RESUMO
The advancement of CRISPR-based gene editing tools into biotherapeutics offers the potential for cures to genetic disorders and for new treatment paradigms for even common diseases. Arguably, the most important component of a CRISPR-based medicine is the guide RNA, which is generally large (>100-mer) synthetic RNA composed of a "tracr" and "spacer" region, the latter of which dictates the on-target editing site as well as potential undesired off-target edits. Aiming to advance contemporary capabilities for gRNA characterization to ensure the spacer region is of high fidelity, top-down mass spectrometry was herein implemented to provide direct and quantitative assessments of highly modified gRNA. In addition to sequencing the spacer region and pinpointing modifications, top-down mass spectra were utilized to quantify single-base spacer substitution impurities down to <1% and to decipher highly dissimilar spacers. To accomplish these results in an automated fashion, we devised custom software capable of sequencing and quantifying impurities in gRNA spacers. Notably, we developed automated tools that enabled the quantification of single-base substitutions, including advanced isotopic pattern matching for C > U and U > C substitutions, and created a de novo sequencing strategy to facilitate the identification and quantification of gRNA impurities with highly dissimilar spacer regions.
RESUMO
Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.
Assuntos
Hipercolesterolemia Familiar Homozigota , Nanopartículas , Animais , Edição de Genes/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fígado/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Lipoproteínas LDL/metabolismoRESUMO
BACKGROUND: VERVE-101 is an investigational in vivo CRISPR base-editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower low-density lipoprotein cholesterol. We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of nonhuman primates and a murine F1 progeny study. METHODS: Cynomolgus monkeys were given a single intravenous infusion of a vehicle control (n=10) or VERVE-101 at a dose of 0.75 mg/kg (n=4) or 1.5 mg/kg (n=22) with subsequent follow-up up to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male nonhuman primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu). RESULTS: Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 (proprotein convertase subtilisin/kexin type 9) of 67% and 83% and reductions of low-density lipoprotein cholesterol of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days after dosing. Liver safety monitoring noted a transient rise in alanine aminotransferase and aspartate aminotransferase concentrations after infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male nonhuman primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals. CONCLUSIONS: VERVE-101 was well tolerated in nonhuman primates and led to 83% lower blood PCSK9 protein and 69% lower low-density lipoprotein cholesterol with durable effects up to 476 days after dosing. These results have supported the initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.
Assuntos
Edição de Genes , Pró-Proteína Convertase 9 , Animais , Feminino , Humanos , Masculino , Camundongos , LDL-Colesterol/genética , LDL-Colesterol/metabolismo , Primatas/genética , Primatas/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/uso terapêutico , Sêmen/metabolismo , Edição de Genes/métodos , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Aterosclerose/genética , Aterosclerose/terapiaRESUMO
Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.
Assuntos
Sistemas CRISPR-Cas , LDL-Colesterol/sangue , Edição de Genes , Modelos Animais , Pró-Proteína Convertase 9/genética , Adenina/metabolismo , Animais , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Mutação com Perda de Função , Macaca fascicularis/sangue , Macaca fascicularis/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/metabolismo , Fatores de TempoRESUMO
Adherence to medication regimens is a major barrier to effective treatment in many disease areas, notably in dementia which causes cognitive impairment that reduces patients' awareness of non-adherence and their ability to manage medication. The development of oral dosage forms that can be infrequently dosed, and therefore improve adherence rate and facilitate direct observed therapy, has been a goal for decades. We describe the first demonstration of an oral formulation that achieves >7-day gastric retention and sustained pharmacokinetics in the challenging dog model. Gastric retention requires physical resistance of the dosage form to gastric emptying forces, which are known to be stronger in dogs than in humans, making successful gastric retention in dogs a stringent test for predicting human translatability. This formulation of memantine hydrochloride is the first oral dosage form that achieves multi-day drug release with near zero-order kinetics and efficient delivery. In the dog model, relative memantine bioavailability approaches 100% with sustained plasma levels of memantine over seven days and profiles that can be tuned by varying components of the formulation. A single gastric resident dosage form achieves an AUC equivalent to 7 daily treatments with the marketed daily capsule, with a Cmax that is no higher than the daily product. PK modeling predicts that the gastroretentive formulation will maintain therapeutic blood levels in humans when administered once weekly. The formulation methodology presented here is applicable to many water soluble drugs and may enable the development of long-acting oral therapies for a wide variety of conditions.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Memantina/administração & dosagem , Administração Oral , Animais , Disponibilidade Biológica , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Cães , Esquema de Medicação , Antagonistas de Aminoácidos Excitatórios/sangue , Antagonistas de Aminoácidos Excitatórios/farmacocinética , Interações Alimento-Droga , Humanos , Masculino , Adesão à Medicação , Memantina/sangue , Memantina/farmacocinética , Modelos Biológicos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
Multigram drug depot systems for extended drug release could transform our capacity to effectively treat patients across a myriad of diseases. For example, tuberculosis (TB) requires multimonth courses of daily multigram doses for treatment. To address the challenge of prolonged dosing for regimens requiring multigram drug dosing, we developed a gastric resident system delivered through the nasogastric route that was capable of safely encapsulating and releasing grams of antibiotics over a period of weeks. Initial preclinical safety and drug release were demonstrated in a swine model with a panel of TB antibiotics. We anticipate multiple applications in the field of infectious diseases, as well as for other indications where multigram depots could impart meaningful benefits to patients, helping maximize adherence to their medication.
Assuntos
Antituberculosos/uso terapêutico , Sistemas de Liberação de Medicamentos , Estômago/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Antituberculosos/farmacologia , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Doxiciclina/uso terapêutico , Sistemas de Liberação de Medicamentos/economia , Liberação Controlada de Fármacos , Humanos , SuínosRESUMO
INTRODUCTION: The development of oral sustained release dosage forms has been a longstanding goal due to the potential for ease of administration, improved pharmacokinetics, reduced dosing frequency, and improved adherence. The benefits of multiday single-dose drug delivery are evident in the success and patient adoption of injected and implanted dosage forms. However, in the space of oral medications, all current commercially available gastric resident dosage forms, and most in development, are limited to gastric residence of less than 1 day. AREAS COVERED: Reviews of systems to extend gastric residence reveal that 1 day or more residence has been an unmet challenge. New dosage forms are in development that seek to address many of the key physiological and design challenges of long-term gastric retention beyond 24 h and up to a week or longer. The present analysis highlights the design, material considerations and implications of unfolding dosage form systems with ultra-long-term gastric residence. EXPERT OPINION: The development of oral dosage forms providing sustained release of high potency medications over days or weeks could transform care, significantly decrease patient burden in chronic disease management and improve outcomes.
Assuntos
Sistemas de Liberação de Medicamentos , Estômago , Administração Oral , Preparações de Ação Retardada , Formas de Dosagem , HumanosRESUMO
The efficacy of antiretroviral therapy is significantly compromised by medication non-adherence. Long-acting enteral systems that can ease the burden of daily adherence have not yet been developed. Here we describe an oral dosage form composed of distinct drug-polymer matrices which achieved week-long systemic drug levels of the antiretrovirals dolutegravir, rilpivirine and cabotegravir in a pig. Simulations of viral dynamics and patient adherence patterns indicate that such systems would significantly reduce therapeutic failures and epidemiological modelling suggests that using such an intervention prophylactically could avert hundreds of thousands of new HIV cases. In sum, weekly administration of long-acting antiretrovirals via a novel oral dosage form is a promising intervention to help control the HIV epidemic worldwide.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Piridonas/administração & dosagem , Rilpivirina/administração & dosagem , Administração Oral , Animais , Fármacos Anti-HIV/farmacocinética , Fármacos Anti-HIV/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Modelos Teóricos , Oxazinas , Cooperação do Paciente , Piperazinas , Estudo de Prova de Conceito , Piridonas/farmacocinética , Piridonas/uso terapêutico , Rilpivirina/farmacocinética , Rilpivirina/uso terapêutico , SuínosRESUMO
Acute idiopathic hyperammonemia in an adult patient is a life-threatening condition often resulting in a rapid progression to irreversible cerebral edema and death. While ammonia-scavenging therapies lower blood ammonia levels, in comparison, clearance of waste nitrogen from the brain may be delayed. Therefore, we used magnetic resonance spectroscopy (MRS) to monitor cerebral glutamine levels, the major reservoir of ammonia, in a gastric bypass patient with hyperammonemic coma undergoing therapy with N-carbamoyl glutamate and the ammonia-scavenging agents, sodium phenylacetate and sodium benzoate. Improvement in mental status mirrored brain glutamine levels, as coma persisted for 48h after plasma ammonia normalized. We hypothesize that the slower clearance for brain glutamine levels accounts for the delay in improvement following initiation of treatment in cases of chronic hyperammonemia. We propose MRS to monitor brain glutamine as a noninvasive approach to be utilized for diagnostic and therapeutic monitoring purposes in adult patients presenting with idiopathic hyperammonemia.
Assuntos
Encéfalo/diagnóstico por imagem , Coma/tratamento farmacológico , Glutamina/metabolismo , Hiperamonemia/tratamento farmacológico , Espectroscopia de Ressonância Magnética/métodos , Encéfalo/metabolismo , Coma/etiologia , Feminino , Derivação Gástrica/efeitos adversos , Glutamatos/uso terapêutico , Humanos , Hiperamonemia/complicações , Hiperamonemia/diagnóstico por imagem , Hiperamonemia/metabolismo , Pessoa de Meia-Idade , Fenilacetatos/uso terapêutico , Benzoato de Sódio/uso terapêutico , Resultado do TratamentoRESUMO
Efforts at elimination of scourges, such as malaria, are limited by the logistic challenges of reaching large rural populations and ensuring patient adherence to adequate pharmacologic treatment. We have developed an oral, ultra-long-acting capsule that dissolves in the stomach and deploys a star-shaped dosage form that releases drug while assuming a geometry that prevents passage through the pylorus yet allows passage of food, enabling prolonged gastric residence. This gastric-resident, drug delivery dosage form releases small-molecule drugs for days to weeks and potentially longer. Upon dissolution of the macrostructure, the components can safely pass through the gastrointestinal tract. Clinical, radiographic, and endoscopic evaluation of a swine large-animal model that received these dosage forms showed no evidence of gastrointestinal obstruction or mucosal injury. We generated long-acting formulations for controlled release of ivermectin, a drug that targets malaria-transmitting mosquitoes, in the gastric environment and incorporated these into our dosage form, which then delivered a sustained therapeutic dose of ivermectin for up to 14 days in our swine model. Further, by using mathematical models of malaria transmission that incorporate the lethal effect of ivermectin against malaria-transmitting mosquitoes, we demonstrated that this system will boost the efficacy of mass drug administration toward malaria elimination goals. Encapsulated, gastric-resident dosage forms for ultra-long-acting drug delivery have the potential to revolutionize treatment options for malaria and other diseases that affect large populations around the globe for which treatment adherence is essential for efficacy.
Assuntos
Antimaláricos/administração & dosagem , Sistemas de Liberação de Medicamentos , Ivermectina/administração & dosagem , Malária/tratamento farmacológico , Estômago/efeitos dos fármacos , Administração Oral , Animais , Cápsulas , Culicidae , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Endoscopia , Análise de Elementos Finitos , Humanos , Malária/transmissão , Modelos Teóricos , Polímeros/química , SuínosRESUMO
Cardio-oncology (the cardiovascular care of cancer patients) has developed as a new translational and clinical field based on the expanding repertoire of mechanism-based cancer therapies. Although these therapies have changed the natural course of many cancers, several may also lead to cardiovascular complications. Many new anticancer drugs approved over the past decade are "targeted" kinase inhibitors that interfere with intracellular signaling contributing to tumor progression. Unexpected cardiovascular and cardiometabolic effects of patient treatment with these inhibitors have provided unique insights into the role of kinases in human cardiovascular biology. Today, an ever-expanding number of cancer therapies targeting novel kinases and other specific cellular and metabolic pathways are being developed and tested in oncology clinical trials. Some of these drugs may affect the cardiovascular system in detrimental ways and others perhaps in beneficial ways. We propose that the numerous ongoing oncology clinical trials are an opportunity for closer collaboration between cardiologists and oncologists to study the cardiovascular and cardiometabolic changes caused by the modulation of these pathways in patients. In this regard, cardio-oncology represents an opportunity and a novel platform for basic and translational investigation and can serve as a potential avenue for optimization of anticancer therapies and for cardiovascular research and drug discovery.
Assuntos
Antineoplásicos/administração & dosagem , Doenças Cardiovasculares/tratamento farmacológico , Sistemas de Liberação de Medicamentos/tendências , Descoberta de Drogas/tendências , Neoplasias/tratamento farmacológico , Medicina de Precisão/tendências , Animais , Antineoplásicos/metabolismo , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Humanos , Terapia de Alvo Molecular/tendências , Neoplasias/diagnóstico , Neoplasias/metabolismo , Medicina de Precisão/métodos , Inibidores de Proteínas Quinases/administração & dosagemRESUMO
Devices resident in the stomach-used for a variety of clinical applications including nutritional modulation for bariatrics, ingestible electronics for diagnosis and monitoring, and gastric-retentive dosage forms for prolonged drug delivery-typically incorporate elastic polymers to compress the devices during delivery through the oesophagus and other narrow orifices in the digestive system. However, in the event of accidental device fracture or migration, the non-degradable nature of these materials risks intestinal obstruction. Here, we show that an elastic, pH-responsive supramolecular gel remains stable and elastic in the acidic environment of the stomach but can be dissolved in the neutral-pH environment of the small and large intestines. In a large animal model, prototype devices with these materials as the key component demonstrated prolonged gastric retention and safe passage. These enteric elastomers should increase the safety profile for a wide range of gastric-retentive devices.
Assuntos
Sistemas de Liberação de Medicamentos , Elastômeros , Polímeros/química , Estômago/efeitos dos fármacos , Animais , Eletrônica , Esôfago/efeitos dos fármacos , Trânsito Gastrointestinal/fisiologia , Géis/química , Humanos , Concentração de Íons de Hidrogênio , Suínos , Comprimidos , Tecnologia FarmacêuticaRESUMO
Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in 'leaky' channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach.
Assuntos
Canais de Cálcio/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Compostos Nitrosos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Homeostase , Camundongos , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca(2+) leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca(2+) release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca(2+) and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles.
Assuntos
Aclimatação/fisiologia , Cálcio/metabolismo , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Termogênese/fisiologia , Tecido Adiposo Marrom/metabolismo , Animais , Temperatura Baixa , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais Iônicos/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Musculares/genética , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Estremecimento/fisiologia , Proteína Desacopladora 1RESUMO
The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death.
Assuntos
Morte Súbita Cardíaca/etiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Epilepsia/genética , Epilepsia/metabolismo , Heterozigoto , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Mutação , Mutação de Sentido Incorreto , Polimorfismo Genético , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
During exercise, defects in calcium (Ca2+) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca2+ release channel required for excitation-contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in "leaky" channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca2+-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca2+ signaling, muscle damage, and impaired exercise capacity.
Assuntos
Adaptação Fisiológica , Canais de Cálcio/metabolismo , Exercício Físico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologiaRESUMO
Over the past century, understanding the mechanisms underlying muscle fatigue and weakness has been the focus of much investigation. However, the dominant theory in the field, that lactic acidosis causes muscle fatigue, is unlikely to tell the whole story. Recently, dysregulation of sarcoplasmic reticulum (SR) Ca(2+) release has been associated with impaired muscle function induced by a wide range of stressors, from dystrophy to heart failure to muscle fatigue. Here, we address current understandings of the altered regulation of SR Ca(2+) release during chronic stress, focusing on the role of the SR Ca(2+) release channel known as the type 1 ryanodine receptor.
Assuntos
Hipertermia Maligna/genética , Músculo Esquelético/fisiopatologia , Doenças Musculares/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cálcio/metabolismo , Tolerância ao Exercício , Humanos , Fadiga Muscular , Doenças Musculares/tratamento farmacológico , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/metabolismo , Estresse MecânicoRESUMO
Voltage-dependent Ca(2+) channel function (Ca(v)1.2, L-type Ca(2+) channel) is required for cardiac excitation-contraction (E-C) coupling. Ca(v)1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Ca(v)1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Ca(v)1.2 function, it is still unclear whether Ca(v)1.2 subunits are PKG substrates. We have identified phosphorylation sites within the alpha(1c) and beta(2a) subunits that are phosphorylated by PKGIalpha in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG-specific manner, in intact HEK cells heterologously expressing alpha(1c) and beta(2a) subunits. Using phospho-epitope-specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with alpha(1c) and beta(2a) subunits caused an inhibition of Ca(v)1.2 whole-cell current. PKG-mediated inhibition of Ca(v)1.2 current was significantly reduced by coexpression of an alanine-substituted Ca(v)1.2 beta(2a) subunit (Ser(496)). Our results identify a molecular mechanism by which cGMP-PKG regulates Ca(v)1.2 phosphorylation and function.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Subunidades Proteicas/metabolismo , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo L/genética , Linhagem Celular , Células Cultivadas , Eletrofisiologia , Regulação da Expressão Gênica , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação , Subunidades Proteicas/genética , Ratos , Ratos Wistar , Serina/metabolismo , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
The long history of lead poisoning provides many lessons about the process by which scientific knowledge is translated into public health policy. In the United States, lead was added to paint and to gasoline in enormous quantities long after medical evidence clearly showed that excessive lead exposure caused considerable morbidity in the population. This article discusses some of the factors that contributed to the slow pace of efforts to address this problem, including the ubiquity and magnitude of lead exposure during much of the twentieth century, which produced a distorted notion about the blood lead level that can be considered "normal"; the prevailing model of disease during this period, notably the novelty of the concept of subclinical disease; the fact that childhood lead poisoning affected mostly families that were politically and economically disenfranchised, fostering a "blame the victim" attitude; and that controlling lead exposure would have impeded efforts to achieve other desirable goals, illustrating the role that value trade-offs often play in policy decisions.