Boldine prevents diabetes-induced skeletal muscle dysfunction by inhibiting large-pore channels

Boldine prevents diabetes-induced skeletal muscle dysfunction by inhibiting large-pore channels | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Boldine prevents diabetes-induced skeletal muscle dysfunction by inhibiting large-pore channels Walter Vásquez, Andrea Lira, Felipe Troncoso, Hermes Sandoval, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9284067/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Background Diabetes mellitus is associated with skeletal muscle dysfunction, including reduced strength, impaired perfusion, lipid accumulation, and inflammation. Activation of large-pore channels increases membrane permeability and inflammatory signaling. Boldine, an alkaloid from Peumus boldus , inhibits these channels and exhibits antioxidant and anti-inflammatory properties. This study evaluated whether boldine prevents diabetes-induced skeletal muscle alterations and explored underlying mechanisms. Methods Diabetes was induced in male C57BL/6J mice using streptozotocin (40 mg/kg/day for 5 days), followed by boldine treatment (50 mg/kg/day, 4 weeks). Muscle strength, resting membrane potential, and gastrocnemius perfusion were assessed. Lipid accumulation, capillary density, and NLRP3 mRNA were analyzed. Human myoblasts (AB1167) under low or high glucose with or without boldine were evaluated for membrane permeability, intracellular Ca²⁺, nitric oxide, inflammasome-related gene expression, and PPARγ reactivity. Results Diabetic mice exhibited reduced muscle strength, membrane depolarization, and ~ 20% lower basal perfusion, all prevented by boldine. Lipid accumulation increased to 52.4 ± 3.6% in diabetic muscle and decreased to 15.2 ± 4.1% with boldine (control: 3.1 ± 1.3%; p < 0.05). NLRP3 mRNA levels increased 17.7-fold and was reduced by ~ 50% with treatment. In vitro, high glucose increased ethidium uptake, Ca²⁺, nitric oxide production, inflammasome-related gene levels, and nuclear PPARγ localization; all were attenuated by boldine. Conclusions Boldine preserves skeletal muscle function, vascular reactivity, and metabolic homeostasis in diabetes, preventing lipid accumulation and inflammasome activation. These effects involve inhibition of large-pore channels, reducing membrane permeability and Ca²⁺-dependent inflammatory signaling, highlighting their role as therapeutic targets in diabetes-induced muscle dysfunction. PPARγ lipid accumulation inflammation myopathy sarcolemma permeability hemichannel blocker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Diabetes mellitus (DM) is a globally prevalent chronic disease, characterized by insufficient insulin production or reduced capacity for its effective use [ 1 ]. One significant complication caused by DM is skeletal muscle myopathy (SMM), which is particularly relevant given that skeletal muscle is crucial for glucose uptake and its deterioration profoundly impacts whole-body glucose homeostasis [ 2 ]. SMM manifests as muscle atrophy, decreased regeneration capacity, and fat infiltration, with adult skeletal muscle satellite cells (muscle progenitors) being especially vulnerable to the diabetic environment [ 3 ]. Under normal conditions, satellite cells remain quiescent, but upon stimuli such as injuries, they proliferate into myoblasts committed to the myogenic lineage, eventually fusing into myotubes and mature myofibers [ 4 ]. However, high glucose exposure promotes adipogenic differentiation in skeletal muscle-derived stem cells, leading to ectopic intramuscular fat accumulation [ 5 ]. Recent studies also implicate fibroadipogenic progenitors in this process, though the underlying molecular mechanisms are still poorly understood [ 6 ]. Notably, the inactivation of connexin 43 (Cx43) and Cx45 expression in skeletal myofibers prevented lipid accumulation in a murine dysferlinopathy model, suggesting a key role for connexin-formed hemichannels in muscle fat infiltration and dysfunction [ 7 ]. Connexins (Cxs) and pannexin1 (Panx1) form large-pore channels called hemichannels in the plasma membrane [ 8 ]. Cx39, Cx43, and Cx45 are expressed in skeletal muscle during progenitor cell proliferation and muscle fiber formation [ 9 ], and their aberrant expression as well as the upregulation of Panx1 hemichannels (Panx1 HCs) have been shown to contribute to various neuromuscular diseases [ 10 ]. Boldine, an alkaloid from Peumus boldus , is known to block three large-pore channels [ 11 ], and previous work has shown that it restores normal differentiation and improves muscle function in a dysferlinopathy model [ 12 ]. Similar protection has been observed in mice with dysferlinopathy treated with pulverized boldo leaves [ 13 ]. In this study, we propose that large-pore channels are key elements in the pathological mechanism of SMM in streptozotocin-induced diabetes and in high glucose-induced aberrant adipogenic commitment during myogenic differentiation. We investigated whether boldine could prevent these diabetic myopathy features, including lipid accumulation, impaired muscle function, and local inflammation, thereby preserving skeletal muscle integrity in diabetic animals. These experiments aim to advance the therapeutic potential of boldine in diabetes-associated skeletal muscle dysfunction. MATERIALS AND METHODS Reagents . Dulbecco’s modified Eagle medium (DMEM), ethidium bromide (Etd + ), and 4-Bromo A23187 were acquired from Sigma-Aldrich (St. Louis, MO, USA), and dithiothreitol (DTT), Fura-2AM, DAF-FM, SB203580, A740003, oil Red O, horse serum, and anti-PPAR-γ were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cy2- and Cy3-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The hydrochloride form of boldine was prepared as described previously [ 11 ]. Streptozotocin (STZ) and acetylcholine was purchased from Sigma-Aldrich (St. Louis, MO, USA). (R)-2-(4-chlorophenyl)-2-oxo-1-phenylethyl quinoline-2-carboxylate (C 24 H 16 ClNO 3 ; MW 401.85 g.mol-1), called D4, was synthetized by Edelris s.a.s. Medicinal Keymistry (Lyon, France). Animals Male wildtype (WT) C57Bl/6 mice were used. Diabetes was induced after a 6-hour fasting period, followed by a single intraperitoneal injection of STZ, 40 mg/kg/day for 5 days. To confirm the development of diabetes, blood samples were collected from the tail two days after STZ administration. Mice were considered diabetic if their blood glucose levels were ≥ 180 mg/dL. Glycaemia was measured using a portable glucometer (Accutrend sensor, Roche, Basel, Switzerland). All protocols were approved by the Bioethics Committee at Universidad de Valparaíso (CBC 85-2023) in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments. All efforts were made to minimize animal suffering and to reduce the number of animals used, and in vitro alternative techniques were implemented when possible. Three experimental groups were included: Control (n = 4), STZ-induced diabetes (STZ, n = 4), and STZ-induced diabetes treated with boldine (STZ + Boldine, n = 4). Diabetes was induced by intraperitoneal injections of STZ (40 mg/kg/day for 5 consecutive days). Boldine was administered orally at a dose of 50 mg/kg/day for four weeks by mixing the compound with peanut butter, which was voluntarily consumed by the animals. Control and STZ mice received peanut butter without boldine as vehicle control. All experimental groups were euthanized at the same age to avoid age-related confounding effects. We have previously shown that membrane permeability as well as the cross-sectional area of skeletal myofibers and glycemia levels of control mice are not affected by boldine [ 14 ], indicating that long term treatment with boldine does not significantly affect skeletal muscles. Therefore, we did not include an additional group of control mice treated with boldine. Forelimb muscle strength. Muscle strength was assessed using a digital force transducer (GPM-100; Melquest, Toyama, Japan) with a triangular metal bar. Mice grasped the bar with their forepaws and were pulled backward until release. Peak force was recorded for each mouse, which underwent three consecutive trials per session, and the highest values used for analysis. Measurements were performed at the end of the treatment period in the three experimental groups (Control, STZ, and STZ + Boldine). All measurements were performed back-to-back by a group-blinded experimenter. Blood perfusion Muscle blood flow was assessed in vivo using Laser Speckle Contrast Imaging (LSCI) technology (Pericam® PSI-HR system, Perimed, Stockholm, Sweeden). Adult mice were anesthetized with 4% isoflurane. The skin over the right gastrocnemius muscle was removed, and microvascular blood flow was recorded from four predefined circular regions of interest (ROIs). The protocol included a 3–4 min baseline recording, followed by a bolus of acetylcholine (10 µM) to induce endothelium-dependent vasodilation. Perfusion was monitored continuously, and time-of-interest windows (40 seconds) were extracted for basal and post-acetylcholine conditions. Data collection and analysis were conducted by two independent investigators (FT and HS). To minimize bias, experiments were performed in a randomized, back-to-back fashion, simultaneously including mice from all experimental groups. Data analysis was performed in a blinded manner. Changes in blood flow are reported in raw perfusion units as indicated previously [ 13 ]. Isolation of mouse skeletal myofibers : Myofibers were isolated from flexor digitorum brevis (FDB) muscles as previously described. FDB muscles were dissected, incubated in 0.2% collagenase type I, and then gently triturated to disperse single myofibers. Dissociated myofibers were washed and suspended in Krebs solution (in mM: 145 NaCl, 5 KCl, 3 CaCl 2 , 1 MgCl 2 , 5.6 glucose, 10 HEPES-Na, pH 7.4) with 10 µM N-benzyl-p-toluene sulphonamide (BTS) to inhibit contractions and reduce damage. Resting membrane potential (RMP) RMP was recorded from myofibers of flexor digitorum brevis muscles in a whole-cell configuration at 25°C. RMP recording was performed on fibers kept in culture for maximum 48 h before the RMP dropped as a consequence of denervation [ 15 ]. The pipette was a borosilicate electrode filled with 3 M KCl, and the bath solution was a Krebs-bicarbonate buffered solution at pH 7.4. Pipette resistance was approximately 50 Ω. All experiments were performed using an Olympus IX 51 inverted microscope, the Axopatch 1-D amplifier, and the Digidata 1322 digitizer as well as Clampex 9.1 acquisition programs. Data were analyzed using Clampfit 2.1. Myoblast cultures. The human myoblast cell line (AB1167) that had been previously described [ 12 ] was cultured in DMEM media containing low glucose concentration (LG; 8 mM). We used 8 mM glucose as the control condition, given that this concentration represents postprandial blood glucose levels in healthy individuals (≤ 7.8 mM) [ 16 ]. In addition, cells were cultured in DMEM containing a high glucose concentration (HG; 25 mM), with or without 50 µM boldine. When cultures reached ~ 70% of confluence, the media was replaced by differentiation media (DMEM 8 or 25 mM plus 5% horse serum) to promote the fusion of myoblasts and myotube formation. Differentiation medium was renewed every 48 h, and boldine (50 µM) was added at each medium renewal. Evaluation of Etd + uptake . This was carried out as previously described [ 14 ]. Briefly, myoblast plated onto plastic culture dishes were washed twice with Krebs solution. For time-lapse measurements, myoblasts were incubated in Krebs solution containing 5 µM Etd + . The intensity of Etd + fluorescence was first recorded for 5 min in regions of interest by using a water immersion Olympus 51W1I upright microscope (Japan). Images were captured with a Retiga 13001 fast cooled monochromatic digital camera (12-bit; QImaging, Canada) every 15 s during 5 min, and image processing was performed offline with ImageJ software (National Institutes of Health). Intracellular Ca 2+ signal . Intracellular Ca 2+ signals were evaluated in immortalized human myoblast loaded with FURA 2. To load FURA 2, myoblasts were incubated with 5 µM FURA 2-AM in DMEM without serum at 37°C for 30 min and then washed three times. Then, the Ca 2+ signals were evaluated using a Nikon Eclipse Ti microscope, and fluorescence emitted upon excitation at two wavelengths (340 and 380 nm) was calculated along with the ratio of the recorded emissions (340/380 ratio). Immunofluorescence analysis. Samples were fixed with 4% paraformaldehyde, incubated overnight at 4°C with diluted primary anti-PPAR-γ antibodies, followed by washes with PBS. Secondary antibodies conjugated to Cy2 or Cy3 were then applied for 1 hour. Samples were rinsed, mounted with fluoromount G (containing DAPI), and visualized on glass slides. Oil red O stain. Transverse cryosections of tibialis anterior muscle and cultured myoblasts were fixed with 4% paraformaldehyde in the presence of 180 mM CaCl₂. Oil Red O stock solution was diluted (3:2 with distilled water) to prepare the working solution, which was applied to samples for 30 min. Excess stains were removed, and samples were rinsed with water to visualize lipid droplets. We performed quantitative analysis of oil red O staining by calculating the stained area fraction using Python 3.12.11 OpenCV (version 4.9.0), NumPy (version 1.26.4), and Matplotlib libraries (version 3.9.2). Reverse transcription polymerase chain reaction (PCR). Total RNA was isolated from tissue or cells using TRIzol following the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). Two microgram aliquots of total RNA were transcribed to cDNA using MMLV-reverse transcriptase (Fermentas, USA), and mRNA levels were evaluated by PCR amplification (GoTaq Flexi DNA polymerase; Promega, USA) The oligos used were the following: NLRP3: S 5´-GCTGGCATCTGGGGAAACCT-´3, AS 5´GCCCTTCTGGGGAGGATAGT-´3; CASP1 S 5´GAAAAGCCATGGCCGACAAG-´3, AS 5´-GCCCCTTTCGGAATAACGGA-´3. 18S: S 5′-TCAAGAACGAAAGTCGGAGG-′3, AS 5′-GGACATCTAAGGGCATCACA-′3. Statistical Analysis. Quantitative variables are presented as mean ± SEM. Considering the data distribution, we used parametric or non-parametric tests, as appropriate, using the Shapiro–Wilk normality test. For multiple group comparisons during perfusion analysis, data are presented as mean values calculated from three different subjects per group. Multiple comparisons were performed using ordinary one-way ANOVA, followed by multiple comparisons by controlling the false discovery rate with the Benjamini and Hochberg method. In other experiments, data were analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test and an appropriate normality test. p < 0.05 was considered a statistically significant difference. Data and statistical analyses were performed using the Microsoft Excel database and GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). RESULTS Boldine improves muscle strength and resting membrane potential (RMP) in diabetic mice To evaluate whether boldine preserves skeletal muscle function in diabetic mice, forelimb grip strength was measured at the end of the treatment period. STZ-treated mice exhibited significantly reduced grip strength compared to Controls (Fig. 1 A), consistent with diabetes-induced muscle weakness. Notably, boldine treatment prevented this functional decline, and grip strength values were comparable to those observed in Control mice (Fig. 1 A). To further assess muscle function at the cellular level, we measured the RMP of freshly isolated skeletal muscle fibers. Fibers from STZ mice exhibited significant depolarization compared to Control fibers, which was prevented by 4 weeks of treatment with boldine (Fig. 1 B). All measurements derived from mice described below were performed after 4 weeks of treatment with boldine and age matched control animals. Boldine maintains the acetylcholine-induced increase in blood perfusion We next examined whether the loss of muscle strength in diabetic mice was associated with impaired endothelium-dependent microvascular perfusion, and whether boldine could restore this function. To do so, we assessed blood perfusion in the right gastrocnemius muscle (Fig. 2 A– 2 C). At baseline, myofibers of STZ-induced diabetic mice exhibited ~ 20% lower microvascular perfusion expressed as units of perfusion compared to Control myofibers (160.1 ± 17.2 versus 199.1 ± 13.8 units, respectively, p = 0.053). Notably, this decline was not observed in STZ-induced diabetic mice treated with boldine (184.6 ± 14.3 perfusion units, p = 0.46). We then evaluated the response to acetylcholine (ACh), an endothelium-dependent vasodilator. As expected, ACh elicited a robust increase in perfusion in Control mice that tended to be biphasic. In contrast, diabetic mice showed a blunted ACh-mediated response, denoting endothelial dysfunction. Boldine restored ACh-induced increases in perfusion (Fig. 2 C), which is in line with the improvement in muscle function observed (Fig. 1 A). To investigate whether the boldine-induced improvement in endothelium-dependent tissue perfusion was accompanied by structural remodeling of the microvasculature, capillary density was assessed in gastrocnemius muscle sections by immunodetection of CD31, a specific marker of endothelial cells. Capillary-to-fiber ratio was used as an index of microvascular density and angiogenic capacity. STZ-induced diabetic mice exhibited a marked reduction in capillaries per muscle fiber compared with Control mice (Fig. 2 D, E), indicating impaired skeletal muscle blood irrigation in the diabetic state. In contrast, boldine treatment significantly restored capillary density in diabetic muscles, as evidenced by an increased number of CD31⁺ structures per fiber relative to untreated STZ mice (Fig. 2 D, E). Collectively, these results indicate that boldine promotes microvascular remodeling in diabetic skeletal muscle and suggest that its beneficial effects on blood perfusion are associated with improved endothelial function and enhanced angiogenic responses. Boldine prevents lipid accumulation and up-regulation of NLRP3 mRNA levels in skeletal myofibers of diabetic mice To determine whether boldine could prevent the lipid accumulation observed in vitro in myoblasts exposed to high glucose, we analyzed cryosections of tibialis anterior muscles. Muscle sections were stained with Oil Red O to visualize neutral lipid droplets. In Controls, virtually no Oil Red O–positive fibers were detected (Fig. 3 , top panel). In contrast, myofibers of skeletal muscle from STZ-induced diabetic mice exhibited a marked increase in intracellular lipid accumulation (52.4 ± 3.6%), as evidenced by the presence of numerous red-stained myofibers (Fig. 3 A). Boldine treatment markedly reduced intracellular lipid accumulation in diabetic mice (Fig. 3 A) to similar levels as those observed in Controls (Boldine: 15.2 ± 4.1% and Controls: 3.1 ± 1.3%, p > 0.05). These results indicate that boldine prevents lipid infiltration into skeletal muscle fibers in diabetic mice. The inflammasome in skeletal myofibers has been shown to be activated by hyperglycemia [ 16 ]. To determine whether this alteration is also present in vivo , we quantified NLRP3 mRNA expression in skeletal muscle from diabetic mice by qPCR. Skeletal muscle from STZ-treated mice exhibited a marked upregulation of NLRP3 expression (17.74 ± 2.81-fold vs. Control; Fig. 3 C), consistent with inflammasome activation under diabetic conditions. Boldine treatment significantly attenuated NLRP3 overexpression, reducing transcript levels by approximately 50%. However, NLRP3 expression remained significantly elevated compared to Controls (9.41 ± 2.56-fold vs. Controls, p < 0.05). Together, these findings indicate that diabetes induces inflammasome activation in skeletal muscles, which is partially reversed by boldine. High glucose induces an increase in membrane permeability through large-pore channel-dependent mechanisms In a previous study, we found that HG increases membrane permeability to Etd + in myofibers [ 14 ]. Now, we investigated whether HG could increase the activity of large-pore channels in myoblasts. Cells were cultured with media containing LG (8 mM), HG (25 mM), or HG plus 50 µM boldine. Cell membranes showed low Etd⁺ permeability due to minimal hemichannel opening. Glucose at 8 mM within postprandial levels does not alter myoblast permeability or differentiation [ 16 ]. In contrast, pathological hemichannel upregulation increases Etd⁺ uptake, which becomes fluorescent upon nucleic acid binding. Here, we found that HG increases fluorescence intensity over time that was not promoted by LG (control condition), reflecting progressive Etd + uptake (Fig. 4 A and B). Notably, boldine prevents elevated Etd + uptake generated by HG alone (Fig. 4 A and B) and, hence, cells differentiated under HG exhibited a significantly increased Etd⁺ uptake rate compared to those maintained in LG or HG supplemented with boldine (Fig. 4 C). Importantly, this effect was not attributable to hyperosmolarity, as exposure to equiosmolar mannitol (25 mM) did not alter Etd⁺ uptake (Fig. 4 C), indicating that the above-described changes observed in cells exposed to HG are not the consequence of hyperosmolarity. To define the contributions of candidate large-pore channels to Etd⁺ uptake, we employed pharmacological inhibitors targeting HCs and the P2X7 receptor (P2X7R). We used D4, which selectively blocks Cx45 and Cx43 HCs [ 14 ], as well as A740003, which is a selective P2X7R antagonist [ 17 ]. Both inhibitors completely abolished the HG-induced increase in Etd⁺ uptake observed in myoblasts (Fig. 4 D), demonstrating that Cx HCs and P2X7R signaling are required for this response. In contrast, acute inhibition of Panx1 hemichannels with 10 Panx1 [ 18 ] did not result in a significant reduction of Etd⁺ uptake (Fig. 4 D), arguing against a major role for Panx1 channels. Collectively, these results indicate that the HG-induced increase in membrane permeability is mediated predominantly by Cx HCs rather than Panx1 CHs. High glucose activates large-pore channels through elevated intracellular Ca²⁺ and NO-dependent mechanisms Activation of Cx43 HCs and P2X7Rs has been linked to intracellular Ca²⁺ elevation and purinergic signaling [ 19 , 20 ]. Given that increased intracellular Ca²⁺ enhances Cx43 HC open probability [ 21 ], we tested whether Ca²⁺ mobilization from intracellular stores contributed to HG-induced increase in membrane permeability. Myoblasts cultured under HG conditions were preincubated for 5 min with MRS2179 (10 µM), which is a selective P2Y1 receptor antagonist that prevents IP₃-mediated Ca²⁺ release [ 22 ]. Although MRS2179 tended to reduce Etd⁺ uptake, this effect did not reach statistical significance (Fig. 5 A), indicating that P2Y1-mediated Ca²⁺ mobilization from intracellular stores does not play a major role in HG-induced membrane permeabilization. Previous studies have demonstrated that p38 MAPK enhances Cx43 HC activity in response to pro-inflammatory stimuli [ 23 ]. To assess the acute involvement of this pathway in HC regulation while minimizing secondary signaling effects, cells were preincubated for only 5 min with pharmacological inhibitors prior to Etd⁺ uptake measurements. Inhibition of p38 MAPK with SB202190 (10 µM) reduced Etd⁺ uptake to levels comparable to those observed under LG conditions (Fig. 5 A), implicating p38 MAPK signaling in HG-induced membrane permeabilization. To further determine the contribution of intracellular Ca²⁺ signaling, myoblasts were loaded with BAPTA-AM (5 µM) to chelate cytosolic Ca²⁺. This intervention significantly reduced Etd⁺ uptake (Fig. 5 A), demonstrating that elevated intracellular Ca²⁺ is required for HG-induced increases in membrane permeability. Consistently, La³⁺, a broad inhibitor of Cx HCs and P2X7R [ 24 ], normalized Etd⁺ uptake in HG-treated cells up to LG levels (Fig. 5 A), further supporting a role for large-pore channels in mediating hyperglycemia-induced permeabilization. Intracellular Ca²⁺ dynamics were then directly assessed using Fura-2 AM in myoblasts differentiated under LG, HG, or HG supplemented with boldine (Fig. 5 B). Cells exposed to HG displayed a significantly elevated basal Ca²⁺ signal relative to cells exposed to LG, whereas boldine fully prevented the increase exhibited by cells exposed to HG (Fig. 5 B). Quantitatively, the mean basal Ca²⁺ was highest in the HG group (1.0800 ± 0.0036), while values in LG and HG + Boldine were comparable (1.030 ± 0.005 and 1.040 ± 0.002, respectively) and significantly lower than HG alone. Increased cytoplasmic Ca²⁺ levels are known to promote oxidative and nitrosative stress through the generation of reactive oxygen and nitrogen species [ 25 ]. In addition, S-nitrosylation of Cx43 [ 26 ] in cysteine residue 271 [ 27 ] has been shown to increase Cx HC activity. To evaluate whether redox-dependent regulation contributes to HC activation during myoblast differentiation under HG-conditions, cells were treated with the sulfhydryl reducing agent DTT (10 mM). The acute application of DTT rapidly and significantly decreased Etd⁺ uptake, reaching a stable minimum within seconds (Fig. 5 C), consistent with reduced membrane permeability. These results indicate that Cx43 HC opening under HG involves redox-sensitive mechanisms. Given that HG increases nitric oxide synthase expression and nitric oxide (NO) production in cell cultures [ 28 ], we tested whether AB1167 cells treated with HG presented higher NO production. We found a significant increase in NO levels in cells cultured in HG, but not in cells incubated in LG or HG plus boldine (Fig. 5 D and E). These results suggest that S-nitrosylation of Cx43 HCs might contribute to increased Edt + uptake induced by HG. Boldine prevents increases in mRNA levels of inflammasome components triggered by high glucose Because HG is known to activate the inflammasome in skeletal muscle cells [ 14 ], we examined whether myoblasts differentiated under HG for 7 days showed evidence of inflammasome activation. Cells differentiated in HG exhibited higher mRNA levels of NLRP3 and caspase-1 compared to cells differentiated in LG (Fig. 6 A, B). Notably, boldine completely prevented the increase in mRNA levels of NLRP3 and significantly prevented increases in caspase 1 mRNA levels (Fis. 6A, B). These findings indicate that HG promotes inflammasome-related gene expression during myoblast differentiation. Boldine prevents high glucose–induced adipogenic drift during myogenic differentiation Skeletal muscles in adult diabetic men show intramuscular triglyceride accumulation [ 29 ], which could result in the decline of muscular strength, as observed in other pathologies [ 12 ]. Here, we evaluated whether immortalized human myoblasts exposed to HG could differentiate to adipocytes as a possible explanation for intramuscular fat accumulation [ 29 ]. After 7 days of differentiation, we found that HG elevated the nuclear content of the adipogenic transcription factor PPARγ, where ~ 45% presented nuclear PPARγ reactivity (Fig. 7 A and B). Notably, this response was prevented with boldine treatment (50 µM) (Fig. 7 A and B). In addition, we observed the presence of triglyceride accumulation in several myoblasts cultured in differentiation media with HG (red droplets). Boldine treatment completely prevented this phenomenon (Fig. 7 C). Thus, boldine blocked the aberrant differentiation of myoblasts to fat-containing cells. DISCUSSION STZ treatment increased HC activity in skeletal muscles, accompanied by higher levels of inflammasome components, lipid accumulation, reduced muscle force, and impaired blood perfusion. High glucose similarly increased HC activity in myoblasts, elevated mRNA levels of inflammasome components, and promoted lipid accumulation. Boldine prevented these alterations in both models and preserved muscle function. Diabetes-induced skeletal muscle weakness has been consistently observed in rodent models [ 30 ], an effect that was confirmed in this study showing reduced forelimb grip strength in STZ-induced diabetic mice. Importantly, boldine treatment fully prevented this decline. This functional impairment is closely associated with a significant depolarization of RMP in skeletal myofibers from diabetic mice, which is indicative of reduced membrane excitability. Such RMP depolarization has also been described for other muscle pathologies, such as denervation [ 15 ] and sepsis [ 31 ], where connexin HCs contribute to excitability loss. Early studies similarly noted reduced RMP in genetically diabetic and alloxan-induced diabetic mice, with these changes correlating with blood glucose levels and disease duration [ 32 ]. Our group previously demonstrated that diabetes induces de novo expression and membrane redistribution of Cx39, Cx43, and Cx45 in skeletal muscle, a pathological pattern that boldine treatment effectively reversed [ 14 ]. It is crucial to note that the genetic deletion of Cx43 and Cx45 in skeletal muscles significantly prevents diabetes-induced muscle atrophy [ 14 ], strongly implicating Cx HCs in both the structural and functional decline of skeletal muscles under hyperglycemic conditions. Our current findings reinforce this evidence, showing that boldine not only prevents reductions in RMP and contractile strength but also preserves muscle mass and excitability by inhibiting HC activity, thereby targeting a unifying mechanism underlying diabetic myopathy. Beyond reduced contractility and excitability, diabetic muscles display a marked inflammatory profile, particularly involving inflammasome activation. HG glucose-mediated inflammasome activation has been well-documented in diabetic patients, who exhibit elevated plasma levels of IL-1β and IL-18. In vitro studies, including our previous report, have shown greater NLRP3 levels in skeletal myofibers treated with HG [ 14 ]. In vivo studies in STZ-induced diabetic mice and db/db mice have also demonstrated upregulations of NLRP3 and caspase-1 [ 33 ]. Our data further demonstrated that blocking connexin HC with boldine can significantly prevent increases in mRNA levels of NLRP3 in diabetic mice, as well as increases in mRNA of NLRP3 and caspase-1 in myoblasts differentiated in HG, suggesting a direct involvement of connexin HCs in inflammasome activation in this context. Another prominent hallmark of diabetic myopathy is intramuscular lipid accumulation. Consistent with previous findings in diabetic rats [ 29 ], the STZ-induced diabetic mice used in the present work exhibited a marked lipid accumulation in tibialis anterior muscle fibers. Boldine treatment significantly reduced the number of lipid-positive myofibers in diabetic mice, reaching levels observed in non-diabetic Controls. This effect aligns with prior work in a dysferlinopathy model, where boldine administration prevented intramuscular fat accumulation and reduced PPARγ expression [ 12 ]. Our in vitro findings further demonstrated that boldine prevents HG–induced lipid accumulation in human myoblasts undergoing myogenic differentiation, extending its protective role against pathological ectopic fat deposition. In parallel, the restoration of muscle perfusion and microvascular network organization observed in vivo in boldine-treated diabetic mice highlights its systemic vasoprotective actions, which would likely contribute to improving muscle integrity and functional capacity. To understand the underlying molecular mechanisms, our study revealed that HG induces an aberrant adipogenic commitment in myoblasts, a process found to involve large-pore channel activation by a mechanism sensitive to boldine [ 34 ]. Boldine prevented HG-induced increases in myoblast membrane permeability, an effect also observed with acute boldine pre-incubation, indicating a direct channel-blocking action. By using more specific blockers, we identified Cx HCs and P2X7Rs as the main mediators of this permeability increase. In contrast, blocking Panx1 HCs and P2Y1Rs caused non-significant effects. P2Y1Rs undergo desensitization under conditions of sustained extracellular ATP release [ 35 ], which may explain these results. The findings support a positive feedback mechanism in which ATP released through Cx HCs activates P2X7Rs, promoting Ca²⁺ entry. Given that P2X7Rs, as well as Cx43 and Cx45 HCs are permeable to Ca²⁺ [ 20 , 36 , 37 ], this rise in intracellular Ca²⁺ further enhances Cx43 HC activity [ 21 ]. In line with the critical role of intracellular free-Ca²⁺, HG significantly increased basal cytoplasmic Ca²⁺ signals, which were effectively reduced by the Ca²⁺ chelator BAPTA. Additionally, p38 MAPK activation, which previously was linked to Cx43 HC activity in inflammatory contexts, was found to boost Etd + uptake in human myoblasts [ 23 ]. Furthermore, high NO production in diabetes, caused by HG-induced upregulation of NO synthase [ 28 ], would explain the elevated NO levels detected in HG-exposed myoblasts. NO enhances Cx43 HC activity through S-nitrosylation of cysteine residues [ 26 , 27 ], and the reversal of HG-induced permeability by DTT supports the involvement of S-nitrosylation in heightening Cx43 HC activity detected in myoblasts induced to undergo muscle differentiate in HG. Elevated Cx HC activity also contributes to increased reactive oxygen species (ROS) production, which can be mitigated by HC inhibitors or antioxidants. Since Cx43 HCs are permeable to Ca²⁺ [ 38 ], blocking these channels may prevent the activation of Ca²⁺-dependent pathways responsible for ROS generation. Boldine possesses intrinsic antioxidant properties [ 34 ], suggesting that its dual action (i.e., blockade of Cx HCs and reduction of oxidative stress) offers a comprehensive therapeutic approach to diabetes-related myopathy. HG-induced adipogenic differentiation of muscle-derived stem cells may be directly linked to the greater ROS production observed in this study [ 5 ]. Elevated oxidative stress has been shown to transform myoblasts into brown adipocytes by reducing MyoD expression via NF-κB activation [ 39 ]. Our findings that boldine prevented HG-induced increases in PPARγ levels and lipid accumulation in myoblasts are consistent with this mechanism. Given that boldine significantly reduces fat infiltration in dysferlinopathy models [ 40 ], we propose that connexin HC represents a common therapeutic target to limit fat infiltration in muscle diseases characterized by metabolic or inflammatory stress, including diabetes, obesity, dysferlinopathy and Duchenne muscular dystrophy [ 6 ]. In conclusion, our data identified large-pore channels as key drivers of diabetes-associated skeletal muscle dysfunction and demonstrated that boldine prevents muscle function alteration, vascular reactivity, and metabolic and inflammatory homeostasis in vivo and in vitro . These findings highlight the potential efficacy of large-pore channel inhibition as a disease-modifying strategy, positioning boldine as a promising therapeutic candidate for diabetic myopathy. Abbreviations ATP: Adenosine triphosphate Ca²⁺: Calcium ion CD31: Cluster of differentiation 31 Cx: Connexin Cx43: Connexin 43 Cx45: Connexin 45 Etd⁺: Ethidium bromide HCs: Hemichannels HG: High glucose LG: Low glucose NLRP3: NOD-like receptor family pyrin domain-containing 3 NO: Nitric oxide P2X7R: Purinergic receptor P2X7 P2Y1R: Purinergic receptor P2Y1 Panx1: Pannexin 1 PPARγ: Peroxisome proliferator-activated receptor gamma RMP: Resting membrane potential STZ: Streptozotocin Declarations Ethics approval and consent to participate: All animal procedures were approved by the Bioethics Committee of the Universidad de Valparaíso (Approval No. CBC 85-2023). Experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. Data Availability: All data supporting the findings of this study are included in the manuscript and its Supporting Information files. Additional datasets are available from the corresponding author, Prof. Juan C. Sáez, upon reasonable request and subject to institutional and ethical approval. Competing interests: The authors declare no competing interests . Funding: This work was partially funded by the following grants from ANID: 1231523 (to J.C.S.), Millenium Institute ICN2025_026 CINV (to J.C.S.), 1240295 (C.E.), GI2301146 (to C.E.), ACT210057 (to X. F.), as well as Doctoral fellowships from ANID (to W.V. and A.L.). Authors’ contributions: W.V. and J.C.S. conceived and designed research. W.V., A.L., F.T., H.S., and L.A.C. performed experiments. W.V., A.L., F.T., H.S., and L.A.C. analyzed data. W.V., C.E., and J.C.S. interpreted results of experiments. W.V., A.L., F.T., H.S., and L.A.C. prepared figures. W.V. drafted manuscript. J.C.S., X.F.F., and C.E. edited and revised manuscript. All authors approved final version of manuscript. Acknowledgments: The experimental data in this paper were drawn from a thesis submitted in partial fulfillment of the requirements for the Doctorate in Biological Sciences, with a mention in Physiological Science (W.V.) at Pontificia Universidad Católica de Chile.The authors used ChatGPT (OpenAI, GPT-4) for language editing and manuscript refinement. The tool was used in a manner that does not conflict with APS ethical policies and the authors take full responsibility for the content. References Antar SA, Ashour NA, Sharaky M, Khattab M, Ashour NA, Zaid RT et al (2023) Diabetes mellitus: Classification, mediators, and complications; a gate to identify potential targets for the development of new effective treatments. 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In STZ-diabetic mice, de novo expression of large-pore channels in skeletal muscles contributes to reducing the resting membrane potential of myofibers, as well as reducing the blood perfusion of muscles, muscle weakness, PPARɤ activation and the accumulation of intramuscular fat. Boldine inhibits large-pore channel activity and, consequently, prevents the above alterations, preserving skeletal muscle function in vivo . Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major Revision 15 May, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers invited by journal 03 Apr, 2026 Editor assigned by journal 02 Apr, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9284067","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617170739,"identity":"0e6a05b3-07f6-4eca-9452-ece2656ab77c","order_by":0,"name":"Walter Vásquez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYFAD9gYQaUGKFp4DIFKCFC0SCURqMTh+xvDjj7LDcvIz3xh+YKghRsuZHGNpnnOHjRln5xhLMBwjQovZgbQEaca2w4nN0jlmDIwNxGg5/yz558+2w/VtkmeI1XIj+ZgEb9vhBB4JHiK12N94fMya51y64QyetGKJBGL8Itmf2HzzR5m1vHz74Y0fPtTYENYCAWxQOoFYDQgto2AUjIJRMAqwAQBHqjOk2BrRnQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4113-8172","institution":"Pontificia Universidad Católica de Chile: Pontificia Universidad Catolica de Chile","correspondingAuthor":true,"prefix":"","firstName":"Walter","middleName":"","lastName":"Vásquez","suffix":""},{"id":617170740,"identity":"21f6f1e0-bd99-4cf2-825a-d60083feab9f","order_by":1,"name":"Andrea Lira","email":"","orcid":"","institution":"Universidad Andrés Bello: Universidad Andres Bello","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Lira","suffix":""},{"id":617170741,"identity":"e396fc16-4780-40d6-8a1a-5f4a4bb59fdb","order_by":2,"name":"Felipe Troncoso","email":"","orcid":"","institution":"Universidad del Bio-Bio - Sede Chillan","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"","lastName":"Troncoso","suffix":""},{"id":617170742,"identity":"1612105b-8ea1-4568-a29e-9bdac6a12271","order_by":3,"name":"Hermes Sandoval","email":"","orcid":"","institution":"Universidad del Bio-Bio - Sede Chillan","correspondingAuthor":false,"prefix":"","firstName":"Hermes","middleName":"","lastName":"Sandoval","suffix":""},{"id":617170743,"identity":"e8a7315e-4160-479e-9ad7-e0d8732a72b5","order_by":4,"name":"Luis A. Cea","email":"","orcid":"","institution":"Universidad Autonoma de Chile","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"A.","lastName":"Cea","suffix":""},{"id":617170744,"identity":"9a26f9a5-f35a-426a-950d-40aa3f7af349","order_by":5,"name":"Xavier F. Figueroa","email":"","orcid":"","institution":"Pontificia Universidad Católica de Chile: Pontificia Universidad Catolica de Chile","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"F.","lastName":"Figueroa","suffix":""},{"id":617170745,"identity":"66d55086-96f8-4612-aea6-1adddbb494e4","order_by":6,"name":"Carlos Escudero","email":"","orcid":"","institution":"Universidad del Bio-Bio - Sede Chillan","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Escudero","suffix":""},{"id":617170746,"identity":"8871ebec-5ea9-4ec7-8d59-b9caa1ee58d2","order_by":7,"name":"Juan C. Sáez","email":"","orcid":"","institution":"Universidad de Valparaíso: Universidad de Valparaiso","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"C.","lastName":"Sáez","suffix":""}],"badges":[],"createdAt":"2026-03-31 20:14:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9284067/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9284067/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106724583,"identity":"0ea76a0d-543a-4468-a7b2-465743a114b6","added_by":"auto","created_at":"2026-04-12 18:28:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoldine preserves forelimb grip strength and membrane potential in diabetic mice.\u003cbr\u003e\nA)\u003c/strong\u003e Forelimb grip strength was measured using a force transducer before (Pre) and after (Post) the treatment period in wild-type (Control), STZ-induced diabetic (STZ), and diabetic mice treated with boldine (STZ + Boldine). STZ mice showed a reduction in grip strength following diabetes induction, whereas the force values of boldine-treated mice were comparable to their baseline measurements and to Controls. \u003cstrong\u003eB)\u003c/strong\u003e Resting membrane potential (RMP) of isolated skeletal muscle fibers from Control, STZ, and STZ + Boldine mice. Fibers from STZ mice displayed significant depolarization compared to Controls, which was prevented by boldine treatment. Data are presented as mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, Tukey’s test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/3ec254bb67395c9c1490ddc8.png"},{"id":106517835,"identity":"c9dbd4c3-56b7-47b6-8b70-22082e6b0d0b","added_by":"auto","created_at":"2026-04-09 12:14:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":705902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlood perfusion of hindlimbs and microvasculature are preserved by boldine in diabetic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eRepresentative images of blood perfusion in the\u003cstrong\u003e \u003c/strong\u003eright gastrocnemius muscle of wild-type (Control), STZ-induced diabetic mice (STZ), and boldine-treated STZ mice (STZ + Boldine) in the absence (Basal) or presence of endothelial-mediated vasodilator acetylcholine (Ach, 10 mM). Perfusion units from 0 to 300, as indicated in the scale. \u003cstrong\u003eB) \u003c/strong\u003eRepresentative traces of blood perfusion as indicated in A. Highlighted area indicate perfusion after Ach stimulation. \u003cstrong\u003eC)\u003c/strong\u003e Average of perfusion unit in basal (Controls, STZ and STZ + Boldine) conditions and same groups in presence of Ach. \u003cstrong\u003eD)\u003c/strong\u003e Representative immunofluorescence images of gastrocnemius muscle cross-sections from wild-types (Controls), STZ-induced diabetic mice (STZ), and boldine-treated STZ mice (STZ + Boldine) stained for CD31 (red), an endothelial cell marker. Scale bar: 50 μm. \u003cstrong\u003eE)\u003c/strong\u003eQuantification of CD31⁺ capillaries per field shows a reduced capillary density in STZ-induced diabetic mice compared to Controls, with recovery in boldine-treated STZ mice. Data are presented as mean ± SEM. n = 4.* p \u0026lt; 0.05, ** p \u0026lt; 0.01, Tukey test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/e53d5c747bf8b7e9aa5a000f.png"},{"id":106517836,"identity":"c91cee4f-2db4-4a87-a603-570617e05517","added_by":"auto","created_at":"2026-04-09 12:14:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":414835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoldine prevents lipid accumulation and increases in NLRP3 mRNA levels in skeletal muscle of diabetic mice.\u003cbr\u003e\n \u003c/strong\u003eA) Representative images of tibialis anterior cryosections stained with Oil Red O from wild-type (Control), streptozotocin-induced diabetic (STZ), and diabetic mice treated with Boldine (STZ + Boldine). Lipid accumulation is evident in the STZ group, while minimal staining is observed in Control and STZ + Boldine mice. Scale bar: 50 µm.\u003cstrong\u003e B)\u003c/strong\u003eQuantification of the percentage of Oil Red O–positive myofibers per field. Data represent mean ± SEM (n = 4 mice per group). *p \u0026lt; 0.05, **p \u0026lt; 0.01, Tukey’s test. \u003cstrong\u003eC)\u003c/strong\u003e NLRP3 were evaluated by qPCR. n=4. **p\u0026lt;0.01 vs Control, Tukey test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/054b2880ded855af6a421a1a.png"},{"id":106725833,"identity":"3541509e-b996-42fb-96de-2f02c9356ed5","added_by":"auto","created_at":"2026-04-12 18:34:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentiation to muscle fate in high glucose increases membrane permeability of myoblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e The Etd\u003csup\u003e+\u003c/sup\u003e uptake assay was performed in myoblasts treated with 8 mM glucose (LG), 25 mM glucose (HG), HG plus 50 µM boldine, or LG plus 25 mM Mannitol. The graph represents the fluorescence intensity of Etd\u003csup\u003e+\u003c/sup\u003e uptake over time. Each point represents the average of ≥15 cells analyzed. \u003cstrong\u003eB)\u003c/strong\u003e Representative photographs of Etd\u003csup\u003e+\u003c/sup\u003e fluorescence (Red) provide a view of a field at the end of an experiment in each condition. \u003cstrong\u003eC)\u003c/strong\u003e Etd\u003csup\u003e+\u003c/sup\u003e uptake rate of myoblasts treated with different conditions. **p\u0026lt;0.01, Tukey´s Test. Data were obtained from four independent experiments with four repeats each (≥15 cells analyzed for each repeat).\u003cstrong\u003e D) \u003c/strong\u003eAfter 7 days, dye uptake was performed with different blockers: 50 µM boldine (Cx and Panx1 HCs); 100 nM D4 (Cx HC blocker); 200 µM \u003csup\u003e10\u003c/sup\u003ePanx1 peptide (Panx1 HC blocker); 10 µM A740003 (P2X7R blocker)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/37469eb3e76d57017827015b.png"},{"id":106517838,"identity":"129f012e-819d-4945-8835-d74346015aeb","added_by":"auto","created_at":"2026-04-09 12:14:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":205396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh glucose increases basal cytoplasmic Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e signal and generates high levels of nitric oxide.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMyoblasts (AB1167) were induced to acquire commitment for muscle differentiation at different concentrations of extracellular glucose. After 7 days, dye uptake was performed. \u003cstrong\u003eA)\u003c/strong\u003e The Etd\u003csup\u003e+\u003c/sup\u003e assay was performed in myoblast treated with HG alone or in myoblasts preincubated for 5 min with the following agents: 10 µM SB202190 (p38), 10 µM MRS2179 (P2Y1R), or 10 mM DTT (Cystine reducer). Some myoblast cultures were loaded with 10 µM BAPTA-AM (BAPTA: Ca\u003csup\u003e2+\u003c/sup\u003e chelator) before evaluating Etd\u003csup\u003e+\u003c/sup\u003e uptake. The effect of 200 µM La\u003csup\u003e3+\u003c/sup\u003e on the uptake of myoblast culture in HG (La\u003csup\u003e3+\u003c/sup\u003e + HG) was also evaluated. n=4. *p\u0026lt;0.05, **p\u0026lt;0.01 Tukey´s Test. Data were obtained from four independent experiments with four repeats each (≥15 cells analyzed for each repeat). \u003cstrong\u003eB) . \u003c/strong\u003eCa\u003csup\u003e2+\u003c/sup\u003e signal denoted as Fura-2 ratio (340/380 nm excitation) of myoblasts treated with different glucose concentrations. B) Basal intracellular Ca²⁺ signal (Fura-2 ratio, F/F₀) in cells treated with low glucose (LG), high glucose (HG) or HG plus 50 µM boldine (HG + Boldine). (n=4 with ≥15 cells recorder in each case). *p\u0026lt;0.05, **p\u0026lt;0.01 Tukey´s Test. \u003cstrong\u003eC)\u003c/strong\u003e Timelapse of Etd\u003csup\u003e+\u003c/sup\u003e uptake in myoblast treated with HG, before and after applying 10 mM DTT (arrow).\u0026nbsp; \u003cstrong\u003eD)\u003c/strong\u003e Representative micrographs of the basal level of nitric oxide (NO) in myoblast cultured in each condition. \u003cstrong\u003eE)\u003c/strong\u003e Bar graph representing the average of DAF fluorescence by myoblast cells treated as mentioned above. n=3. ***p\u0026lt;0.001, Tukey´s Test. (≥15 cells analyzed for each repeat).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/05bd65656631d7cf594c5bec.png"},{"id":106517839,"identity":"ac12afb9-2187-4c73-890b-6fe3a48616ed","added_by":"auto","created_at":"2026-04-09 12:14:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":34367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoldine prevents \u0026nbsp;increases in mRNA levels of inflammasome components.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMyoblasts were cultured in low (LG) or high (HG) glucose concentrations (8 and 25 mM, respectively), or HG plus boldine (HG + Boldine) in medium that induces the acquisition of skeletal muscle commitment. After 7 days in culture, the mRNA levels of \u003cstrong\u003eA)\u003c/strong\u003e NLRP3, and \u003cstrong\u003eB)\u003c/strong\u003e caspase-1 were evaluated by qPCR. n=3. *p\u0026lt;0.05 vs LG, Tukey test. Dashed lines indicate the mean of control values.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/d45484e90ac35d3cf848c38b.png"},{"id":106517840,"identity":"a8484c4f-e8b2-44ad-be51-7d1c296cdb39","added_by":"auto","created_at":"2026-04-09 12:14:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":437936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoldine prevents the acquisition of adipogenic commitment and differentiation to adipocyte-like cells of myoblast induced to acquire myogenic commitment in high glucose. \u003c/strong\u003eMyoblasts were incubated in low (LG) and high glucose (HG) conditions (8 and 25 mM, respectively) in the medium that induces differentiation along with myogenic differentiation medium in HG plus 50 µM boldine (HG + Boldine). After 7 days, they were fixed for immunofluorescence. \u003cstrong\u003eA) \u003c/strong\u003eAnalysis against PPAR (fuchsia) and nuclear staining with DAPI (blue). n=4 cell cultures.\u003cstrong\u003e B)\u003c/strong\u003eQuantification of PPARγ positive nuclei expressed as percentage (% PPARγ + nuclei) from 10 fields like in (A). n=4 cell cultures. ****p\u0026lt;0.0001, Tukey´s Test. \u003cstrong\u003eC)\u003c/strong\u003e Detection of triglyceride accumulation with oil Red O staining (arrow). n=4 cell cultures. Scale bar: 10 µm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/6be170a64ef158b5a8a2b658.png"},{"id":106959591,"identity":"0c7ca9d1-5fe5-4329-9acd-da1de5a0cd5f","added_by":"auto","created_at":"2026-04-15 09:11:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3086398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/28cb1df4-7af2-4836-9e17-0047a4cf1b4b.pdf"},{"id":106517833,"identity":"8a5bcee0-91b0-49e3-96fe-9d463f86c3a6","added_by":"auto","created_at":"2026-04-09 12:14:10","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":234212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn myoblasts, high glucose increases large-pore channel activity, elevating cytoplasmic Ca²⁺ concentration and nitric oxide generation, which contribute to increasing the activity of large-pore channels (including Cx-formed hemichannels and P2X7Rs), raising the levels of inflammasome components, and promoting lipid accumulation. In STZ-diabetic mice, \u003cem\u003ede novo\u003c/em\u003e expression of large-pore channels in skeletal muscles contributes to reducing the resting membrane potential of myofibers, as well as reducing the blood perfusion of muscles, muscle weakness, PPARɤ activation and the accumulation of intramuscular fat. Boldine inhibits large-pore channel activity and, consequently, prevents the above alterations, preserving skeletal muscle function \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-9284067/v1/ed2f82c2f2bda4caf29ebc55.png"}],"financialInterests":"","formattedTitle":"Boldine prevents diabetes-induced skeletal muscle dysfunction by inhibiting large-pore channels","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDiabetes mellitus (DM) is a globally prevalent chronic disease, characterized by insufficient insulin production or reduced capacity for its effective use [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One significant complication caused by DM is skeletal muscle myopathy (SMM), which is particularly relevant given that skeletal muscle is crucial for glucose uptake and its deterioration profoundly impacts whole-body glucose homeostasis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. SMM manifests as muscle atrophy, decreased regeneration capacity, and fat infiltration, with adult skeletal muscle satellite cells (muscle progenitors) being especially vulnerable to the diabetic environment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder normal conditions, satellite cells remain quiescent, but upon stimuli such as injuries, they proliferate into myoblasts committed to the myogenic lineage, eventually fusing into myotubes and mature myofibers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, high glucose exposure promotes adipogenic differentiation in skeletal muscle-derived stem cells, leading to ectopic intramuscular fat accumulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recent studies also implicate fibroadipogenic progenitors in this process, though the underlying molecular mechanisms are still poorly understood [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, the inactivation of connexin 43 (Cx43) and Cx45 expression in skeletal myofibers prevented lipid accumulation in a murine dysferlinopathy model, suggesting a key role for connexin-formed hemichannels in muscle fat infiltration and dysfunction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Connexins (Cxs) and pannexin1 (Panx1) form large-pore channels called hemichannels in the plasma membrane [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Cx39, Cx43, and Cx45 are expressed in skeletal muscle during progenitor cell proliferation and muscle fiber formation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and their aberrant expression as well as the upregulation of Panx1 hemichannels (Panx1 HCs) have been shown to contribute to various neuromuscular diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoldine, an alkaloid from \u003cem\u003ePeumus boldus\u003c/em\u003e, is known to block three large-pore channels [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and previous work has shown that it restores normal differentiation and improves muscle function in a dysferlinopathy model [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similar protection has been observed in mice with dysferlinopathy treated with pulverized boldo leaves [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we propose that large-pore channels are key elements in the pathological mechanism of SMM in streptozotocin-induced diabetes and in high glucose-induced aberrant adipogenic commitment during myogenic differentiation. We investigated whether boldine could prevent these diabetic myopathy features, including lipid accumulation, impaired muscle function, and local inflammation, thereby preserving skeletal muscle integrity in diabetic animals. These experiments aim to advance the therapeutic potential of boldine in diabetes-associated skeletal muscle dysfunction.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cem\u003eReagents\u003c/em\u003e. Dulbecco\u0026rsquo;s modified Eagle medium (DMEM), ethidium bromide (Etd\u003csup\u003e+\u003c/sup\u003e), and 4-Bromo A23187 were acquired from Sigma-Aldrich (St. Louis, MO, USA), and dithiothreitol (DTT), Fura-2AM, DAF-FM, SB203580, A740003, oil Red O, horse serum, and anti-PPAR-γ were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cy2- and Cy3-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The hydrochloride form of boldine was prepared as described previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Streptozotocin (STZ) and acetylcholine was purchased from Sigma-Aldrich (St. Louis, MO, USA). (R)-2-(4-chlorophenyl)-2-oxo-1-phenylethyl quinoline-2-carboxylate (C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eClNO\u003csub\u003e3\u003c/sub\u003e; MW 401.85 g.mol-1), called D4, was synthetized by Edelris s.a.s. Medicinal Keymistry (Lyon, France).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAnimals\u003c/strong\u003e \u003cp\u003eMale wildtype (WT) C57Bl/6 mice were used. Diabetes was induced after a 6-hour fasting period, followed by a single intraperitoneal injection of STZ, 40 mg/kg/day for 5 days. To confirm the development of diabetes, blood samples were collected from the tail two days after STZ administration. Mice were considered diabetic if their blood glucose levels were \u0026ge;\u0026thinsp;180 mg/dL. Glycaemia was measured using a portable glucometer (Accutrend sensor, Roche, Basel, Switzerland). All protocols were approved by the Bioethics Committee at Universidad de Valpara\u0026iacute;so (CBC 85-2023) in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments. All efforts were made to minimize animal suffering and to reduce the number of animals used, and \u003cem\u003ein vitro\u003c/em\u003e alternative techniques were implemented when possible.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThree experimental groups were included: Control (n\u0026thinsp;=\u0026thinsp;4), STZ-induced diabetes (STZ, n\u0026thinsp;=\u0026thinsp;4), and STZ-induced diabetes treated with boldine (STZ\u0026thinsp;+\u0026thinsp;Boldine, n\u0026thinsp;=\u0026thinsp;4). Diabetes was induced by intraperitoneal injections of STZ (40 mg/kg/day for 5 consecutive days). Boldine was administered orally at a dose of 50 mg/kg/day for four weeks by mixing the compound with peanut butter, which was voluntarily consumed by the animals. Control and STZ mice received peanut butter without boldine as vehicle control. All experimental groups were euthanized at the same age to avoid age-related confounding effects. We have previously shown that membrane permeability as well as the cross-sectional area of skeletal myofibers and glycemia levels of control mice are not affected by boldine [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], indicating that long term treatment with boldine does not significantly affect skeletal muscles. Therefore, we did not include an additional group of control mice treated with boldine.\u003c/p\u003e \u003cp\u003e \u003cem\u003eForelimb muscle strength.\u003c/em\u003e Muscle strength was assessed using a digital force transducer (GPM-100; Melquest, Toyama, Japan) with a triangular metal bar. Mice grasped the bar with their forepaws and were pulled backward until release. Peak force was recorded for each mouse, which underwent three consecutive trials per session, and the highest values used for analysis. Measurements were performed at the end of the treatment period in the three experimental groups (Control, STZ, and STZ\u0026thinsp;+\u0026thinsp;Boldine). All measurements were performed back-to-back by a group-blinded experimenter.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBlood perfusion\u003c/strong\u003e \u003cp\u003eMuscle blood flow was assessed \u003cem\u003ein vivo\u003c/em\u003e using Laser Speckle Contrast Imaging (LSCI) technology (Pericam\u0026reg; PSI-HR system, Perimed, Stockholm, Sweeden). Adult mice were anesthetized with 4% isoflurane. The skin over the right gastrocnemius muscle was removed, and microvascular blood flow was recorded from four predefined circular regions of interest (ROIs). The protocol included a 3\u0026ndash;4 min baseline recording, followed by a bolus of acetylcholine (10 \u0026micro;M) to induce endothelium-dependent vasodilation. Perfusion was monitored continuously, and time-of-interest windows (40 seconds) were extracted for basal and post-acetylcholine conditions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eData collection and analysis were conducted by two independent investigators (FT and HS). To minimize bias, experiments were performed in a randomized, back-to-back fashion, simultaneously including mice from all experimental groups. Data analysis was performed in a blinded manner. Changes in blood flow are reported in raw perfusion units as indicated previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIsolation of mouse skeletal myofibers\u003c/em\u003e: Myofibers were isolated from flexor digitorum brevis (FDB) muscles as previously described. FDB muscles were dissected, incubated in 0.2% collagenase type I, and then gently triturated to disperse single myofibers. Dissociated myofibers were washed and suspended in Krebs solution (in mM: 145 NaCl, 5 KCl, 3 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 5.6 glucose, 10 HEPES-Na, pH 7.4) with 10 \u0026micro;M N-benzyl-p-toluene sulphonamide (BTS) to inhibit contractions and reduce damage.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eResting membrane potential (RMP)\u003c/strong\u003e \u003cp\u003eRMP was recorded from myofibers of flexor digitorum brevis muscles in a whole-cell configuration at 25\u0026deg;C. RMP recording was performed on fibers kept in culture for maximum 48 h before the RMP dropped as a consequence of denervation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The pipette was a borosilicate electrode filled with 3 M KCl, and the bath solution was a Krebs-bicarbonate buffered solution at pH 7.4. Pipette resistance was approximately 50 Ω. All experiments were performed using an Olympus IX 51 inverted microscope, the Axopatch 1-D amplifier, and the Digidata 1322 digitizer as well as Clampex 9.1 acquisition programs. Data were analyzed using Clampfit 2.1.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMyoblast cultures.\u003c/em\u003e The human myoblast cell line (AB1167) that had been previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] was cultured in DMEM media containing low glucose concentration (LG; 8 mM). We used 8 mM glucose as the control condition, given that this concentration represents postprandial blood glucose levels in healthy individuals (\u0026le;\u0026thinsp;7.8 mM) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, cells were cultured in DMEM containing a high glucose concentration (HG; 25 mM), with or without 50 \u0026micro;M boldine. When cultures reached\u0026thinsp;~\u0026thinsp;70% of confluence, the media was replaced by differentiation media (DMEM 8 or 25 mM plus 5% horse serum) to promote the fusion of myoblasts and myotube formation. Differentiation medium was renewed every 48 h, and boldine (50 \u0026micro;M) was added at each medium renewal.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEvaluation of Etd\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003euptake\u003c/em\u003e. This was carried out as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, myoblast plated onto plastic culture dishes were washed twice with Krebs solution. For time-lapse measurements, myoblasts were incubated in Krebs solution containing 5 \u0026micro;M Etd\u003csup\u003e+\u003c/sup\u003e. The intensity of Etd\u003csup\u003e+\u003c/sup\u003e fluorescence was first recorded for 5 min in regions of interest by using a water immersion Olympus 51W1I upright microscope (Japan). Images were captured with a Retiga 13001 fast cooled monochromatic digital camera (12-bit; QImaging, Canada) every 15 s during 5 min, and image processing was performed offline with ImageJ software (National Institutes of Health).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIntracellular Ca\u003c/em\u003e \u003csup\u003e \u003cem\u003e2+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003esignal\u003c/em\u003e. Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e signals were evaluated in immortalized human myoblast loaded with FURA 2. To load FURA 2, myoblasts were incubated with 5 \u0026micro;M FURA 2-AM in DMEM without serum at 37\u0026deg;C for 30 min and then washed three times. Then, the Ca\u003csup\u003e2+\u003c/sup\u003e signals were evaluated using a Nikon Eclipse Ti microscope, and fluorescence emitted upon excitation at two wavelengths (340 and 380 nm) was calculated along with the ratio of the recorded emissions (340/380 ratio).\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunofluorescence analysis.\u003c/em\u003e Samples were fixed with 4% paraformaldehyde, incubated overnight at 4\u0026deg;C with diluted primary anti-PPAR-γ antibodies, followed by washes with PBS. Secondary antibodies conjugated to Cy2 or Cy3 were then applied for 1 hour. Samples were rinsed, mounted with fluoromount G (containing DAPI), and visualized on glass slides.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOil red O stain.\u003c/em\u003e Transverse cryosections of tibialis anterior muscle and cultured myoblasts were fixed with 4% paraformaldehyde in the presence of 180 mM CaCl₂. Oil Red O stock solution was diluted (3:2 with distilled water) to prepare the working solution, which was applied to samples for 30 min. Excess stains were removed, and samples were rinsed with water to visualize lipid droplets. We performed quantitative analysis of oil red O staining by calculating the stained area fraction using Python 3.12.11 OpenCV (version 4.9.0), NumPy (version 1.26.4), and Matplotlib libraries (version 3.9.2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eReverse transcription polymerase chain reaction (PCR).\u003c/em\u003e Total RNA was isolated from tissue or cells using TRIzol following the manufacturer\u0026rsquo;s instructions (Invitrogen, Waltham, MA, USA). Two microgram aliquots of total RNA were transcribed to cDNA using MMLV-reverse transcriptase (Fermentas, USA), and mRNA levels were evaluated by PCR amplification (GoTaq Flexi DNA polymerase; Promega, USA)\u003c/p\u003e \u003cp\u003eThe oligos used were the following: NLRP3: S 5\u0026acute;-GCTGGCATCTGGGGAAACCT-\u0026acute;3, AS 5\u0026acute;GCCCTTCTGGGGAGGATAGT-\u0026acute;3; CASP1 S 5\u0026acute;GAAAAGCCATGGCCGACAAG-\u0026acute;3, AS 5\u0026acute;-GCCCCTTTCGGAATAACGGA-\u0026acute;3. 18S: S 5\u0026prime;-TCAAGAACGAAAGTCGGAGG-\u0026prime;3, AS 5\u0026prime;-GGACATCTAAGGGCATCACA-\u0026prime;3.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analysis.\u003c/em\u003e Quantitative variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Considering the data distribution, we used parametric or non-parametric tests, as appropriate, using the Shapiro\u0026ndash;Wilk normality test. For multiple group comparisons during perfusion analysis, data are presented as mean values calculated from three different subjects per group. Multiple comparisons were performed using ordinary one-way ANOVA, followed by multiple comparisons by controlling the false discovery rate with the Benjamini and Hochberg method. In other experiments, data were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s multiple-comparison test and an appropriate normality test. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a statistically significant difference. Data and statistical analyses were performed using the Microsoft Excel database and GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBoldine improves muscle strength and resting membrane potential (RMP) in diabetic mice\u003c/h2\u003e \u003cp\u003eTo evaluate whether boldine preserves skeletal muscle function in diabetic mice, forelimb grip strength was measured at the end of the treatment period. STZ-treated mice exhibited significantly reduced grip strength compared to Controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), consistent with diabetes-induced muscle weakness. Notably, boldine treatment prevented this functional decline, and grip strength values were comparable to those observed in Control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess muscle function at the cellular level, we measured the RMP of freshly isolated skeletal muscle fibers. Fibers from STZ mice exhibited significant depolarization compared to Control fibers, which was prevented by 4 weeks of treatment with boldine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). All measurements derived from mice described below were performed after 4 weeks of treatment with boldine and age matched control animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBoldine maintains the acetylcholine-induced increase in blood perfusion\u003c/h3\u003e\n\u003cp\u003eWe next examined whether the loss of muscle strength in diabetic mice was associated with impaired endothelium-dependent microvascular perfusion, and whether boldine could restore this function. To do so, we assessed blood perfusion in the right gastrocnemius muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). At baseline, myofibers of STZ-induced diabetic mice exhibited\u0026thinsp;~\u0026thinsp;20% lower microvascular perfusion expressed as units of perfusion compared to Control myofibers (160.1 \u0026plusmn; 17.2 versus 199.1 \u0026plusmn; 13.8 units, respectively, p\u0026thinsp;=\u0026thinsp;0.053). Notably, this decline was not observed in STZ-induced diabetic mice treated with boldine (184.6 \u0026plusmn; 14.3 perfusion units, p\u0026thinsp;=\u0026thinsp;0.46).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then evaluated the response to acetylcholine (ACh), an endothelium-dependent vasodilator. As expected, ACh elicited a robust increase in perfusion in Control mice that tended to be biphasic. In contrast, diabetic mice showed a blunted ACh-mediated response, denoting endothelial dysfunction. Boldine restored ACh-induced increases in perfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), which is in line with the improvement in muscle function observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo investigate whether the boldine-induced improvement in endothelium-dependent tissue perfusion was accompanied by structural remodeling of the microvasculature, capillary density was assessed in gastrocnemius muscle sections by immunodetection of CD31, a specific marker of endothelial cells. Capillary-to-fiber ratio was used as an index of microvascular density and angiogenic capacity. STZ-induced diabetic mice exhibited a marked reduction in capillaries per muscle fiber compared with Control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E), indicating impaired skeletal muscle blood irrigation in the diabetic state. In contrast, boldine treatment significantly restored capillary density in diabetic muscles, as evidenced by an increased number of CD31⁺ structures per fiber relative to untreated STZ mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Collectively, these results indicate that boldine promotes microvascular remodeling in diabetic skeletal muscle and suggest that its beneficial effects on blood perfusion are associated with improved endothelial function and enhanced angiogenic responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBoldine prevents lipid accumulation and up-regulation of NLRP3 mRNA levels in skeletal myofibers of diabetic mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether boldine could prevent the lipid accumulation observed \u003cem\u003ein vitro\u003c/em\u003e in myoblasts exposed to high glucose, we analyzed cryosections of tibialis anterior muscles. Muscle sections were stained with Oil Red O to visualize neutral lipid droplets. In Controls, virtually no Oil Red O\u0026ndash;positive fibers were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, top panel). In contrast, myofibers of skeletal muscle from STZ-induced diabetic mice exhibited a marked increase in intracellular lipid accumulation (52.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6%), as evidenced by the presence of numerous red-stained myofibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Boldine treatment markedly reduced intracellular lipid accumulation in diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) to similar levels as those observed in Controls (Boldine: 15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% and Controls: 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results indicate that boldine prevents lipid infiltration into skeletal muscle fibers in diabetic mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe inflammasome in skeletal myofibers has been shown to be activated by hyperglycemia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To determine whether this alteration is also present \u003cem\u003ein vivo\u003c/em\u003e, we quantified NLRP3 mRNA expression in skeletal muscle from diabetic mice by qPCR. Skeletal muscle from STZ-treated mice exhibited a marked upregulation of NLRP3 expression (17.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81-fold vs. Control; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), consistent with inflammasome activation under diabetic conditions. Boldine treatment significantly attenuated NLRP3 overexpression, reducing transcript levels by approximately 50%. However, NLRP3 expression remained significantly elevated compared to Controls (9.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56-fold vs. Controls, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Together, these findings indicate that diabetes induces inflammasome activation in skeletal muscles, which is partially reversed by boldine.\u003c/p\u003e\n\u003ch3\u003eHigh glucose induces an increase in membrane permeability through large-pore channel-dependent mechanisms\u003c/h3\u003e\n\u003cp\u003eIn a previous study, we found that HG increases membrane permeability to Etd\u003csup\u003e+\u003c/sup\u003e in myofibers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Now, we investigated whether HG could increase the activity of large-pore channels in myoblasts. Cells were cultured with media containing LG (8 mM), HG (25 mM), or HG plus 50 \u0026micro;M boldine. Cell membranes showed low Etd⁺ permeability due to minimal hemichannel opening. Glucose at 8 mM within postprandial levels does not alter myoblast permeability or differentiation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In contrast, pathological hemichannel upregulation increases Etd⁺ uptake, which becomes fluorescent upon nucleic acid binding. Here, we found that HG increases fluorescence intensity over time that was not promoted by LG (control condition), reflecting progressive Etd\u003csup\u003e+\u003c/sup\u003e uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Notably, boldine prevents elevated Etd\u003csup\u003e+\u003c/sup\u003e uptake generated by HG alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B) and, hence, cells differentiated under HG exhibited a significantly increased Etd⁺ uptake rate compared to those maintained in LG or HG supplemented with boldine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Importantly, this effect was not attributable to hyperosmolarity, as exposure to equiosmolar mannitol (25 mM) did not alter Etd⁺ uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating that the above-described changes observed in cells exposed to HG are not the consequence of hyperosmolarity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo define the contributions of candidate large-pore channels to Etd⁺ uptake, we employed pharmacological inhibitors targeting HCs and the P2X7 receptor (P2X7R). We used D4, which selectively blocks Cx45 and Cx43 HCs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], as well as A740003, which is a selective P2X7R antagonist [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Both inhibitors completely abolished the HG-induced increase in Etd⁺ uptake observed in myoblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), demonstrating that Cx HCs and P2X7R signaling are required for this response. In contrast, acute inhibition of Panx1 hemichannels with \u003csup\u003e10\u003c/sup\u003ePanx1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] did not result in a significant reduction of Etd⁺ uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), arguing against a major role for Panx1 channels. Collectively, these results indicate that the HG-induced increase in membrane permeability is mediated predominantly by Cx HCs rather than Panx1 CHs.\u003c/p\u003e\n\u003ch3\u003eHigh glucose activates large-pore channels through elevated intracellular Ca²⁺ and NO-dependent mechanisms\u003c/h3\u003e\n\u003cp\u003eActivation of Cx43 HCs and P2X7Rs has been linked to intracellular Ca\u0026sup2;⁺ elevation and purinergic signaling [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Given that increased intracellular Ca\u0026sup2;⁺ enhances Cx43 HC open probability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we tested whether Ca\u0026sup2;⁺ mobilization from intracellular stores contributed to HG-induced increase in membrane permeability. Myoblasts cultured under HG conditions were preincubated for 5 min with MRS2179 (10 \u0026micro;M), which is a selective P2Y1 receptor antagonist that prevents IP₃-mediated Ca\u0026sup2;⁺ release [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although MRS2179 tended to reduce Etd⁺ uptake, this effect did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), indicating that P2Y1-mediated Ca\u0026sup2;⁺ mobilization from intracellular stores does not play a major role in HG-induced membrane permeabilization. Previous studies have demonstrated that p38 MAPK enhances Cx43 HC activity in response to pro-inflammatory stimuli [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To assess the acute involvement of this pathway in HC regulation while minimizing secondary signaling effects, cells were preincubated for only 5 min with pharmacological inhibitors prior to Etd⁺ uptake measurements. Inhibition of p38 MAPK with SB202190 (10 \u0026micro;M) reduced Etd⁺ uptake to levels comparable to those observed under LG conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), implicating p38 MAPK signaling in HG-induced membrane permeabilization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further determine the contribution of intracellular Ca\u0026sup2;⁺ signaling, myoblasts were loaded with BAPTA-AM (5 \u0026micro;M) to chelate cytosolic Ca\u0026sup2;⁺. This intervention significantly reduced Etd⁺ uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), demonstrating that elevated intracellular Ca\u0026sup2;⁺ is required for HG-induced increases in membrane permeability. Consistently, La\u0026sup3;⁺, a broad inhibitor of Cx HCs and P2X7R [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], normalized Etd⁺ uptake in HG-treated cells up to LG levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), further supporting a role for large-pore channels in mediating hyperglycemia-induced permeabilization.\u003c/p\u003e \u003cp\u003eIntracellular Ca\u0026sup2;⁺ dynamics were then directly assessed using Fura-2 AM in myoblasts differentiated under LG, HG, or HG supplemented with boldine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Cells exposed to HG displayed a significantly elevated basal Ca\u0026sup2;⁺ signal relative to cells exposed to LG, whereas boldine fully prevented the increase exhibited by cells exposed to HG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Quantitatively, the mean basal Ca\u0026sup2;⁺ was highest in the HG group (1.0800\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0036), while values in LG and HG\u0026thinsp;+\u0026thinsp;Boldine were comparable (1.030\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 and 1.040\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, respectively) and significantly lower than HG alone.\u003c/p\u003e \u003cp\u003eIncreased cytoplasmic Ca\u0026sup2;⁺ levels are known to promote oxidative and nitrosative stress through the generation of reactive oxygen and nitrogen species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, S-nitrosylation of Cx43 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] in cysteine residue 271 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] has been shown to increase Cx HC activity. To evaluate whether redox-dependent regulation contributes to HC activation during myoblast differentiation under HG-conditions, cells were treated with the sulfhydryl reducing agent DTT (10 mM). The acute application of DTT rapidly and significantly decreased Etd⁺ uptake, reaching a stable minimum within seconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), consistent with reduced membrane permeability. These results indicate that Cx43 HC opening under HG involves redox-sensitive mechanisms.\u003c/p\u003e \u003cp\u003eGiven that HG increases nitric oxide synthase expression and nitric oxide (NO) production in cell cultures [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], we tested whether AB1167 cells treated with HG presented higher NO production. We found a significant increase in NO levels in cells cultured in HG, but not in cells incubated in LG or HG plus boldine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E). These results suggest that S-nitrosylation of Cx43 HCs might contribute to increased Edt\u003csup\u003e+\u003c/sup\u003e uptake induced by HG.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBoldine prevents increases in mRNA levels of inflammasome components triggered by high glucose\u003c/h2\u003e \u003cp\u003eBecause HG is known to activate the inflammasome in skeletal muscle cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we examined whether myoblasts differentiated under HG for 7 days showed evidence of inflammasome activation. Cells differentiated in HG exhibited higher mRNA levels of NLRP3 and caspase-1 compared to cells differentiated in LG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Notably, boldine completely prevented the increase in mRNA levels of NLRP3 and significantly prevented increases in caspase 1 mRNA levels (Fis. 6A, B). These findings indicate that HG promotes inflammasome-related gene expression during myoblast differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBoldine prevents high glucose–induced adipogenic drift during myogenic differentiation\u003c/h3\u003e\n\u003cp\u003eSkeletal muscles in adult diabetic men show intramuscular triglyceride accumulation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which could result in the decline of muscular strength, as observed in other pathologies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Here, we evaluated whether immortalized human myoblasts exposed to HG could differentiate to adipocytes as a possible explanation for intramuscular fat accumulation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter 7 days of differentiation, we found that HG elevated the nuclear content of the adipogenic transcription factor PPARγ, where ~\u0026thinsp;45% presented nuclear PPARγ reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). Notably, this response was prevented with boldine treatment (50 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). In addition, we observed the presence of triglyceride accumulation in several myoblasts cultured in differentiation media with HG (red droplets). Boldine treatment completely prevented this phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Thus, boldine blocked the aberrant differentiation of myoblasts to fat-containing cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSTZ treatment increased HC activity in skeletal muscles, accompanied by higher levels of inflammasome components, lipid accumulation, reduced muscle force, and impaired blood perfusion. High glucose similarly increased HC activity in myoblasts, elevated mRNA levels of inflammasome components, and promoted lipid accumulation. Boldine prevented these alterations in both models and preserved muscle function.\u003c/p\u003e \u003cp\u003eDiabetes-induced skeletal muscle weakness has been consistently observed in rodent models [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], an effect that was confirmed in this study showing reduced forelimb grip strength in STZ-induced diabetic mice. Importantly, boldine treatment fully prevented this decline. This functional impairment is closely associated with a significant depolarization of RMP in skeletal myofibers from diabetic mice, which is indicative of reduced membrane excitability. Such RMP depolarization has also been described for other muscle pathologies, such as denervation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and sepsis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], where connexin HCs contribute to excitability loss. Early studies similarly noted reduced RMP in genetically diabetic and alloxan-induced diabetic mice, with these changes correlating with blood glucose levels and disease duration [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our group previously demonstrated that diabetes induces \u003cem\u003ede novo\u003c/em\u003e expression and membrane redistribution of Cx39, Cx43, and Cx45 in skeletal muscle, a pathological pattern that boldine treatment effectively reversed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It is crucial to note that the genetic deletion of Cx43 and Cx45 in skeletal muscles significantly prevents diabetes-induced muscle atrophy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], strongly implicating Cx HCs in both the structural and functional decline of skeletal muscles under hyperglycemic conditions. Our current findings reinforce this evidence, showing that boldine not only prevents reductions in RMP and contractile strength but also preserves muscle mass and excitability by inhibiting HC activity, thereby targeting a unifying mechanism underlying diabetic myopathy.\u003c/p\u003e \u003cp\u003eBeyond reduced contractility and excitability, diabetic muscles display a marked inflammatory profile, particularly involving inflammasome activation. HG glucose-mediated inflammasome activation has been well-documented in diabetic patients, who exhibit elevated plasma levels of IL-1β and IL-18. \u003cem\u003eIn vitro\u003c/em\u003e studies, including our previous report, have shown greater NLRP3 levels in skeletal myofibers treated with HG [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e studies in STZ-induced diabetic mice and db/db mice have also demonstrated upregulations of NLRP3 and caspase-1 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our data further demonstrated that blocking connexin HC with boldine can significantly prevent increases in mRNA levels of NLRP3 in diabetic mice, as well as increases in mRNA of NLRP3 and caspase-1 in myoblasts differentiated in HG, suggesting a direct involvement of connexin HCs in inflammasome activation in this context.\u003c/p\u003e \u003cp\u003eAnother prominent hallmark of diabetic myopathy is intramuscular lipid accumulation. Consistent with previous findings in diabetic rats [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the STZ-induced diabetic mice used in the present work exhibited a marked lipid accumulation in tibialis anterior muscle fibers. Boldine treatment significantly reduced the number of lipid-positive myofibers in diabetic mice, reaching levels observed in non-diabetic Controls. This effect aligns with prior work in a dysferlinopathy model, where boldine administration prevented intramuscular fat accumulation and reduced PPARγ expression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our \u003cem\u003ein vitro\u003c/em\u003e findings further demonstrated that boldine prevents HG\u0026ndash;induced lipid accumulation in human myoblasts undergoing myogenic differentiation, extending its protective role against pathological ectopic fat deposition. In parallel, the restoration of muscle perfusion and microvascular network organization observed \u003cem\u003ein vivo\u003c/em\u003e in boldine-treated diabetic mice highlights its systemic vasoprotective actions, which would likely contribute to improving muscle integrity and functional capacity.\u003c/p\u003e \u003cp\u003eTo understand the underlying molecular mechanisms, our study revealed that HG induces an aberrant adipogenic commitment in myoblasts, a process found to involve large-pore channel activation by a mechanism sensitive to boldine [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Boldine prevented HG-induced increases in myoblast membrane permeability, an effect also observed with acute boldine pre-incubation, indicating a direct channel-blocking action. By using more specific blockers, we identified Cx HCs and P2X7Rs as the main mediators of this permeability increase. In contrast, blocking Panx1 HCs and P2Y1Rs caused non-significant effects. P2Y1Rs undergo desensitization under conditions of sustained extracellular ATP release [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which may explain these results. The findings support a positive feedback mechanism in which ATP released through Cx HCs activates P2X7Rs, promoting Ca\u0026sup2;⁺ entry. Given that P2X7Rs, as well as Cx43 and Cx45 HCs are permeable to Ca\u0026sup2;⁺ [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], this rise in intracellular Ca\u0026sup2;⁺ further enhances Cx43 HC activity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn line with the critical role of intracellular free-Ca\u0026sup2;⁺, HG significantly increased basal cytoplasmic Ca\u0026sup2;⁺ signals, which were effectively reduced by the Ca\u0026sup2;⁺ chelator BAPTA. Additionally, p38 MAPK activation, which previously was linked to Cx43 HC activity in inflammatory contexts, was found to boost Etd\u003csup\u003e+\u003c/sup\u003e uptake in human myoblasts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, high NO production in diabetes, caused by HG-induced upregulation of NO synthase [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], would explain the elevated NO levels detected in HG-exposed myoblasts. NO enhances Cx43 HC activity through S-nitrosylation of cysteine residues [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and the reversal of HG-induced permeability by DTT supports the involvement of S-nitrosylation in heightening Cx43 HC activity detected in myoblasts induced to undergo muscle differentiate in HG.\u003c/p\u003e \u003cp\u003eElevated Cx HC activity also contributes to increased reactive oxygen species (ROS) production, which can be mitigated by HC inhibitors or antioxidants. Since Cx43 HCs are permeable to Ca\u0026sup2;⁺ [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], blocking these channels may prevent the activation of Ca\u0026sup2;⁺-dependent pathways responsible for ROS generation. Boldine possesses intrinsic antioxidant properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], suggesting that its dual action (i.e., blockade of Cx HCs and reduction of oxidative stress) offers a comprehensive therapeutic approach to diabetes-related myopathy.\u003c/p\u003e \u003cp\u003eHG-induced adipogenic differentiation of muscle-derived stem cells may be directly linked to the greater ROS production observed in this study [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Elevated oxidative stress has been shown to transform myoblasts into brown adipocytes by reducing MyoD expression via NF-κB activation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our findings that boldine prevented HG-induced increases in PPARγ levels and lipid accumulation in myoblasts are consistent with this mechanism. Given that boldine significantly reduces fat infiltration in dysferlinopathy models [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], we propose that connexin HC represents a common therapeutic target to limit fat infiltration in muscle diseases characterized by metabolic or inflammatory stress, including diabetes, obesity, dysferlinopathy and Duchenne muscular dystrophy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our data identified large-pore channels as key drivers of diabetes-associated skeletal muscle dysfunction and demonstrated that boldine prevents muscle function alteration, vascular reactivity, and metabolic and inflammatory homeostasis \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. These findings highlight the potential efficacy of large-pore channel inhibition as a disease-modifying strategy, positioning boldine as a promising therapeutic candidate for diabetic myopathy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATP: Adenosine triphosphate\u003cbr\u003e\u0026nbsp;Ca\u0026sup2;⁺: Calcium ion\u003cbr\u003e\u0026nbsp;CD31: Cluster of differentiation 31\u003cbr\u003e\u0026nbsp;Cx: Connexin\u003cbr\u003e\u0026nbsp;Cx43: Connexin 43\u003cbr\u003e\u0026nbsp;Cx45: Connexin 45\u003cbr\u003e\u0026nbsp;Etd⁺: Ethidium bromide\u0026nbsp;\u003cbr\u003e\u0026nbsp;HCs: Hemichannels\u003cbr\u003e\u0026nbsp;HG: High glucose\u003cbr\u003e\u0026nbsp;LG: Low glucose\u003cbr\u003e\u0026nbsp;NLRP3: NOD-like receptor family pyrin domain-containing 3\u003cbr\u003e\u0026nbsp;NO: Nitric oxide\u003cbr\u003e\u0026nbsp;P2X7R: Purinergic receptor P2X7\u003cbr\u003e\u0026nbsp;P2Y1R: Purinergic receptor P2Y1\u003cbr\u003e\u0026nbsp;Panx1: Pannexin 1\u003cbr\u003e\u0026nbsp;PPAR\u0026gamma;: Peroxisome proliferator-activated receptor gamma\u003cbr\u003e\u0026nbsp;RMP: Resting membrane potential\u003cbr\u003e\u0026nbsp;STZ: Streptozotocin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eAll animal procedures were approved by the Bioethics Committee of the Universidad de Valparaíso (Approval No. CBC 85-2023). Experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll data supporting the findings of this study are included in the manuscript and its Supporting Information files. Additional datasets are available from the corresponding author, Prof. Juan C. Sáez, upon reasonable request and subject to institutional and ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was partially funded by the following grants from ANID: 1231523 (to J.C.S.), Millenium Institute ICN2025_026 CINV (to J.C.S.), 1240295 (C.E.), GI2301146 (to C.E.), ACT210057 (to X. F.), as well as Doctoral fellowships from ANID (to W.V. and A.L.). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions:\u0026nbsp;\u003c/strong\u003eW.V. and J.C.S. conceived and designed research. W.V., A.L., F.T., H.S., and L.A.C. performed experiments. W.V., A.L., F.T., H.S., and L.A.C. analyzed data. W.V., C.E., and J.C.S. interpreted results of experiments. W.V., A.L., F.T., H.S., and L.A.C. prepared figures. W.V. drafted manuscript. J.C.S., X.F.F., and C.E. edited and revised manuscript. All authors approved final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe experimental data in this paper were drawn from a thesis submitted in partial fulfillment of the requirements for the Doctorate in Biological Sciences, with a mention in Physiological Science (W.V.) at Pontificia Universidad Católica de Chile.The authors used ChatGPT (OpenAI, GPT-4) for language editing and manuscript refinement. The tool was used in a manner that does not conflict with APS ethical policies and the authors take full responsibility for the content.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntar SA, Ashour NA, Sharaky M, Khattab M, Ashour NA, Zaid RT et al (2023) Diabetes mellitus: Classification, mediators, and complications; a gate to identify potential targets for the development of new effective treatments. 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BMC Cell Biol 17(Suppl 1):15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12860-016-0096-6\u003c/span\u003e\u003cspan address=\"10.1186/s12860-016-0096-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PPARγ, lipid accumulation, inflammation, myopathy, sarcolemma permeability, hemichannel blocker","lastPublishedDoi":"10.21203/rs.3.rs-9284067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9284067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDiabetes mellitus is associated with skeletal muscle dysfunction, including reduced strength, impaired perfusion, lipid accumulation, and inflammation. Activation of large-pore channels increases membrane permeability and inflammatory signaling. Boldine, an alkaloid from \u003cem\u003ePeumus boldus\u003c/em\u003e, inhibits these channels and exhibits antioxidant and anti-inflammatory properties. This study evaluated whether boldine prevents diabetes-induced skeletal muscle alterations and explored underlying mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDiabetes was induced in male C57BL/6J mice using streptozotocin (40 mg/kg/day for 5 days), followed by boldine treatment (50 mg/kg/day, 4 weeks). Muscle strength, resting membrane potential, and gastrocnemius perfusion were assessed. Lipid accumulation, capillary density, and NLRP3 mRNA were analyzed. Human myoblasts (AB1167) under low or high glucose with or without boldine were evaluated for membrane permeability, intracellular Ca\u0026sup2;⁺, nitric oxide, inflammasome-related gene expression, and PPARγ reactivity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDiabetic mice exhibited reduced muscle strength, membrane depolarization, and ~\u0026thinsp;20% lower basal perfusion, all prevented by boldine. Lipid accumulation increased to 52.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6% in diabetic muscle and decreased to 15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% with boldine (control: 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NLRP3 mRNA levels increased 17.7-fold and was reduced by ~\u0026thinsp;50% with treatment. In vitro, high glucose increased ethidium uptake, Ca\u0026sup2;⁺, nitric oxide production, inflammasome-related gene levels, and nuclear PPARγ localization; all were attenuated by boldine.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBoldine preserves skeletal muscle function, vascular reactivity, and metabolic homeostasis in diabetes, preventing lipid accumulation and inflammasome activation. These effects involve inhibition of large-pore channels, reducing membrane permeability and Ca\u0026sup2;⁺-dependent inflammatory signaling, highlighting their role as therapeutic targets in diabetes-induced muscle dysfunction.\u003c/p\u003e","manuscriptTitle":"Boldine prevents diabetes-induced skeletal muscle dysfunction by inhibiting large-pore channels","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 12:14:05","doi":"10.21203/rs.3.rs-9284067/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2026-05-15T04:55:01+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-04-06T13:17:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-03T13:20:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T12:51:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2026-03-31T16:12:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91ce6738-6b29-420b-b4f8-14596c7af203","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Major Revision","date":"2026-05-15T04:55:01+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T09:28:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 12:14:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9284067","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9284067","identity":"rs-9284067","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}