Airway remodeling (AR) is a hallmark of chronic respiratory diseases (CRDs) and involves progressive structural alterations of the airway wall, leading to wall thickening and airway narrowing. Multiple cell types contribute to AR, including lung fibroblasts, which - under the influence of transforming growth factor type β (TGF-β) - upregulate pro-fibrotic markers, undergo phenotypic transition into myofibroblasts, and increase their contractile activity. Current treatment guidelines for CRDs lack effective therapies directly targeting AR. Recent reports suggest that the transient receptor potential ankyrin 1 (TRPA1) ion channel may be implicated in the mechanisms underlying AR. To explore this, we designed and synthesized 1,3,5-triazine-based derivatives (compounds 7 and 8), which exhibit agonistic activity towards TRPA1. In this study, we evaluated their pharmacological activity in TGF-β-stimulated human lung fibroblasts (MRC-5) by assessing calcium influx, pro-fibrotic markers expression, and contractile properties. Both compounds induced a robust calcium influx in MRC-5 cells, while the TRPA1 inhibitors and TGF-β stimulation attenuated this response. The tested 1,3,5-triazine-based TRPA1 activators decreased the expression of selected pro-fibrotic markers, such as α-smooth muscle actin (α-SMA), transgelin and collagen type I, reduced myofibroblasts number, and impaired the contractility of TGF-β-stimulated MRC-5 cells. Notably, the anti-fibrotic effects of the 1,3,5-triazine-based TRPA1 activators exceeded those of ASP-7663, a well-characterized TRPA1 agonist. Our findings expand current knowledge on TRPA1 modulation in AR and provide a novel perspective for drug development in CRDs.

Abstract Necrotic death of pancreatic acinar cells leads to premature activation of digestive enzymes and is a central driver of inflammation in acute pancreatitis (AP). In contrast, apoptosis is a non-lytic, regulated form of cell death that prevents the release of intracellular content and is therefore considered less damaging. The induction of the intrinsic apoptotic pathway is tightly regulated by the balance between pro- and anti-apoptotic members of the BCL2 protein family. Pharmacological inhibitors of anti-apoptotic BCL2 proteins – BH3 mimetics – have thus emerged as tools to therapeutically shift cell fate from necrosis to apoptosis. Early-generation BH3 mimetics such as HA14-1 and BH3I-2′ used to validate this concept were hindered by limited potency and significant off-target effects, including disruption of calcium homeostasis. More selective and potent second-generation agents – particularly Navitoclax (ABT-263) and Venetoclax (ABT-199) – have overcome these limitations, with Venetoclax now approved for clinical use in haematological malignancies. Here, we explore the therapeutic potential of BCL2 inhibitors in pancreatic diseases, with a focus on AP and a comparison to chronic pancreatitis (CP). In AP models, BCL2 inhibition – especially with Venetoclax – redirects cell death from necrosis to apoptosis, preserving intracellular ATP, attenuating aberrant Ca 2+ signalling, and reducing tissue damage and inflammation, without overt toxicity to healthy acinar cells in the absence of injurious stimuli. In contrast, in CP, a disease marked by fibrosis and prevailing apoptotic loss of parenchyma, BCL2 inhibition provides no benefit and may exacerbate acinar atrophy and fibrogenesis. We discuss the mechanistic basis of these divergent effects, including the role of BCL2 proteins in cell death regulation and calcium dynamics. Finally, we highlight future directions, including the rationale for clinical testing of Venetoclax in AP, and discuss cautionary considerations for its use in CP, particularly in patients with coexisting conditions such as leukaemia, who may already be receiving BH3 mimetics.

Pancreatic fibrosis is a key pathological feature of chronic pancreatitis and pancreatic cancer, driven by the persistent activation of pancreatic stellate cells. These cells, normally quiescent, undergo profound phenotypic changes in response to environmental cues, yet the interplay between mechanical forces and metabolic reprogramming during this transition remains poorly understood. As the stromal microenvironment actively communicates with epithelial and vascular compartments, understanding this mechano-metabolic signalling axis is critical for uncovering novel mechanisms of tissue remodelling. To investigate the biomechanical and biochemical alterations during stellate cell activation, we employed atomic force microscopy and Raman spectroscopy to measure changes in cell stiffness, morphology, and molecular composition. These data were complemented by transcriptomic analyses to evaluate gene expression profiles related to lipid metabolism and autophagy. Quantitative statistical tests, including ANOVA and Kruskal-Wallis tests with appropriate post hoc corrections, were applied. Activation of human pancreatic stellate cells led to progressive cytoskeletal remodelling, increased cellular stiffness, and a flattened morphology. Raman spectroscopy revealed an expansion of the cytoplasmic area, changes in nucleic acid signal, and significant increases in lipid content, particularly in unsaturated lipids and triacylglycerols. Gene expression analysis demonstrated upregulation of lipid elongation and desaturation pathways, along with enhanced autophagy, suggesting a coordinated metabolic adaptation. These changes support the myofibroblast-like phenotype and may influence intercellular signalling by altering extracellular matrix composition, mechanical tension, and the release of signalling molecules that affect the surrounding microenvironment. Our findings reveal that pancreatic stellate cell activation involves a tightly coupled shift in mechanical and metabolic states, highlighting an integrated signalling process that may modulate stromal–vascular and stromal–epithelial communication. This mechano-metabolic axis represents a potential therapeutic target in fibrotic and neoplastic pancreatic diseases, where aberrant stromal signalling contributes to disease progression.

The mammalian diffuse stellate cell system comprises retinoid-storing cells capable of remarkable transformations from a quiescent to an activated myofibroblast-like phenotype. Activated pancreatic stellate cells (PSCs) attract attention owing to the pivotal role they play in development of tissue fibrosis in chronic pancreatitis and pancreatic cancer. However, little is known about the actual role of PSCs in the normal pancreas. These enigmatic cells have recently been shown to respond to physiological stimuli in a manner that is markedly different from their neighbouring pancreatic acinar cells (PACs). Here, we demonstrate the capacity of PSCs to generate nitric oxide (NO), a free radical messenger mediating, for example, inflammation and vasodilatation. We show that production of cytosolic NO in PSCs is unambiguously related to cytosolic Ca(2+) signals. Only stimuli that evoke Ca(2+) signals in the PSCs elicit consequent NO generation. We provide fresh evidence for the striking difference between signalling pathways in PSCs and adjacent PACs, because PSCs, in contrast to PACs, generate substantial Ca(2+)-mediated and NOS-dependent NO signals. We also show that inhibition of NO generation protects both PSCs and PACs from necrosis. Our results highlight the interplay between Ca(2+) and NO signalling pathways in cell-cell communication, and also identify a potential therapeutic target for anti-inflammatory therapies.

Acute pancreatitis (AP), a severe inflammatory disorder of the pancreas, lacks effective pharmacological treatment. The disease is primarily driven by necrosis of pancreatic acinar cells (PACs), which intensifies inflammation and organ injury. This study explores the potential of BCL2 inhibitors, specifically Navitoclax and Venetoclax, to shift cell death pathways from necrosis to apoptosis and thereby mitigate disease severity. Ex vivo models using cerulein or ethanol/palmitoleic acid (EtOH/POA) showed that both inhibitors significantly reduced necrosis, increased apoptosis, and improved PAC viability and ATP levels. In mouse models of AP, both drugs promoted apoptosis and decreased tissue necrosis, with Venetoclax showing superior efficacy and safety. Venetoclax markedly reduced disease severity in two AP models without affecting healthy tissue or inducing thrombocytopenia. In contrast, Navitoclax caused apoptosis even in healthy tissue and triggered thrombocytopenia. Additionally, both drugs attenuated pathological Ca2+ responses in PACs and upregulated the expression of Ca²⁺-binding proteins S100A8/A9 and the chemokine CCL8. The latter may mediate enhanced apoptotic clearance and limit secondary necrosis, supporting the therapeutic shift from necrosis to apoptosis. Proteomic analyses revealed extensive drug-induced remodeling. In the short-term AP model, both inhibitors altered expression of proteins linked to intracellular compartments and extracellular signaling, reflecting cellular adaptation. In CP, Navitoclax upregulated ECM and lysosomal proteins while downregulating ribosomal components—indicating intensified fibrosis and suppressed protein synthesis. Venetoclax had milder effects and did not worsen fibrosis. Despite Navitoclax’s efficacy toward activated pancreatic stellate cells in vitro, it exacerbated fibrosis and tissue atrophy in CP in vivo, likely due to ongoing parenchymal damage and stellate cell activation. Together, these findings demonstrate that selective BCL2 inhibition with Venetoclax promotes apoptosis, reduces necrosis, and improves outcomes in AP, supporting its repurposing as a therapeutic strategy. However, BCL2 inhibition does not benefit CP and may aggravate fibrosis, underscoring the need for context-specific approaches.

Excessive Hedgehog (Hh) signaling activity contributes to fibrosis in multiple organs. However, its role in pancreatic stellate cell (PSC) activation and fibrosis development during chronic pancreatitis (CP) remains elusive. We show that GLI2 is one of the top-ranked effectors in the pancreas of CP patients and is highly expressed in activated PSCs. PSC-specific deletion of Gli2, but not Smo, significantly reduces fibrosis and the severity of the mouse CP, indicating that GLI2 in PSCs can be driven by non-canonical fashion during CP. In culture-activated primary PSCs, early nuclear translocation and increased GLI2 expression are observed promptly following in vitro culture. Whereas GLI2 inhibition reduces PSC activation, SMO inhibition dose not consistently affect changes in GLI2 levels or PSC activation. TGF-β1 promotes GLI2 activation and expression, while these processes and resultant PSC activation are reversed by TGF-β1/SMAD3 inhibition. Altogether, these findings demonstrate the activation of the non-canonical Hh pathway in PSCs during CP and highlight that GLI2 represents a promising therapeutic target for CP.

With the challenge of limited early stage detection and a resulting five-year survival rate of only 13%, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Replacing the high-cost and time-consuming grading of pancreatic samples by pathologists with automated diagnostic approaches can revolutionize PDAC detection and thus accelerate patient admission into the clinical setting for treatment. To address this unmet diagnostic need and facilitate the shift of tissue screening toward automated systems, we combined stain-free histology-specifically, Fourier-transform infrared (FT-IR) imaging-with machine learning. The obtained stain-free model was trained to distinguish between normal, benign, and malignant areas in analyzed specimens using hematoxylin and eosin stained pancreatic tissues isolated from KC (KrasG12D/+; Pdx1-Cre) or KPC mice (KrasG12D/+; Trp53R172H/+; Pdx1-Cre). Due to the pancreas-specific mosaic expression of the mutant Kras and Trp53 genes, changes in pancreatic tissues of this mouse model of PDAC closely mirror the gradual transformation of normal pancreatic epithelia into (pre)malignant structures. Thus, this mouse model provides a reliable representation of human disease progression, which we tracked in our study with a Random Forest classifier to achieve accurate detection at the cellular level. This approach yielded a comprehensive model that distinguishes normal pancreatic tissues from pathological features such as pancreatic intraepithelial neoplasia (PanIN), cancerous regions, hemorrhages, and collagen fibers, as well as a streamlined model designed to rapidly identify normal tissues versus pathologically altered regions, including PanINs. These models offer highly accurate diagnostic tools for the early detection of pancreatic malignancies, thus significantly improving the chance for timely therapeutic intervention against PDAC.

VDAC1, a large conductance channel in the outer mitochondrial membrane, plays a crucial role in mitochondrial physiology. VDAC1 supports cellular metabolism and survival by serving as a mitochondrial Ca2+-uptake and ATP-exit system. Conversely, VDAC1 also contributes to apoptosis by forming oligomeric pores mediating cytochrome c release. Recently, Oflaz et al., EMBO J, 2025, identified the Ca2+-binding protein Annexin A5 as a dynamic, Ca2+-dependent switch that enhances VDAC1’s Ca2+-transport function while at the same time preventing pro-apoptotic VDAC1 oligomer formation.

Purpose: Given the increased use of polyethylene glycol (PEG) in refining the therapeutic activity of medicines, our research focuses on explaining the potential mechanism of immune reactions associated with this polymer. We aim to investigate the interaction of different types of PEG with mouse and human immune cells, thereby contributing to understanding PEG interactions with the immune system and verifying the proinflammatory activity of the tested polymers. Patients and Methods: Mouse macrophage and neutrophil cell lines, human peripheral blood mononuclear cells, and polymorphonuclear cells isolated from healthy donors were exposed to various PEGs. ROS, NO, and cytokine production were analyzed using fluorescence intensity, absorbance, or cytometric measurements. Toll-like receptor (TLR) signaling was verified using HEK-bluereporter cell lines. Finally, neutrophil trap formation was studied using immunofluorescence labeling, and calcium imaging was performed using a Ca2+-sensitive indicator and fluorescence microscope. Results: Our findings show that specific PEG and mPEG are not toxic to tested mouse and human cells. However, they exert proinflammatory activity against human immune cells, as evidenced by the increased secretion of proinflammatory cytokines, such as IFN-a2, IFN-gamma, TNF-alpha, MCP-1, IL-8, IL-17A, and IL-23. This phenomenon is independent of PEG signaling via TLR. Additionally, mPEG10 induced the formation of neutrophil extracellular traps and intracellular calcium signaling. Conclusion: Our finding suggests that some PEG types have proinflammatory activity against human immune cells, manifesting in the upregulated production of cytokines and neutrophils trap releasing.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca2+ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN(+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin.

Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations-KrasG12D and Trp53R172H-present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since Ca2+ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular Ca2+ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited Ca2+ signals of different frequencies in KPC cells compared to control cells. Finally, Ca2+ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent.

Although severe abdominal pain is the main symptom of acute pancreatitis, its mechanisms are poorly understood. An emerging body of literature evidence indicates that neurogenic inflammation might play a major role in modulating the perception of pain from the pancreas. Neurogenic inflammation is the result of a crosstalk between injured pancreatic tissue and activated neurons, which leads to an auto-amplification loop between inflammation and pain during the progression of acute pancreatitis. In this review, we summarize recent findings on the role of neuropeptides, ion channels, and the endocannabinoid system in acute pancreatitis-related pain. We also highlight potential therapeutic strategies that could be applied for managing severe pain in this disease.

Abstract Background Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations. Methods EVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis. Results We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFβ/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue. Conclusions In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration. Graphical Abstract

Disorders such as pancreatic or hepatic fibrosis are a cruel reminder that disruption of the delicate physiological balance could result in severe pathological consequences. Fibrosis is usually associated with chronic diseases and manifests itself as excessive deposition of the extracellular matrix, which gradually leads to the replacement of the cellular components by fibrotic lesions, significantly compromising normal tissue functions. The main cellular mediators of fibrosis are different populations of tissue fibroblasts, predominantly hepatic and pancreatic stellate cells in the liver and pancreas, respectively. These cells undergo a phenotypic switch in response to (bio)chemical or physical stimuli and acquire a myofibroblast-like phenotype characterised by increased contractile and adhesive properties, elevated expression of certain cytoskeletal and membrane proteins, and prominent production of extracellular matrix components. In the past few decades, a substantial scientific effort has been undertaken to investigate the pathogenesis of fibrosis. Here, cellular mechanisms of hepatic and pancreatic fibrosis, their aetiological factors, associated diseases and prospective therapies are discussed. New therapies against fibrosis are likely to be focused on regulation of hepatic/pancreatic stellate cell physiology as well as normalisation of the organ mechanostasis.

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-β-activated PSCs, compare the expression of Ca2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca2+-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis.

Acute pancreatitis (AP) is a common digestive disease without specific treatment, and its pathogenesis features multiple deleterious amplification loops dependent on translation, triggered by cytosolic Ca2+ ([Ca2+](i)) overload; however, the underlying mechanisms in Ca2+ overload of AP remains incompletely understood. Here we show that microRNA-26a (miR-26a) inhibits pancreatic acinar cell (PAC) store-operated Ca2+ entry (SOCE) channel expression, Ca2+ overload, and AP. We find that major SOCE channels are post-transcriptionally induced in PACs during AP, whereas miR-26a expression is reduced in experimental and human AP and correlated with AP severity. Mechanistically, miR-26a simultaneously targets Trpc3 and Trpc6 SOCE channels and attenuates physiological oscillations and pathological elevations of [Ca2+](i) in PACs. MiR-26a deficiency increases SOCE channel expression and [Ca2+](i) overload, and significantly exacerbates AP. Conversely, global or PAC-specific overexpression of miR-26a in mice ameliorates pancreatic edema, neutrophil infiltration, acinar necrosis, and systemic inflammation, accompanied with remarkable improvements on pathological determinants related with [Ca2+](i) overload. Moreover, pancreatic or systemic administration of an miR-26a mimic to mice significantly alleviates experimental AP. These findings reveal a previously unknown mechanism underlying AP pathogenesis, establish a critical role for miR-26a in Ca2+ signaling in the exocrine pancreas, and identify a potential target for the treatment of AP.

Obesity-related acute pancreatitis (AP) is characterized by increasing prevalence worldwide and worse clinical outcomes compared to AP of other etiologies. Chaiqin chengqi decoction (CQCQD), a Chinese herbal formula, has long been used for the clinical management of AP but its therapeutic actions and the underlying mechanisms have not been fully elucidated. This study has investigated the pharmacological mechanisms of CQCQD in a novel mouse model of obesity-related alcohol-induced AP (OA-AP). The mouse OA-AP model was induced by a high-fat diet for 12 weeks and subsequently two intraperitoneal injections of ethanol, CQCQD was administered 2 h after the first injection of ethanol. The severity of OA-AP was assessed and correlated with changes in transcriptomic profiles and network pharmacology in the pancreatic and adipose tissues, and further docking analysis modeled the interactions between compounds of CQCQD and their key targets. The results showed that CQCQD significantly reduced pancreatic necrosis, alleviated systemic inflammation, and decreased the parameters associated with multi-organ dysfunction. Transcriptomics and network pharmacology analysis, as well as further experimental validation, have shown that CQCQD induced Nrf2/HO-1 antioxidant protein response and decreased Akt phosphorylation in the pancreatic and adipose tissues. In vitro, CQCQD protected freshly isolated pancreatic acinar cells from H2O2-elicited oxidative stress and necrotic cell death. The docking results of AKT1 and the active compounds related to AKT1 in CQCQD showed high binding affinity. In conclusion, CQCQD ameliorates the severity of OA-AP by activating of the antioxidant protein response and down-regulating of the PI3K/Akt signaling pathway in the pancreas and visceral adipose tissue.

microRNA-378a (miR-378a) is one of the most highly expressed microRNAs in the heart. However, its role in the human cardiac tissue has not been fully understood. It was observed that miR-378a protects cardiomyocytes from hypertrophic growth by regulation of IGF1R and the expression of downstream kinases. Increased levels of miR-378a were reported in the serum of Duchenne muscular dystrophy (DMD) patients and female carriers of DMD gene-associated mutations with developed cardiomyopathy. In order to shed more light on the role of miR-378a in human cardiomyocytes and its potential involvement in DMD-related cardiomyopathy, we generated two human induced pluripotent stem cell (hiPSC) models; one with deletion of miR-378a and the second one with deletion of DMD exon 50 leading to the DMD phenotype. Our results indicate that lack of miR-378a does not influence the pluripotency of hiPSC and their ability to differentiate into cardiomyocytes (hiPSC-CM). miR-378a-deficient hiPSC-CM exhibited, however, significantly bigger size compared to the isogenic control cells, indicating the role of this miRNA in the hypertrophic growth of human cardiomyocytes. In accordance, the level of NFATc3, phosphoAKT, phosphoERK and ERK was higher in these cells compared to the control counterparts. A similar effect was achieved by silencing miR-378a with antagomirs. Of note, the percentage of cells with nuclear localization of NFATc3 was higher in miR-378a-deficient hiPSC-CM. Analysis of electrophysiological properties and Ca2+ oscillations revealed the decrease in the spike slope velocity and lower frequency of calcium spikes in miR-378a-deficient hiPSC-CM. Interestingly, the level of miR-378a increased gradually during cardiac differentiation of hiPSC. Of note, it was low until day 15 in differentiating DMD-deficient hiPSC-CM and then rose to a similar level as in the isogenic control counterparts. In summary, our findings confirmed the utility of hiPSC-based models for deciphering the role of miR-378a in the control and diseased human cardiomyocytes.