RESUMO
Collectrin (Tmem27), an angiotensin-converting enzyme 2 homologue, is a chaperone of amino acid transporters in the kidney and endothelium. Global collectrin knockout (KO) mice have hypertension, endothelial dysfunction, exaggerated salt sensitivity, and diminished renal blood flow. This phenotype is associated with altered nitric oxide and superoxide balance and increased proximal tubule (PT) Na+/H+ exchanger isoform 3 (NHE3) expression. Collectrin is located on the X chromosome where genome-wide association population studies have largely been excluded. In the present study, we generated PT-specific collectrin KO (PT KO) mice to determine the precise contribution of PT collectrin in blood pressure homeostasis. We also examined the association of human TMEM27 single-nucleotide polymorphisms with blood pressure traits in 11,926 Hispanic Community Health Study/Study of Latinos (HCHS/SOL) Hispanic/Latino participants. PT KO mice exhibited hypertension, and this was associated with increased baseline NHE3 expression and diminished lithium excretion. However, PT KO mice did not display exaggerated salt sensitivity or a reduction in renal blood flow compared with control mice. Furthermore, PT KO mice exhibited enhanced endothelium-mediated dilation, suggesting a compensatory response to systemic hypertension induced by deficiency of collectrin in the PT. In HCHS/SOL participants, we observed sex-specific single-nucleotide polymorphism associations with diastolic blood pressure. In conclusion, loss of collectrin in the PT is sufficient to induce hypertension, at least in part, through activation of NHE3. Importantly, our model supports the notion that altered renal blood flow may be a determining factor for salt sensitivity. Further studies are needed to investigate the role of the TMEM27 locus on blood pressure and salt sensitivity in humans.NEW & NOTEWORTHY The findings of our study are significant in several ways: 1) loss of an amino acid chaperone in the proximal tubule is sufficient to cause hypertension, 2) the results in global and proximal tubule-specific collectrin knockout mice support the notion that vascular dysfunction is required for salt sensitivity or that impaired renal tubule function causes hypertension but is not sufficient to cause salt sensitivity, and 3) our study is the first to implicate a role of collectrin in human hypertension.
Assuntos
Pressão Sanguínea , Hipertensão , Túbulos Renais Proximais , Glicoproteínas de Membrana , Animais , Feminino , Humanos , Masculino , Camundongos , Pressão Sanguínea/fisiologia , Estudo de Associação Genômica Ampla , Hispânico ou Latino/genética , Hipertensão/genética , Túbulos Renais Proximais/metabolismo , Camundongos Knockout , Cloreto de Sódio na Dieta/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genéticaRESUMO
BACKGROUND: GSTM1 encodes glutathione S-transferase µ-1 (GSTM1), which belongs to a superfamily of phase 2 antioxidant enzymes. The highly prevalent GSTM1 deletion variant is associated with kidney disease progression in human cohorts: the African American Study of Kidney Disease and Hypertension and the Atherosclerosis Risk in Communities (ARIC) Study. METHODS: We generated a Gstm1 knockout mouse line to study its role in a CKD model (involving subtotal nephrectomy) and a hypertension model (induced by angiotensin II). We examined the effect of intake of cruciferous vegetables and GSTM1 genotypes on kidney disease in mice as well as in human ARIC study participants. We also examined the importance of superoxide in the mediating pathways and of hematopoietic GSTM1 on renal inflammation. RESULTS: Gstm1 knockout mice displayed increased oxidative stress, kidney injury, and inflammation in both models. The central mechanism for kidney injury is likely mediated by oxidative stress, because treatment with Tempol, an superoxide dismutase mimetic, rescued kidney injury in knockout mice without lowering BP. Bone marrow crosstransplantation revealed that Gstm1 deletion in the parenchyma, and not in bone marrow-derived cells, drives renal inflammation. Furthermore, supplementation with cruciferous broccoli powder rich in the precursor to antioxidant-activating sulforaphane significantly ameliorated kidney injury in Gstm1 knockout, but not wild-type mice. Similarly, among humans (ARIC study participants), high consumption of cruciferous vegetables was associated with fewer kidney failure events compared with low consumption, but this association was observed primarily in participants homozygous for the GSTM1 deletion variant. CONCLUSIONS: Our data support a role for the GSTM1 enzyme in the modulation of oxidative stress, inflammation, and protective metabolites in CKD.
Assuntos
Brassicaceae , Dieta , Deleção de Genes , Glutationa Transferase/genética , Insuficiência Renal Crônica/genética , Verduras , Animais , Modelos Animais de Doenças , Feminino , Glutationa Transferase/fisiologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Insuficiência Renal Crônica/prevenção & controleRESUMO
The mitochondrial unfolded protein response (UPRmt) is a cytoprotective signaling pathway triggered by mitochondrial dysfunction. UPRmt activation upregulates chaperones, proteases, antioxidants, and glycolysis at the gene level to restore proteostasis and cell energetics. Activating transcription factor 5 (ATF5) is a proposed mediator of the mammalian UPRmt. Herein, we hypothesized pharmacological UPRmt activation may protect against cardiac ischemia-reperfusion (I/R) injury in an ATF5-dependent manner. Accordingly, in vivo administration of the UPRmt inducers oligomycin or doxycycline 6 h before ex vivo I/R injury (perfused heart) was cardioprotective in wild-type but not global Atf5-/- mice. Acute ex vivo UPRmt activation was not cardioprotective, and loss of ATF5 did not impact baseline I/R injury without UPRmt induction. In vivo UPRmt induction significantly upregulated many known UPRmt-linked genes (cardiac quantitative PCR and Western blot analysis), and RNA-Seq revealed an UPRmt-induced ATF5-dependent gene set, which may contribute to cardioprotection. This is the first in vivo proof of a role for ATF5 in the mammalian UPRmt and the first demonstration that UPRmt is a cardioprotective drug target.NEW & NOTEWORTHY Cardioprotection can be induced by drugs that activate the mitochondrial unfolded protein response (UPRmt). UPRmt protection is dependent on activating transcription factor 5 (ATF5). This is the first in vivo evidence for a role of ATF5 in the mammalian UPRmt.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Doxiciclina/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Oligomicinas/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fatores Ativadores da Transcrição/deficiência , Fatores Ativadores da Transcrição/genética , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
Controversy surrounds the molecular identity of mitochondrial K+ channels that are important for protection against cardiac ischemia-reperfusion injury. Although KNa1.2 (sodium-activated potassium channel encoded by Kcn2) is necessary for cardioprotection by volatile anesthetics, electrophysiological evidence for a channel of this type in mitochondria is lacking. The endogenous physiological role of a potential mito-KNa1.2 channel is also unclear. In this study, single channel patch-clamp of 27 independent cardiac mitochondrial inner membrane (mitoplast) preparations from wild-type (WT) mice yielded 6 channels matching the known ion sensitivity, ion selectivity, pharmacology, and conductance properties of KNa1.2 (slope conductance, 138 ± 1 pS). However, similar experiments on 40 preparations from Kcnt2-/- mice yielded no such channels. The KNa opener bithionol uncoupled respiration in WT but not Kcnt2-/- cardiomyocytes. Furthermore, when oxidizing only fat as substrate, Kcnt2-/- cardiomyocytes and hearts were less responsive to increases in energetic demand. Kcnt2-/- mice also had elevated body fat, but no baseline differences in the cardiac metabolome. These data support the existence of a cardiac mitochondrial KNa1.2 channel, and a role for cardiac KNa1.2 in regulating metabolism under conditions of high energetic demand.-Smith, C. O., Wang, Y. T., Nadtochiy, S. M., Miller, J. H., Jonas, E. A., Dirksen, R. T., Nehrke, K., Brookes, P. S. Cardiac metabolic effects of KNa1.2 channel deletion and evidence for its mitochondrial localization.
RESUMO
Stimulation of the cytosolic NAD+ dependent deacetylase SIRT1 is cardioprotective against ischemia-reperfusion (IR) injury. NAD+ precursors including nicotinamide mononucleotide (NMN) are thought to induce cardioprotection via SIRT1. Herein, while NMN protected perfused hearts against IR (functional recovery: NMN 42⯱â¯7% vs. vehicle 11⯱â¯3%), this protection was insensitive to the SIRT1 inhibitor splitomicin (recovery 47⯱â¯8%). Although NMN-induced cardioprotection was absent in Sirt3-/- hearts (recovery 9⯱â¯5%), this was likely due to enhanced baseline injury in Sirt3-/- (recovery 6⯱â¯2%), since similar injury levels in WT hearts also blunted the protective efficacy of NMN. Considering alternative cardiac effects of NMN, and the requirement of glycolysis for NAD+, we hypothesized NMN may confer protection in part via direct stimulation of cardiac glycolysis. In primary cardiomyocytes, NMN induced cytosolic and extracellular acidification and elevated lactate. In addition, [U-13C]glucose tracing in intact hearts revealed that NMN stimulated glycolytic flux. Consistent with a role for glycolysis in NMN-induced protection, hearts perfused without glucose (palmitate as fuel source), or hearts perfused with galactose (no ATP from glycolysis) exhibited no benefit from NMN (recovery 11⯱â¯4% and 15⯱â¯2% respectively). Acidosis during early reperfusion is known to be cardioprotective (i.e., acid post-conditioning), and we also found that NMN was cardioprotective when delivered acutely at reperfusion (recovery 39⯱â¯8%). This effect of NMN was not additive with acidosis, suggesting overlapping mechanisms. We conclude that the acute cardioprotective benefits of NMN are mediated in part via glycolytic stimulation, with the downstream protective mechanism involving enhanced ATP synthesis during ischemia and/or enhanced acidosis during reperfusion.
Assuntos
Miocárdio/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Sirtuína 1/genética , Sirtuína 3/genética , Acidose/genética , Acidose/metabolismo , Acidose/patologia , Ácidos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cardiotônicos/farmacologia , Glucose/metabolismo , Glicólise/genética , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NAD/metabolismo , Naftalenos/farmacologia , Mononucleotídeo de Nicotinamida/farmacologia , Pironas/farmacologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
2-Hydroxyglutarate (2-HG) is a hypoxic metabolite with potentially important epigenetic signaling roles. The mechanisms underlying 2-HG generation are poorly understood, but evidence suggests a potential regulatory role for the sirtuin family of lysine deacetylases. Thus, we hypothesized that the acetylation status of the major 2-HG-generating enzymes [lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH)] may govern their 2-HG-generating activity. In vitro acetylation of these enzymes, with confirmation by western blotting, mass spectrometry, reversibility by recombinant sirtuins and an assay for global lysine occupancy, yielded no effect on 2-HG-generating activity. In addition, while elevated 2-HG in hypoxia is associated with the activation of lysine deacetylases, we found that mice lacking mitochondrial SIRT3 exhibited hyperacetylation and elevated 2-HG. These data suggest that there is no direct link between enzyme acetylation and 2-HG production. Furthermore, our observed effects of in vitro acetylation on the canonical activities of IDH, MDH and LDH appeared to contrast with previous findings wherein acetyl-mimetic lysine mutations resulted in the inhibition of these enzymes. Overall, these data suggest that a causal relationship should not be assumed between acetylation of metabolic enzymes and their activities, canonical or otherwise.
Assuntos
Glutaratos/metabolismo , Lisina/metabolismo , Mitocôndrias Cardíacas/enzimologia , Proteínas Mitocondriais/genética , Processamento de Proteína Pós-Traducional , Sirtuína 3/genética , Acetilação , Animais , Hipóxia Celular , Ensaios Enzimáticos , Células HEK293 , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Cinética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Sirtuína 3/deficiênciaRESUMO
BACKGROUND: Anesthetic preconditioning (APC) is a clinically important phenomenon in which volatile anesthetics (VAs) protect tissues such as heart against ischemic injury. The mechanism of APC is thought to involve K channels encoded by the Slo gene family, and the authors showed previously that slo-2 is required for APC in Caenorhabditis elegans. Thus, the authors hypothesized that a slo-2 ortholog may mediate APC-induced cardioprotection in mammals. METHODS: A perfused heart model of ischemia-reperfusion injury, a fluorescent assay for K flux, and mice lacking Slo2.1 (Slick), Slo2.2 (Slack), or both (double knockouts, Slo2.x dKO) were used to test whether these channels are required for APC-induced cardioprotection and for cardiomyocyte or mitochondrial K transport. RESULTS: In wild-type (WT) hearts, APC improved post-ischemia-reperfusion functional recovery (APC = 39.5 ± 3.7% of preischemic rate × pressure product vs. 20.3 ± 2.3% in controls, means ± SEM, P = 0.00051, unpaired two-tailed t test, n = 8) and lowered infarct size (APC = 29.0 ± 4.8% of LV area vs. 51.4 ± 4.5% in controls, P = 0.0043, n = 8). Protection by APC was absent in hearts from Slo2.1 mice (% recovery APC = 14.6 ± 2.6% vs. 16.5 ± 2.1% in controls, P = 0.569, n = 8 to 9, infarct APC = 52.2 ± 5.4% vs. 53.5 ± 4.7% in controls, P = 0.865, n = 8 to 9). APC protection was also absent in Slo2.x dKO hearts (% recovery APC = 11.0 ± 1.7% vs. 11.9 ± 2.2% in controls, P = 0.725, n = 8, infarct APC = 51.6 ± 4.4% vs. 50.5 ± 3.9% in controls, P = 0.855, n = 8). Meanwhile, Slo2.2 hearts responded similar to WT (% recovery APC = 41.9 ± 4.0% vs. 18.0 ± 2.5% in controls, P = 0.00016, n = 8, infarct APC = 25.2 ± 1.3% vs. 50.8 ± 3.3% in controls, P < 0.000005, n = 8). Furthermore, VA-stimulated K transport seen in cardiomyocytes or mitochondria from WT or Slo2.2 mice was absent in Slo2.1 or Slo2.x dKO. CONCLUSION: Slick (Slo2.1) is required for both VA-stimulated K flux and for the APC-induced cardioprotection.
Assuntos
Anestésicos Inalatórios/uso terapêutico , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Canais de Potássio/genética , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Células HEK293 , Humanos , Isoflurano/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canais de Potássio Ativados por Sódio , Tálio/metabolismoRESUMO
An all-fiber optic catheter-based polarization-sensitive optical coherence tomography system is demonstrated. A novel multiplexing method was used to illuminate the sample, splitting the light from a 58.5 kHz Fourier-domain mode-locked laser such that two different polarization states, alternated in time, are generated by two semiconductor optical amplifiers. A 2.3 mm forward-view cone-scanning catheter probe was designed, fabricated, and used to acquire sample scattering intensity and phase retardation images. The system was first verified with a quarter-wave plate and then by obtaining intensity and phase retardation images of high-birefringence plastic, human skin in vivo, and untreated and thermally ablated porcine myocardium ex vivo. The system can potentially in vivo image of the cardiac wall to aid radio-frequency ablation therapy for cardiac arrhythmias.
Assuntos
Ablação por Cateter/instrumentação , Tomografia de Coerência Óptica/instrumentação , Animais , Arritmias Cardíacas/terapia , Birrefringência , Desenho de Equipamento , Humanos , Lasers , Monitorização Fisiológica/instrumentação , Fibras Ópticas , SuínosRESUMO
Defibrillation shocks from implantable cardioverter defibrillators can be lifesaving but can also damage cardiac tissues via electroporation. This study characterizes the spatial distribution and extent of defibrillation shock-induced electroporation with and without a 45-min postshock period for cell membranes to recover. Langendorff-perfused rabbit hearts (n = 31) with and without a chronic left ventricular (LV) myocardial infarction (MI) were studied. Mean defibrillation threshold (DFT) was determined to be 161.4 ± 17.1 V and 1.65 ± 0.44 J in MI hearts for internally delivered 8-ms monophasic truncated exponential (MTE) shocks during sustained ventricular fibrillation (>20 s, SVF). A single 300-V MTE shock (twice determined DFT voltage) was used to terminate SVF. Shock-induced electroporation was assessed by propidium iodide (PI) uptake. Ventricular PI staining was quantified by fluorescent imaging. Histological analysis was performed using Masson's Trichrome staining. Results showed PI staining concentrated near the shock electrode in all hearts. Without recovery, PI staining was similar between normal and MI groups around the shock electrode and over the whole ventricles. However, MI hearts had greater total PI uptake in anterior (P < 0.01) and posterior (P < 0.01) LV epicardial regions. Postrecovery, PI staining was reduced substantially, but residual staining remained significant with similar spacial distributions. PI staining under SVF was similar to previously studied paced hearts. In conclusion, electroporation was spatially correlated with the active region of the shock electrode. Additional electroporation occurred in the LV epicardium of MI hearts, in the infarct border zone. Recovery of membrane integrity postelectroporation is likely a prolonged process. Short periods of SVF did not affect electroporation injury.
Assuntos
Desfibriladores Implantáveis/efeitos adversos , Cardioversão Elétrica/efeitos adversos , Eletroporação , Traumatismos Cardíacos/etiologia , Infarto do Miocárdio/complicações , Miócitos Cardíacos/patologia , Fibrilação Ventricular/terapia , Animais , Estimulação Cardíaca Artificial , Membrana Celular/patologia , Permeabilidade da Membrana Celular , Modelos Animais de Doenças , Cardioversão Elétrica/instrumentação , Fibrose , Traumatismos Cardíacos/patologia , Traumatismos Cardíacos/fisiopatologia , Ventrículos do Coração/lesões , Ventrículos do Coração/patologia , Masculino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Coelhos , Recuperação de Função Fisiológica , Fatores de Tempo , Fibrilação Ventricular/etiologia , Fibrilação Ventricular/patologia , Fibrilação Ventricular/fisiopatologiaRESUMO
Radiofrequency ablation (RFA) is an important standard therapy for cardiac arrhythmias, but direct monitoring of tissue treatment is currently lacking. We demonstrate an RFA catheter integrated with polarization sensitive optical coherence tomography (PSOCT) for directly monitoring the RFA process in real time. The integrated RFA/OCT catheter was modified from a standard clinical RFA catheter and includes a miniature forward-viewing cone-scanning OCT probe. The PSOCT system was validated with a quarter-wave plate while the RFA function of the integrated catheter was validated by comparing lesion sizes with those made with an unmodified RFA catheter. Additionally, the integrated catheter guided catheter-tissue apposition and monitored RFA lesion formation in cardiac tissue in real time. The results show that catheter-tissue contact can be characterized by observing the features of the blood and tissue in the acquired OCT images and that RFA lesion formation can be confirmed by monitoring the change in phase retardance in the acquired PSOCT images. This system demonstrates the feasibility of an integrated RFA/OCT catheter to deliver RF energy and image the cardiac wall simultaneously and justifies further research into use of this technology to aid RFA therapy for cardiac arrhythmias.
RESUMO
Succinate accumulates during ischemia, and its oxidation at reperfusion drives injury. The mechanism of ischemic succinate accumulation is controversial and is proposed to involve reversal of mitochondrial complex II. Herein, using stable-isotope-resolved metabolomics, we demonstrate that complex II reversal is possible in hypoxic mitochondria but is not the primary succinate source in hypoxic cardiomyocytes or ischemic hearts. Rather, in these intact systems succinate primarily originates from canonical Krebs cycle activity, partly supported by aminotransferase anaplerosis and glycolysis from glycogen. Augmentation of canonical Krebs cycle activity with dimethyl-α-ketoglutarate both increases ischemic succinate accumulation and drives substrate-level phosphorylation by succinyl-CoA synthetase, improving ischemic energetics. Although two-thirds of ischemic succinate accumulation is extracellular, the remaining one-third is metabolized during early reperfusion, wherein acute complex II inhibition is protective. These results highlight a bifunctional role for succinate: its complex-II-independent accumulation being beneficial in ischemia and its complex-II-dependent oxidation being detrimental at reperfusion.
Assuntos
Ciclo do Ácido Cítrico , Isquemia Miocárdica/metabolismo , Ácido Succínico/metabolismo , Animais , Ácido Aspártico/metabolismo , Autofagia , Complexo II de Transporte de Elétrons/metabolismo , Metabolismo Energético , Glicogenólise , Glicólise , Masculino , Camundongos Endogâmicos C57BL , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transaminases/metabolismoRESUMO
Human cardiac myocytes derived from pluripotent stem cells (hCM) have invigorated interest in genetic disease mechanisms and cardiac safety testing; however, the technology to fully assess electrophysiological function in an assay that is amenable to high throughput screening has lagged. We describe a fully contactless system using optical pacing with an infrared (IR) laser and multi-site high fidelity fluorescence imaging to assess multiple electrophysiological parameters from hCM monolayers in a standard 96-well plate. Simultaneous multi-site action potentials (FluoVolt) or Ca2+ transients (Fluo4-AM) were measured, from which high resolution maps of conduction velocity and action potential duration (APD) were obtained in a single well. Energy thresholds for optical pacing were determined for cell plating density, laser spot size, pulse width, and wavelength and found to be within ranges reported previously for reliable pacing. Action potentials measured using FluoVolt and a microelectrode exhibited the same morphology and rate of depolarization. Importantly, we show that this can be achieved accurately with minimal damage to hCM due to optical pacing or fluorescence excitation. Finally, using this assay we demonstrate that hCM exhibit reproducible changes in repolarization and impulse conduction velocity for Flecainide and Quinidine, two well described reference compounds. In conclusion, we demonstrate a high fidelity electrophysiological screening assay that incorporates optical pacing with IR light to control beating rate of hCM monolayers.
Assuntos
Raios Infravermelhos , Miócitos Cardíacos/fisiologia , Eletrofisiologia Cardíaca , Células Cultivadas , Humanos , Microeletrodos , Microscopia de Fluorescência , Óptica e Fotônica , Técnicas de Patch-ClampRESUMO
BACKGROUND: The relationship between changes in endocardial cushion and resultant congenital heart diseases (CHD) has yet to be established. It has been shown that increased regurgitant flow early in embryonic heart development leads to endocardial cushion defects, but it remains unclear how abnormal endocardial cushions during the looping stages might affect the fully septated heart. The goal of this study was to reproducibly alter blood flow in vivo and then quantify the resultant effects on morphology of endocardial cushions in the looping heart and on CHDs in the septated heart. METHODS: Optical pacing was applied to create regurgitant flow in embryonic hearts, and optical coherence tomography (OCT) was utilized to quantify regurgitation and morphology. Embryonic quail hearts were optically paced at 3 Hz (180 bpm, well above intrinsic rate 60-110 bpm) at stage 13 of development (3-4 weeks human) for 5 min. Pacing fatigued the heart and led to at least 1 h of increased regurgitant flow. Resultant morphological changes were quantified with OCT imaging at stage 19 (cardiac looping-4-5 weeks human) or stage 35 (4 chambered heart-8 weeks human). RESULTS: All paced embryos imaged at stage 19 displayed structural changes in cardiac cushions. The amount of regurgitant flow immediately after pacing was inversely correlated with cardiac cushion size 24-h post pacing (P value < .01). The embryos with the most regurgitant flow and smallest cushions after pacing had a decreased survival rate at 8 days (P < .05), indicating that those most severe endocardial cushion defects were lethal. Of the embryos that survived to stage 35, 17/18 exhibited CHDs including valve defects, ventricular septal defects, hypoplastic ventricles, and common AV canal. CONCLUSION: The data illustrate a strong inverse relationship in which regurgitant flow precedes abnormal and smaller cardiac cushions, resulting in the development of CHDs.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Comunicação Atrioventricular/etiologia , Cardiopatias Congênitas/embriologia , Animais , Modelos Animais de Doenças , Comunicação Atrioventricular/diagnóstico , Comunicação Atrioventricular/embriologia , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/fisiopatologia , Organogênese , Codorniz , Tomografia de Coerência ÓpticaRESUMO
Novel clinical treatments to target peripheral nerves are being developed which primarily use electrical current. Recently, infrared (IR) light was shown to inhibit peripheral nerves with high spatial and temporal specificity. Here, for the first time, we demonstrate that IR can selectively and reversibly inhibit small-diameter axons at lower radiant exposures than large-diameter axons. We provide a mathematical rationale, and then demonstrate it experimentally in individual axons of identified neurons in the marine mollusk Aplysia californica, and in axons within the vagus nerve of a mammal, the musk shrew Suncus murinus. The ability to selectively, rapidly, and reversibly control small-diameter sensory fibers may have many applications, both for the analysis of physiology, and for treating diseases of the peripheral nervous system, such as chronic nausea, vomiting, pain, and hypertension. Moreover, the mathematical analysis of how IR affects the nerve could apply to other techniques for controlling peripheral nerve signaling.
Assuntos
Axônios/fisiologia , Axônios/efeitos da radiação , Raios Infravermelhos , Animais , Aplysia , Fenômenos Eletrofisiológicos/efeitos da radiação , Raios Infravermelhos/efeitos adversos , Masculino , Neurônios/fisiologia , Neurônios/efeitos da radiação , Transmissão Sináptica/efeitos da radiação , Nervo VagoRESUMO
Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart.
Assuntos
Coração/diagnóstico por imagem , Raios Infravermelhos , Lasers , Contração Miocárdica , Potenciais de Ação/efeitos da radiação , Animais , Cálcio/análise , Embrião não Mamífero , Coração/embriologia , Coração/efeitos da radiação , Modelos Animais , Contração Miocárdica/efeitos da radiação , CodornizRESUMO
Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.
Assuntos
Citoproteção , Complexo I de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Células-Tronco/patologia , Superóxidos/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Drosophila/efeitos dos fármacos , Drosophila/metabolismo , Coração/efeitos dos fármacos , Hiperplasia , Intestinos/citologia , Camundongos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Perfusão , Ratos , Células-Tronco/efeitos dos fármacos , Tunicamicina/farmacologiaRESUMO
Cardiac conduction maturation is an important and integral component of heart development. Optical mapping with voltage-sensitive dyes allows sensitive measurements of electrophysiological signals over the entire heart. However, accurate measurements of conduction velocity during early cardiac development is typically hindered by low signal-to-noise ratio (SNR) measurements of action potentials. Here, we present a novel image processing approach based on least squares optimizations, which enables high-resolution, low-noise conduction velocity mapping of smaller tubular hearts. First, the action potential trace measured at each pixel is fit to a curve consisting of two cumulative normal distribution functions. Then, the activation time at each pixel is determined based on the fit, and the spatial gradient of activation time is determined with a two-dimensional (2D) linear fit over a square-shaped window. The size of the window is adaptively enlarged until the gradients can be determined within a preset precision. Finally, the conduction velocity is calculated based on the activation time gradient, and further corrected for three-dimensional (3D) geometry that can be obtained by optical coherence tomography (OCT). We validated the approach using published activation potential traces based on computer simulations. We further validated the method by adding artificially generated noise to the signal to simulate various SNR conditions using a curved simulated image (digital phantom) that resembles a tubular heart. This method proved to be robust, even at very low SNR conditions (SNR = 2-5). We also established an empirical equation to estimate the maximum conduction velocity that can be accurately measured under different conditions (e.g. sampling rate, SNR, and pixel size). Finally, we demonstrated high-resolution conduction velocity maps of the quail embryonic heart at a looping stage of development.
RESUMO
We demonstrate an ultrathin flexible cone-scanning forward-viewing OCT probe which can fit through the working channel of a flexible ureteroscope for renal pelvis imaging. The probe is fabricated by splicing a 200 µm section of core-less fiber and a 150 µm section of gradient-index (GRIN) fiber to the end of a single mode (SM) fiber. The probe is designed for common-path OCT imaging where the back-reflection of the GRIN fiber/air interface is used as the reference signal. Optimum sensitivity was achieved with a 2 degree polished probe tip. A correlation algorithm was used to correct image distortion caused by non-uniform rotation of the probe. The probe is demonstrated by imaging human skin in vivo and porcine renal pelvis ex vivo and is suitable for imaging the renal pelvis in vivo for cancer staging.
RESUMO
Cardiac electrophysiology plays a critical role in the development and function of the heart. Studies of early embryonic electrical activity have lacked a viable point stimulation technique to pace in vitro samples. Here, optical pacing by high-precision infrared stimulation is used to pace excised embryonic hearts, allowing electrophysiological parameters to be quantified during pacing at varying rates with optical mapping. Combined optical pacing and optical mapping enables electrophysiological studies in embryos under more physiological conditions and at varying heart rates, allowing detection of abnormal conduction and comparisons between normal and pathological electrical activity during development in various models.
RESUMO
Optical mapping (OM) of cardiac electrical activity conventionally collects information from a three-dimensional (3-D) surface as a two-dimensional (2-D) projection map. When applied to measurements of the embryonic heart, this method ignores the substantial and complex curvature of the heart surface, resulting in significant errors when calculating conduction velocity, an important electrophysiological parameter. Optical coherence tomography (OCT) is capable of imaging the 3-D structure of the embryonic heart and accurately characterizing the surface topology. We demonstrate an integrated OCT/OM imaging system capable of simultaneous conduction mapping and 3-D structural imaging. From these multimodal data, we obtained 3-D activation maps and corrected conduction velocity maps of early embryonic quail hearts. 3-D correction eliminates underestimation bias in 2-D conduction velocity measurements, therefore enabling more accurate measurements with less experimental variability. The integrated system will also open the door to correlate the structure and electrophysiology, thereby improving our understanding of heart development.