultrafiltração , Água Corporal , Impedância Elétrica , Humanos , PosturaRESUMO
Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.
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Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio/normas , Medições Luminescentes/normas , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/sangue , COVID-19/imunologia , COVID-19/virologia , Teste Sorológico para COVID-19/instrumentação , Calibragem , Proteínas de Ligação ao GTP/química , Genes Reporter , Humanos , Imunoconjugados/química , Limite de Detecção , Luciferases/genética , Luciferases/metabolismo , Testes Imediatos , SARS-CoV-2/genéticaRESUMO
We investigated the contribution of the intracellular calcium (Ca (i) (2+) ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca (i) (2+) transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE((R))). Our data show that the Ca (i) (2+) transient, like the hyperpolarization-activated "funny current" (I (f)) and the ACh-sensitive potassium current (I (K,ACh)), is an important determinant of ACh-mediated pacemaker slowing. When I (f) and I (K,ACh) were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 micro M ACh was still able to reduce pacemaker frequency by 72%. In these I (f) and I (K,ACh)-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca (i) (2+) transient decay (r (2) = 0.98) and slow diastolic Ca (i) (2+) rise (r (2) = 0.73). Inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (5 microM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca (i) (2+) transient and reduced the sarcoplasmic reticulum (SR) Ca(2+) content, all in a concentration-dependent fashion. At 1 microM ACh, the spontaneous activity and Ca (i) (2+) transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 microM) and I (K,ACh) was inhibited by tertiapin (100 nM). Also, inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (25 microM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca (i) (2+) affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca (i) (2+) transient via a cAMP-dependent signaling pathway. Inhibition of the Ca (i) (2+) transient contributes to pacemaker slowing and inhibits Ca (i) (2+) -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca (i) (2+) transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.
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Acetilcolina/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Receptores Muscarínicos/metabolismo , Nó Sinoatrial/metabolismo , Animais , Ácido Egtázico/análogos & derivados , Agonistas Muscarínicos , Técnicas de Patch-Clamp , Coelhos , Rianodina , Retículo Sarcoplasmático/metabolismo , Nó Sinoatrial/citologiaRESUMO
Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH(i)) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na(+)-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2(a,b)(-/-) mice (deficient in the main isoforms Ae2a, Ae2b(1), and Ae2b(2)). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2(a,b)(-/-) mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2(a,b)(-/-) mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts--but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2(a,b)(-/-) mice.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Antiporters/fisiologia , Osso e Ossos/anormalidades , Osteoclastos/fisiologia , Osteopetrose/genética , Animais , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Osteoclastos/metabolismo , Proteínas SLC4A , Crânio/anormalidadesRESUMO
A 28-year-old female was admitted 2 years after gastric bypass limb distalization because of severe weight loss, fatigue, chronic diarrhea, massive edema, and a serum albumin of 10 g/L without proteinuria. A diagnosis of severe energy and protein malnutrition was made, and enteral tube feeding was started in combination with pancreatic enzyme supplementation every 3 h. Within 24 h after the start of tube feeding, she developed severe hyperammonemia. Tube feeding was stopped immediately, and this led to a normalization of serum ammonia levels within 8 h. When tube feeding was resumed, albeit at a lower rate and with preventive measures taken, hyperammonemia occurred again. The underlying causes and treatments of hyperammonemia during tube feeding are discussed.
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Nutrição Enteral/efeitos adversos , Derivação Gástrica/efeitos adversos , Hiperamonemia/etiologia , Desnutrição Proteico-Calórica/terapia , Adulto , Feminino , Humanos , Desnutrição Proteico-Calórica/etiologiaRESUMO
BACKGROUND: Severe protein malnutrition, with a serum albumin < 25 g/L, is one of the complications that may develop after bariatric surgery. It is associated with increased morbidity and mortality and requires timely diagnosis and appropriate treatment to prevent rapid clinical deterioration. However, evidence-based recommendations for a specific treatment approach are currently not available. The present study describes the efficacy of a newly developed treatment regimen for post-bariatric patients presenting with severe hypoalbuminemia. METHODS: A single-centre, retrospective analysis of eleven post-bariatric patients presenting with severe hypoalbuminemia, treated with continuous 24 h nasal-jejunal tube feeding of a medium chain triglyceride (MCT) formulation in combination with pancreatic enzyme supplementation every 3 h. RESULTS: Duration of tube feeding ranged from 25 to 156 days (median 64 days) and pancreatic enzyme was supplemented for 22-195 days (median 75 days). An increase in serum albumin levels of 5 g/L and 10 g/L was achieved after a median period of 20 (range 6-26 days) and 36 days (range 21-57 days), respectively. Albumin levels were > 35 g/L after a median period of 58 days (range 44-171 days). CONCLUSION: In this case series, a continuous 24-h nasal-jejunal MCT tube feed combined with frequent pancreatic enzyme supplementation was effective in all patients presenting with severe post-bariatric hypoalbuminemia and was not associated with adverse effects.
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Cirurgia Bariátrica/efeitos adversos , Nutrição Enteral , Fármacos Gastrointestinais/uso terapêutico , Pancreatina/uso terapêutico , Desnutrição Proteico-Calórica/terapia , Adulto , Idoso , Terapia de Reposição de Enzimas , Feminino , Humanos , Hipoalbuminemia/etiologia , Hipoalbuminemia/terapia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/terapia , Desnutrição Proteico-Calórica/etiologia , Estudos RetrospectivosRESUMO
Adequate glucocorticoid replacement in patients with primary or secondary adrenal insufficiency is essential to maintain general well-being. Little is known about the effects of bariatric surgery on glucocorticoid absorption. This study evaluates glucocorticoid absorption before and after bariatric surgery, with assessment of plasma cortisol profiles in five patients receiving glucocorticoid replacement therapy for primary (n = 1) or secondary (n = 4) adrenal insufficiency. One patient underwent sleeve gastrectomy (SG), one a one-anastomosis gastric bypass (mini-GB), and three a Roux-en-Y gastric bypass (RYGB). Pharmacokinetic calculations were based on plasma cortisol measurements performed during the first 6 hours after ingestion of the morning dose. Plasma cortisol profiles were very similar before and after surgery; only minor differences were observed. After SG, plasma peak cortisol concentration and cortisol area under the curve (AUC) were higher by 23% and 24%, respectively, and time to peak cortisol was 10 minutes shorter. The mini-GB had no marked effect on pharmacokinetic parameters. In the three patients who underwent RYGB, AUC changes ranged from -12% to 20%. In conclusion, in this small number of patients with adrenal insufficiency, plasma cortisol profiles were similar before and after bariatric surgery. However, in view of individual differences in response to different types of surgery, we recommend postoperative cortisol profiling to guide appropriate glucocorticoid dose adjustment.
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BACKGROUND: Pharmacokinetic data suggest that the risk of metformin-associated lactic acidosis (MALA) may be increased after Roux-en-Y gastric bypass (RYGB) surgery. The aim of this study was to examine the impact of metformin on plasma lactate levels before and after RYGB surgery. METHODS: Retrospective study of plasma lactate levels before and 3 months after RYGB surgery in patients with type 2 diabetes mellitus (T2DM) not using metformin (MET-0, N = 58), on a stable dose (MET-S, N = 138), or on a decreasing dose (MET-D, N = 85) of metformin. RESULTS: Preoperatively, lactate levels were similar in patients on metformin (1.8 ± 0.05 mmol/L) and those not on metformin (1.7 ± 0.08 mmol/L), P = 0.21. Three months postoperatively, lactate levels had decreased in all groups (P < 0.001) to 1.3 ± 0.07 (SE), 1.4 ± 0.05, and 1.2 ± 0.05 mmol/l in MET-0, MET-S, and MET-D, respectively. Lactate levels differed between the groups (P = 0.03), with the lowest level in MET-D. The number of patients with hyperlactatemia (lactate > 2 mmol/l) decreased from 31 to 14%, from 22 to 8.6%, and from 26 to 4.7% in MET-S, MET-0, and MET-D, respectively. CONCLUSION: Mild hyperlactatemia (lactate > 2 mmol/l) is common in morbidly obese patients with T2DM. It is probably related to increase lactate production by adipocytes. Lactate levels decreased after RYGB-induced weight loss, irrespective of the use of metformin. We therefore conclude that there is no need for routinely lowering of the metformin dose after uncomplicated RYGB surgery, as long as normal renal function is preserved.
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Acidose Láctica/induzido quimicamente , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/efeitos adversos , Ácido Láctico/sangue , Metformina/efeitos adversos , Obesidade Mórbida/cirurgia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/cirurgia , Feminino , Derivação Gástrica/efeitos adversos , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Metformina/uso terapêutico , Pessoa de Meia-Idade , Obesidade Mórbida/sangue , Obesidade Mórbida/tratamento farmacológico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, is increasingly used in obese patients with type 2 diabetes mellitus (T2DM) in doses of up to 3.0 mg/day because of its attractive pharmacological profile. It is currently not known how to proceed with this medication during fasting for surgery. Discontinuation is likely to result in hyperglycaemia, while continuation might lead to hypoglycaemia, but, in view of its mode of action, continuation of GLP-1 analogs is likely to be safe. However, as evidence-based guidelines on GLP-1 management during perioperative fasting are not available, the safety of either policy needs to be confirmed on an individual basis. We therefore decided to perform a preoperative assessment of the glucose response to fasting during continuation of GLP-1 before giving a recommendation in individual cases. So far, 12 severely obese T2DM patients scheduled for bariatric surgery have been evaluated preoperatively by measuring glucose and insulin levels during a 32-hour fast with continuation of liraglutide. Hypoglycaemia was not observed. This suggests that liraglutide in doses of up to 3.0 mg can be safely continued during surgery without risking hypoglycaemia. LEARNING POINTS: Evidence-based guidelines on how to proceed with GLP-1 analogs during perioperative fasting are currently not available.Our observations suggest that liraglutide in doses of up to 3.0 mg/day can be safely continued during perioperative fasting for 32 hours.Further evaluation is needed for fasting periods longer than 32 hours.
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The endocrine function of the heart was shown by the identification of a potent inhibitor of renal tubular NaCl reabsorbtion, atrial natriuretic peptide/factor (ANP), in atrial extracts. Since then, Nppa, the gene encoding ANP, has become an important tool for molecular and developmental biologists. Expression of Nppa is an early and specific marker for the differentiating working myocardium of the atria and ventricles of the developing heart. Around the time of birth, ventricular Nppa expression is down-regulated, to be reactivated in response to a variety of cardiovascular disorders. Many aspects of the regulatory pathways that control Nppa gene expression during cardiac development or disease have been revealed. However, several fundamental issues remain to be resolved. Particularly, the regulatory mechanisms underlying the ventricular Nppa expression in the embryonic heart and the reactivation in the failing ventricle remain unclear. Here we review current knowledge on the transcriptional regulation of the Nppa gene.
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Fator Natriurético Atrial/genética , Doenças Cardiovasculares/metabolismo , Desenvolvimento Fetal/fisiologia , Regulação da Expressão Gênica , Coração/embriologia , Contração Miocárdica/fisiologia , Animais , Biomarcadores/análise , Diferenciação Celular , Hipóxia Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Transgênicos , Transcrição GênicaRESUMO
OBJECTIVE: In patients with heart disease, the transition from compensatory hypertrophy to heart failure (HF) is associated with altered calcium handling. Up-regulated Na(+)/H(+)-exchanger (NHE-1) activity underlies increased [Na(+)](i) and disturbance of cellular calcium handling in HF. We hypothesize that chronic inhibition of NHE-1 activity prevents the hypertrophic response, cellular remodeling, and development of HF. METHODS: Rabbits received a control or cariporide (inhibitor of NHE-1) diet for 3 months, starting after induction of combined volume and pressure overload. Age-matched animals served as control. Development of HF was examined echocardiographically and electrocardiographically after 3 months. [Na(+)](i), [Ca(2+)](i), pH(i), and action potentials were measured in left ventricular midmural myocytes with SBFI, indo-1, SNARF, and di-4-anepps. Sarcoplasmic reticulum calcium content was calculated from the response of [Ca(2+)](i) to rapid cooling. Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. RESULTS: Chronic treatment of rabbits with the specific Na(+)/H(+)-exchanger activity inhibitor cariporide greatly attenuated development of hypertrophy and entirely abolished development of HF; the heart/body weight ratio increased only little, no change in lung weight occurred, left ventricular dimensions and fractional shortening changed mildly, ascites was not present, QT duration did not increase, and sudden death did not occur. Chronic cariporide treatment also prevented cellular electrical and ionic remodeling. Myocyte dimensions were unaltered, action potentials were not prolonged, cytoplasmic sodium and NHE-1 activity did not increase, cytoplasmic and SR calcium handling remained undisturbed, and no increase of the incidence of calcium after-transient dependent delayed after depolarizations (DADs) occurred. CONCLUSION: We conclude that enhanced activity of NHE-1 underlies cardiac cellular electrical and ionic remodeling in experimental heart failure, and that chronic dietary treatment with cariporide attenuates hypertrophy, development of HF, and cellular remodeling.
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Cardiomegalia/prevenção & controle , Guanidinas/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Sulfonas/uso terapêutico , Potenciais de Ação , Animais , Cálcio/metabolismo , Cardiomegalia/diagnóstico , Cardiomegalia/metabolismo , Citoplasma/metabolismo , Ecocardiografia , Eletrocardiografia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/metabolismo , Masculino , Coelhos , Retículo Sarcoplasmático/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Remodelação VentricularRESUMO
BACKGROUND: Sphingosine-1-phosphate plays vital roles in cardiomyocyte physiology, myocardial ischemia-reperfusion injury, and ischemic preconditioning. The function of the cardiomyocyte sphingosine-1-phosphate receptor 1 (S1P1) in vivo is unknown. METHODS AND RESULTS: Cardiomyocyte-restricted deletion of S1P1 in mice (S1P1 (α) (MHCC) (re)) resulted in progressive cardiomyopathy, compromised response to dobutamine, and premature death. Isolated cardiomyocytes from S1P1 (α) (MHCC) (re) mice revealed reduced diastolic and systolic Ca(2+) concentrations that were secondary to reduced intracellular Na(+) and caused by suppressed activity of the sarcolemmal Na(+)/H(+) exchanger NHE-1 in the absence of S1P1. This scenario was successfully reproduced in wild-type cardiomyocytes by pharmacological inhibition of S1P1 or sphingosine kinases. Furthermore, Sarcomere shortening of S1P1 (α) (MHCC) (re) cardiomyocytes was intact, but sarcomere relaxation was attenuated and Ca(2+) sensitivity increased, respectively. This went along with reduced phosphorylation of regulatory myofilament proteins such as myosin light chain 2, myosin-binding protein C, and troponin I. In addition, S1P1 mediated the inhibitory effect of exogenous sphingosine-1-phosphate on ß-adrenergic-induced cardiomyocyte contractility by inhibiting the adenylate cyclase. Furthermore, ischemic precondtioning was abolished in S1P1 (α) (MHCC) (re) mice and was accompanied by defective Akt activation during preconditioning. CONCLUSIONS: Tonic S1P1 signaling by endogenous sphingosine-1-phosphate contributes to intracellular Ca(2+) homeostasis by maintaining basal NHE-1 activity and controls simultaneously myofibril Ca(2+) sensitivity through its inhibitory effect on adenylate cyclase. Cardioprotection by ischemic precondtioning depends on intact S1P1 signaling. These key findings on S1P1 functions in cardiac physiology may offer novel therapeutic approaches to cardiac diseases.
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Cálcio/metabolismo , Cardiomiopatias/genética , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/metabolismo , Receptores de Lisoesfingolipídeo/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Potenciais de Ação , Adenilil Ciclases/metabolismo , Animais , Western Blotting , Miosinas Cardíacas/metabolismo , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/metabolismo , Proteínas de Transporte/metabolismo , Ecocardiografia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Tomografia por Emissão de Pósitrons , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Sarcômeros/metabolismo , Receptores de Esfingosina-1-Fosfato , Troponina I/metabolismoRESUMO
OBJECTIVE: Hypertrophied myocytes of failing hearts have prolonged action potential durations. It is unknown how the swelling-activated Cl(-) current (I(Cl,swell)) affects the abnormal AP configuration. METHODS: We studied I(Cl,swell) in ventricular myocytes isolated from failing and age-matched normal rabbit hearts. We applied whole-cell patch-clamp methodology and activated I(Cl,swell) by lowering tonicity of the superfusate. RESULTS: Neither with ruptured-patch nor with amphotericin B perforated-patch, whole-cell clamp we found I(Cl,swell) active under isotonic conditions in either the normal or the hypertrophied failing heart (HFH) myocytes. I(Cl,swell) caused AP shortening and resting membrane potential (V(m)) depolarization in an osmotic gradient-dependent fashion. However, in the HFH myocytes swelling-induced AP changes were significantly smaller, even though the cells underwent the same relative change in planar cell surface area. Voltage-clamp experiments revealed that in HFH myocytes I(Cl,swell) current density was approximately 50% reduced. CONCLUSION: Reduced I(Cl,swell) densities in HFH myocytes cause limited AP shortening and V(m) depolarization upon swelling of the cells.
Assuntos
Canais de Cloreto/metabolismo , Insuficiência Cardíaca/metabolismo , Potenciais de Ação , Animais , Tamanho Celular , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Soluções Isotônicas/farmacologia , Masculino , Potenciais da Membrana , Concentração Osmolar , Técnicas de Patch-Clamp , CoelhosRESUMO
BACKGROUND: In animal models of heart failure (HF), heart rate decreases due to an increase in intrinsic cycle length (CL) of the sinoatrial node (SAN). Pacemaker activity of SAN cells is complex and modulated by the membrane clock, i.e., the ensemble of voltage gated ion channels and electrogenic pumps and exchangers, and the Ca(2+) clock, i.e., the ensemble of intracellular Ca(2+) ([Ca(2+)]i) dependent processes. HF in SAN cells results in remodeling of the membrane clock, but few studies have examined its effects on [Ca(2+)]i homeostasis. METHODS: SAN cells were isolated from control rabbits and rabbits with volume and pressure overload-induced HF. [Ca(2+)]i concentrations, and action potentials (APs) and Na(+)-Ca(2+) exchange current (INCX) were measured using indo-1 and patch-clamp methodology, respectively. RESULTS: The frequency of spontaneous [Ca(2+)]i transients was significantly lower in HF SAN cells (3.0 ± 0.1 (n = 40) vs. 3.4 ± 0.1 Hz (n = 45); mean ± SEM), indicating that intrinsic CL was prolonged. HF slowed the [Ca(2+)]i transient decay, which could be explained by the slower frequency and reduced sarcoplasmic reticulum (SR) dependent rate of Ca(2+) uptake. Other [Ca(2+)]i transient parameters, SR Ca(2+) content, INCX density, and INCX-[Ca(2+)]i relationship were all unaffected by HF. Combined AP and [Ca(2+)]i recordings demonstrated that the slower [Ca(2+)]i transient decay in HF SAN cells may result in increased INCX during the diastolic depolarization, but that this effect is likely counteracted by the HF-induced increase in intracellular Na(+). ß-adrenergic and muscarinic stimulation were not changed in HF SAN cells, except that late diastolic [Ca(2+)]i rise, a prominent feature of the Ca(2+) clock, is lower during ß-adrenergic stimulation. CONCLUSIONS: HF SAN cells have a slower [Ca(2+)]i transient decay with limited effects on pacemaker activity. Reduced late diastolic [Ca(2+)]i rise during ß-adrenergic stimulation may contribute to an impaired increase in intrinsic frequency in HF SAN cells.