RESUMO
Cardiac structural changes associated with dilated cardiomyopathy (DCM) include cardiomyocyte hypertrophy and myocardial fibrosis. Connective tissue growth factor (CTGF) has been associated with tissue remodeling and is highly expressed in failing hearts. Our aim was to test if inhibition of CTGF would alter the course of cardiac remodeling and preserve cardiac function in the protein kinase Cε (PKCε) mouse model of DCM. Transgenic mice expressing constitutively active PKCε in cardiomyocytes develop cardiac dysfunction that was evident by 3 months of age, and that progressed to cardiac fibrosis, heart failure, and increased mortality. Beginning at 3 months of age, PKCε mice were treated with a neutralizing monoclonal antibody to CTGF (FG-3149) for an additional 3 months. CTGF inhibition significantly improved left ventricular (LV) systolic and diastolic functions in PKCε mice, and slowed the progression of LV dilatation. Using gene arrays and quantitative PCR, the expression of many genes associated with tissue remodeling was elevated in PKCε mice, but significantly decreased by CTGF inhibition. However total collagen deposition was not attenuated. The observation of significantly improved LV function by CTGF inhibition in PKCε mice suggests that CTGF inhibition may benefit patients with DCM. Additional studies to explore this potential are warranted.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Envelhecimento/patologia , Animais , Anticorpos Neutralizantes/farmacologia , Cardiomiopatia Dilatada/patologia , Colágeno/metabolismo , Diástole/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sístole/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Up-regulation and activation of PYK2, a member of the FAK family of protein tyrosine kinases, is involved in the pathogenesis of left ventricular (LV) remodeling and heart failure (HF). PYK2 activation can be prevented by CRNK, the C-terminal domain of PYK2. We previously demonstrated that adenoviral-mediated CRNK gene transfer improved survival and LV function, and slowed LV remodeling in a rat model of coronary artery ligation-induced HF. We now interrogate whether cardiomyocyte-specific, transgenic CRNK expression prevents LV remodeling and HF in a mouse model of dilated cardiomyopathy (DCM) caused by constitutively active Protein Kinase Cε (caPKCε). Transgenic (TG; FVB/N background) mice were engineered to express rat CRNK under control of the α-myosin heavy chain promoter, and crossed with FVB/N mice with cardiomyocyte-specific expression of caPKCε to create double TG mice. LV structure, function, and gene expression were evaluated in all 4 groups (nonTG FVB/N; caPKCε(+/-); CRNK(+/-); and caPKCε×CRNK (PXC) double TG mice) at 1, 3, 6, 9 and 12mo of age. CRNK expression followed a Mendelian distribution, and CRNK mice developed and survived normally through 12mo. Cardiac structure, function and selected gene expression of CRNK mice were similar to nonTG littermates. CRNK had no effect on caPKCε expression and vice versa. PYK2 was up-regulated ~6-fold in caPKCε mice, who developed a non-hypertrophic, progressive DCM with reduced systolic (Contractility Index=151±5 vs. 90±4s(-1)) and diastolic (Tau=7.5±0.5 vs. 14.7±1.3ms) function, and LV dilatation (LV Remodeling Index (LVRI)=4.2±0.1 vs. 6.0±0.3 for FVB/N vs. caPKCε mice, respectively; P<0.05 for each at 12mo). In double TG PXC mice, CRNK expression significantly prolonged survival, improved contractile function (Contractile Index=115±8s(-1); Tau=9.5±1.0ms), and reduced LV remodeling (LVRI=4.9±0.1). Cardiomyocyte-specific expression of CRNK improves contractile function and slows LV remodeling in a mouse model of DCM.
Assuntos
Cardiomiopatia Dilatada/genética , Quinase 2 de Adesão Focal/genética , Miócitos Cardíacos/metabolismo , Transgenes , Função Ventricular/fisiologia , Remodelação Ventricular , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Quinase 2 de Adesão Focal/deficiência , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Longevidade , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C-épsilon/deficiência , Proteína Quinase C-épsilon/genética , Estrutura Terciária de ProteínaRESUMO
Integrins are adhesive, signaling, and mechanotransduction proteins. Talin (Tln) activates integrins and links it to the actin cytoskeleton. Vertebrates contain two talin genes, tln1 and tln2. How Tln1 and Tln2 function in cardiac myocytes (CMs) is unknown. Tln1 and Tln2 expression were evaluated in the normal embryonic and adult mouse heart as well as in control and failing human adult myocardium. Tln1 function was then tested in the basal and mechanically stressed myocardium after cardiomyocyte-specific excision of the Tln1 gene. During embryogenesis, both Tln forms are highly expressed in CMs, but in the mature heart Tln2 becomes the main Tln isoform, localizing to the costameres. Tln1 expression is minimal in the adult CM. With pharmacological and mechanical stress causing hypertrophy, Tln1 is up-regulated in CMs and is specifically detected at costameres, suggesting its importance in the compensatory response to CM stress. In human failing heart, CM Tln1 also increases compared with control samples from normal functioning myocardium. To directly test Tln1 function in CMs, we generated CM-specific Tln1 knock-out mice (Tln1cKO). Tln1cKO mice showed normal basal cardiac structure and function but when subjected to pressure overload showed blunted hypertrophy, less fibrosis, and improved cardiac function versus controls. Acute responses of ERK1/2, p38, Akt, and glycogen synthase kinase 3 after mechanical stress were strongly blunted in Tln1cKO mice. Given these results, we conclude that Tln1 and Tln2 have distinct functions in the myocardium. Our data show that reduction of CM Tln1 expression can lead to improved cardiac remodeling following pressure overload.
Assuntos
Cardiomegalia/metabolismo , Miocárdio/metabolismo , Talina/biossíntese , Adulto , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Feminino , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Fisiológico/genética , Talina/genética , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cardiovascular toxicity is one of the more common causes of attrition in preclinical and clinical drug development. Preclinical cardiovascular safety assessment involves numerous in vitro and in vivo endpoints which are being continually reviewed and improved to lower the incidence of cardiovascular toxicity that manifests only after the initiation of clinical trials. An example of notable preclinical toxicity is necrosis in the papillary muscle of the left ventricle in dogs that is induced by exaggerated pharmacological effects of vasodilators or positive inotropic/vasodilating off-target drug effects. Two distinct, small-molecule inhibitors that target an intracellular kinase, Compound A and Compound B, were profiled in 2-week dose-range finding and 4-week toxicity studies. Serum cardiac troponin (cTnI) was evaluated after a single dose and after 2-week and 4-week repeat dose studies with each kinase inhibitor. Acute effects on hemodynamic (heart rate, blood pressures, left ventricular contractility) and electrocardiographic (QTcV, PR, QRS intervals) endpoints by each inhibitor were assessed in an anesthetized dog cardiovascular model. Cardiovascular degeneration/necrosis with and without fibrosis was observed in dogs and correlated to increases in serum cTnI in repeat-dose toxicity studies. At the same doses used in toxicologic assessments, both kinase inhibitors produced sustained increases in heart rate, left ventricular contractility, and cardiac output, and decreases in mean arterial pressure. Cardiac pathology findings associated with these 2 kinase inhibitors were accompanied not only by cardiac troponin elevations but also associated with hemodynamic changes, highlighting the importance of the link of the physiologic-toxicologic interplay in cardiovascular safety assessment.
Assuntos
Sistema Cardiovascular , Contração Miocárdica , Animais , Cães , Hemodinâmica , Frequência Cardíaca , Necrose , Troponina/farmacologiaRESUMO
The strategic and targeted use of an anesthetized canine cardiovascular model early in drug discovery enables a comprehensive cardiovascular and electrophysiological assessment of potential safety liabilities and guides compound selection prior to initiation of chronic toxicological studies. An ideal model would enable exposure-response relationships to guide safety margin calculations, have a low threshold to initiate, and have quick delivery of decision quality data. We have aimed to profile compounds with diverse mechanism of actions (MoAs) of "non-QT" cardiovascular drug effects and evaluate the ability of nonclinical in vivo cardiovascular models to detect clinically reported effects. The hemodynamic effects of 11 drugs (atropine, itraconazole, atenolol, ivabradine, milrinone, enalaprilat, fasudil, amlodipine, prazosin, amiloride, and hydrochlorothiazide) were profiled in an anesthetized dog cardiovascular model. Derived parameters included: heart rate, an index of left ventricular contractility, mean arterial pressure, systemic vascular resistance, and cardiac output. Species specific plasma protein data was generated (human, dog) and utilized to calculate free drug concentrations. Using the anesthetized dog cardiovascular model, 10 of the 11 drugs displayed the predicted changes in CV parameters based on their primary MoAs and corresponding clinically described effects. Interestingly but not unexpected, 1 of 11 failed to display their predicted CV pattern which is likely due to a delay in pharmacodynamic effect that is beyond the duration of the experimental model (hydrochlorothiazide). The analysis from the current study supports the strategic use of the anesthetized dog model early in the drug discovery process for a comprehensive cardiovascular evaluation with good translation to human.
Assuntos
Ventrículos do Coração , Hemodinâmica , Cães , Animais , Humanos , Avaliação Pré-Clínica de Medicamentos , Frequência Cardíaca , Preparações Farmacêuticas , Hidroclorotiazida/farmacologia , Pressão SanguíneaRESUMO
This editorial prefaces the annual themed issue on safety pharmacology (SP) methods which has been published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). Here we highlight content derived from the 2023 Safety Pharmacology Society (SPS) meeting held in Brussels, Belgium. The meeting generated 138 abstracts, reproduced in the current volume of JPTM. As in prior years, the manuscripts reflect various areas of innovation in SP including in silico modeling of stroke volume, cardiac output and systemic vascular resistance, computational approaches that compare drug-induced proarrhythmic sensitivity of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), an evaluation of the utility of the corrected J-Tpeak and Tpeak-to-Tend parameters from the ECG as potential proarrhythmia biomarkers, and the applicability of nonclinical concentration-QTc (C-QTc) modeling of data derived from the conduct of the in vivo QTc study as a component of the core battery of safety pharmacology studies.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Eletrocardiografia/efeitos dos fármacos , Eletrocardiografia/métodos , Sistema Cardiovascular/efeitos dos fármacos , Simulação por ComputadorRESUMO
BACKGROUND: Heart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective tissue growth factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited. METHODS AND RESULTS: Left ventricle (LV) tissue from patients undergoing cardiac transplantation for ischemic (ICM; n = 20) and dilated (DCM; n = 20) cardiomyopathies and from nonfailing (NF; n = 20) donor hearts were examined. Paired samples (n = 15) from patients undergoing LV assist device (LVAD) implantation as "bridge to transplant" (34-1,145 days) also were analyzed. There was more interstitial fibrosis in both ICM and DCM compared with NF hearts. Hydroxyproline concentration was also significantly increased in DCM compared with NF samples. The expression of CTGF, transforming growth factor (TGF) ß1, collagen (COL) 1-α1, COL3-α1, matrix metalloproteinase (MMP) 2, and MMP9 mRNA in ICM and DCM were also significantly elevated compared with NF samples. Although TGF-ß1, CTGF, COL1-α1, and COL3-α1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading. CONCLUSIONS: CTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.
Assuntos
Cardiomiopatia Dilatada/patologia , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Transplante de Coração , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fibrose , Insuficiência Cardíaca/diagnóstico , Transplante de Coração/tendências , Humanos , Remodelação Ventricular/genéticaRESUMO
This editorial prefaces the annual themed issue on safety pharmacology (SP) methods published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). We highlight here the content derived from the recent 2022 Safety Pharmacology Society (SPS) and Canadian Society of Pharmacology and Therapeutics (CSPT) joint meeting held in Montreal, Quebec, Canada. The meeting also generated 179 abstracts (reproduced in the current volume of JPTM). As in previous years the manuscripts reflect various areas of innovation in SP including a comparison of the sensitivity of cross-over and parallel study designs for QTc assessment, use of human-induced pluripotent stem cell (hi-PSC) neuronal cell preparations for use in neuropharmacological safety screening, and hiPSC derived cardiac myocytes in assessing inotropic adversity. With respect to the latter, we anticipate the emergence of a large data set of positive and negative controls that will test whether the imperative to miniaturize, humanize and create a high throughput process is offset by any loss of precision and accuracy.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Farmacologia , Humanos , Canadá , Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Farmacologia/métodos , Congressos como AssuntoRESUMO
INTRODUCTION: Secondary pharmacology profiling is routinely applied in pharmaceutical drug discovery to investigate the pharmaceutical effects of a drug at molecular targets distinct from (off-target) the intended therapeutic molecular target (on-target). Data from a randomized, placebo-controlled clinical trial, the APPROVe (Adenomatous Polyp Prevention on VIOXX, rofecoxib) trial, raised significant concerns about COX-2 inhibition as a primary or secondary target, shaping the screening and decision-making processes of some pharmaceutical companies. COX-2 is often included in off-target screens due to cardiovascular (CV) safety concerns about secondary interactions with this target. Several potential mechanisms of COX-2-mediated myocardial infarctions have been considered including, effects on platelet stickiness/aggregation, vasal tone and blood pressure, and endothelial cell activation. In the present study, we focused on each of these mechanisms as potential effects of COX-2 inhibitors, to find evidence of mechanism using various in vitro and in vivo preclinical models. METHODS: Compounds tested in the study, with a range of COX-2 selectivity, included rofecoxib, celecoxib, etodolac, and meloxicam. Compounds were screened for inhibition of COX-2 vs COX-1 enzymatic activity, ex vivo platelet aggregation (using whole blood from multiple species), ex vivo canine femoral vascular ring model, in vitro human endothelial cell activation (with and without COX-2 induction), and in vivo cardiovascular assessment (anesthetized dog). RESULTS: The COX-2 binding assessment generally confirmed the COX-2 selectivity previously reported. COX-2 inhibitors did not have effects on platelet function (spontaneous aggregation or inhibition of aggregation), cardiovascular parameters (mean arterial pressure, heart rate, and left ventricular contractility), or endothelial cell activation. However, rofecoxib uniquely produced an endothelial mediated constriction response in canine femoral arteries. CONCLUSION: Our data suggest that rofecoxib-related cardiovascular events in humans are not predicted by COX-2 potency or selectivity. In addition, the vascular ring model suggested possible adverse cardiovascular effects by COX-2 inhibitors, although these effects were not seen in vivo studies. These results may also suggest that COX-2 inhibition alone is not responsible for rofecoxib-mediated adverse cardiovascular outcomes.
Assuntos
Doenças Cardiovasculares , Anel Vascular , Animais , Cães , Humanos , Inibidores de Ciclo-Oxigenase 2/efeitos adversos , Ciclo-Oxigenase 2 , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/tratamento farmacológico , Fatores de Risco , Fatores de Risco de Doenças Cardíacas , Preparações Farmacêuticas , Anti-Inflamatórios não Esteroides/efeitos adversosRESUMO
OBJECTIVE: Focal adhesion kinase-related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. METHODS AND RESULTS: Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas). CONCLUSION: We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.
Assuntos
Movimento Celular/fisiologia , Proteína Substrato Associada a Crk/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Proteínas Tirosina Quinases/metabolismo , Adenoviridae/genética , Animais , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Cateterismo/efeitos adversos , Células Cultivadas , Quinase 2 de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Animais , Fosforilação/fisiologia , Ligação Proteica/fisiologia , RNA Interferente Pequeno/farmacologia , RatosRESUMO
The 2021 Annual Safety Pharmacology (SP) Society (SPS) meeting was held virtually October 4-8, 2021 due to the continuing COVID-19 global pandemic. This themed issue of J Pharmacol Toxicol Methods comprises articles arising from the meeting. As in previous years the manuscripts reflect various areas of innovation in SP including a perspective on aging and its impact on drug attrition during safety assessments, an integrated assessment of respiratory, cardiovascular and animal activity of in vivo nonclinical studies, development of a dynamic QT-rate correction method in primates, evaluation of the "comprehensive in vitro proarrhythmia assay" (CiPA) ion channel protocol to the automated patch clamp, and best practices regarding the conduct of hERG electrophysiology studies and an analysis of secondary pharmacology assays by the FDA. The meeting also generated 85 abstracts (reproduced in the current volume of J Pharmacol Toxicol Methods). It appears that the validation of methods remains a challenge in SP. Nevertheless, the continued efforts to mine approaches to detection of proarrhythmia liability remains a baffling obsession given the ability of Industry to completely prevent drugs entering into clinical study only to be found to have proarrhythmic properties, with no reports of such for at least ten years. Perhaps it is time to move on from CiPA and find genuine problems to solve?
Assuntos
COVID-19 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Indóis , Canais Iônicos , PropionatosRESUMO
OBJECTIVE: To examine whether interference with FRNK targeting to focal adhesions (FAs) affects its inhibitory activity and tyrosine phosphorylation. METHODS AND RESULTS: Focal adhesion kinase and its autonomously expressed C-terminal inhibitor, focal adhesion kinase-related nonkinase (FRNK), regulate vascular smooth muscle cell (VSMC) signaling and migration. FRNK-paxillin binding was reduced by a point mutation in its FA targeting domain (L341S-FRNK). Green fluorescent protein-tagged wild type and L341S-FRNK were then adenovirally expressed in VSMCs. L341S-FRNK targeted to VSMC FAs, despite previous studies in other cell types. L341S-FRNK affected FA binding kinetics (assessed by total internal reflection fluorescnece [TIRF] microscopy and fluorescence recovery after photobleaching [FRAP]) and reduced its steady-state paxillin interaction (determined by coimmunoprecipitation). Both wt-FRNK and L341S-FRNK lowered basal and angiotensin II-stimulated focal adhesion kinase, paxillin, and extracellular signal-regulated kinase 1/2 phosphorylation. However, the degree of inhibition was significantly reduced by L341S-FRNK. L341S-FRNK also demonstrated significantly greater migratory activity compared with wt-FRNK-expressing VSMCs. Angiotensin II-induced Y168 phosphorylation was Src dependent, as evident by a significant reduction in Y168 phosphorylation by the Src family kinase inhibitor PP2 is 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Surprisingly, Y168 phosphorylation was unaffected by its targeting. Furthermore, Y232 phosphorylation increased approximately 3-fold in L341S-FRNK, which was less sensitive to PP2. CONCLUSIONS: FRNK inhibition of VSMC migration requires both FA targeting and Y168 phosphorylation by Src family kinases. FRNK-Y232 phosphorylation occurs outside of FAs, probably by a PP2-insensitive kinase.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Miócitos de Músculo Liso/fisiologia , Paxilina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Movimento Celular/fisiologia , Músculo Liso Vascular , Fosforilação , Mutação Puntual , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Ratos , Transdução de SinaisRESUMO
INTRODUCTION: A successful integration of automated blood sampling (ABS) into the telemetry instrumented canine cardiovascular model is presented in this study. This combined model provides an efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in dog while providing a complete Pharmacokinetic/Pharmacodynamic (PK/PD) profile for discovery compounds without handling artifacts, reducing the need for a separate pharmacokinetic study. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points. A series of four use cases utilizing four different test compounds and analytical endpoints are described to illustrate some of the potential applications of the technique. RESULTS: In the four presented use cases, automated blood sampling in telemetry instrumented dogs provides simultaneous cardiovascular (heart rate, arterial blood pressure, and left ventricular pressure), electrophysiological assessment (QTc, PR, and QRS intervals), body temperature, and animal activity, while collecting multiple blood samples for drug analysis. CONCLUSION: The combination of automated blood sampling with cardiovascular telemetry monitoring is a novel capability designed to support safety pharmacology cardiovascular assessment of discovery molecules. By combining telemetry and high-fidelity ABS, the model provides an enhanced PK/PD understanding of drug-induced hemodynamic and electrocardiographic effects of discovery compounds in conscious beagles in the same experimental session. Importantly, the model can reduce the need for a separate pharmacokinetic study (positive reduction 3R impact), reduces compound syntheses requirements, and shorten development timelines. Furthermore, implementation of this approach has also improved animal welfare by reducing the animal handling during a study, thereby reducing stress and associated data artifacts (positive refinement 3R impact).
Assuntos
Sistema Cardiovascular , Telemetria , Animais , Pressão Sanguínea , Cães , Eletrocardiografia , Frequência Cardíaca , Macaca fascicularis , MasculinoRESUMO
INTRODUCTION: A successful integration of automated blood sampling (ABS) into the telemetry instrumented canine cardiovascular model is presented in this study. This combined model provides an efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in dog while providing a complete Pharmacokinetic/Pharmacodynamic (PK/PD) profile for discovery compounds without handling artifacts, reducing the need for a separate pharmacokinetic study. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points. A series of four use cases utilizing four different test compounds and analytical endpoints are described to illustrate some of the potential applications of the technique. RESULTS: In the four presented use cases, automated blood sampling in telemetry instrumented dogs provides simultaneous cardiovascular (heart rate, arterial blood pressure, and left ventricular pressure), electrophysiological assessment (QTc, PR, and QRS intervals), body temperature, and animal activity, while collecting multiple blood samples for drug analysis. CONCLUSION: The combination of automated blood sampling with cardiovascular telemetry monitoring is a novel capability designed to support safety pharmacology cardiovascular assessment of discovery molecules. By combining telemetry and high-fidelity ABS, the model provides an enhanced PK/PD understanding of drug-induced hemodynamic and electrocardiographic effects of discovery compounds in conscious beagles in the same experimental session. Importantly, the model can reduce the need for a separate pharmacokinetic study (positive reduction 3R impact), reduces compound syntheses requirements, and shorten development timelines. Furthermore, implementation of this approach has also improved animal welfare by reducing the animal handling during a study, thereby reducing stress and associated data artifacts (positive refinement 3R impact).
Assuntos
Sistema Cardiovascular , Telemetria , Animais , Pressão Sanguínea , Cães , Eletrocardiografia , Frequência Cardíaca , MasculinoRESUMO
INTRODUCTION: This manuscript presents a successful integration of multi-timepoint biomarker blood sampling (e.g., cytokines) in a conscious dog cardiovascular study using automated blood sampling via vascular access ports in telemetry instrumented dogs. In addition to determining plasma exposure of the test compound, the assessment of biomarkers of interest allows for more comprehensive preclinical evaluation on a traditional conscious dog cardiovascular (CV) telemetry study especially for immunology and immune-oncology molecules. This model system provides a rapid and efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in large species that are commonly used for preclinical safety evaluations while collecting multiple blood samples for drug and cytokine analysis. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (ABS) (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points for cytokine analysis. Four beagles received low-dose lipopolysaccharide solution (LPS) (0.1 and 0.5 µg/mL). The following cytokines were measured by Milliplex® map Canine Cytokine Magnetic Bead Panel: Interleukin (IL) 2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, TNF-α, MCP-1, KC-like, GM-CSF, IFN gamma, and IP10. RESULTS: Low dose LPS administration induced a pronounced dose-dependent, transient release of key inflammatory cytokines (IL-2, IL-6, IL-10, TNF-α, MCP-1, and KC-like). Cytokine responses were similar to other canine and human endotoxin models. LPS administration led to an increase in body temperature, heart rate, and mean arterial pressure, as well as a decrease in QTcV interval. CONCLUSION: Successful incorporation of cytokine analysis in telemetry instrumented dogs with vascular access ports allows for translational PK/PD modeling of both efficacy and safety of compounds in the immunology as well as the immune-oncology therapeutic areas designed to modulate the immune system. Remote collection of blood samples simultaneously with CV endpoints is a significant enhancement for assessment of biomarkers that are sensitive to animal handling and excitement associated with room disturbances which are obligatory with manual blood collection. Furthermore, implementing this approach has also refined our animal welfare procedure by reducing the handling during a study and thereby reducing stress (positive refinement 3R impact).
Assuntos
Cães , Fatores Imunológicos , Telemetria , Animais , Temperatura Corporal , Sistema Cardiovascular , Citocinas , Frequência Cardíaca , Fatores Imunológicos/análise , MasculinoRESUMO
Successful implementation of automated blood sampling (ABS) into a telemetry instrumented canine cardiovascular model provides simultaneous cardiovascular assessment of novel compounds while collecting multiple blood samples for analysis of drug level, cytokines, and biomarkers. Purpose-bred male Beagle dogs (n = 36) were instrumented with a dual-pressure telemetry transmitter and vascular access port. Modifications to acclimation practices, surgical procedures, and housing were required for implementation of ABS in our established cardiovascular canine telemetry colony. These modifications have increased the use and reproducibility of the model by combining early pharmacokinetic and cardiovascular studies, thus achieving both refinement and reduction from a 3R perspective. In addition, the modified model can shorten timelines and reduce the compound requirement in early stages of drug development. This telemetry-ABS model provides an efficient means to quickly identify potential effects on key cardiovascular parameters in a large animal species and to obtain a more complete pharmacokinetic-pharmacodynamic profile for discovery compounds.
Assuntos
Modelos Cardiovasculares , Telemetria , Animais , Pressão Sanguínea , Cães , Eletrocardiografia , Frequência Cardíaca , Masculino , Reprodutibilidade dos TestesRESUMO
A ventricular myocyte experiences changes in length and load during every beat of the heart and has the ability to remodel cell shape to maintain cardiac performance. Specifically, myocytes elongate in response to increased diastolic strain by adding sarcomeres in series, and they thicken in response to continued systolic stress by adding filaments in parallel. Myocytes do this while still keeping the resting sarcomere length close to its optimal value at the peak of the length-tension curve. This review focuses on the little understood mechanisms by which direction of growth is matched in a physiologically appropriate direction. We propose that the direction of strain is detected by differential phosphorylation of proteins in the costamere, which then transmit signaling to the Z-disc for parallel or series addition of thin filaments regulated via the actin capping processes. In this review, we link mechanotransduction to the molecular mechanisms for regulation of myocyte length and width.
Assuntos
Miocárdio/metabolismo , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Estresse Mecânico , Animais , Humanos , Modelos Biológicos , Desenvolvimento Muscular/fisiologia , Miocárdio/enzimologia , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologiaRESUMO
INTRODUCTION: The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative differentiates torsadogenic risk of 28 drugs affecting ventricular repolarization based on multiple in vitro human derived ionic currents. However, a standardized prospective assessment of the electrophysiologic effects of these drugs in an integrated in vivo preclinical cardiovascular model is lacking. This study questioned whether QTc interval prolongation in a preclinical in vivo model could detect clinically reported QTc prolongation and assign torsadogenic risk for ten CiPA drugs. METHODS: An acute intravenous administered ascending dose anesthetized dog cardiovascular model was used to assess QTc prolongation along with other electrocardiographic (PR, QRS intervals) and hemodynamic (heart rate, blood pressures, left ventricular contractility) parameters at plasma concentrations spanning and exceeding clinical exposures. hERG current block potency was characterized using IC50 values from automated patch clamp. RESULTS: All eight drugs eliciting clinical QTc prolongation also delayed repolarization in anesthetized dogs at plasma concentrations within four-fold clinical exposures. In vitro QTc safety margins (defined based on clinical Cmax values/plasma concentrations eliciting statistically significant QTc prolongation in dogs) were lower for high vs intermediate torsadogenic risk drugs. In comparison, hERG IC10 values represented as total drug concentrations were better predictors of preclinical QTc prolongation than hERG IC50 values. CONCLUSION: There was good concordance for QTc prolongation in the anesthetized dog model and clinical torsadogenic risk assignment. QTc assessment in the anesthetized dog remains a valuable part of a more comprehensive preclinical integrated risk assessment for delayed repolarization and torsadogenic risk as part of a global cardiovascular evaluation.
Assuntos
Antiarrítmicos/farmacologia , Síndrome do QT Longo/tratamento farmacológico , Torsades de Pointes/tratamento farmacológico , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Eletrocardiografia , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Síndrome do QT Longo/induzido quimicamente , Masculino , Modelos Cardiovasculares , Estudos Prospectivos , Medição de Risco , Torsades de Pointes/induzido quimicamenteRESUMO
The design of a novel transduction complex has permitted the introduction of protein-quantum dot conjugates into the cytoplasm of living cells. Appropriate subcellular localization of quantum dot-conjugated cardiac troponin C to the myofibrils and a nuclear peptide to the nucleus was attained in living cardiac myocytes using this approach. This new methodology opens the possibility for live tracking of exogenous proteins and study of protein dynamics.
Assuntos
Núcleo Celular/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Pontos Quânticos , Troponina C/metabolismo , Animais , Microscopia Confocal/métodos , Miofibrilas/metabolismo , Ratos , Troponina C/farmacologiaRESUMO
INTRODUCTION: The goal of this study was to determine whether assessment of myocardial contractility and hemodynamics in an anesthetized dog model, could consistently detect drug-induced changes in the inotropic state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. METHODS: Derived parameters included: diastolic, systolic and mean arterial BP, peak systolic LVP, HR, end-diastolic LVP, and LVdP/dtmax as the primary contractility index. RESULTS: These results demonstrate that statistically significant increases (amrinone and pimobendan) and decreases (atenolol and itraconazole) in left ventricular dP/dtmax were observed at clinically relevant exposures. DISCUSSION: The analysis from the current study supports the strategic use of the anesthetized dog model early in the drug Discovery process for a comprehensive cardiovascular evaluation that can include left ventricular dP/dtmax with good translation to human.