Juan C. Saez

Dysferlinopathies are progressive muscular dystrophies caused by mutations in the DYSF gene, leading to defective membrane repair, chronic inflammation, lipid accu-mulation, and muscle degeneration. No approved therapies currently halt disease progression. This study evaluated the effects of daily oral administration of pulverized Boldo (Peumus boldus) leaves, a nutraceutical with reported bioactive properties, in symptomatic blAJ mice, a model of dysferlinopathy. Mice received Boldo for four weeks, and muscle function, vascular performance, histology, and molecular markers were assessed. Boldo treated mice exhibited significantly improved grip strength and restored endothelium dependent vasodilation. Gastrocnemius muscle perfusion and capillary density increased after treatment. Histological analysis revealed prevention of myofiber atrophy, a reduction in centrally nucleated fibers, and improved tissue architecture. Lipid accumulation observed in untreated blAJ mice was absent with Boldo. At the cellular level, Boldo normalized sarcolemmal membrane permeability and decreased mRNA levels of inflammasome components (NLRP3, ASC, IL-1β), suggesting anti-inflammatory activity. These results indicate that Boldo improves vascular and muscle integrity in dysferlinopathy and support its potential as a com-plementary therapeutic strategy.

Glucocorticoids are frequently used as anti-inflammatory and immunosuppressive agents. However, high doses and/or prolonged use induce undesired secondary effects such as muscular atrophy. Recently, de novo expression of connexin43 and connexin45 hemichannels (Cx43 HCs and Cx45 HCs, respectively) has been proposed to play a critical role in the mechanism underlying myofiber atrophy induced by dexamethasone (Dex: a synthetic glucocorticoid), but their involvement in specific muscle changes promoted by Dex remains poorly understood. Moreover, treatments that could prevent the undesired effects of glucocorticoids on skeletal muscles remain unknown. In the present work, a 7-day Dex treatment in adult mice was found to induce weight loss and skeletal muscle changes including expression of functional Cx43/Cx45 HCs, elevated atrogin immunoreactivity, atrophy, oxidative stress and mitochondrial dysfunction. All these undesired effects were absent in muscles of mice simultaneously treated with Dex and vitamin E (VitE). Moreover, VitE was found to rapidly inhibit the activity of Cx HCs in freshly isolated myofibers of Dex treated mice. Exposure to alkaline pH induced free radical generation only in HeLa cells expressing Cx43 or Cx45 where Ca²⁺ was present in the extracellular milieu, response that was prevented by VitE. Besides, VitE and two other anti-oxidant compounds, Tempol and Resveratrol, were found to inhibit Cx43 HCs in HeLa cells transfectants. Thus, we propose that in addition to their intrinsic anti-oxidant potency, some antioxidants could be used to reduce expression and/or opening of Cx HCs and consequently reduce the undesired effect of glucocorticoids on skeletal muscles.

Chagas disease affects low-income nations with health consequences that impact the economy of those countries. Interestingly, inhibitors of channels formed by proteins of the gap junction family, such as suramin and boldine, exhibit trypanocidal activity. Gap junction proteins are integral membrane proteins present in both vertebrates and invertebrates that participate in cellular communication. These proteins form gap junction channels, which connect the cytoplasm of neighboring cells or non-junctional channels that connect the intra- and extracellular milieu. Interestingly, Trypanosoma cruzi modulates the expression of proteins of the gap junction family or modify the activity of the channels formed by these proteins in host cells. Moreover, Lucifer yellow microinjected into fibroblast was incorporated into associated trypanosomes of Trypanosoma musculi, suggesting the possibility of direct communication via gap junction channels between them. In this chapter, we summarized the current knowledge about the possible roles of gap junction family proteins in Chagas disease.

In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: “In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43”. In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1β), 3) fibrosis (immune detection of type III collagen; Col III) and 4) activity of RhoA/ROCK (amount of phosphorylated MYPT1; p-MYPT1). The ratio Uprot/UCrea, SBP, oxidative stress, inflammation, amount of Cx43 and p-MYPT1 remained high 2 weeks after suspending AngII treatment in rats treated for 4 weeks with AngII. These responses were not observed in rats treated with AngII for less than 4 weeks, in which all measurements returned spontaneously close to the control values after suspending AngII treatment. Rats treated with AngII for 6 weeks and co-treated for the last 4 weeks with Fasudil, an inhibitor of ROCK, showed high SBP but did not present renal damage or increased amount of renal Cx43. Therefore, renal damage induced by AngII correlates with the activation of RhoA/ROCK and the increase in Cx43 amounts and can be prevented by inhibitors of this pathway.

Connexin43 (Cx43), pannexin1 (Panx1) and P2X₇ receptor (P2X₇R) are expressed in kidneys and are known to constitute a feedforward mechanism leading to inflammation in other tissues. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remain unknown. In the present work, we found that MES-13 cells, from a cell line derived from mesangial cells, stimulated with angiotensin II (AngII) developed oxidative stress (OS, thiobarbituric acid reactive species (TBARS) and generated pro-inflammatory cytokines (ELISA; IL-1β and TNF-α). The membrane permeability increased progressively several hours before the latter outcome, which was a response prevented by Losartan, indicating the involvement of AT1 receptors. Western blot analysis showed that the amount of phosphorylated MYPT (a substrate of RhoA/ROCK) and Cx43 increased progressively and in parallel in cells treated with AngII, a response followed by an increase in the amount in Panx1 and P2X₇R. Greater membrane permeability was partially explained by opening of Cx43 hemichannels (Cx43 HCs) and Panx1 channels (Panx1 Chs), as well as P2X₇Rs activation by extracellular ATP, which was presumably released via Cx HCs and Panx1 Chs. Additionally, inhibition of RhoA/ROCK blocked the progressive increase in membrane permeability, and the remaining response was explained by the other non-selective channels. The rise of activity in the RhoA/ROCK-dependent pathway, as well as in Cx HCs, P2X₇R, and to a minor extent in Panx1 Chs led to higher amounts of TBARS and pro-inflammatory cytokines. We propose that AngII-induced mesangial cell damage could be effectively inhibited by concomitantly inhibiting the RhoA/ROCK-dependent pathway and one or more non-selective channel(s) activated through this pathway.

Precursor cells of skeletal muscles express connexins 39, 43 and 45 and pannexin1. In these cells, most connexins form two types of membrane channels, gap junction channels and hemichannels, whereas pannexin1 forms only hemichannels. All these channels are low-resistance pathways permeable to ions and small molecules that coordinate developmental events. During late stages of skeletal muscle differentiation, myofibers become innervated and stop expressing connexins but still express pannexin1 hemichannels that are potential pathways for the ATP release required for potentiation of the contraction response. Adult injured muscles undergo regeneration, and connexins are reexpressed and form membrane channels. In vivo, connexin reexpression occurs in undifferentiated cells that form new myofibers, favoring the healing process of injured muscle. However, differentiated myofibers maintained in culture for 48 h or treated with proinflammatory cytokines for less than 3 h also reexpress connexins and only form functional hemichannels at the cell surface. We propose that opening of these hemichannels contributes to drastic changes in electrochemical gradients, including reduction of membrane potential, increases in intracellular free Ca²⁺ concentration and release of diverse metabolites (e.g., NAD⁺ and ATP) to the extracellular milieu, contributing to multiple metabolic and physiologic alterations that characterize muscles undergoing atrophy in several acquired and genetic human diseases. Consequently, inhibition of connexin hemichannels expressed by injured or denervated skeletal muscles might reduce or prevent deleterious changes triggered by conditions that promote muscle atrophy.