Publications by authors named "Antonella Liantonio"

57 Publications

Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Front Cell Dev Biol 2021 26;9:635063. Epub 2021 Feb 26.

Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca signaling. So far there is no cure for TAM and the mechanisms through which or gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca influx through SOCE. To explore how Ca homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca handling (. Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early () and late () differentiation markers together with mitochondrial markers (. We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.
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http://dx.doi.org/10.3389/fcell.2021.635063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952532PMC
February 2021

Corrigendum to "Ion Channels Involvement in Neurodevelopmental Disorders" [Neuroscience 440C (2020) 337-359].

Neuroscience 2020 Sep 29;443:233. Epub 2020 Jul 29.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Italy. Electronic address:

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http://dx.doi.org/10.1016/j.neuroscience.2020.07.026DOI Listing
September 2020

Ion Channels Involvement in Neurodevelopmental Disorders.

Neuroscience 2020 08 28;440:337-359. Epub 2020 May 28.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Italy. Electronic address:

Inherited and sporadic mutations in genes encoding for brain ion channels, affecting membrane expression or biophysical properties, have been associated with neurodevelopmental disorders characterized by epilepsy, cognitive and behavioral deficits with significant phenotypic and genetic heterogeneity. Over the years, the screening of a growing number of patients and the functional characterization of newly identified mutations in ion channels genes allowed to recognize new phenotypes and to widen the clinical spectrum of known diseases. Furthermore, advancements in understanding disease pathogenesis at atomic level or using patient-derived iPSCs and animal models have been pivotal to orient therapeutic intervention and to put the basis for the development of novel pharmacological options for drug-resistant disorders. In this review we will discuss major improvements and critical issues concerning neurodevelopmental disorders caused by dysfunctions in brain sodium, potassium, calcium, chloride and ligand-gated ion channels.
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http://dx.doi.org/10.1016/j.neuroscience.2020.05.032DOI Listing
August 2020

Kv1.1 Channelopathies: Pathophysiological Mechanisms and Therapeutic Approaches.

Int J Mol Sci 2020 Apr 22;21(8). Epub 2020 Apr 22.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", 70125 Bari, Italy.

Kv1.1 belongs to the subfamily of voltage-gated potassium channels and acts as a critical regulator of neuronal excitability in the central and peripheral nervous systems. is the only gene that has been associated with episodic ataxia type 1 (EA1), an autosomal dominant disorder characterized by ataxia and myokymia and for which different and variable phenotypes have now been reported. The iterative characterization of channel defects at the molecular, network, and organismal levels contributed to elucidating the functional consequences of mutations and to demonstrate that ataxic attacks and neuromyotonia result from cerebellum and motor nerve alterations. Dysfunctions of the Kv1.1 channel have been also associated with epilepsy and knock-out mouse is considered a model of sudden unexpected death in epilepsy. The tissue-specific association of Kv1.1 with other Kv1 members, auxiliary and interacting subunits amplifies Kv1.1 physiological roles and expands the pathogenesis of Kv1.1-associated diseases. In line with the current knowledge, Kv1.1 has been proposed as a novel and promising target for the treatment of brain disorders characterized by hyperexcitability, in the attempt to overcome limited response and side effects of available therapies. This review recounts past and current studies clarifying the roles of Kv1.1 in and beyond the nervous system and its contribution to EA1 and seizure susceptibility as well as its wide pharmacological potential.
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http://dx.doi.org/10.3390/ijms21082935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215777PMC
April 2020

Functional Study of Novel Bartter's Syndrome Mutations in ClC-Kb and Rescue by the Accessory Subunit Barttin Toward Personalized Medicine.

Front Pharmacol 2020 17;11:327. Epub 2020 Mar 17.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

Type III and IV Bartter syndromes (BS) are rare kidney tubulopathies caused by loss-of-function mutations in the and genes coding respectively for the ClC-Kb chloride channels and accessory subunit barttin. ClC-K channels are expressed in the Henle's loop, distal convoluted tubule, and cortical collecting ducts of the kidney and contribute to chloride absorption and urine concentration. In our Italian cohort, we identified two new mutations in , G167V and G289R, in children affected by BS and previously reported genetic variants, A242E, a chimeric gene and the deletion of the whole . All the patients had hypokalemia and metabolic alkalosis, increased serum renin and aldosterone levels and were treated with a symptomatic therapy. In order to define the molecular mechanisms responsible for BS, we co-expressed ClC-Kb wild type and channels with point mutations with barttin in HEK 293 cells and characterized chloride currents through the patch-clamp technique. In addition, we attempted to revert the functional defect caused by BS mutations through barttin overexpression. G167V and A242E channels showed a drastic current reduction compared to wild type, likely suggesting compromised expression of mutant channels at the plasma membrane. Conversely, G289R channel was similar to wild type raising the doubt that an additional mutation in another gene or other mechanisms could account for the clinical phenotype. Interestingly, increasing ClC-K/barttin ratio augmented G167V and A242E mutants' chloride current amplitudes towards wild type levels. These results confirm a genotype-phenotype correlation in BS and represent a preliminary proof of concept that molecules functioning as molecular chaperones can restore channel function in expression-defective ClC-Kb mutants.
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http://dx.doi.org/10.3389/fphar.2020.00327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092721PMC
March 2020

Cisplatin-Induced Skeletal Muscle Dysfunction: Mechanisms and Counteracting Therapeutic Strategies.

Int J Mol Sci 2020 Feb 13;21(4). Epub 2020 Feb 13.

Department of Pharmacy-Drug Sciences, University of Bari, 70125 Bari, Italy.

Among the severe side effects induced by cisplatin chemotherapy, muscle wasting is the most relevant one. This effect is a major cause for a clinical decline of cancer patients, since it is a negative predictor of treatment outcome and associated to increased mortality. However, despite its toxicity even at low doses, cisplatin remains the first-line therapy for several types of solid tumors. Thus, effective pharmacological treatments counteracting or minimizing cisplatin-induced muscle wasting are urgently needed. The dissection of the molecular pathways responsible for cisplatin-induced muscle dysfunction gives the possibility to identify novel promising therapeutic targets. In this context, the use of animal model of cisplatin-induced cachexia is very useful. Here, we report an update of the most relevant researches on the mechanisms underlying cisplatin-induced muscle wasting and on the most promising potential therapeutic options to preserve muscle mass and function.
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http://dx.doi.org/10.3390/ijms21041242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072891PMC
February 2020

Elucidating the Contribution of Skeletal Muscle Ion Channels to Amyotrophic Lateral Sclerosis in search of new therapeutic options.

Sci Rep 2019 02 28;9(1):3185. Epub 2019 Feb 28.

Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, 70125, Bari, Italy.

The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1 mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.
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http://dx.doi.org/10.1038/s41598-019-39676-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395744PMC
February 2019

Ion Channels in Drug Discovery and Safety Pharmacology.

Methods Mol Biol 2018 ;1800:313-326

Department of Pharmacy - Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

Ion channels are membrane proteins involved in almost all physiological processes, including neurotransmission, muscle contraction, pace-making activity, secretion, electrolyte and water balance, immune response, and cell proliferation. Due to their broad distribution in human body and physiological roles, ion channels are attractive targets for drug discovery and safety pharmacology. Over the years ion channels have been associated to many genetic diseases ("channelopathies"). For most of these diseases the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a number of patients. The search for the development of new and more specific therapeutic approaches is therefore strongly pursued. At the same time acquired channelopathies or dangerous side effects (such as proarrhythmic risk) can develop as a consequence of drugs unexpectedly targeting ion channels. Several noncardiovascular drugs are known to block cardiac ion channels, leading to potentially fatal delayed ventricular repolarization. Thus, the search of reliable preclinical cardiac safety testing in early stage of drug discovery is mandatory. To fulfill these needs, both ion channels drug discovery and toxicology strategies are evolving toward comprehensive research approaches integrating ad hoc designed in silico predictions and experimental studies for a more reliable and quick translation of results to the clinic side.Here we discuss two examples of how the combination of in silico methods and patch clamp experiments can help addressing drug discovery and safety issues regarding ion channels.
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http://dx.doi.org/10.1007/978-1-4939-7899-1_15DOI Listing
February 2019

[Bartter syndrome, severe rare orphan kidney disease: a step towards therapy through pharmacogenetic and epidemiological studies].

G Ital Nefrol 2018 May;35(3)

Dipartimento di Farmacia - Scienze del Farmaco, Università di Bari, Italia.

Bartter syndromes (BS) types 1-5 are rare salt-losing tubulopathies presenting with overlapping clinical phenotypes including marked salt wasting and hypokalemia leading to polyuria, polydipsia, volume contraction, muscle weakness and growth retardation. These diseases are due to an impairment of sodium, potassium, chloride reabsorption caused by mutations in genes encoding for ion channel or transporters expressed in specific nephron tubule segments. Particularly, BS type 3 is a clinically heterogeneous form caused by mutations in CLCNKB gene which encodes the ClC-Kb chloride channel involved in NaCl reabsorption in the renal tubule. Specific therapy for BS is lacking and the only pharmacotherapy up today available is purely symptomatic and characterized by limiting side effects. The improvement of our understanding of the phenotype/genotype correlation and of the precise pathogenic mechanisms associated with BS type 3 as well as the pharmacological characterization of ClC-K chloride channels are fundamental to design therapies tailored upon patients' mutation. This mini review focused on recent studies representing relevant forward steps in the field as well as noteworthy examples of how basic and clinical research can cooperate to gain insight into the pathophysiology of this renal channelopathy, paving the way for a personalized therapy.
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May 2018

Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

FASEB J 2018 02 3;32(2):1025-1043. Epub 2018 Jan 3.

Pharmacology Unit, Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

Muscle fibers lacking dystrophin undergo a long-term alteration of Ca homeostasis, partially caused by a leaky Ca release ryanodine (RyR) channel. S48168/ARM210, an RyR calcium release channel stabilizer (a Rycal compound), is expected to enhance the rebinding of calstabin to the RyR channel complex and possibly alleviate the pathologic Ca leakage in dystrophin-deficient skeletal and cardiac muscle. This study systematically investigated the effect of S48168/ARM210 on the phenotype of mdx mice by means of a first proof-of-concept, short (4 wk), phase 1 treatment, followed by a 12-wk treatment (phase 2) performed in parallel by 2 independent laboratories. The mdx mice were treated with S48168/ARM210 at two different concentrations (50 or 10 mg/kg/d) in their drinking water for 4 and 12 wk, respectively. The mice were subjected to treadmill sessions twice per week (12 m/min for 30 min) to unmask the mild disease. This testing was followed by in vivo forelimb and hindlimb grip strength and fatigability measurement, ex vivo extensor digitorum longus (EDL) and diaphragm (DIA) force contraction measurement and histologic and biochemical analysis. The treatments resulted in functional (grip strength, ex vivo force production in DIA and EDL muscles) as well as histologic improvement after 4 and 12 wk, with no adverse effects. Furthermore, levels of cellular biomarkers of calcium homeostasis increased. Therefore, these data suggest that S48168/ARM210 may be a safe therapeutic option, at the dose levels tested, for the treatment of Duchenne muscular dystrophy (DMD).-Capogrosso, R. F., Mantuano, P., Uaesoontrachoon, K., Cozzoli, A., Giustino, A., Dow, T., Srinivassane, S., Filipovic, M., Bell, C., Vandermeulen, J., Massari, A. M., De Bellis, M., Conte, E., Pierno, S., Camerino, G. M., Liantonio, A., Nagaraju, K., De Luca, A. Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.
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http://dx.doi.org/10.1096/fj.201700182RRRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888399PMC
February 2018

Growth hormone secretagogues hexarelin and JMV2894 protect skeletal muscle from mitochondrial damages in a rat model of cisplatin-induced cachexia.

Sci Rep 2017 10 12;7(1):13017. Epub 2017 Oct 12.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "A. Moro", Bari, Italy.

Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia.
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http://dx.doi.org/10.1038/s41598-017-13504-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638899PMC
October 2017

Paving the way for Bartter syndrome type 3 drug discovery: a hope from basic research.

J Physiol 2017 08 7;595(16):5403-5404. Epub 2017 Jul 7.

Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

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http://dx.doi.org/10.1113/JP274645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556148PMC
August 2017

Pharmacovigilance database search discloses ClC-K channels as a novel target of the AT receptor blockers valsartan and olmesartan.

Br J Pharmacol 2017 07 26;174(13):1972-1983. Epub 2017 Apr 26.

Department of Pharmacy - Drug Sciences, University of Bari 'Aldo Moro', Bari, Italy.

Background And Purpose: Human ClC-K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCNKB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCNKB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC-K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database.

Experimental Approach: We searched for drugs having a Bartter-like syndrome as a reported side effect, with the assumption that BS could be causatively related to the block of ClC-K channels. The ability of the selected BS-causing drugs to bind and block ClC-K channels was then validated through an integrated experimental and computational approach based on patch clamp electrophysiology in HEK293 cells and molecular docking simulations.

Key Results: Valsartan and olmesartan were able to block ClC-Ka channels and the molecular requirements for effective inhibition of these channels have been identified.

Conclusion And Implications: These results suggest additional mechanisms of action for these sartans further to their primary AT receptor antagonism and propose these compounds as leads for designing new potent ClC-K ligands.
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http://dx.doi.org/10.1111/bph.13794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466521PMC
July 2017

Growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin-induced cachexia.

J Cachexia Sarcopenia Muscle 2017 Jun 10;8(3):386-404. Epub 2017 Mar 10.

Department of Pharmacy - Drug Sciences, University of Bari, Via Orabona 4, 70125, Bari, Italy.

Background: Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose-limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium-dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS-R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood.

Methods: By a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast-twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin-induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention.

Results: Cisplatin-treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up-regulation of atrogin1/Murf-1 genes and a down-regulation of Pgc1-a gene, all indexes of muscle atrophy, and by a two-fold increase in resting intracellular calcium, [Ca ] , compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store-operated calcium entry were ~50% significantly reduced in cisplatin-treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in vivo (forelimb force and muscle volume) outcomes in cachectic animals. Administration of hexarelin or JMV2894 markedly reduced the cisplatin-induced alteration of calcium homeostasis by both common as well as drug-specific mechanisms of action. This effect correlated with muscle function preservation as well as amelioration of various atrophic indexes, thus supporting the functional impact of GHS activity on calcium homeostasis.

Conclusions: Our findings provide a direct evidence that a dysregulation of calcium homeostasis plays a key role in cisplatin-induced model of cachexia gaining insight into the etiopathogenesis of this form of muscle wasting. Furthermore, our demonstration that GHS administration efficaciously prevents cisplatin-induced calcium homeostasis alteration contributes to elucidate the mechanism of action through which GHS could potentially ameliorate chemotherapy-associated cachexia.
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http://dx.doi.org/10.1002/jcsm.12185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703021PMC
June 2017

JMV2894, a novel growth hormone secretagogue, accelerates body mass recovery in an experimental model of cachexia.

Endocrine 2017 Oct 28;58(1):106-114. Epub 2016 Nov 28.

Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy.

Oncologic patients subjected to chemotherapy frequently present aphagia, malnutrition, and cachexia. The purpose of this study was to investigate whether selected growth hormone secretagogues including hexarelin, JMV2894 and JMV2951 could antagonize body weight loss and wasting induced by cisplatin administration in rats. The three growth hormone secretagogues behaved as full agonists of the growth hormone secretagogues receptor both in terms of ability to stimulate calcium mobilization in Chinese hamster ovary cells and stimulation of growth hormone release in neonatal rats. Adult rats were (i) treated with vehicle throughout (controls), or (ii) treated with cisplatin (days 1-3) and a growth hormone secretagogues or vehicle, (days 1-12). Body weight and food consumption were measured daily. Although all growth hormone secretagogues caused initial transient acute increases in food intake, the total amount of food eaten by controls and growth hormone secretagogues treated groups over the 12 experimental days was not significantly different. All groups pre-treated with cisplatin lost up to 5-10 % body weight in the first 4 days; they subsequently gained weight at a rate comparable with controls. Interestingly, rats which received JMV2894 demonstrated a faster gain in body weight than any other growth hormone secretagogues treated group and at the end of the protocol reached a weight similar to that of controls. JMV2894 did not stimulate perirenal and epididymal fat accumulation but reduced MuRF mRNA levels in skeletal muscles. In conclusion, our findings demonstrate that JMV2894 antagonizes cisplatin induced weight loss in rats and may prove useful in antagonizing cachexia associated with cancer and chemotherapy in humans.
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http://dx.doi.org/10.1007/s12020-016-1184-2DOI Listing
October 2017

Statin-induced myotoxicity is exacerbated by aging: A biophysical and molecular biology study in rats treated with atorvastatin.

Toxicol Appl Pharmacol 2016 09 1;306:36-46. Epub 2016 Jul 1.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, Bari, Italy. Electronic address:

Statin-induced skeletal muscle damage in rats is associated to the reduction of the resting sarcolemmal chloride conductance (gCl) and ClC-1 chloride channel expression. These drugs also affect the ClC-1 regulation by increasing protein kinase C (PKC) activity, which phosphorylate and close the channel. Also the intracellular resting calcium (restCa) level is increased. Similar alterations are observed in skeletal muscles of aged rats, suggesting a higher risk of statin myotoxicity. To verify this hypothesis, we performed a 4-5-weeks atorvastatin treatment of 24-months-old rats to evaluate the ClC-1 channel function by the two-intracellular microelectrodes technique as well as transcript and protein expression of different genes sensitive to statins by quantitative real-time-PCR and western blot analysis. The restCa was measured using FURA-2 imaging, and histological analysis of muscle sections was performed. The results show a marked reduction of resting gCl, in agreement with the reduced ClC-1 mRNA and protein expression in atorvastatin-treated aged rats, with respect to treated adult animals. The observed changes in myocyte-enhancer factor-2 (MEF2) expression may be involved in ClC-1 expression changes. The activity of PKC was also increased and further modulate the gCl in treated aged rats. In parallel, a marked reduction of the expression of glycolytic and mitochondrial enzymes demonstrates an impairment of muscle metabolism. No worsening of restCa or histological features was found in statin-treated aged animals. These findings suggest that a strong reduction of gCl and alteration of muscle metabolism coupled to muscle atrophy may contribute to the increased risk of statin-induced myopathy in the elderly.
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http://dx.doi.org/10.1016/j.taap.2016.06.032DOI Listing
September 2016

ATP Sensitive Potassium Channels in the Skeletal Muscle Function: Involvement of the KCNJ11(Kir6.2) Gene in the Determination of Mechanical Warner Bratzer Shear Force.

Front Physiol 2016 10;7:167. Epub 2016 May 10.

Department of Veterinary Medicine, University of Bari Aldo Moro Bari, Italy.

The ATP-sensitive K(+)-channels (KATP) are distributed in the tissues coupling metabolism with K(+) ions efflux. KATP subunits are encoded by KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), and ABCC9 (SUR2) genes, alternative RNA splicing give rise to SUR variants that confer distinct physiological properties on the channel. An high expression/activity of the sarco-KATP channel is observed in various rat fast-twitch muscles, characterized by elevated muscle strength, while a low expression/activity is observed in the slow-twitch muscles characterized by reduced strength and frailty. Down-regulation of the KATP subunits of fast-twitch fibers is found in conditions characterized by weakness and frailty. KCNJ11 gene knockout mice have reduced glycogen, lean phenotype, lower body fat, and weakness. KATP channel is also a sensor of muscle atrophy. The KCNJ11 gene is located on BTA15, close to a QTL for meat tenderness, it has also a role in glycogen storage, a key mechanism of the postmortem transformation of muscle into meat. The role of KCNJ11 gene in muscle function may underlie an effect of KCNJ11 genotypes on meat tenderness, as recently reported. The fiber phenotype and genotype are important in livestock production science. Quantitative traits including meat production and quality are influenced both by environment and genes. Molecular markers can play an important role in the genetic improvement of animals through breeding strategies. Many factors influence the muscle Warner-Bratzler shear force including breed, age, feeding, the biochemical, and functional parameters. The role of KCNJ11gene and related genes on muscle tenderness will be discussed in the present review.
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http://dx.doi.org/10.3389/fphys.2016.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862255PMC
May 2016

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery.

Front Pharmacol 2016 10;7:121. Epub 2016 May 10.

Department of Pharmacy - Drug Sciences, University of Bari "Aldo Moro" Bari, Italy.

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.
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http://dx.doi.org/10.3389/fphar.2016.00121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861771PMC
May 2016

Kidney CLC-K chloride channels inhibitors: structure-based studies and efficacy in hypertension and associated CLC-K polymorphisms.

J Hypertens 2016 May;34(5):981-92

aDipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari, Bari bIstituto di Biofisica, CNR, Genova, Italy *Antonella Liantonio and Paola Imbrici contributed equally to the writing of this article.

Objective: Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential. Here, we assessed the feasibility of using CLC-K blockers in hypertension and aimed at enhancing drug inhibitory affinity.

Methods And Results: We demonstrated that acute in-vivo administration of MT-189 to spontaneously hypertensive rats (SHR) caused a reduction of blood pressure and defined the CLC-K/barttin gene expression pattern in kidney of SHR in comparison with normotensive Wistar-Kyoto rats. Based on MT-189, we designed and tested a new series of benzofuran derivatives on CLC-K chloride channels heterologously expressed in HEK293 cells. These studies enabled us to elucidate the causative molecular relationship for obtaining the most potent and selective inhibitor (SRA-36) described so far, with an IC50 of 6.6 ± 1 μmol/l. The biophysical and pharmacological characterization of A447T CLC-Ka and Y315F CLC-Ka, both polymorphisms associated with hypertension, showed that SRA-36 is an efficacious inhibitor of the chloride currents sustained by these polymorphisms. Molecular docking studies allowed hypothesizing an inhibition mechanism for the considered ligands, laying the foundations for the rational design of new and more effective CLC-K inhibitors.

Conclusion: The SRA-36 molecule represents a new potential therapeutic option for hypertension.
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http://dx.doi.org/10.1097/HJH.0000000000000876DOI Listing
May 2016

Effects of Nandrolone in the Counteraction of Skeletal Muscle Atrophy in a Mouse Model of Muscle Disuse: Molecular Biology and Functional Evaluation.

PLoS One 2015 11;10(6):e0129686. Epub 2015 Jun 11.

Section of Pharmacology, Dept. of Pharmacy & Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129686PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466268PMC
May 2016

On the metabolically active form of metaglidasen: improved synthesis and investigation of its peculiar activity on peroxisome proliferator-activated receptors and skeletal muscles.

ChemMedChem 2015 Mar 29;10(3):555-65. Epub 2015 Jan 29.

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari "Aldo Moro", 70126 Bari (Italy).

Metaglidasen is a fibrate-like drug reported as a selective modulator of peroxisome proliferator-activated receptor γ (PPARγ), able to lower plasma glucose levels in the absence of the side effects typically observed with thiazolidinedione antidiabetic agents in current use. Herein we report an improved synthesis of metaglidasen's metabolically active form halofenic acid (R)-2 and that of its enantiomer (S)-2. The activity of the two stereoisomers was carefully examined on PPARα and PPARγ subtypes. As expected, both showed partial agonist activity toward PPARγ; the investigation of PPARα activity, however, led to unexpected results. In particular, (S)-2 was found to act as a partial agonist, whereas (R)-2 behaved as an antagonist. X-ray crystallographic studies with PPARγ were carried out to gain more insight on the molecular-level interactions and to propose a binding mode. Given the adverse effects provoked by fibrate drugs on skeletal muscle function, we also investigated the capacity of (R)-2 and (S)-2 to block conductance of the skeletal muscle membrane chloride channel. The results showed a more beneficial profile for (R)-2, the activity of which on skeletal muscle function, however, should not be overlooked in the ongoing clinical trials studying its long-term effects.
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http://dx.doi.org/10.1002/cmdc.201402462DOI Listing
March 2015

Calcium homeostasis is altered in skeletal muscle of spontaneously hypertensive rats: cytofluorimetric and gene expression analysis.

Am J Pathol 2014 Oct 30;184(10):2803-15. Epub 2014 Jul 30.

Section of Pharmacology, Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function.
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http://dx.doi.org/10.1016/j.ajpath.2014.06.020DOI Listing
October 2014

Angiotensin II modulates mouse skeletal muscle resting conductance to chloride and potassium ions and calcium homeostasis via the AT1 receptor and NADPH oxidase.

Am J Physiol Cell Physiol 2014 Oct 30;307(7):C634-47. Epub 2014 Jul 30.

Unit of Pharmacology, Department of Pharmacy and Drug Sciences, University of Bari "A. Moro," Bari, Italy; and

Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50 = 0.06 μM) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2 antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine and the NADPH-oxidase (NOX) inhibitor apocynin also antagonized ANG II effects on resting ionic conductances; the ANG II-dependent gK increase was blocked by iberiotoxin, an inhibitor of calcium-activated potassium channels. ANG II also lowered the threshold for myofiber and muscle contraction. Both ANG II and the AT1 agonist L162,313 increased the intracellular calcium transients, measured by fura-2, with a two-step pattern. These latter effects were not observed in the presence of losartan and of the phospholipase C inhibitor U73122 and the in absence of extracellular calcium, disclosing a Gq-mediated calcium entry mechanism. The data show for the first time that the AT1-mediated ANG II pathway, also involving NOX and ROS, directly modulates ion channels and calcium homeostasis in adult myofibers.
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http://dx.doi.org/10.1152/ajpcell.00372.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187056PMC
October 2014

I-J loop involvement in the pharmacological profile of CLC-K channels expressed in Xenopus oocytes.

Biochim Biophys Acta 2014 Nov 26;1838(11):2745-56. Epub 2014 Jul 26.

Istituto di Biofisica, CNR, Via De Marini 6, 16149 Genoa, Italy. Electronic address:

CLC-K chloride channels and their subunit, barttin, are crucial for renal NaCl reabsorption and for inner ear endolymph production. Mutations in CLC-Kb and barttin cause Bartter syndrome. Here, we identified two adjacent residues, F256 and N257, that when mutated hugely alter in Xenopus oocytes CLC-Ka's biphasic response to niflumic acid, a drug belonging to the fenamate class, with F256A being potentiated 37-fold and N257A being potently blocked with a KD~1μM. These residues are localized in the same extracellular I-J loop which harbors a regulatory Ca(2+) binding site. This loop thus can represent an ideal and CLC-K specific target for extracellular ligands able to modulate channel activity. Furthermore, we demonstrated the involvement of the barttin subunit in the NFA potentiation. Indeed the F256A mutation confers onto CLC-K1 a transient potentiation induced by NFA which is found only when CLC-K1/F256A is co-expressed with barttin. Thus, in addition to the role of barttin in targeting and gating, the subunit participates in the pharmacological modulation of CLC-K channels and thus represents a further target for potential drugs.
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http://dx.doi.org/10.1016/j.bbamem.2014.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331650PMC
November 2014

Targeting kidney CLC-K channels: pharmacological profile in a human cell line versus Xenopus oocytes.

Biochim Biophys Acta 2014 Oct 24;1838(10):2484-91. Epub 2014 May 24.

Dipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy. Electronic address:

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca(2+) is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks.
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http://dx.doi.org/10.1016/j.bbamem.2014.05.017DOI Listing
October 2014

Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

Pflugers Arch 2014 Dec 20;466(12):2215-28. Epub 2014 Mar 20.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, 70125, Bari, Italy.

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.
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http://dx.doi.org/10.1007/s00424-014-1495-1DOI Listing
December 2014

Effects of pleiotrophin overexpression on mouse skeletal muscles in normal loading and in actual and simulated microgravity.

PLoS One 2013 28;8(8):e72028. Epub 2013 Aug 28.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, Bari, Italy.

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756024PMC
May 2014

Growth hormone secretagogues exert differential effects on skeletal muscle calcium homeostasis in male rats depending on the peptidyl/nonpeptidyl structure.

Endocrinology 2013 Oct 8;154(10):3764-75. Epub 2013 Jul 8.

Section of Pharmacology, Department of Pharmacy-Drug Sciences, University of Bari, Via Orabona, 4, Campus, I-70125 Bari, Italy.

The orexigenic and anabolic effects induced by ghrelin and the synthetic GH secretagogues (GHSs) are thought to positively contribute to therapeutic approaches and the adjunct treatment of a number of diseases associated with muscle wasting such as cachexia and sarcopenia. However, many questions about the potential utility and safety of GHSs in both therapy and skeletal muscle function remain unanswered. By using fura-2 cytofluorimetric technique, we determined the acute effects of ghrelin, as well as of peptidyl and nonpeptidyl synthetic GHSs on calcium homeostasis, a critical biomarker of muscle function, in isolated tendon-to-tendon male rat skeletal muscle fibers. The synthetic nonpeptidyl GHSs, but not peptidyl ghrelin and hexarelin, were able to significantly increase resting cytosolic calcium [Ca²⁺]i. The nonpeptidyl GHS-induced [Ca²⁺]i increase was independent of GHS-receptor 1a but was antagonized by both thapsigargin/caffeine and cyclosporine A, indicating the involvement of the sarcoplasmic reticulum and mitochondria. Evaluation of the effects of a pseudopeptidyl GHS and a nonpeptidyl antagonist of the GHS-receptor 1a together with a drug-modeling study suggest the conclusion that the lipophilic nonpeptidyl structure of the tested compounds is the key chemical feature crucial for the GHS-induced calcium alterations in the skeletal muscle. Thus, synthetic GHSs can have different effects on skeletal muscle fibers depending on their molecular structures. The calcium homeostasis dysregulation specifically induced by the nonpeptidyl GHSs used in this study could potentially counteract the beneficial effects associated with these drugs in the treatment of muscle wasting of cachexia- or other age-related disorders.
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http://dx.doi.org/10.1210/en.2013-1334DOI Listing
October 2013

Paracrine effects of IGF-1 overexpression on the functional decline due to skeletal muscle disuse: molecular and functional evaluation in hindlimb unloaded MLC/mIgf-1 transgenic mice.

PLoS One 2014 3;8(6):e65167. Epub 2013 Jun 3.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari Aldo Moro, Bari, Italy.

Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065167PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670938PMC
January 2015

An olive oil-derived antioxidant mixture ameliorates the age-related decline of skeletal muscle function.

Age (Dordr) 2014 Feb 30;36(1):73-88. Epub 2013 May 30.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari "Aldo Moro", Via Orabona 4-campus, 70125, Bari, Italy,

Age-related skeletal muscle decline is characterized by the modification of sarcolemma ion channels important to sustain fiber excitability and to prevent metabolic dysfunction. Also, calcium homeostasis and contractile function are impaired. In the aim to understand whether these modifications are related to oxidative damage and can be reverted by antioxidant treatment, we examined the effects of in vivo treatment with an waste water polyphenolic mixture (LACHI MIX HT) supplied by LACHIFARMA S.r.l. Italy containing hydroxytirosol (HT), gallic acid, and homovanillic acid on the skeletal muscles of 27-month-old rats. After 6-week treatment, we found an improvement of chloride ClC-1 channel conductance, pivotal for membrane electrical stability, and of ATP-dependent potassium channel activity, important in coupling excitability with fiber metabolism. Both of them were analyzed using electrophysiological techniques. The treatment also restored the resting cytosolic calcium concentration, the sarcoplasmic reticulum calcium release, and the mechanical threshold for contraction, an index of excitation-contraction coupling mechanism. Muscle weight and blood creatine kinase levels were preserved in LACHI MIX HT-treated aged rats. The antioxidant activity of LACHI MIX HT was confirmed by the reduction of malondialdehyde levels in the brain of the LACHI MIX HT-treated aged rats. In comparison, the administration of purified HT was less effective on all the parameters studied. Although muscle function was not completely recovered, the present study provides evidence of the beneficial effects of LACHI MIX HT, a natural compound, to ameliorate skeletal muscle functional decline due to aging-associated oxidative stress.
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http://dx.doi.org/10.1007/s11357-013-9544-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889891PMC
February 2014