Publications by authors named "Jean-François Rolland"

25 Publications

  • Page 1 of 1

Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy.

PLoS One 2021 12;16(1):e0245397. Epub 2021 Jan 12.

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

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients' condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245397PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802948PMC
January 2021

Parallel All-Optical Assay to Study Use-Dependent Functioning of Voltage-Gated Ion Channels in a Miniaturized Format.

SLAS Discov 2021 Mar 17;26(3):460-469. Epub 2020 Dec 17.

AXXAM S.p.A, Bresso (Milan), Italy.

Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.
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http://dx.doi.org/10.1177/2472555220976083DOI Listing
March 2021

Optogenetics and Optical Tools in Automated Patch Clamping.

Methods Mol Biol 2021 ;2188:311-330

Sophion Bioscience A/S, Ballerup, Denmark.

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single cell transduction, and subcellular pathways.Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light activated ion channels and photoactivated ligands.
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http://dx.doi.org/10.1007/978-1-0716-0818-0_16DOI Listing
March 2021

Editorial: From Peptide and Protein Toxins to Ion Channel Structure/Function and Drug Design.

Front Pharmacol 2020 25;11:548366. Epub 2020 Sep 25.

Department of Molecular Neuroimmune Signaling, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

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http://dx.doi.org/10.3389/fphar.2020.548366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546396PMC
September 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

A long-term treatment with taurine prevents cardiac dysfunction in mdx mice.

Transl Res 2019 02 28;204:82-99. Epub 2018 Sep 28.

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

Taurine is an amino acid abundantly present in heart and skeletal muscle. Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to skeletal muscle wasting and heart failure. An altered taurine metabolism has been described in dystrophic animals and short-term taurine administration exerts promising amelioration of early muscular alterations in the mdx mouse model of DMD. To reinforce the therapeutic and nutraceutical taurine potential in DMD, we evaluated the effects of a long-term treatment on cardiac and skeletal muscle function of mdx mice in a later disease stage. Taurine was administered in drinking water (1 g/kg/day) to wt and mdx mice for 6 months, starting at 6 months of age. Ultrasonography evaluation of heart and hind limb was performed, in parallel with in vivo and ex vivo functional tests and biochemical, histological and gene expression analyses. 12-month-old mdx mice showed a significant worsening of left ventricular function parameters (shortening fraction, ejection fraction, stroke volume), which were significantly counteracted by the taurine treatment. In parallel, histologic signs of damage were reduced by taurine along with the expression of proinflammatory myocardial IL-6. Interestingly, no effects were observed on hind limb volume and percentage of vascularization or on in vivo and ex vivo muscle functional parameters, suggesting a tissue-specific action of taurine in relation to the disease phase. A trend toward increase in taurine was found in heart and quadriceps from treated animals, paralleled by a slight decrease in mdx mice plasma. Our study provides evidences that taurine can prevent late heart dysfunction in mdx mice, further corroborating the interest on this amino acid toward clinical trials.
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http://dx.doi.org/10.1016/j.trsl.2018.09.004DOI Listing
February 2019

Data on protein abundance alteration induced by chronic exercise in mdx mice model of Duchenne muscular dystrophy and potential modulation by apocynin and taurine.

Data Brief 2018 Jun 19;18:555-575. Epub 2018 Mar 19.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

Here we present original data related to the research paper entitled "Proteome analysis in dystrophic mdx mouse muscle reveals a drastic alteration of Key Metabolic and Contractile Proteins after chronic exercise and the potential modulation by anti-oxidant compounds" (Gamberi et al., 2018) [1]. The dystrophin-deficient mdx mouse is the most common animal model for Duchenne muscular dystrophy. The mdx mice phenotype of the disorder is milder than in human sufferers and it can be worsened by chronic treadmill exercise. Apocynin and taurine are two antioxidant compounds proved to be beneficial on some pathology related parameters (Schröder and Schoser, 2009) [2]. This article reports the detailed proteomic data on protein abundance alterations, in tibialis anterior muscle of mdx mice, induced by chronic exercise protocol. A selected group of mdx mice was also treated with apocynin and taurine during this protocol. Detailed MS data, comparison between mdx wild type, exercised mdx wild type, and complete analysis of spot variation are provided. Furthermore, in wild type mice subjected to the same exercise protocol, the abundance of key proteins, resulted modified in exercised mdx, were analyzed by western blot.
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http://dx.doi.org/10.1016/j.dib.2018.03.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996268PMC
June 2018

Effect of a long-term treatment with metformin in dystrophic mdx mice: A reconsideration of its potential clinical interest in Duchenne muscular dystrophy.

Biochem Pharmacol 2018 08 21;154:89-103. Epub 2018 Apr 21.

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

The pharmacological stimulation of AMP-activated protein kinase (AMPK) via metabolic enhancers has been proposed as potential therapeutic strategy for Duchenne muscular dystrophy (DMD). Metformin, a widely-prescribed anti-hyperglycemic drug which activates AMPK via mitochondrial respiratory chain, has been recently tested in DMD patients in synergy with nitric oxide (NO)-precursors, with encouraging results. However, preclinical data supporting the use of metformin in DMD are still poor, and its actions on skeletal muscle appear controversial. Therefore, we investigated the effects of a long-term treatment with metformin (200 mg/kg/day in drinking water, for 20 weeks) in the exercised mdx mouse model, characterized by a severe mechanical-metabolic maladaptation. Metformin significantly ameliorated histopathology in mdx gastrocnemius muscle, in parallel reducing TGF-β1 with a recovery score (r.s) of 106%; this was accompanied by a decreased plasma matrix-metalloproteinase-9 (r.s. 43%). In addition, metformin significantly increased mdx diaphragm twitch and tetanic tension ex vivo (r.s. 44% and 36%, respectively), in spite of minor effects on in vivo weakness. However, no clear protective actions on dystrophic muscle metabolism were observed, as shown by the poor metformin effect on AMPK activation measured by western blot, on the expression of mechanical-metabolic response genes analyzed by qPCR, and by the lack of fast-to-slow fiber-type-shift assessed by SDH staining in tibialis anterior muscle. Similar results were obtained in the milder phenotype of sedentary mdx mice. The lack of metabolic effects could be, at least partly, due to metformin inability to increase low mdx muscle levels of l-arginine, l-citrulline and taurine, found by HPLC. Our findings encourage to explore alternative, metabolism-independent mechanisms of action to differently repurpose metformin in DMD, supporting its therapeutic combination with NO-sources.
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http://dx.doi.org/10.1016/j.bcp.2018.04.022DOI Listing
August 2018

Dual Action of Mexiletine and Its Pyrroline Derivatives as Skeletal Muscle Sodium Channel Blockers and Anti-oxidant Compounds: Toward Novel Therapeutic Potential.

Front Pharmacol 2017 12;8:907. Epub 2018 Jan 12.

Unit of Pharmacology, Department of Pharmacy-Drug Science, University of Bari Aldo Moro, Bari, Italy.

Mexiletine (Mex) has been recently appointed as an orphan-drug in myotonic-syndromes, being a potent use-dependent blocker of skeletal-muscle sodium channels (Na1.4). Available evidences about a potential anti-oxidant effect of Mex and its tetramethyl-pyrroline-derivatives , suggest the possibility to further enlarge the therapeutic potential of Mex-like compounds in myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative stress. In line with this and based on our previous structure-activity-relationship studies, we synthesized new compounds with a tetramethyl-pyrroline-ring on the amino-group of both Mex (VM11) and of its potent use-dependent isopropyl-derivative (CI16). The compounds were tested for their ability to block native Na1.4 and to exert cyto-protective effects against oxidative-stress injury in myoblasts. Voltage-clamp-recordings on adult myofibers were performed to assess the tonic and use-dependent block of peak sodium-currents (I) by VM11 and CI16, as well as Mex, VM11 and CI16 were 3 and 6-fold more potent than Mex in producing a tonic-block of peak sodium-currents (I), respectively. Interestingly, CI16 showed a 40-fold increase of potency with respect to Mex during high-frequency stimulation (10-Hz), resulting the strongest use-dependent Mex-like compound so far. The derivatives also behaved as inactivated channel blockers, however the voltage dependent block was modest. The experimental data fitted with the molecular-modeling simulation based on previously proposed interaction of main pharmacophores with Na1.4 binding-site. CI16 and VM11 were then compared to Mex and its isopropyl derivative (Me5) for the ability to protect CC-cells from HO-cytotoxicity in the concentration range effective on Na1.4. Mex and Me5 showed a moderate cyto-protective effect in the presence of HO, Importantly, CI16 and VM11 showed a remarkable cyto-protection at concentrations effective for use-dependent block of Na1.4. This effect was comparable to that of selected anti-oxidant drugs proved to exert protective effect in preclinical models of progressive myopathies such as muscular dystrophies. Then, the tetramethyl-pyrroline compounds have increased therapeutic profile as sodium channel blockers and an interesting cyto-protective activity. The overall profile enlarges therapeutic potential from channelopathies to myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative-stress, i.e., muscular dystrophies.
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http://dx.doi.org/10.3389/fphar.2017.00907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770958PMC
January 2018

Proteome analysis in dystrophic mdx mouse muscle reveals a drastic alteration of key metabolic and contractile proteins after chronic exercise and the potential modulation by anti-oxidant compounds.

J Proteomics 2018 01 28;170:43-58. Epub 2017 Sep 28.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy. Electronic address:

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. In the present study, we describe, the pattern of differentially abundant spots that is associated to the worsening of dystrophy phenotype induced by chronic exercise. Our proteomic analysis pointed out 34 protein spots with different abundance between sedentary and exercised mdx mice. These proteins belong mostly to glucose metabolism, energy production and sarcomere structure categories. Interestingly exercise induced an increase of typical fast twitch fiber proteins (Troponin T fast skeletal muscle, Troponin I fast skeletal muscle and Myozenin-1) combined with an increase of several glycolytic enzymes. Concerning energy transfer, Adenylate kinase, showed a marked decrease when compared with non-exercised mdx. The decline of this enzyme correlates with increased Creatin kinase enzyme, suggesting that a compensatory energy metabolism mechanism could be activated in mdx mouse skeletal muscle following exercise. In addition, we analysed muscles from exercised mdx mice treated with two natural anti-oxidant compounds, apocynin and taurine, that in our previous study, were proved to be beneficial on some pathology related parameters, and we showed that these compounds can counteract exercise-induced changes in the abundance of several proteins.

Significance: Mdx mouse model of Duchenne muscular dystrophy shows a phenotype of the disorder milder than in human sufferers. This phenotype can be worsened by a different protocols of chronic exercise. These protocols can mimic the muscle progressive damage observed in humans, can allow studying the effects of inadequate training on dystrophic muscles and have been largely used to assess the ability of a drug to reduce the damage induced by exercise. In this study, we describe for the first time, the pattern of protein variation associated with the worsening of dystrophy phenotype induced by chronic exercise. Our proteomic analysis pointed out 34 protein spots with different amount between sedentary and exercised mdx mice. These proteins belong mostly to glucose metabolism, energy production and sarcomere structure categories and their variation indicates that mdx exercised muscle are not able to carry out the metabolic changes associated to fast-to-slow transition typically observed in aerobically trained muscle.
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http://dx.doi.org/10.1016/j.jprot.2017.09.009DOI Listing
January 2018

Contractile efficiency of dystrophic mdx mouse muscle: in vivo and ex vivo assessment of adaptation to exercise of functional end points.

J Appl Physiol (1985) 2017 Apr 5;122(4):828-843. Epub 2017 Jan 5.

Section of Pharmacology, Department of Pharmacy and Drug Sciences, University of Bari "Aldo Moro," Bari, Italy;

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8, and 12 wk of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (WT) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles were lower in mdx compared with WT mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to WT muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised WT muscle. The severe histopathology and the high levels of muscular TGF-β1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Therefore, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients. We focused on the adaptation/maladaptation of dystrophic mdx mouse muscles to a standard protocol of exercise to provide guidance in the development of more effective drug and physical therapies in Duchenne muscular dystrophy. The mdx muscles showed a modest functional adaptation to chronic exercise, but it was not sufficient to overcome the progressive in vivo weakness, nor to counter signs of muscle damage. Therefore, a complex involvement of multiple systems underlies the maladaptive response of dystrophic muscle.
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http://dx.doi.org/10.1152/japplphysiol.00776.2015DOI Listing
April 2017

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

Identification of selective inhibitors of the potassium channel Kv1.1-1.2((3)) by high-throughput virtual screening and automated patch clamp.

ChemMedChem 2012 Oct 30;7(10):1775-83. Epub 2012 Mar 30.

Max Planck Institute for Biophysical Chemistry, Computational Biomolecular Dynamics Group, Am Fassberg 11, 37077 Göttingen, Germany.

Two voltage-dependent potassium channels, Kv1.1 (KCNA1) and Kv1.2 (KCNA2), are found to co-localize at the juxtaparanodal region of axons throughout the nervous system and are known to co-assemble in heteromultimeric channels, most likely in the form of the concatemer Kv1.1-1.2((3)) . Loss of the myelin sheath, as is observed in multiple sclerosis, uncovers the juxtaparanodal region of nodes of Ranvier in myelinated axons leading to potassium conductance, resulting in loss of nerve conduction. The selective blocking of these Kv channels is therefore a promising approach to restore nerve conduction and function. In the present study, we searched for novel inhibitors of Kv1.1-1.2((3)) by combining a virtual screening protocol and electrophysiological measurements on a concatemer Kv1.1-1.2((3)) stably expressed in Chinese hamster ovary K1 (CHO-K1) cells. The combined use of four popular virtual screening approaches (eHiTS, FlexX, Glide, and Autodock-Vina) led to the identification of several compounds as potential inhibitors of the Kv1.1-1.2((3)) channel. From 89 electrophysiologically evaluated compounds, 14 novel compounds were found to inhibit the current carried by Kv1.1-1.2((3)) channels by more than 80 % at 10 μM. Accordingly, the IC(50) values calculated from concentration-response curve titrations ranged from 0.6 to 6 μM. Two of these compounds exhibited at least 30-fold higher potency in inhibition of Kv1.1-1.2((3)) than they showed in inhibition of a set of cardiac ion channels (hERG, Nav1.5, and Cav1.2), resulting in a profile of selectivity and cardiac safety. The results presented herein provide a promising basis for the development of novel selective ion channel inhibitors, with a dramatically lower demand in terms of experimental time, effort, and cost than a sole high-throughput screening approach of large compound libraries.
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http://dx.doi.org/10.1002/cmdc.201100600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525944PMC
October 2012

Structural nucleotide analogs are potent activators/inhibitors of pancreatic β cell KATP channels: an emerging mechanism supporting their use as antidiabetic drugs.

J Pharmacol Exp Ther 2012 Feb 25;340(2):266-76. Epub 2011 Oct 25.

Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Via Orabona No. 4, I-70126 Bari, Italy.

The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic β cell ATP-sensitive K+ (KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic β cell KATP channel and the Kir6.2ΔC36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by intraperitoneal glucose tolerance test and on fasting glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP (IC₅₀ = 10.1 × 10⁻⁹ M) and Kir6.2ΔC36 (IC₅₀ = 9.6 × 10⁻⁹ M) channels, which induced depolarization. In contrast, the 2-phenyl analog was a potent opener (drug concentration needed to enhance the current by 50% = 0.04 × 10⁻⁹ M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl > 2-benzyl > 2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg/kg) and 2-phenyl analogs (2-30 mg/kg) reduced and enhanced the glucose areas under the curves, respectively, after glucose loading in mice. These compounds did not affect the fasting glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which confer the KATP channel blocking action with glucose-lowering effects and the opening action with increased glucose levels, respectively. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel antidiabetic drugs.
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http://dx.doi.org/10.1124/jpet.111.185835DOI Listing
February 2012

Multiple pathological events in exercised dystrophic mdx mice are targeted by pentoxifylline: outcome of a large array of in vivo and ex vivo tests.

J Appl Physiol (1985) 2009 Apr 8;106(4):1311-24. Epub 2009 Jan 8.

Unit of Pharmacology, Department of Pharmaco-biology, Faculty of Pharmacy, University of Bari, Bari, Italy.

The phosphodiesterases inhibitor pentoxifylline gained attention for Duchenne muscular dystrophy therapy for its claimed anti-inflammatory, antioxidant, and antifibrotic action. A recent finding also showed that pentoxifylline counteracts the abnormal overactivity of a voltage-independent calcium channel in myofibers of dystrophic mdx mice. The possible link between workload, altered calcium homeostasis, and oxidative stress pushed toward a more detailed investigation. Thus a 4- to 8-wk treatment with pentoxifylline (50 mg x kg(-1) x day(-1) ip) was performed in mdx mice, undergoing or not a chronic exercise on treadmill. In vivo, the treatment partially increased forelimb strength and enhanced resistance to treadmill running in exercised animals. Ex vivo, pentoxifylline restored the mechanical threshold, an electrophysiological index of calcium homeostasis, and reduced resting cytosolic calcium in extensor digitorum longus muscle fibers. Mn quenching and patch-clamp technique confirmed that this effect was paralleled by a drug-induced reduction of membrane permeability to calcium. The treatment also significantly enhanced isometric tetanic tension in mdx diaphragm. The plasma levels of creatine kinase and reactive oxygen species were both significantly reduced in treated-exercised animals. Dihydroethidium staining, used as an indicator of reactive oxygen species production, showed that pentoxifylline significantly reduced the exercise-induced increase in fluorescence in the mdx tibialis anterior muscle. A significant decrease in connective tissue area and profibrotic cytokine transforming growth factor-beta(1) was solely found in tibialis anterior muscle. In both diaphragm and gastrocnemius muscle, a significant increase in neural cell adhesion molecule-positive area was instead observed. This data supports the interest toward pentoxifylline and allows insight in the level of cross talk between pathogenetic events in workloaded dystrophic muscle.
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http://dx.doi.org/10.1152/japplphysiol.90985.2008DOI Listing
April 2009

Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

Neurobiol Dis 2008 Nov 23;32(2):243-53. Epub 2008 Jul 23.

Unit of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Italy.

Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.
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http://dx.doi.org/10.1016/j.nbd.2008.07.009DOI Listing
November 2008

Overactivity of exercise-sensitive cation channels and their impaired modulation by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline.

Neurobiol Dis 2006 Dec 28;24(3):466-74. Epub 2006 Sep 28.

Unit of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari, Italy.

Cell-attached patch-clamp recordings on native striated myofibers from adult dystrophic mdx mice revealed a higher occurrence and open probability compared to non-dystrophic wild-type myofibers of a 30 pS voltage-insensitive Ca2+-permeable channel, inhibited by Gd3+, streptomycin and ruthenium red. Myofibers from in vivo exercised animals had higher channel occurrence and/or open probability. Insulin-like growth factor 1 (3.3 nM) induced and/or enhanced channel activity, via PI3 kinase, in wild-type but not in mdx myofibers. Interestingly, in both genotypes the current was silenced by db-cAMP or pentoxifylline, a phosphodiesterase inhibitor. The channel activity/occurrence in pentoxifylline-treated exercised mdx (50 mg/kg/day i.p. for 4-8 weeks) overlapped that of exercised wild-type mice. Thus, a growth factor-sensitive current, likely due to a TRP channel, is activated in vivo by exercise in native striated fibers; its deregulation in the absence of dystrophin may contribute to Ca2+ homeostasis alteration. The possibility to pharmacologically counteract abnormal channel activity discloses important therapeutic application.
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http://dx.doi.org/10.1016/j.nbd.2006.08.010DOI Listing
December 2006

First evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant.

Neuromuscul Disord 2006 Apr 15;16(4):237-48. Epub 2006 Mar 15.

Unit of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari, Italy.

BN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4-6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-beta1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 microM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types.
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http://dx.doi.org/10.1016/j.nmd.2006.01.013DOI Listing
April 2006

Fiber type-related changes in rat skeletal muscle calcium homeostasis during aging and restoration by growth hormone.

Neurobiol Dis 2006 Feb 8;21(2):372-80. Epub 2005 Sep 8.

Division of Pharmacology, Department of Pharmaco-Biology, Faculty of Pharmacy, University of Bari, Bari I-70125, Italy.

The mechanisms by which aging induces muscle impairment are not well understood yet. We studied the impact of aging on Ca2+ homeostasis in the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL) muscles of aged rats by using the fura-2 fluorescent probe. In both muscles aging increases the resting cytosolic calcium concentration ([Ca2+]i). This effect was independent on calcium influx since a reduced resting permeability of sarcolemma to divalent cations was observed in aged muscles likely due to a reduced activity of leak channels. Importantly the effects of aging on resting [Ca2+]i, fiber diameter, mechanical threshold and sarcolemmal resting conductances were less pronounced in the soleus muscle, suggesting that muscle impairment may be less dependent on [Ca2+]i in the slow-twitch muscle. The treatment of aged rats with growth hormone restored the resting [Ca2+]i toward adult values in both muscles. Thus, an increase of resting [Ca2+]i may contribute to muscle weakness associated with aging and may be considered for developing new therapeutic strategies in the elderly.
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http://dx.doi.org/10.1016/j.nbd.2005.07.012DOI Listing
February 2006

A multidisciplinary evaluation of the effectiveness of cyclosporine a in dystrophic mdx mice.

Am J Pathol 2005 Feb;166(2):477-89

Sezione di Farmacologia, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università degli Studi di Bari, Via Orabona 4, Campus, 70125 Bari, Italy.

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1602333PMC
http://dx.doi.org/10.1016/S0002-9440(10)62270-5DOI Listing
February 2005

Atypical Ca2+-induced Ca2+ release from a sarco-endoplasmic reticulum Ca2+-ATPase 3-dependent Ca2+ pool in mouse pancreatic beta-cells.

J Physiol 2004 Aug 24;559(Pt 1):141-56. Epub 2004 Jun 24.

Unité d'Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, Brussels, Belgium.

The contribution of Ca(2+) release from intracellular stores to the rise in the free cytosolic Ca(2+) concentration ([Ca(2+)](c)) triggered by Ca(2+) influx was investigated in mouse pancreatic beta-cells. Depolarization of beta-cells by 45 mm K(+) (in the presence of 15 mm glucose and 0.1 mm diazoxide) evoked two types of [Ca(2+)](c) responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca(2+)](c) peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca(2+)](c) and voltage-dependent Ca(2+) current established that the TCP did not result from a larger Ca(2+) current. Abolition of the TCP by thapsigargin and its absence in sarco-endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) knockout mice show that it is caused by Ca(2+) mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of beta-cells but required a rise in [Ca(2+)](c) pointing to a Ca(2+)-induced Ca(2+) release (CICR). This CICR did not involve inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified beta-cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP(3)R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K(+), but plays little role in response to glucose in non-obese mouse pancreatic beta-cells.
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http://dx.doi.org/10.1113/jphysiol.2004.067454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1665062PMC
August 2004

SERCA3 ablation does not impair insulin secretion but suggests distinct roles of different sarcoendoplasmic reticulum Ca(2+) pumps for Ca(2+) homeostasis in pancreatic beta-cells.

Diabetes 2002 Nov;51(11):3245-53

Unité d'Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, Brussels, Belgium.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.
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http://dx.doi.org/10.2337/diabetes.51.11.3245DOI Listing
November 2002

G protein-independent activation of an inward Na(+) current by muscarinic receptors in mouse pancreatic beta-cells.

J Biol Chem 2002 Oct 2;277(41):38373-80. Epub 2002 Aug 2.

Unité d'Endocrinologie et Métabolisme, University of Louvain, Faculty of Medicine, UCL 55.30, Avenue Hippocrate 55, B-1200 Brussels, Belgium.

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.
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http://dx.doi.org/10.1074/jbc.M203888200DOI Listing
October 2002

Feedback control of the ATP-sensitive K(+) current by cytosolic Ca(2+) contributes to oscillations of the membrane potential in pancreatic beta-cells.

Diabetes 2002 Feb;51(2):376-84

Unité d'Endocrinologie et Métabolisme, University of Louvain, Brussels, Belgium.

During glucose stimulation, pancreatic beta-cells display membrane potential oscillations that correspond to intermittent Ca(2+) influx, leading to oscillations of the cytosolic free calcium concentration ([Ca(2+)](c)) and insulin secretion. The role of ATP-sensitive K(+) (K(+)-ATP) channels in the control of these oscillations was investigated by measuring the K(+)-ATP current (I(KATP)) with the perforated mode of the patch-clamp technique. No oscillations of I(KATP) were observed when glucose-stimulated beta-cells were kept hyperpolarized, thus with low and stable [Ca(2+)](c). However, increasing [Ca(2+)](c) by Ca(2+) influx (depolarizing pulses) or Ca(2+) mobilization (acetylcholine) transiently augmented I(KATP). This effect was abolished by tolbutamide, attenuated by increasing the glucose concentration in the medium, and prevented by abrogation of the [Ca(2+)](c) rise, which demonstrates that the current is really I(KATP) and that its increase is Ca(2+)-dependent. Injection of a current of a similar amplitude to that of the Ca(2+)-induced increase in I(KATP) was sufficient to repolarize glucose-stimulated beta-cells. These results suggest that, in the absence of [Ca(2+)](c) oscillations, no metabolic oscillations affect I(KATP) in pancreatic beta-cells. In contrast, [Ca(2+)](c) oscillations evoke I(KATP) oscillations. This mechanism may constitute the feedback loop controlling the glucose-induced oscillating electrical activity in beta-cells.
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http://dx.doi.org/10.2337/diabetes.51.2.376DOI Listing
February 2002