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1.
Anesth Analg ; 136(5): 941-948, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37058731

RESUMO

BACKGROUND: Early detection and quantification of perioperative hemorrhage remains challenging. Peripheral intravenous waveform analysis (PIVA) is a novel method that uses a standard intravenous catheter to detect interval hemorrhage. We hypothesize that subclinical blood loss of 2% of the estimated blood volume (EBV) in a rat model of hemorrhage is associated with significant changes in PIVA. Secondarily, we will compare PIVA association with volume loss to other static, invasive, and dynamic markers. METHODS: Eleven male Sprague Dawley rats were anesthetized and mechanically ventilated. A total of 20% of the EBV was removed over ten 5 minute-intervals. The peripheral intravenous pressure waveform was continuously transduced via a 22-G angiocatheter in the saphenous vein and analyzed using MATLAB. Mean arterial pressure (MAP) and central venous pressure (CVP) were continuously monitored. Cardiac output (CO), right ventricular diameter (RVd), and left ventricular end-diastolic area (LVEDA) were evaluated via transthoracic echocardiogram using the short axis left ventricular view. Dynamic markers such as pulse pressure variation (PPV) were calculated from the arterial waveform. The primary outcome was change in the first fundamental frequency (F1) of the venous waveform, which was assessed using analysis of variance (ANOVA). Mean F1 at each blood loss interval was compared to the mean at the subsequent interval. Additionally, the strength of the association between blood loss and F1 and each other marker was quantified using the marginal R2 in a linear mixed-effects model. RESULTS: PIVA derived mean F1 decreased significantly after hemorrhage of only 2% of the EBV, from 0.17 to 0.11 mm Hg, P = .001, 95% confidence interval (CI) of difference in means 0.02 to 0.10, and decreased significantly from the prior hemorrhage interval at 4%, 6%, 8%, 10%, and 12%. Log F1 demonstrated a marginal R2 value of 0.57 (95% CI 0.40-0.73), followed by PPV 0.41 (0.28-0.56) and CO 0.39 (0.26-0.58). MAP, LVEDA, and systolic pressure variation displayed R2 values of 0.31, and the remaining predictors had R2 values ≤0.2. The difference in log F1 R2 was not significant when compared to PPV 0.16 (95% CI -0.07 to 0.38), CO 0.18 (-0.06 to 0.04), or MAP 0.25 (-0.01 to 0.49) but was significant for the remaining markers. CONCLUSIONS: The mean F1 amplitude of PIVA was significantly associated with subclinical blood loss and most strongly associated with blood volume among the markers considered. This study demonstrates feasibility of a minimally invasive, low-cost method for monitoring perioperative blood loss.


Assuntos
Pressão Arterial , Volume Sanguíneo , Masculino , Animais , Ratos , Ratos Sprague-Dawley , Pressão Sanguínea , Hemorragia/diagnóstico , Hemodinâmica
2.
Pediatr Res ; 93(6): 1539-1545, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36042330

RESUMO

BACKGROUND: Peripheral intravenous analysis (PIVA) has been shown to be more sensitive than central venous pressure (CVP) for detecting hemorrhage and volume overload. We hypothesized that PIVA is superior to CVP for detecting right ventricular (RV) failure in a rat model of respiratory arrest. METHODS: Eight Wistar rats were studied in accordance with the ARRIVE guidelines. CVP, mean arterial pressure (MAP), and PIVA were recorded. Respiratory arrest was achieved with IV Rocuronium. PIVA utilizes Fourier transform to quantify the amplitude of the peripheral venous waveform, expressed as the "f1 amplitude". RV diameter was measured with transthoracic echocardiography. RESULTS: RV diameter increased from 0.34 to 0.54 cm during arrest, p = 0.001, and returned to 0.33 cm post arrest, p = 0.97. There was an increase in f1 amplitude from 0.07 to 0.38 mmHg, p = 0.01 and returned to 0.08 mmHg, p = 1.0. MAP decreased from 119 to 67 mmHg, p = 0.004 and returned to 136 mmHg, p = 0.50. There was no significant increase in CVP from 9.3 mmHg at baseline to 10.5 mmHg during respiratory arrest, p = 0.91, and recovery to 8.6 mmHg, p = 0.81. CONCLUSIONS: This study highlights the utility of PIVA to detect RV failure in small-caliber vessels, comparable to peripheral veins in the human pediatric population. IMPACT: Right ventricular failure remains a diagnostic challenge, particularly in pediatric patients with small vessel sizes limiting invasive intravascular monitor use. Intravenous analysis has shown promise in detecting hypovolemia and volume overload. Intravenous analysis successfully detects right ventricular failure in a rat respiratory arrest model. Intravenous analysis showed utility despite utilizing small peripheral venous access and therefore may be applicable to a pediatric population. Intravenous analysis may be helpful in differentiating various types of shock.


Assuntos
Insuficiência Cardíaca , Insuficiência Respiratória , Humanos , Criança , Animais , Ratos , Ratos Wistar , Pressão Venosa Central , Ecocardiografia , Infusões Intravenosas
3.
JCI Insight ; 7(3)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-34990403

RESUMO

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.


Assuntos
Calsequestrina/genética , Regulação da Expressão Gênica , Frequência Cardíaca/fisiologia , Células de Purkinje/patologia , RNA/genética , Taquicardia Ventricular/diagnóstico , Animais , Calsequestrina/biossíntese , Linhagem Celular , Modelos Animais de Doenças , Camundongos Knockout , Células de Purkinje/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologia
4.
Semin Cardiothorac Vasc Anesth ; 25(1): 11-18, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32957831

RESUMO

BACKGROUND: Assessing intravascular hypovolemia due to hemorrhage remains a clinical challenge. Central venous pressure (CVP) remains a commonly used monitor in surgical and intensive care settings for evaluating blood loss, despite well-described pitfalls of static pressure measurements. The authors investigated an alternative to CVP, intravenous waveform analysis (IVA) as a method for detecting blood loss and examined its correlation with echocardiography. METHODS: Seven anesthetized, spontaneously breathing male Sprague Dawley rats with right internal jugular central venous and femoral arterial catheters underwent hemorrhage. Mean arterial pressure (MAP), heart rate, CVP, and IVA were assessed and recorded. Hemorrhage was performed until each rat had 25% estimated blood volume removed. IVA was obtained using fast Fourier transform and the amplitude of the fundamental frequency (f1) was measured. Transthoracic echocardiography was performed utilizing a parasternal short axis image of the left ventricle during hemorrhage. MAP, CVP, and IVA were compared with blood removed and correlated with left ventricular end diastolic area (LVEDA). RESULTS: All 7 rats underwent successful hemorrhage. MAP and f1 peak amplitude obtained by IVA showed significant changes with hemorrhage. MAP and f1 peak amplitude also significantly correlated with LVEDA during hemorrhage (R = 0.82 and 0.77, respectively). CVP did not significantly change with hemorrhage, and there was no significant correlation between CVP and LVEDA. CONCLUSIONS: In this study, f1 peak amplitude obtained by IVA was superior to CVP for detecting acute, massive hemorrhage. In addition, f1 peak amplitude correlated well with LVEDA on echocardiography. Translated clinically, IVA might provide a viable alternative to CVP for detecting hemorrhage.


Assuntos
Pressão Venosa Central/fisiologia , Ecocardiografia/métodos , Hemorragia/complicações , Hipovolemia/complicações , Hipovolemia/diagnóstico , Animais , Modelos Animais de Doenças , Hemorragia/fisiopatologia , Hipovolemia/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley
5.
Hypertension ; 68(2): 418-26, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27271310

RESUMO

UNLABELLED: Splanchnic venous pooling is a major hemodynamic determinant of orthostatic hypotension, but is not specifically targeted by pressor agents, the mainstay of treatment. We developed an automated inflatable abdominal binder that provides sustained servo-controlled venous compression (40 mm Hg) and can be activated only on standing. We tested the efficacy of this device against placebo and compared it to midodrine in 19 autonomic failure patients randomized to receive either placebo, midodrine (2.5-10 mg), or placebo combined with binder on separate days in a single-blind, crossover study. Systolic blood pressure (SBP) was measured seated and standing before and 1-hour post medication; the binder was inflated immediately before standing. Only midodrine increased seated SBP (31±5 versus 9±4 placebo and 7±5 binder, P=0.003), whereas orthostatic tolerance (defined as area under the curve of upright SBP [AUCSBP]) improved similarly with binder and midodrine (AUCSBP, 195±35 and 197±41 versus 19±38 mm Hg×minute for placebo; P=0.003). Orthostatic symptom burden decreased with the binder (from 21.9±3.6 to 16.3±3.1, P=0.032) and midodrine (from 25.6±3.4 to 14.2±3.3, P<0.001), but not with placebo (from 19.6±3.5 to 20.1±3.3, P=0.756). We also compared the combination of midodrine and binder with midodrine alone. The combination produced a greater increase in orthostatic tolerance (AUCSBP, 326±65 versus 140±53 mm Hg×minute for midodrine alone; P=0.028, n=21) and decreased orthostatic symptoms (from 21.8±3.2 to 12.9±2.9, P<0.001). In conclusion, servo-controlled abdominal venous compression with an automated inflatable binder is as effective as midodrine, the standard of care, in the management of orthostatic hypotension. Combining both therapies produces greater improvement in orthostatic tolerance. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00223691.


Assuntos
Hipotensão Ortostática , Dispositivos de Compressão Pneumática Intermitente , Midodrina , Circulação Esplâncnica , Idoso , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Determinação da Pressão Arterial/métodos , Feminino , Humanos , Hipotensão Ortostática/diagnóstico , Hipotensão Ortostática/fisiopatologia , Hipotensão Ortostática/terapia , Masculino , Midodrina/administração & dosagem , Midodrina/efeitos adversos , Monitorização Fisiológica/métodos , Circulação Esplâncnica/efeitos dos fármacos , Circulação Esplâncnica/fisiologia , Resultado do Tratamento , Vasoconstritores/administração & dosagem , Vasoconstritores/efeitos adversos
6.
Shock ; 46(4): 447-52, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27070329

RESUMO

BACKGROUND: Unrecognized hemorrhage and unguided resuscitation is associated with increased perioperative morbidity and mortality. The authors investigated peripheral venous waveform analysis (PIVA) as a method for quantitating hemorrhage as well as iatrogenic fluid overload during resuscitation. METHODS: The authors conducted a prospective study on Yorkshire Pigs (n = 8) undergoing hemorrhage, autologous blood return, and administration of balanced crystalloid solution beyond euvolemia. Intra-arterial blood pressure, electrocardiogram, and pulse oximetry were applied to each subject. Peripheral venous pressure was measured continuously through an upper extremity standard peripheral IV catheter and analyzed with LabChart. The primary outcome was comparison of change in the first fundamental frequency (f1) of PIVA with standard and invasive monitoring and shock index (SI). RESULTS: Hemorrhage, return to euvolemia, and iatrogenic fluid overload resulted in significantly non-zero slopes of f1 amplitude. There were no significant differences in heart rate or mean arterial pressure, and a late change in SI. For the detection of hypovolemia the PIVA f1 amplitude change generated an receiver operator curves (ROC) curve with an area under the curve (AUC) of 0.93; heart rate AUC = 0.61; mean arterial pressure AUC = 0.48, and SI AUC = 0.72. For hypervolemia the f1 amplitude generated an ROC curve with an AUC of 0.85, heart rate AUC = 0.62, mean arterial pressure AUC = 0.63, and SI AUC = 0.65. CONCLUSIONS: In this study, PIVA demonstrated a greater sensitivity for detecting acute hemorrhage, return to euvolemia, and iatrogenic fluid overload compared with standard monitoring and SI. PIVA may provide a low-cost, minimally invasive monitoring solution for monitoring and resuscitating patients with perioperative hemorrhage.


Assuntos
Análise de Fourier , Hemorragia/diagnóstico , Hipovolemia/diagnóstico , Doença Iatrogênica , Animais , Pressão Arterial/fisiologia , Volume Sanguíneo/fisiologia , Frequência Cardíaca/fisiologia , Hemorragia/fisiopatologia , Hipovolemia/fisiopatologia , Masculino , Estudos Prospectivos , Ressuscitação/efeitos adversos , Suínos
7.
IEEE Trans Biomed Circuits Syst ; 10(4): 912-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26863670

RESUMO

Bioelectrical impedance analysis (BIA) is a noninvasive and commonly used method for the assessment of body composition including body water. We designed a small, portable and wireless multi-frequency impedance spectrometer based on the 12 bit impedance network analyzer AD5933 and a precision wide-band constant current source for tetrapolar whole body impedance measurements. The impedance spectrometer communicates via Bluetooth with mobile devices (smart phone or tablet computer) that provide user interface for patient management and data visualization. The export of patient measurement results into a clinical research database facilitates the aggregation of bioelectrical impedance analysis and biolectrical impedance vector analysis (BIVA) data across multiple subjects and/or studies. The performance of the spectrometer was evaluated using a passive tissue equivalent circuit model as well as a comparison of body composition changes assessed with bioelectrical impedance and dual-energy X-ray absorptiometry (DXA) in healthy volunteers. Our results show an absolute error of 1% for resistance and 5% for reactance measurements in the frequency range of 3 kHz to 150 kHz. A linear regression of BIA and DXA fat mass estimations showed a strong correlation (r(2)=0.985) between measures with a maximum absolute error of 6.5%. The simplicity of BIA measurements, a cost effective design and the simple visual representation of impedance data enables patients to compare and determine body composition during the time course of a specific treatment plan in a clinical or home environment.


Assuntos
Composição Corporal/fisiologia , Espectroscopia Dielétrica/métodos , Absorciometria de Fóton , Adulto , Idoso , Estatura , Peso Corporal , Telefone Celular , Espectroscopia Dielétrica/instrumentação , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Tecnologia sem Fio
9.
Lab Chip ; 13(23): 4663-73, 2013 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-24126415

RESUMO

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.


Assuntos
Catecolaminas/análise , Células Cromafins/metabolismo , Cromatografia Líquida de Alta Pressão , Técnicas Eletroquímicas , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Cálcio/metabolismo , Carbacol/farmacologia , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Dimetilpolisiloxanos , Eletrodos , Cinética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Platina/química , Cloreto de Potássio/farmacologia , Silício/química , Estimulação Química
10.
J Mol Cell Cardiol ; 60: 8-15, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23570978

RESUMO

Myocardial infarction (MI) is a major risk for ventricular arrhythmia. Pause-triggered ventricular arrhythmia can be caused by increased myofilament Ca binding due to sarcomeric mutations or Ca-sensitizing compounds. Myofilament Ca sensitivity is also increased after MI. Here we hypothesize that MI increases risk for pause-triggered ventricular arrhythmias, which can be prevented by myofilament Ca-desensitization and contractile uncoupling. To test this hypothesis, we generated a murine chronic MI model using male B6SJLF1/J mice (n=40) that underwent permanent ligation of the left anterior descending coronary artery. 4 weeks post MI, cardiac structure, function and myofilament Ca sensitivity were evaluated. Pause-dependent arrhythmia susceptibility was quantified in isolated hearts with pacing trains of increasing frequency, followed by a pause and an extra stimulus. Coronary ligation resulted in a mean infarct size of 39.6±5.7% LV and fractional shortening on echocardiography was reduced by 40% compared to non-infarcted controls. Myofilament Ca sensitivity was significantly increased in post MI hearts (pCa50: Control=5.66±0.03; MI=5.84±0.05; P<0.01). Exposure to the Ca desensitizer/contractile uncoupler blebbistatin (BLEB, 3 µM) reduced myofilament Ca sensitivity of MI hearts to that of control hearts and selectively reduced the frequency of post-pause ectopic beats (MI 0.12±0.04 vs MI+BLEB 0.01±0.005 PVC/pause; P=0.02). BLEB also reduced the incidence of ventricular tachycardia in chronic MI hearts from 59% to 10% (P<0.05). We conclude that chronic MI hearts exhibit increased myofilament Ca sensitivity and pause-triggered ventricular arrhythmias, which can be prevented by blebbistatin. Decreasing myofilament Ca sensitivity may be a strategy to reduce arrhythmia burden after MI.


Assuntos
Cálcio/metabolismo , Contração Miocárdica , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miofibrilas/metabolismo , Taquicardia Ventricular/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Masculino , Camundongos , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miofibrilas/patologia , Taquicardia Ventricular/patologia
11.
PLoS One ; 8(4): e60986, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23593369

RESUMO

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+)]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+)]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Colforsina/farmacologia , Vasos Coronários/fisiologia , Espaço Intracelular/metabolismo , Nucleotídeos Cíclicos/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Vasos Coronários/efeitos dos fármacos , Proteínas de Choque Térmico HSP20/metabolismo , Histamina/farmacologia , Espaço Intracelular/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Contração Muscular/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Paxilina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Sus scrofa
12.
J Biol Chem ; 287(47): 39613-25, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23033485

RESUMO

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/tratamento farmacológico , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/metabolismo , Naftiridinas/química , Naftiridinas/farmacologia , Fenetilaminas/efeitos adversos , Bloqueadores dos Canais de Potássio/efeitos adversos , Piridinas/química , Piridinas/farmacologia , Sulfonamidas/efeitos adversos , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Proteínas Musculares/genética , Miocárdio/metabolismo , Miocárdio/patologia , Fenetilaminas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Coelhos , Relação Estrutura-Atividade , Sulfonamidas/farmacologia
13.
Circ Res ; 111(2): 170-9, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22647877

RESUMO

RATIONALE: Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear. OBJECTIVE: To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart. METHODS AND RESULTS: Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with postpause action potential prolongation, afterdepolarizations, and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca-sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction. CONCLUSIONS: Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, action potential prolongation, and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.


Assuntos
Arritmias Cardíacas/metabolismo , Cálcio/fisiologia , Citosol/metabolismo , Homeostase/genética , Líquido Intracelular/metabolismo , Miofibrilas/metabolismo , Potenciais de Ação/genética , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Citosol/patologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Miofibrilas/genética , Ligação Proteica/genética , Regulação para Cima/genética
14.
J Biomech ; 45(5): 888-94, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22176709

RESUMO

Aortic valve (AV) disease is often characterized by the formation of calcific nodules within AV leaflets that alter functional biomechanics. In vitro, formation of these nodules is associated with osteogenic differentiation and/or increased contraction and apoptosis of AV interstitial cells (AVICs), leading to growth of calcium phosphate crystal structures. In several other cell types, increased intracellular Ca(2+) has been shown to be an important part in activation of osteogenic differentiability. However, elevated intracellular Ca(2+) is known to mediate cell contraction, and has also been shown to lead to apoptosis in many cell types. Therefore, a rise in intracellular Ca(2+) may precede cellular changes that lead to calcification, and fibroblasts similar to AVICs have been shown to exhibit increases in intracellular Ca(2+) in response to mechanical strain. In this study, we hypothesized that strain induces intracellular Ca(2+) accumulation through stretch-activated calcium channels. We were also interested in assessing possible correlations between intracellular Ca(2+) increases and apoptosis in AVICs. To test our hypothesis, cultured porcine AVICs were used to assess correlates between strain, intracellular Ca(2+), and apoptosis. Ca(2+) sensitive fluorescent dyes were utilized to measure real-time intracellular Ca(2+) changes in strained AVICs. Ca(2+) changes were then correlated with AVIC apoptosis using flow cytometric Annexin V apoptosis assays. These data indicate that strain-dependent accumulation of intracellular Ca(2+) is correlated with apoptosis in AVICs. We believe that these findings indicate early mechanotransductive events that may initiate AV calcification pathways.


Assuntos
Valva Aórtica/citologia , Valva Aórtica/metabolismo , Apoptose/fisiologia , Cálcio/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Doenças das Valvas Cardíacas/patologia , Animais , Fenômenos Biomecânicos/fisiologia , Canais de Cálcio/metabolismo , Fosfatos de Cálcio/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Doenças das Valvas Cardíacas/metabolismo , Mecanotransdução Celular/fisiologia , Osteogênese/fisiologia , Estresse Mecânico , Suínos , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
15.
PLoS One ; 6(12): e28935, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22216144

RESUMO

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Miocárdio/citologia , Polímeros/farmacologia , Células-Tronco/citologia , Animais , Sequência de Bases , Primers do DNA , Humanos , Imuno-Histoquímica , Espectroscopia de Ressonância Magnética , Espécies Reativas de Oxigênio/metabolismo
16.
Biophys J ; 99(10): 3113-8, 2010 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-21081057

RESUMO

We studied the transmembrane potential and magnetic fields from electrical activity at the apex of the isolated rabbit heart experimentally using optical mapping and superconducting quantum interference device microscopy, and theoretically using monodomain and bidomain models. The cardiac apex has a complex spiral fiber architecture that plays an important role in the development and propagation of action currents during stimulation at the apex. This spiral fiber orientation contains both radial electric currents that contribute to the electrocardiogram and electrically silent circular currents that cannot be detected by the electrocardiogram but are detectable by their magnetic field, B(z). In our experiments, the transmembrane potential, V(m), was first measured optically and then B(z) was measured with a superconducting quantum interference device microscope. Based on a simple model of the spiral structure of the apex, V(m) was expected to exhibit circular wave front patterns and B(z) to reflect the circular component of the action currents. Although the circular V(m) wave fronts were detected, the B(z) maps were not as simple as expected. However, we observed a pattern consistent with a tilted axis for the apex spiral fiber geometry. We were able to simulate similar patterns in both a monodomain model of a tilted stack of rings of dipole current and a bidomain model of a tilted stack of spiraled cardiac tissue that was stimulated at the apex. The fact that the spatial pattern of the magnetic data was more complex than the simple circles observed for V(m) suggests that the magnetic data contain information that cannot be found electrically.


Assuntos
Eletrofisiologia/métodos , Coração/fisiologia , Magnetismo , Potenciais da Membrana/fisiologia , Animais , Modelos Biológicos , Coelhos , Função Ventricular/fisiologia
17.
Anal Chem ; 80(8): 2728-33, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18351750

RESUMO

A microfabricated titration calorimeter having nanowatt sensitivity is presented. The device is achieved by modifying a commercial, suspended-membrane, thin-film thermopile infrared sensor. Chemical reactions are studied by placing a 50.0 nL droplet of one reagent directly on the sensor and injecting nanoliter droplets of a second reagent through a micropipette by means of a pressure-driven droplet injector with 1% reliability in volume delivery. External thermal noise is minimized by a two-layer thermal shielding system. Evaporation is prevented by positioning the micropipette through a tiny hole in a cover glass, sealed by a drop of oil. The device is calibrated using two acid-base reactions: H2SO4 + HEPES buffer, and NaOH + HCl. The measured power sensitivity is 2.90(4) V/W, giving a detection limit of 22 nW. The 1/e time constant for a single injection is 1.1 s. The day-to-day power sensitivity is reproducible to approximately 2%. A computational model of the sensor reproduces the power sensitivity within 10% and the time constant within 20%. For a 50 nL sample and 0.8-1.5 nL titrant injection volumes, the heat uncertainty of 44 nJ corresponds to a 3sigma detection limit of 132 nJ, or the binding energy associated with 2.9 pM of IgG-protein A complex.


Assuntos
Calorimetria/instrumentação , Calorimetria/métodos , Nanotecnologia/métodos , Calibragem , Temperatura Alta , Raios Infravermelhos , Modelos Teóricos , Nanotecnologia/instrumentação , Sensibilidade e Especificidade
18.
Am J Physiol Heart Circ Physiol ; 294(5): H2352-62, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18359893

RESUMO

In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Adaptação Fisiológica , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Estimulação Cardíaca Artificial , Citosol/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/enzimologia , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Tempo
19.
Biomed Microdevices ; 10(3): 347-54, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18214684

RESUMO

The metabolic activity of cells can be monitored by measuring the pH in the extracellular environment. Microfabrication and microfluidic technologies allow the sensor size and the extracellular volumes to be comparable to single cells. A glass substrate with thin film pH sensitive IrO( x ) electrodes was sealed to a replica-molded polydimethylsiloxane (PDMS) microfluidic network with integrated valves. The device, termed NanoPhysiometer, allows the trapping of single cardiac myocytes and the measurement of the pH in a detection volume of 0.36 nL. For wild-type (WT) single cardiac myocytes an acidification rate of 6.45 +/- 0.38 mpH/min was measured in comparison to 19.5 +/- 0.38 mpH/min for very long chain Acyl-CoA dehydrogenase (VLCAD) deficient mice in 0.8 mM of Ca(2+). VLCAD deficiency is a fatty acid oxidation disease leading to cardiomyopathy and arrhythmias. The acidification rate increased to 11.96 +/- 1.33 mpH/min for WT and to 32.0 +/- 4.64 mpH/min for VLCAD -/- in 1.8 mM of Ca(2+). The NanoPhysiometer concept can be extended to study ischemia/reperfusion injury or disorders of other biological systems to identify strategies for treatment and possible pharmacological targets.


Assuntos
Separação Celular/instrumentação , Eletroquímica/instrumentação , Concentração de Íons de Hidrogênio , Eletrodos Seletivos de Íons , Técnicas Analíticas Microfluídicas/instrumentação , Miócitos Cardíacos/química , Nanotecnologia/instrumentação , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Cultivadas , Eletroquímica/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Microquímica/instrumentação , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Miócitos Cardíacos/metabolismo , Nanotecnologia/métodos
20.
Biosens Bioelectron ; 22(7): 1303-10, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16860556

RESUMO

In this paper we describe a new approach to measure pH differences in microfluidic devices and demonstrated acidification rate measurements in on-chip cell culture systems with nl wells. We use two miniaturized identical iridium oxide (IrOx) thin film electrodes (20 micromx400 microm), one as a quasi-reference electrode, the other as a sensing electrode, placed in two confluent compartments on chip. The IrOx electrodes were deposited onto microfabricated platinum (Pt) electrodes simultaneously using electrodeposition. Incorporating the electrodes into a microfluidic device allowed us to expose each electrode to a different solution with a pH difference of one pH unit maintaining a confluent connection between the electrodes. In this configuration, we obtained a reproducible voltage difference between the two IrOx thin film electrodes, which corresponds to the electrode sensitivities of -70 mV/pH at 22 degrees C. In order to measure the acidification rate of cells in nl cell culture volumes we placed one IrOx thin film electrode in the perfusion channel as a quasi-reference electrode and the other in the cell culture volume. We obtained an acidification rate of 0.19+/-0.02 pH/min for fibroblast cells using a stop flow protocol. These results show that we can use two identical miniaturized microfabricated IrOx electrodes to measure pH differences to monitor the metabolic activity of cell cultures on chip. Furthermore, our approach can also be applied in biosensor or bioanalytical applications.


Assuntos
Técnicas Biossensoriais/instrumentação , Fibroblastos/metabolismo , Irídio , Metabolismo , Nanotecnologia , Animais , Linhagem Celular , Eletrodos , Concentração de Íons de Hidrogênio , Camundongos
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