Antonietta Mele

Right ventricular diastolic abnormalities in systemic sclerosis. Relation to left ventricular involvement and pulmonary hypertension

OBJECTIVES To investigate right ventricular diastolic function in systemic sclerosis (SSc) and its relation to clinical features of the disease. METHODS Seventy seven unselected SSc patients and 33 healthy subjects were submitted to echocardiography and echo Doppler study to assess left and right systolic as well diastolic function and to estimate maximal arterial systolic pulmonary pressure (PAP). In addition, the patients were investigated to define the SSc subset and the extent of skin and internal organ involvement. RESULTS An abnormal right ventricular filling, as expressed by an inverted tricuspidal (Tr) E/A ratio (Tr E/A ratio <1), was detected in 31 of the 77 SSc patients (40%) and in 0 of the 36 controls ( p<0.001 ). All the 31 patients with an inverted Tr E/A ratio were found to have a PAP > 30 mm Hg. Twenty resulted to have an inverted mitral (Mit) E/A ratio (Mit E/A ratio <1), indicating an abnormal left ventricular filling. In multiple regression analysis, Tr E/A ratio resulted to be independently correlated to both PAP (r= −0.35;p<0.003) and Mit E/A ratio (r=0.39;p<0.001). CONCLUSIONS This study points out an impaired right ventricular filling in a significant percentage of SSc patients whatever the subset. This alteration is independently correlated to both PAP and left ventricular filling abnormalities.

Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K+-deficient rats.

Carbonic‐anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch‐clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+‐activated‐K+ (BK) channel of K+‐deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10− 6M)>BFT(DE50=5.93x10− 5M)>ETX(DE50=1.17x10− 4M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra‐molecular hydrogen bond with calculated inter‐atomic distances ranging between 1.82 A° and 3.01 A° and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery

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.

Phenotype-dependent functional and pharmacological properties of BK channels in skeletal muscle: Effects of microgravity

We investigated the involvement of calcium-activated potassium channel (BK) in skeletal muscle phenotype determination and response to acetazolamide, a BK opener. The BKs of slow-twitching soleus (SOL) and fast-twitching flexor digitorum brevis (FDB) muscles of the rat were investigated by patch-clamp technique. The changes of BK properties following muscle disuse were investigated in the hindlimb-unloaded (HU) rat, an animal model of disuse/microgravity. Two functionally different BKs were found in skeletal muscle. The BK of FDB was sensitive to calcium and to acetazolamide, in contrast the BK of SOL was less sensitive to calcium and was resistant to acetazolamide. After 3-14 days HU, in parallel with the slow-to-fast phenotype transition of the fibers, the BK of SOL acquired properties similar to those of FDB. In skeletal muscle, the BK plays muscle-specific roles contributing to the calcium-dependent phenotype determination/adaptation to disuse. The phenotype specificity of acetazolamide has implications for drug-based therapy of neuromuscular disorders associated to disuse.

Acetazolamide prevents vacuolar myopathy in skeletal muscle of K+‐depleted rats

Background and purpose: Acetazolamide and dichlorphenamide are carbonic anhydrase (CA) inhibitors effective in the clinical condition of hypokalemic periodic paralysis (hypoPP). Whether these drugs prevent vacuolar myopathy, which is a pathogenic factor in hypoPP, is unknown. The effects of these drugs on the efflux of lactate from skeletal muscle were also investigated.

Carbonic anhydrase inhibitors ameliorate the symptoms of hypokalaemic periodic paralysis in rats by opening the muscular Ca2+-activated-K+channels

Carbonic-anhydrase inhibitors are effective in channelopathies possibly by opening the Ca2+-activated-K+ channels. However, the in vivo effects of these drugs in K+-deficient rats, the animal model of familial hypokalaemic periodic paralysis(hypokalaemic-PP), are currently unknown. Measures of insulin-responses, serum electrolytes levels and patch-clamp experiments were therefore performed in K+ -deficient rats treated in vivo with dichlorphenamide (DCP), ethoxzolamide (ETX), hydrochlorthiazide (HCT), methazolamide (MTZ), bendroflumethiazide (BFT) and acetazolamide (ACTZ). Ten days treatments of K+-deficient rats with DCP, BFT, ETX and ACTZ (5.6 mg/kg per day) restored the serum [K+] to control values and prevented the insulin-induced paralysis. In ex vivo experiments, the carbonic-anhydrase inhibitors enhanced the activity of Ca2+-activated-K+ channels with the order of efficacy: ACTZ>BFT>ETX>DCP. In contrast, HCT and MTZ failed to stimulate the Ca2+-activated-K+ channels and to prevent the hypokalaemia and paralysis. At the concentration of 1mg/kg per day, all these drugs failed to ameliorate the hypokalaemic-PP symptoms. The activation of Ca2+-activated-K+ channel in addition to the mild diuretic effect explained the efficacy of ACTZ and DCP in K+ -deficient rats and in familial hypokalaemic-PP.

The KATP channel is a molecular sensor of atrophy in skeletal muscle

The involvement of ATP‐sensitive K+ (KATP) channels in the atrophy of slow‐twitch (MHC‐I) soleus (SOL) and fast‐twitch (MHC‐IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14‐day‐hindlimb‐unloaded (14‐HU) rats, an animal model of disuse, and in vitro in drug‐induced muscle atrophy. Patch‐clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14‐HU rats and controls. A down‐regulation of KATP channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14‐HU rats. The observed changes in KATP currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC‐IIa expression. Half of the SOL fibres of 14‐HU rats had reduced diameter and KATP currents and were labelled by MHC‐I antibodies. Non‐atrophic fibres were labelled by MHC‐IIa (22%) antibodies and had enhanced KATP currents, or were labelled by MHC‐I (28%) antibodies but had normal current. FDB was not affected in 14‐HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long‐term incubation of the control muscles in vitro with the KATP channel blocker glibenclamide (10−6 m) reduced the KATP currents with atrophy and these effects were prevented by the KATP channel opener diazoxide (10−4 m). The in vivo down‐regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the KATP channel as a molecular sensor of atrophy.

Splicing of the rSlo Gene Affects the Molecular Composition and Drug Response of Ca2+-Activated K+ Channels in Skeletal Muscle

The molecular composition and drug responses of calcium-activated K+ (BK) channels of skeletal muscle are unknown. Patch-clamp experiments combined with transcript scanning of the Kcnma1 gene encoding the alpha subunit of the BK channel were performed in rat slow-twitch soleus (Sol) and fast-twitch flexor digitorum brevis (FDB) skeletal muscles. Five splicing products of the Kcnma1 gene were isolated from Sol and FDB: the e17, e22, +29 aa, Slo27 and Slo0 variants. RT-PCR analysis demonstrated that the expression of e22 and Slo0 were 80–90% higher in FDB than Sol, whereas the expression of Slo27 was 60% higher in Sol than FDB, and the +29 aa variant was equally expressed in both muscle types. No beta 1-4 subunits were detected. In Sol, a large BK current with low Ca2+ sensitivity was recorded. The BK channel of Sol also showed a reduced response to BK channel openers, such as NS1619, acetazolamide and related drugs. In FDB, a reduced BK current with high Ca2+ sensitivity and an enhanced drug response was recorded. The total BK RNA content, which was 200% higher in Sol than in FDB, correlated with the BK currents in both muscles. Drug responses primarily correlated with e22 and Slo0 expression levels in FDB and to Slo27 expression in Sol muscle. In conclusion, phenotype-dependent BK channel biophysical and pharmacological properties correlated with the expression levels of the variants in muscles. These findings may be relevant to conditions affecting postural muscles, such as prolonged bed-rest, and to diseases affecting fast-twitch muscles, such as periodic paralysis. Down-regulation or up-regulation of the variants associated with pathological conditions may affect channel composition and drug responses.

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

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.

Reduced expression of Kir6.2/SUR2A subunits explains KATP deficiency in K+-depleted rats.

We investigated on the mechanism responsible for the reduced ATP-sensitive K(+)(K(ATP)) channel activity recorded from skeletal muscle of K(+)-depleted rats. Patch-clamp and gene expression measurements of K(ATP) channel subunits were performed. A down-regulation of the K(ATP) channel subunits Kir6.2(-70%) and SUR2A(-46%) in skeletal muscles of K(+)-depleted rats but no changes in the expression of Kir6.1, SUR1 and SUR2B subunits were observed. A reduced K(ATP) channel currents of -69.5% in K(+)-depleted rats was observed. The Kir6.2/SUR2A-B agonist cromakalim showed similar potency in activating the K(ATP) channels of normokalaemic and K(+)-depleted rats but reduced efficacy in K(+)-depleted rats. The Kir6.2/SUR1-2B agonist diazoxide activated K(ATP) channels in normokalaemic and K(+)-depleted rats with equal potency and efficacy. The down-regulation of the Kir6.2 explains the reduced K(ATP) channel activity in K(+)-depleted rats. The lower expression of SUR2A explains the reduced efficacy of cromakalim; preserved SUR1 expression accounts for the efficacy of diazoxide. Kir6.2/SUR2A deficiency is associated with impaired muscle function in K(+)-depleted rats and in hypoPP.