RESUMO
Mice systemically lacking dipeptidyl peptidase-4 (DPP4) have improved islet health, glucoregulation, and reduced obesity with high-fat diet (HFD) feeding compared to wild-type mice. Some, but not all, of this improvement can be linked to the loss of DPP4 in endothelial cells (ECs), pointing to the contribution of non-EC types. The importance of intra-islet signaling mediated by α to ß cell communication is becoming increasingly clear; thus, our objective was to determine if ß cell DPP4 regulates insulin secretion and glucose tolerance in HFD-fed mice by regulating the local concentrations of insulinotropic peptides. Using ß cell double incretin receptor knockout mice, ß cell- and pancreas-specific Dpp4-/- mice, we reveal that ß cell incretin receptors are necessary for DPP4 inhibitor effects. However, although ß cell DPP4 modestly contributes to high glucose (16.7 mM)-stimulated insulin secretion in isolated islets, it does not regulate whole-body glucose homeostasis.
RESUMO
Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase-positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4-/-) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4-/- mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N-methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.
Assuntos
Hepatite C , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Glucose/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Dipeptidil Peptidase 4/metabolismo , Células Endoteliais/metabolismo , Ribavirina/metabolismo , Hepatócitos/metabolismoRESUMO
BACKGROUND/AIMS: Glaucoma is a chronic disease that requires lifelong monitoring and treatment. However, its control is limited due to discontinuous intraocular pressure (IOP) monitoring related to the practitioners' office hours. Implantable telemetric IOP sensors have made self-measurements possible and provide important information regarding the IOP profiles of patients. However, limited long-term monitoring data are currently available. METHODS: In the ARGOS-01 study, a telemetric IOP sensor was implanted in the ciliary sulcus of six patients with open-angle glaucoma during cataract surgery between 2011 and 2012. This study reports telemetric monitoring data collected by self-tonometry and automated measurements and during outpatient visits, including an analysis of one active patient with several years of follow-up. The long-term safety, tolerability and functionality were assessed in the remaining patients during the last visit. RESULTS: The follow-up period was up to 10 years, in which almost 25 000 IOP measurements were performed. The patients had excellent tolerance of the implanted sensor and did not experience sensor-related discomfort or complications. The active patient reported easy handling of self-tonometry and did not experience long-term restrictions in activities of daily living due to the implanted sensor. Telemetric data provide an insight into patients' measurement routines and IOP fluctuations. CONCLUSION: So far, our data suggest good long-term safety, tolerability and functionality of the implanted sensors up to almost ten years. Such sensors may help facilitate patients' self-measurements of IOP. This disease monitoring method should be investigated further to determine if it helps improve wider patient experience, engagement and visual prognosis for those being treated for complex glaucoma.
Assuntos
Glaucoma de Ângulo Aberto , Glaucoma , Humanos , Pressão Intraocular , Glaucoma de Ângulo Aberto/diagnóstico , Atividades Cotidianas , Reprodutibilidade dos Testes , Glaucoma/diagnóstico , Tonometria Ocular/métodosRESUMO
The small intestine is a highly adaptable organ serving as both a barrier to the external environment and a conduit for nutrient absorption. Enterocytes package dietary triglycerides (TG) into chylomicrons for transport into circulation; the remaining TGs are stored in cytosolic lipid droplets (CLDs). The current study aimed to characterize the impact of diet composition on intestinal lipid handling in male and female wild-type mice. Mice were continued on their grain-based diet (GBD) and switched to either a high-fat, high cholesterol Western-style diet (WD) or a ketogenic diet (KD) for 3 or 5 weeks. KD-fed mice displayed significantly higher plasma TG levels in response to an olive oil gavage than WD- and GBD-fed mice; TG levels were ~2-fold higher in male KD-fed mice than female KD-fed mice. Poloxamer-407 experiments revealed enhanced intestinal-TG secretion rates in male mice fed a KD upon olive oil gavage, whereas secretion rates were unchanged in female mice. Surprisingly, jejunal CLD size and TG mass after oil gavage were similar among the groups. At fasting, TG mass was significantly higher in the jejunum of male KD-fed mice and the duodenum of female KD-fed mice, providing increased substrate for chylomicron formation. In addition to greater fasting intestinal TG stores, KD-fed male mice displayed longer small intestinal lengths, while female mice displayed markedly longer jejunal villi lengths. After 5 weeks of diet, 12 h fasting-2 h refeeding experiments revealed jejunal TG levels were similar between diet groups in male mice; however, in female mice, jejunal TG mass was significantly higher in KD-fed mice compared to GBD- and WD-fed mice. These experiments reveal that KD feeding promotes distinct morphological and functional changes to the murine small intestine compared to the WD diet. Moreover, changes to intestinal lipid handling in response to carbohydrate and protein restriction manifest differently in male and female mice.
Assuntos
Quilomícrons , Enterócitos , Animais , Quilomícrons/metabolismo , Dieta Hiperlipídica , Enterócitos/metabolismo , Feminino , Masculino , Camundongos , Azeite de Oliva/metabolismo , Triglicerídeos/metabolismoRESUMO
Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (r = 0.76, P = 0.03), 4-D echocardiographic infarct volume (r = 0.85, P = 0.008), and surface area (r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI.NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.
Assuntos
Infarto do Miocárdio/diagnóstico por imagem , Ultrassonografia/métodos , Algoritmos , Animais , Débito Cardíaco , Feminino , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Imageamento Tridimensional/métodos , Imageamento Tridimensional/normas , Masculino , Camundongos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Sensibilidade e Especificidade , Ultrassonografia/normasRESUMO
PURPOSE: Nonperfusion of retinal tissue due to arterial occlusion leads inevitably to mostly irreversible retinal damage. Until today no evidence-based treatment exists. Inhalation of 100% oxygen at high atmospheric pressure causes an increased solubility of oxygen in the blood that helps the retinal tissue to survive through diffusion in case of an artery occlusion till vascular recanalization occurs. Hence the purpose of this study is to compare the visual outcome in patients with retinal branch artery obstruction treated with hyperbaric oxygen versus patients treated with hemodilution only. METHODS: Non-randomized, monocentric, retrospective study. Patients with diagnosis of non-arteritic retinal branch artery occlusion (BRAO) treated with hyperbaric oxygen therapy between 1997 and 2017. Exclusion criteria were central retinal artery occlusion, presence of a cilioretinal artery and arteritic cases. The control group was matched based on visual acuity (VA) at admission, age, and delay between symptoms and beginning of clinical care. RESULTS: The control group and the matching oxygen group contained 14 patients each. Initial VA in the matched HBO group was 0.18 ± 0.19 and 0.23 ± 0.19 in the control group (p = 0.57). Final VA at discharge was 0.69 ± 0.29 in the matched oxygen group and 0.32 ± 0.23 in the control group (p = 0.0009). HBO-treated patients had a significant visual increase compared with the control group. The most common comorbidities were arterial hypertension and vascular sclerosis. CONCLUSION: HBO treatment appears to have a beneficial effect on visual outcome in patients with retinal branch artery occlusion. HBO treatment could be a rescue therapy at an early stage of BRAO, especially to bridge the time of a potential reperfusion. However, further, prospective, randomized clinical trials are required to verify this assumption.
Assuntos
Artérias Ciliares/patologia , Oxigenoterapia Hiperbárica/métodos , Retina/patologia , Oclusão da Artéria Retiniana/terapia , Acuidade Visual , Idoso , Feminino , Angiofluoresceinografia/métodos , Seguimentos , Fundo de Olho , Humanos , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismo , Oclusão da Artéria Retiniana/diagnóstico , Oclusão da Artéria Retiniana/metabolismo , Estudos Retrospectivos , Tomografia de Coerência Óptica/métodos , Resultado do TratamentoRESUMO
The health effects of the endocrine disruptor Bisphenol A (BPA) led to its partial replacement with Bisphenol S (BPS) in several products including food containers, toys, and thermal paper receipts. The acute effects of BPS on myocardial contractility are unknown. We perfused mouse hearts from both sexes for 15 min with physiologically relevant doses of BPS or BPA. In females BPS (1 nM) decreased left ventricular systolic pressure by 5 min, whereas BPA (1 nM) effects were delayed to 10 min. BPS effects in male mice were attenuated. In both sexes ER-ß antagonism abolished the effects of BPS. Cardiac myofilament function was not impacted by BPS or BPA in either sex, although there were sex-dependent differences in troponin I phosphorylation. BPS increased phospholamban phosphorylation at S16 only in female hearts, whereas BPA reduced phosphorylation in both sexes. BPA decreased phospholamban phosphorylation at T17 in both sexes while BPS caused dephosphorylation only in females. This is the first study to compare sex differences in the acute myocardial response to physiologically relevant levels of BPS and BPA, and demonstrates a rapid ability of both to depress heart function. This study raises concerns about the safety of BPS as a replacement for BPA.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Receptor beta de Estrogênio/metabolismo , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Fenóis/farmacologia , Sulfonas/farmacologia , Animais , Cálcio/metabolismo , Feminino , Masculino , Camundongos , Miofibrilas/metabolismo , Fosforilação , Fatores Sexuais , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
AIM: Cardiovascular disease (CVD) risk is lower in pre-menopausal females vs age matched males. After menopause risk equals or exceeds that of males. CVD protection of pre-menopausal females is ascribed to high circulating oestrogen levels. Despite experimental evidence that oestrogen are cardioprotective, oestrogen replacement therapy trials have not shown clear benefits. One hypothesis to explain the discrepancy proposed hearts remodel during peri-menopause. Peri-menopasual myocardial changes have never been investigated, nor has the ability of oestrogen to regulate heart function during peri-menopause. METHODS: We injected female mice with 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg/d IP) to cause gradual ovarian failure over 120d and act as a peri-menopausal model RESULTS: Left ventricular function assessed by Langendorff perfusion found no changes in VCD-injected mice at 60 or 120 days compared to intact mice. Cardiac myofilament activity was altered at 60 and 120 days indicating a molecular remodelling in peri-menopause. Myocardial TGF-ß1 increased at 60 days post-VCD treatment along with reduced Akt phosphorylation. Acute activation of oestrogen receptor-α (ERα) or -ß (ERß) depressed left ventricular contractility in hearts from intact mice. ER-regulation of myocardial and myofilament function, and myofilament phosphorylation, were disrupted in the peri-menopausal model. Disruption occurred without alterations in total ERα or ERß expression. CONCLUSIONS: This is the first study to demonstrate remodelling of the heart in a model of peri-menopause, along with a disruption in ER-dependent regulation of the heart. These data indicate that oestrogen replacement therapy initiated after menopause affects a heart that is profoundly different from that found in reproductively intact animals.
Assuntos
Cicloexenos/toxicidade , Menopausa/fisiologia , Miofibrilas/fisiologia , Insuficiência Ovariana Primária/induzido quimicamente , Função Ventricular Esquerda/fisiologia , Compostos de Vinila/toxicidade , Animais , Carcinógenos/toxicidade , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Coração , Camundongos , Miofibrilas/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
OBJECTIVE: The aim of this study was to evaluate the susceptibility of dentin to brushing abrasion using four different toothbrushes (rotating-oscillating, sonic and two types of manual toothbrushes) with the same brushing forces. METHODS: Dentin samples (n = 72) were selected from 72 impacted third molars. Half of the surface of dentin samples was covered with an adhesive tape, creating a protected and a freely exposed area in the same specimen. Brushing was performed with either a: sonic (Sonicare PowerUp, Philips GmbH, Hamburg, Germany), b: oscillating-rotating (Oral B Vitality Precisions Clean, Procter & Gamble, Schwalbach am Taunus, Germany) or two different manual toothbrushes c: flat trim brush head toothbrush (Dr. Best: Original, Glaxo-Smith-Kline, Bühl, Germany) and d: rippled-shaped brush head toothbrush (Blend-a-Dent, Complete V-Interdental, Blend-a-med, Schwalbach, Germany) in a custom made automatic brushing machine. The brushing force was set to 2 N and a whitening toothpaste (RDA = 150) was used. The simulation period was performed over a calculated period to mimic a brushing behavior of two times a day brushing for eight years and six months. Dentin loss was quantitatively determined by profilometry and statistically analyzed by Wilcoxon and Mann-Whitney-U Test (p < 0.05). RESULTS: The mean (standard deviation) surface loss was 21.03 (±1.26) µm for the sonic toothbrush, 15.71 (±0.85) µm for the oscillating-rotating toothbrush, 6.13 (±1.24) µm for the manual toothbrush with flat trim brush head and 2.50 (±0.43) µm for the manual toothbrush with rippled-shaped brush head. Differences between all groups were statistically significant at p<0.05. CONCLUSION: Using the same brushing force and a highly abrasive toothpaste, manual toothbrushes are significantly less abrasive compared to power toothbrushes for an 8.5-year simulation.
Assuntos
Abrasão Dentária/etiologia , Escovação Dentária/efeitos adversos , Dentina/lesões , Humanos , Escovação Dentária/métodosRESUMO
Cardiac myofilaments act as the central contractile apparatus of heart muscle cells. Covalent modification of constituent proteins through phosphorylation is a rapid and powerful mechanism to control myofilament function, and is increasingly seen as a mechanism of disease. While the relationship between protein kinases and cardiac myofilaments has been widely examined, the impact of protein dephosphorylation by protein phosphatases is poorly understood. This review outlines the mechanisms by which the mostly widely expressed protein phosphatases in cardiac myocytes regulate myofilament function, and the emerging role of myofilament-associated protein phosphatases in heart failure. The importance of regulatory subunits and subcellular compartmentalization in determining the functional impact of protein phosphatases on myofilament and myocardial function is also discussed, as are discrepancies about the roles of protein phosphatases in regulating myofilament function. The potential for targeting these molecular messengers in the treatment of heart failure is discussed as a key future direction.
Assuntos
Miofibrilas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Isoenzimas , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas Fosfatases/genética , Fosforilação , Transdução de SinaisRESUMO
Connective Tissue Growth Factor (CTGF, CCN2) is considered to play an important role in cardiac remodelling. We studied whether stretch is a primary stimulus to induce CTGF expression in vivo in rabbit heart, and in vitro in isolated cardiomyocytes and fibroblasts. Twenty weeks of combined volume and pressure overload resulted in eccentric left ventricular (LV) hypertrophy, with increased LV internal diameter (+36 %) and LV weight (+53 %). Myocardial CTGF mRNA and protein levels were substantially increased in the overloaded animals. In isolated adult rabbit cardiomyocytes, cyclic stretch strongly induced CTGF mRNA expression (2.9-fold at 48 h), whereas in cardiac fibroblasts CTGF-induction was transient and modest (1.4-fold after 4 h). Conditioned medium from stretched fibroblasts induced CTGF mRNA expression in non-stretched cardiomyocytes (2.3-fold at 48 h). Our findings indicate that stretch is an important primary trigger for CTGF-induction in the overloaded heart.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Remodelação Ventricular , Animais , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Hemodinâmica , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Miócitos Cardíacos/patologia , RNA Mensageiro/metabolismo , Coelhos , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima , Função Ventricular Esquerda , Pressão VentricularRESUMO
Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser(315). The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both myofilament Ca(2+) sensitivity (pCa(50)) and maximal Ca(2+)-activated tension of contraction (T(max)). In cMyBP-C KO skinned myocytes, PKD increased pCa(50) but did not alter T(max). This suggests that cMyBP-C is not involved in PKD-mediated sensitization of myofilaments to Ca(2+) but is essential for PKD-induced increase in T(max). Furthermore, the phosphorylation of both PKD-Ser(916) and cMyBP-C-Ser(315) was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca(2+) sensitivity and by an increased T(max) through phosphorylation of cMyBP-C.
Assuntos
Proteínas de Transporte/metabolismo , Acoplamento Excitação-Contração , Contração Miocárdica , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Proteínas de Transporte/genética , Estimulação Elétrica , Acoplamento Excitação-Contração/efeitos dos fármacos , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/enzimologia , Fosforilação , Ligação Proteica , Ratos , Ratos Endogâmicos Lew , SerinaRESUMO
Brief periods of ventricular pacing during the early reperfusion phase (pacing-induced postconditioning, PPC) have been shown to reduce infarct size as measured after 2 h of reperfusion. In this study, we investigated (1) whether PPC leads to maintained reduction in infarct size, (2) whether abnormal mechanical load due to asynchronous activation is the trigger for PPC and (3) the signaling pathways that are involved in PPC. Rabbit hearts were subjected to 30 min of coronary occlusion in vivo, followed by 6 weeks of reperfusion. PPC consisted of ten 30-s intervals of left ventricular (LV) pacing, starting at reperfusion. PPC reduced infarct size (TTC staining) normalized to area at risk, from 49.0 +/- 3.3% in control to 22.9 +/- 5.7% in PPC rabbits. In isolated ejecting rabbit hearts, replacing LV pacing by biventricular pacing abolished the protective effect of PPC, whereas ten 30-s periods of high preload provided a protective effect similar to PPC. The protective effect of PPC was neither affected by the adenosine receptor blocker 8-SPT nor by the angiotensin II receptor blocker candesartan, but was abrogated by the cytoskeletal microtubule-disrupting agent colchicine. Blockers of the mitochondrial K(ATP) channel (5HD), PKC (chelerythrine) and PI3-kinase (wortmannin) all abrogated the protection provided by PPC. In the in situ pig heart, PPC reduced infarct size from 35 +/- 4 to 16 +/- 12%, a protection which was abolished by the stretch-activated channel blocker gadolinium. No infarct size reduction was achieved if PPC application was delayed by 5 min or if only five pacing cycles were used. The present study indicates that (1) PPC permanently reduces myocardial injury, (2) abnormal mechanical loading is a more likely trigger for PPC than electrical stimulation or G-coupled receptor stimulation and (3) PPC may share downstream pathways with other modes of cardioprotection.
Assuntos
Estimulação Cardíaca Artificial , Coração/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Algoritmos , Animais , Fenômenos Biomecânicos , Feminino , Hemodinâmica , Técnicas In Vitro , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Coelhos , Suínos , SístoleRESUMO
The beta(3)-adrenoceptors (beta(3)-ARs) have been identified and characterized in the human heart. Specific beta(3)-AR stimulation, unlike beta(1)-AR or beta(2)-AR stimulation, decreases cardiac contractility, partly via the G(i)-NO pathway. However, the precise role of cardiac beta(3)-ARs is not yet completely understood. Indeed, under normal conditions, the beta(3)-AR response is present only to a very low degree in rats and mice. Therefore, we evaluated whether beta(3)-ARs were present and functional in rabbit ventricular cardiomyocytes, and whether the rabbit could serve as a relevant model for the study of cardiac beta(3)-ARs. We used RT-PCR and Western blot to measure the beta(3)-AR transcripts and protein levels in rabbit ventricular cardiomyocytes. We also analysed the effect of beta(3)-AR stimulation using isoproterenol in combination with nadolol or SR 58611A on cardiomyocyte shortening, Ca(2+) transient, L-type Ca(2+) current (I(Ca,L)), delayed rectifier potassium current (I(Ks)) and action potential duration (APD). For the first time, we show that beta(3)-ARs are expressed in rabbit ventricular cardiomyocytes. The mRNA and protein sequences present a high homology to those of rat and human beta(3)-ARs. Furthermore, beta(3)-AR stimulation decreases cardiomyocyte shortening, Ca(2+) transient and I(Ca,L) amplitudes, via a G(i)-NO pathway. Importantly, beta(3)-AR stimulation enhances I(Ks) amplitude and shortens the APD. Taken together, our results indicate that the rabbit provides a relevant model, easily used in laboratories, to study the roles of cardiac beta(3)-ARs in physiological conditions.
Assuntos
Sinalização do Cálcio , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Potenciais de Ação , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Canais de Potássio de Retificação Tardia/metabolismo , Relação Dose-Resposta a Droga , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ventrículos do Coração/metabolismo , Humanos , Isoproterenol/farmacologia , Masculino , Modelos Animais , Dados de Sequência Molecular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Nadolol/farmacologia , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Coelhos , Ratos , Receptores Adrenérgicos beta 3/efeitos dos fármacos , Receptores Adrenérgicos beta 3/genética , Tetra-Hidronaftalenos/farmacologia , Fatores de TempoRESUMO
We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma(-/-)) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca(2+) content and release but not L-type Ca(2+) current (I(Ca,L)), demonstrating PI3Kgamma locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3Kgamma ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca(2+) transients, SR Ca(2+) content, and phospholamban phosphorylation (PLN-PO(4)) by similar amounts to levels observed in untreated PI3Kgamma(-/-) myocytes. Combined PDE3 and PDE4 inhibition caused no further increases in SR function. By contrast, only PDE3 inhibition affected Ca(2+) transients, SR Ca(2+) loads, and PLN-PO(4) levels in PI3Kgamma(-/-) myocytes. On the other hand, inhibition of PDE3 or PDE4 alone did not affect I(Ca,L) in either PI3Kgamma(-/-) or WT cardiomyocytes, whereas simultaneous PDE3 and PDE4 inhibition elevated I(Ca,L) in both groups. Ryanodine receptor (RyR(2)) phosphorylation levels were not different in basal conditions between PI3Kgamma(-/-) and WT myocytes and increased in both groups with PDE inhibition. Our results establish that L-type Ca(2+) channels, RyR(2), and SR Ca(2+) pumps are regulated differently in distinct subcellular compartments by PDE3 and PDE4. In addition, the loss of PI3Kgamma selectively abolishes PDE4 activity, not PDE3, in subcellular compartments containing the SR Ca(2+)-ATPase but not RyR(2) or L-type Ca(2+) channels.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Miócitos Cardíacos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Compartimento Celular/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Inibidores Enzimáticos/farmacologia , Cardiopatias/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Mutantes , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Fosfatidilinositol 3-Quinases/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Ventricular myocytes are continuously exposed to fluid shear in vivo by relative movement of laminar sheets and adjacent cells. Preliminary observations have shown that neonatal myocytes respond to fluid shear by increasing their beating rate, which could have an arrhythmogenic effect under elevated shear conditions. The objective of this study is to investigate the characteristics of the fluid shear response in cultured myocytes and to study selected potential mechanisms. Cultured neonatal rat ventricular myocytes that were spontaneously beating were subjected to low shear rates (5-50/s) in a fluid flow chamber using standard culture medium. The beating rate was measured from digital microscopic recordings. The myocytes reacted to low shear rates by a graded and reversible increase in their spontaneous beating rate of up to 500%. The response to shear was substantially attenuated in the presence of the beta-adrenergic agonist isoproterenol (by 86+/-8%), as well as after incubation with integrin-blocking RGD peptides (by 92+/-8%). The results suggest that the beta-adrenergic signaling pathway and integrin activation, which are known to interact, may play an important role in the response mechanism.
Assuntos
Potenciais de Ação/fisiologia , Relógios Biológicos/fisiologia , Frequência Cardíaca/fisiologia , Mecanotransdução Celular/fisiologia , Miócitos Cardíacos/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Animais Recém-Nascidos , Velocidade do Fluxo Sanguíneo , Células Cultivadas , Ventrículos do Coração/citologia , Humanos , Ratos , Ratos Sprague-Dawley , Resistência ao Cisalhamento , Estresse Mecânico , Função VentricularRESUMO
Targeted deletion of actinin-associated LIM protein (ALP) in mice leads to right ventricular (RV) dysplasia and a mild RV cardiomyopathy. Although the phenotype has been thoroughly characterized, the mechanisms leading from the cytoskeletal defect to the disease are unclear. We hypothesized that ALP deficiency may be associated with (1) changes in regional systolic dysfunction and (2) regional dysregulation of hypertrophic growth, in accordance with the restricted expression of ALP in the outflow tract of the RV. We examined RV regional epicardial systolic strains with respect to end-diastole in ALP knockout (ALPKO) mice and wild-type controls using an open-chest preparation. Strain components were consistently lower in the ALPKO mice than wild-type controls (second principal strain E2: p = 0.05). RV pressure was slightly but not significantly lower in ALPKO mice as well. To assess regional growth, geometric remodeling was analyzed in ALPKO and wild-type mice after 4 weeks of chronic hypoxia (11% oxygen). The average amount of RV wall thickening in response to hypoxia was reduced to 11% in the ALPKO mice compared with 44% in the wild-type controls. In summary, the results are consistent with the view that disruption of ALP is associated with diminished RV contractile function as well as altered hypertrophic remodeling.
Assuntos
Ventrículos do Coração/fisiopatologia , Proteínas dos Microfilamentos/deficiência , Disfunção Ventricular Direita/fisiopatologia , Remodelação Ventricular , Animais , Cardiomiopatias , Coração/fisiopatologia , Ventrículos do Coração/patologia , Hipertrofia/genética , Hipertrofia/patologia , Hipertrofia/fisiopatologia , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Miocárdio/patologia , Disfunção Ventricular Direita/patologia , Remodelação Ventricular/genéticaRESUMO
Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 microm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction.
Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Diástole/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/metabolismo , Animais , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Sarcômeros/fisiologia , Sístole/fisiologiaRESUMO
Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.