RESUMO
Heart failure (HF) remains a huge medical burden worldwide, with aging representing a major risk factor. Here, we report the effects of sacubitril/valsartan, an approved drug for HF with reduced EF, in an experimental model of aging-related HF with preserved ejection fraction (HFpEF). Eighteen-month-old female Fisher 344 rats were treated for 12 weeks with sacubitril/valsartan (60 mg/kg/day) or with valsartan (30 mg/kg/day). Three-month-old rats were used as control. No differential action of sacubitril/valsartan versus valsartan alone, either positive or negative, was observed. The positive effects of both sacubitril/valsartan and valsartan on cardiac hypertrophy was evidenced by a significant reduction of wall thickness and myocyte cross-sectional area. Contrarily, myocardial fibrosis in aging heart was not reduced by any treatment. Doppler echocardiography and left ventricular catheterization evidenced diastolic dysfunction in untreated and treated old rats. In aging rats, both classical and non-classical renin-angiotensin-aldosterone system (RAAS) were modulated. In particular, with respect to untreated animals, both sacubitril/valsartan and valsartan showed a partial restoration of cardioprotective non-classical RAAS. In conclusion, this study evidenced the favorable effects, by both treatments, on age-related cardiac hypertrophy. The attenuation of cardiomyocyte size and hypertrophic response may be linked to a shift towards cardioprotective RAAS signaling. However, diastolic dysfunction and cardiac fibrosis persisted despite of treatment and were accompanied by myocardial inflammation, endothelial activation, and oxidative stress.
Assuntos
Envelhecimento , Aminobutiratos , Compostos de Bifenilo , Combinação de Medicamentos , Insuficiência Cardíaca , Ratos Endogâmicos F344 , Tetrazóis , Valsartana , Animais , Aminobutiratos/farmacologia , Aminobutiratos/uso terapêutico , Compostos de Bifenilo/farmacologia , Valsartana/farmacologia , Valsartana/uso terapêutico , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Feminino , Tetrazóis/farmacologia , Tetrazóis/uso terapêutico , Ratos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Fibrose , Estresse Oxidativo/efeitos dos fármacos , Antagonistas de Receptores de Angiotensina/farmacologia , Antagonistas de Receptores de Angiotensina/uso terapêutico , Volume Sistólico/efeitos dos fármacos , Modelos Animais de Doenças , Neprilisina/antagonistas & inibidores , Neprilisina/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologiaRESUMO
Introduction: During aging, sarcopenia and decline in physiological processes lead to partial loss of muscle strength, atrophy, and increased fatigability. Muscle changes may be related to a reduced intake of essential amino acids playing a role in proteostasis. We have recently shown that branched-chain amino acid (BCAA) supplements improve atrophy and weakness in models of muscle disuse and aging. Considering the key roles that the alteration of Ca2+-related homeostasis and store-operated calcium entry (SOCE) play in several muscle dysfunctions, this study has been aimed at gaining insight into the potential ability of BCAA-based dietary formulations in aged mice on various players of Ca2+ dyshomeostasis. Methods: Seventeen-month-old male C57BL/6J mice received a 12-week supplementation with BCAAs alone or boosted with two equivalents of L-alanine (2-Ala) or with dipeptide L-alanyl-L-alanine (Di-Ala) in drinking water. Outcomes were evaluated on ex vivo skeletal muscles indices vs. adult 3-month-old male C57BL/6J mice. Results: Ca2+ imaging confirmed a decrease in SOCE and an increase of resting Ca2+ concentration in aged vs. adult mice without alteration in the canonical components of SOCE. Aged muscles vs. adult muscles were characterized by a decrease in the expression of ryanodine receptor 1 (RyR1), the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) pump, and sarcalumenin together with an alteration of the expression of mitsugumin 29 and mitsugumin 53, two recently recognized players in the SOCE mechanism. BCAAs, particularly the formulation BCAAs+2-Ala, were able to ameliorate all these alterations. Discussion: These results provide evidence that Ca2+ homeostasis dysfunction plays a role in the functional deficit observed in aged muscle and supports the interest of dietary BCAA supplementation in counteracting sarcopenia-related SOCE dysregulation.
RESUMO
Oral anticoagulant therapy (OAT) for managing atrial fibrillation (AF) encompasses vitamin K antagonists (VKAs, such as warfarin), which was the mainstay of anticoagulation therapy before 2010, and direct-acting oral anticoagulants (DOACs, namely dabigatran etexilate, rivaroxaban, apixaban, edoxaban), approved for the prevention of AF stroke over the last thirteen years. Due to the lower risk of major bleeding associated with DOACs, anticoagulant switching is a common practice in AF patients. Nevertheless, there are issues related to OAT switching that still need to be fully understood, especially for patients in whom AF and heart failure (HF) coexist. Herein, the effective impact of the therapeutic switching from warfarin to DOACs in HF patients with AF, in terms of cardiac remodeling, clinical status, endothelial function and inflammatory biomarkers, was assessed by a machine learning (ML) analysis of a clinical database, which ultimately shed light on the real positive and pleiotropic effects mediated by DOACs in addition to their anticoagulant activity.
Assuntos
Anticoagulantes , Fibrilação Atrial , Insuficiência Cardíaca , Aprendizado de Máquina , Humanos , Fibrilação Atrial/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Anticoagulantes/uso terapêutico , Anticoagulantes/administração & dosagem , Anticoagulantes/farmacologia , Administração Oral , Masculino , Feminino , Idoso , Doença Crônica , Varfarina/uso terapêuticoRESUMO
Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X-linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild-type (WT) H2K-2B4 and the dystrophic (DYS) H2K-SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day-11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium-sensitive outward current compared to WT. Furthermore, day-11 DYS cells showed more positive resting membrane potential (+10 mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.
Assuntos
Distrofia Muscular de Duchenne , Animais , Camundongos , Distrofia Muscular de Duchenne/metabolismo , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Biomarcadores/metabolismo , Canais Iônicos/metabolismo , Desenvolvimento MuscularRESUMO
Myasthenia gravis (MG) is a rare autoimmune disease that causes debilitating muscle weakness due to impaired neuromuscular transmission. Since most (about 80-90%) MG patients present autoantibodies against the acetylcholine receptor, standard medical therapy consists of symptomatic treatment with acetylcholinesterase inhibitors (e.g., pyridostigmine). In addition, considering the autoimmune basis of MG, standard therapy includes immunomodulating agents, such as corticosteroids, azathioprine, cyclosporine A, and cyclophosphamide. New strategies have been proposed for the treatment of MG and include complement blockade (i.e., eculizumab, ravulizumab, and zilucoplan) and neonatal Fc receptor antagonism (i.e., efgartigimod and rozanolixizumab). The aim of this review is to provide a detailed overview of the pre- and post-marketing evidence on the five pharmacological treatments most recently approved for the treatment of MG, by identifying both preclinical and clinical studies registered in clinicaltrials.gov. A description of the molecules currently under evaluation for the treatment of MG is also provided.
Assuntos
Miastenia Gravis , Humanos , Recém-Nascido , Acetilcolinesterase/uso terapêutico , Corticosteroides/uso terapêutico , Autoanticorpos , Miastenia Gravis/tratamento farmacológico , Receptores Colinérgicos/uso terapêutico , Terapias em EstudoRESUMO
Despite being extremely relevant for the protection of prenatal and neonatal health, the developmental toxicity (Dev Tox) is a highly complex endpoint whose molecular rationale is still largely unknown. The lack of availability of high-quality data as well as robust nontesting methods makes its understanding even more difficult. Thus, the application of new explainable alternative methods is of utmost importance, with Dev Tox being one of the most animal-intensive research themes of regulatory toxicology. Descending from TIRESIA (Toxicology Intelligence and Regulatory Evaluations for Scientific and Industry Applications), the present work describes TISBE (TIRESIA Improved on Structure-Based Explainability), a new public web platform implementing four fundamental advancements for in silico analyses: a three times larger dataset, a transparent XAI (explainable artificial intelligence) framework employing a fragment-based fingerprint coding, a novel consensus classifier based on five independent machine learning models, and a new applicability domain (AD) method based on a double top-down approach for better estimating the prediction reliability. The training set (TS) includes as many as 1008 chemicals annotated with experimental toxicity values. Based on a 5-fold cross-validation, a median value of 0.410 for the Matthews correlation coefficient was calculated; TISBE was very effective, with a median value of sensitivity and specificity equal to 0.984 and 0.274, respectively. TISBE was applied on two external pools made of 1484 bioactive compounds and 85 pediatric drugs taken from ChEMBL (Chemical European Molecular Biology Laboratory) and TEDDY (Task-Force in Europe for Drug Development in the Young) repositories, respectively. Notably, TISBE gives users the option to clearly spot the molecular fragments responsible for the toxicity or the safety of a given chemical query and is available for free at https://prometheus.farmacia.uniba.it/tisbe.
Assuntos
Inteligência Artificial , Animais , Recém-Nascido , Criança , Humanos , Reprodutibilidade dos Testes , ConsensoAssuntos
Diabetes Mellitus Tipo 2 , Doença de Lafora , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose , SódioRESUMO
Spinal muscular atrophy (SMA) is a rare neuromuscular disease, with an estimated incidence of about 1 in 10,000 live births. To date, three orphan drugs have been approved for the treatment of SMA: nusinersen, onasemnogene abeparvovec, and risdiplam. The aim of this narrative review was to provide an overview of the pre- and post-marketing evidence on the pharmacological treatments approved for the treatment of SMA by identifying preclinical and clinical studies registered in clinicaltrials.gov and in the EU PAS register from their inception until the 4 January 2023. The preclinical evidence on the drugs approved for SMA allowed a significant acceleration in the experimental phase of these drugs. However, since these drugs had been authorized through accelerated programs, the conduction of post-marketing studies was requested as a condition of their marketing approval to better understand their risk-benefit profiles in real-world settings. As of the 4 January 2023, a total of 69 post-marketing studies concerning the three orphan drugs approved for SMA were identified in clinicaltrials.gov (N = 65; 94.2%) and in the EU PAS register (N = 4; 5.8%). Currently, ongoing studies are primarily aimed at providing evidence concerning the risk-benefit profile of the three drugs in specific populations that were not included in the pivotal trials and to investigate the long-term safety and clinical benefits of these drugs. Real-world data sources collecting information regarding the natural history of the disease and post-marketing surveillance of the available therapies are increasingly becoming essential for generating real-world evidence on this rare disease and its orphan drugs.
RESUMO
Brugada syndrome (BrS) is an inherited cardiac channelopathy first diagnosed in 1992 but still considered a challenging disease in terms of diagnosis, arrhythmia risk prediction, pathophysiology and management. Despite about 20% of individuals carrying pathogenic variants in the SCN5A gene, the identification of a polygenic origin for BrS and the potential role of common genetic variants provide the basis for applying polygenic risk scores for individual risk prediction. The pathophysiological mechanisms are still unclear, and the initial thinking of this syndrome as a primary electrical disease is evolving towards a partly structural disease. This review focuses on the main scientific advancements in the identification of biomarkers for diagnosis, risk stratification, pathophysiology and therapy of BrS. A comprehensive model that integrates clinical and genetic factors, comorbidities, age and gender, and perhaps environmental influences may provide the opportunity to enhance patients' quality of life and improve the therapeutic approach.
RESUMO
The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies.
Assuntos
Proteínas Quinases Ativadas por AMP , Distrofia Muscular de Duchenne , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Introduction: Sodium-glucose cotransporter type 2 inhibitors (SGLT2i), gliflozins, play an emerging role for the treatment of heart failure with reduced left ventricular ejection fraction (HFrEF). Nevertheless, the effects of SGLT2i on ventricular remodeling and function have not been completely understood yet. Explainable artificial intelligence represents an unprecedented explorative option to clinical research in this field. Based on echocardiographic evaluations, we identified some key clinical responses to gliflozins by employing a machine learning approach. Methods: Seventy-eight consecutive diabetic outpatients followed for HFrEF were enrolled in the study. Using a random forests classification, a single subject analysis was performed to define the profile of patients treated with gliflozins. An explainability analysis using Shapley values was used to outline clinical parameters that mostly improved after gliflozin therapy and machine learning runs highlighted specific variables predictive of gliflozin response. Results: The five-fold cross-validation analyses showed that gliflozins patients can be identified with a 0.70 ± 0.03% accuracy. The most relevant parameters distinguishing gliflozins patients were Right Ventricular S'-Velocity, Left Ventricular End Systolic Diameter and E/e' ratio. In addition, low Tricuspid Annular Plane Systolic Excursion values along with high Left Ventricular End Systolic Diameter and End Diastolic Volume values were associated to lower gliflozin efficacy in terms of anti-remodeling effects. Discussion: In conclusion, a machine learning analysis on a population of diabetic patients with HFrEF showed that SGLT2i treatment improved left ventricular remodeling, left ventricular diastolic and biventricular systolic function. This cardiovascular response may be predicted by routine echocardiographic parameters, with an explainable artificial intelligence approach, suggesting a lower efficacy in case of advanced stages of cardiac remodeling.
RESUMO
Introduction: Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Methods: Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). Results: In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-ß1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Discussion: Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.
Assuntos
Hormônio do Crescimento , Fator de Crescimento Insulin-Like I , Distrofia Muscular de Duchenne , Secretagogos , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Fibrose , Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/uso terapêutico , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Secretagogos/metabolismo , Camundongos Endogâmicos mdx , Animais , Camundongos , Masculino , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like I/uso terapêuticoRESUMO
Sarcalumenin (SAR) is a luminal Ca2+ buffer protein with high capacity but low affinity for calcium binding found predominantly in the longitudinal sarcoplasmic reticulum (SR) of fast- and slow-twitch skeletal muscles and the heart. Together with other luminal Ca2+ buffer proteins, SAR plays a critical role in modulation of Ca2+ uptake and Ca2+ release during excitation-contraction coupling in muscle fibers. SAR appears to be important in a wide range of other physiological functions, such as Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) stabilization, Store-Operated-Calcium-Entry (SOCE) mechanisms, muscle fatigue resistance and muscle development. The function and structural features of SAR are very similar to those of calsequestrin (CSQ), the most abundant and well-characterized Ca2+ buffer protein of junctional SR. Despite the structural and functional similarity, very few targeted studies are available in the literature. The present review provides an overview of the role of SAR in skeletal muscle physiology, as well as of its possible involvement and dysfunction in muscle wasting disorders, in order to summarize the current knowledge on SAR and drive attention to this important but still underinvestigated/neglected protein.
Assuntos
Cálcio , Retículo Sarcoplasmático , Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , HumanosRESUMO
Ultrasonography is a safe, non-invasive imaging technique used in several fields of medicine, offering the possibility to longitudinally monitor disease progression and treatment efficacy over time. This is particularly useful when a close follow-up is required, or in patients with pacemakers (not suitable for magnetic resonance imaging). By virtue of these advantages, ultrasonography is commonly used to detect multiple skeletal muscle structural and functional parameters in sports medicine, as well as in neuromuscular disorders, e.g., myotonic dystrophy and Duchenne muscular dystrophy (DMD). The recent development of high-resolution ultrasound devices allowed the use of this technique in preclinical settings, particularly for echocardiographic assessments that make use of specific guidelines, currently lacking for skeletal muscle measurements. In this review, we describe the state of the art for ultrasound skeletal muscle applications in preclinical studies conducted in small rodents, aiming to provide the scientific community with necessary information to support an independent validation of these procedures for the achievement of standard protocols and reference values useful in translational research on neuromuscular disorders.
Assuntos
Distrofia Muscular de Duchenne , Doenças Neuromusculares , Humanos , Músculo Esquelético , Ultrassonografia , EcocardiografiaRESUMO
Cardiorenal syndrome encompasses a spectrum of disorders involving heart and kidney dysfunction, and sharing common risk factors, such as hypertension and diabetes. Clinical studies have shown that patients with and without diabetes may benefit from using sodium-glucose cotransporter 2 inhibitors to reduce the risk of heart failure and ameliorate renal endpoints. Because the underlying mechanisms remain elusive, we investigated the effects of dapagliflozin on the progression of renal damage, using a model of non-diabetic cardiorenal disease. Dahl salt-sensitive rats were fed a high-salt diet for five weeks and then randomized to dapagliflozin or vehicle for the following six weeks. After treatment with dapagliflozin, renal function resulted ameliorated as shown by decrease of albuminuria and urine albumin-to-creatinine ratio. Functional benefit was accompanied by a decreased accumulation of extracellular matrix and a reduced number of sclerotic glomeruli. Dapagliflozin significantly reduced expression of inflammatory and endothelial activation markers such as NF-κB and e-selectin. Upregulation of pro-oxidant-releasing NADPH oxidases 2 and 4 as well as downregulation of antioxidant enzymes were also counteracted by drug treatment. Our findings also evidenced the modulation of both classic and non-classic renin-angiotensin-aldosterone system (RAAS), and effects of dapagliflozin on gene expression of ion channels/transporters involved in renal homeostasis. Thus, in a non-diabetic model of cardiorenal syndrome, dapagliflozin provides renal protection by modulating inflammatory response, endothelial activation, fibrosis, oxidative stress, local RAAS and ion channels.
Assuntos
Síndrome Cardiorrenal , Diabetes Mellitus , Animais , Ratos , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Síndrome Cardiorrenal/tratamento farmacológico , Síndrome Cardiorrenal/metabolismo , Diabetes Mellitus/tratamento farmacológico , Rim/metabolismo , Ratos Endogâmicos DahlRESUMO
The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure-activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity.
Assuntos
Mexiletina , Miotonia , Canal de Sódio Disparado por Voltagem NAV1.4 , Bloqueadores do Canal de Sódio Disparado por Voltagem , Humanos , Mexiletina/farmacologia , Mexiletina/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Miotonia/tratamento farmacológico , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Síndrome , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
In age-related sarcopenia, the gradual loss of skeletal muscle mass, function and strength is underpinned by an imbalanced rate of protein synthesis/breakdown. Hence, an adequate protein intake is considered a valuable strategy to mitigate sarcopenia. Here, we investigated the effects of a 12-week oral supplementation with branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) with recognized anabolic properties, in 17-month-old (AGED) C57BL/6J male mice. BCAAs (2:1:1) were formulated in drinking water, alone or plus two L-Alanine equivalents (2ALA) or dipeptide L-Alanyl-L-Alanine (Di-ALA) to boost BCAAs bioavailability. Outcomes were evaluated on in/ex vivo readouts vs. 6-month-old (ADULT) mice. In vivo hind limb plantar flexor torque was improved in AGED mice treated with BCAAs + Di-ALA or 2ALA (recovery score, R.S., towards ADULT: ≥20%), and all mixtures significantly increased hind limb volume. Ex vivo, myofiber cross-sectional areas were higher in gastrocnemius (GC) and soleus (SOL) muscles from treated mice (R.S. ≥ 69%). Contractile indices of isolated muscles were improved by the mixtures, especially in SOL muscle (R.S. ≥ 20%). The latter displayed higher mTOR protein levels in mice supplemented with 2ALA/Di-ALA-enriched mixtures (R.S. ≥ 65%). Overall, these findings support the usefulness of BCAAs-based supplements in sarcopenia, particularly as innovative formulations potentiating BCAAs bioavailability and effects.
Assuntos
Aminoácidos de Cadeia Ramificada , Sarcopenia , Masculino , Camundongos , Animais , Aminoácidos de Cadeia Ramificada/metabolismo , Sarcopenia/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Suplementos NutricionaisRESUMO
The C57BL/10ScSn-Dmdmdx/J (mdx) mouse model has been used by researchers for decades as a model to study pathology of and develop therapies for Duchenne muscular dystrophy. However, the model is relatively mildly affected compared to the human situation. Recently, the D2.B10-Dmdmdx/J (D2.mdx) mouse model was suggested as a more severely affected and therefore better alternative. While the pathology of this model is indeed more pronounced early in life, it is not progressive, and increasing evidence suggest that it actually partially resolves with age. As such, caution is needed when using this model. However, as preclinical experts of the TREAT-NMD advisory committee for therapeutics (TACT), we frequently encounter study designs that underestimate this caveat. We here provide context for how to best use the two models for preclinical studies at the current stage of knowledge.
Assuntos
Distrofia Muscular de Duchenne , Animais , Camundongos , Humanos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/patologia , Camundongos Endogâmicos C57BL , Comitês Consultivos , Modelos Animais de DoençasRESUMO
Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype-phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.
Assuntos
Epilepsia , Canal de Potássio Kv1.1 , Acetazolamida , Ataxia/tratamento farmacológico , Ataxia/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Células HEK293 , Humanos , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.1/genética , Mutação , PotássioRESUMO
ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of ß-dystroglycan (ß-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle ß-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-ß-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of ß-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-ß-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-ß-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing ß-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing ß-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.