Recent Advances in the Pathogenesis and Drug Action in Periodic Paralyses and Related Channelopathies

The periodic paralysis (PP) are rare autosomal-dominant disorders associated to mutations in the skeletal muscle sodium, calcium, and potassium channel genes characterized by muscle fiber depolarization with un-excitability, episodes of weakness with variations in serum potassium concentrations. Recent advances in thyrotoxic PP and hypokalemic PP (hypoPP) confirm the involvement of the muscle potassium channels in the pathogenesis of the diseases and their role as target of action for drugs of therapeutic interest. The novelty in the gating pore currents theory help to explain the disease symptoms, and open the possibility to more specifically target the disease. It is now known that the fiber depolarization in the hypoPP is due to an unbalance between the novel identified depolarizing gating pore currents (Igp) carried by protons or Na+ ions flowing through aberrant alternative pathways of the mutant subunits and repolarizing inwardly rectifying potassium channel (Kir) currents which also includes the ATP-sensitive subtype. Abnormal activation of the Igp or deficiency in the Kir channels predispose to fiber depolarization. One pharmacological strategy is based on blocking the Igp without affecting normal channel gating. It remains safe and effective the proposal of targeting the KATP, Kir channels, or BK channels by drugs capable to specifically open at nanomolar concentrations the skeletal muscle subtypes with less side effects

Grand Challenge for Ion Channels: an Underexploited Resource for Therapeutics

In the last decade, only a few new ion channel drugs have reached the market according to the Food and Drug Administration. These include nateglinide, a non-sulfonylurea blocker of KATP channel used in type II diabetes; ziconotide, a N-type calcium channel blocker against severe chronic pain; pregabalin, a calcium channel a2δ subunit ligand indicated for neuropathic pain; ranolazine, a blocker of late sodium current for chronic angina pectoris; and lubiprostone, a ClC-2 chloride channel activator for chronic idiopathic constipation. In the meantime, the sodium channel blocker mexiletine, indicated as a class II

Acetazolamide opens the muscular K(Ca)2+ channel: A novel mechanism of action that may explain the therapeutic effect of the drug in hypokalemic periodic paralysis

Acetazolamide is a thiazide derivative clinically used in skeletal muscle disorders related to altered K+ homeostasis such as the periodic paralyses. The mechanism of action responsible for the therapeutic effects of the drug is still unknown, however. In the present work, we investigated the mechanism of action of acetazolamide in the K-deficient diet rat, an animal model of human hypokalemic periodic paralysis (hypoPP). The in vivo administration of 2.8- and 5.6-mg/kg-1/ day-1 concentrations of acetazolamide to K-deficient diet rats prevented paralysis and depolarization of the fibers induced by insulin. In the acetazolamide-treated animals, intense sarcolemma Ca2+-activated K+ channel (K(Ca)2+) activity was recorded. Acetazolamide also restored the serum K+ levels to control values. The concentrations of acetazolamide needed to enhance the K(Ca)2+ current by 50% in vitro were 6.17 and 4.01 x 10-6 M at -60 and +30 mV of membrane potentials, respectively. In normokalemic animals, the thiazide derivative enhanced the K(Ca)2+ current with similar efficacy. Our data demonstrate that the therapeutic effects of acetazolamide in the K-deficient diet rats and possibly in human hypokalemic periodic paralysis patients can be mediated by activation of the K(Ca)2+ channel

In vivo evaluation of antimyotonic efficacy of β-adrenergic drugs in a rat model of myotonia

AbstractThe sodium channel blocker mexiletine is considered the first-line drug in myotonic syndromes, a group of muscle disorders characterized by membrane over-excitability. We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, block voltage-gated sodium channels in a manner reminiscent to mexiletine, whereas salbutamol and nadolol do not. We now developed a pharmacological rat model of myotonia congenita to perform in vivo preclinical test of antimyotonic drugs. Myotonia was induced by i.p. injection of 30 mg/kg of anthracene-9-carboxylic acid (9-AC), a muscle chloride channel blocker, and evaluated by measuring the time of righting reflex (TRR). The TRR was prolonged from <0.5 s in control conditions to a maximum of ∼4 s, thirty minutes after 9-AC injection, then gradually recovered in a few hours. Oral administration of mexiletine twenty minutes after 9-AC injection significantly hampered the TRR prolongation, with an half-maximum efficient dose (ED50) of 12 mg/kg. Both propranolol and clenbuterol produced a dose-dependent antimyotonic effect similar to mexiletine, with ED50 values close to 20 mg/kg. Antimyotonic effects of 40 mg/kg mexiletine and propranolol lasted for 2 h. We also demonstrated, using patch-clamp methods, that both propranolol enantiomers exerted a similar block of skeletal muscle hNav1.4 channels expressed in HEK293 cells. The two enantiomers (15 mg/kg) also showed a similar antimyotonic activity in vivo in the myotonic rat. Among the drugs tested, the R(+)-enantiomer of propranolol may merit further investigation in humans, because it exerts antimyotonic effect in the rat model, while lacking of significant activity on the β-adrenergic pathway. This study provides a new and useful in vivo preclinical model of myotonia congenita in order to individuate the most promising antimyotonic drugs to be tested in humans

Fluvastatin and atorvastatin affect calcium homeostasis of rat skeletal muscle fibers in vivo and in vitro by impairing the sarcoplasmic reticulum/mitochondria Ca2+-release system

The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg-1) and atorvastatin (5 and 10 mg kg-1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca2+]i, up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca2+]i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced caffeine responsiveness. In vitro application of fluvastatin caused changes of [Ca2+]i, resembling the effect obtained after the in vivo administration. Indeed, fluvastatin produced a shift of mechanical threshold for contraction toward negative potentials and an increase of resting [Ca2+]i. By using ruthenium red and cyclosporine A, we determined the sequence of the statin-induced Ca2+ release mechanism. Mitochondria appeared as the cellular structure responsible for the earlier event leading to a subsequent large sarcoplasmic reticulum Ca2+ release. In conclusion, we suggest that calcium homeostasis alteration may be a crucial event for myotoxicity induced by this widely used class of hypolipidemic drug

Investigating a Benzofurane Derivative Binding Site on Human CLC-5

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In vivo longitudinal study of rodent skeletal muscle atrophy using ultrasonography

Muscle atrophy is a widespread ill condition occurring in many diseases, which can reduce quality of life and increase morbidity and mortality. We developed a new method using non-invasive ultrasonography to measure soleus and gastrocnemius lateralis muscle atrophy in the hindlimb-unloaded rat, a well-Accepted model of muscle disuse. Soleus and gastrocnemius volumes were calculated using the conventional truncated-cone method and a newly-designed sinusoidal method. For Soleus muscle, the ultrasonographic volume determined in vivo with either method was linearly correlated to the volume determined ex-vivo from excised muscles as muscle weight-To-density ratio. For both soleus and gastrocnemius muscles, a strong linear correlation was obtained between the ultrasonographic volume and the muscle fiber cross-sectional area determined ex-vivo on muscle cryosections. Thus ultrasonography allowed the longitudinal in vivo evaluation of muscle atrophy progression during hindlimb unloading. This study validates ultrasonography as a powerful method for the evaluation of rodent muscle atrophy in vivo, which would prove useful in disease models and therapeutic trials

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

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

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

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