Diabetes induces modifications in costameric proteins and increases cardiomyocyte stiffness.

Several studies have demonstrated that diabetes mellitus can increase the risk of cardiovascular disease and remains the principal cause of death in these patients. Costameres connect the sarcolemma with the cytoskeleton and extracellular matrix, facilitating the transmission of mechanical forces and cell signaling. They are related to cardiac physiology because individual cardiac cells are connected by intercalated discs that synchronize muscle contraction. Diabetes impacts the nanomechanical properties of cardiomyocytes, resulting in increased cellular and left ventricular stiffness, as evidenced in clinical studies of these patients. The question of whether costameric proteins are affected by diabetes in the heart has not been studied. This work analyzes whether type 1 diabetes mellitus (T1DM) modifies the costameric proteins and coincidentally changes the cellular mechanics in the same cardiomyocytes. The samples were analyzed by immunotechniques using laser confocal microscopy. Significant statistical differences were found in the spatial arrangement of the costameric proteins. However, these differences are not due to their expression. Atomic force microscopy was used to compare intrinsic cellular stiffness between diabetic and normal cardiomyocytes and obtain the first elasticity map sections of diabetic living cardiomyocytes. Data obtained demonstrated that diabetic cardiomyocytes had higher stiffness than control. The present work shows experimental evidence that intracellular changes related to cell-cell and cell-extracellular matrix communication occur, which could be related to cardiac pathogenic mechanisms. These changes could contribute to alterations in the mechanical and electrical properties of cardiomyocytes and, consequently, to diabetic cardiomyopathy.NEW & NOTEWORTHY The structural organization of cardiomyocyte proteins is critical for their efficient functioning as a contractile unit in the heart. This work shows that diabetes mellitus induces significant changes in the spatial organization of costamere proteins, t tubules, and intercalated discs. We obtained the first elasticity map sections of living diabetic cardiomyocytes. The results show statistical differences in the map sections of diabetic and control cardiomyocytes, with diabetic cardiomyocytes being stiffer than normal ones.

Hypoxic Cardioprotection by New Antihypertensive Compounds in High Salt-Diet Hypertensive Rats: Glucose Transport Participation and Its Possible Pathway.

Hypertension (HP) is a health condition that overloads the heart and increases the risk of heart attack and stroke. In an infarction, the lack of oxygen causes an exclusive use of glycolysis, which becomes a crucial source of ATP for the heart with a higher glucose uptake mediated by glucose transporters (GLUTs). Due to the unpleasant effects of antihypertensives, new drugs need to be researched to treat this disease. This study aimed to evaluate the cardioprotective effect of three novel antihypertensive compounds (LQMs, "Laboratorio de Química Medicinal") synthesized from Changrolin under hypoxic conditions with the participation of two primary cardiac GLUT1 and GLUT4 using a high-salt diet HP model. The model used a diet with 10% salt to increase arterial blood pressure in Wistar rats. In isolated cardiomyocytes from these rats, glucose uptake was measured during hypoxia, evaluating the participation of GLUTs with or without the animals' previous treatment with LQM312, 319, and 345 compounds. In silico calculations were performed to understand the affinity of the compounds for the trafficking of GLUTs. Results: Control cells do shift to glucose uptake exclusively in hypoxia (from 1.84 ± 0.09 µg/g/h to 2.67 ± 0.1 µg/g/h). Meanwhile, HP does not change its glucose uptake (from 2.38 ± 0.24 µg/g/h to 2.33 ± 0.26 µg/g/h), which is associated with cardiomyocyte damage. The new compounds lowered the systolic blood pressure (from 149 to 120 mmHg), but only LQM312 and LQM319 improved the metabolic state of hypoxic cardiomyocytes mediated by GLUT1 and GLUT4. In silico studies suggested that Captopril and LQM312 may mimic the interaction with the AMPK γ-subunit. Therefore, these compounds could activate AMPK, promoting the GLUT4 trafficking signaling pathway. These compounds are proposed to be cardioprotective during hypoxia under HP.

Activation of G Protein-Coupled Estrogen Receptor (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway.

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-ß-estradiol (E2, 10 nM) or G-1 (1 µM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 µM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

Cardiac Development Long Non-Coding RNA (CARDEL) Is Activated during Human Heart Development and Contributes to Cardiac Specification and Homeostasis.

Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac differentiation using human pluripotent stem cells (hPSCs) has been used to uncover the intricate gene-network regulation involved in the proper formation and function of the human heart. Here, we searched for uncharacterized cardiac-development genes by combining a temporal evaluation of human cardiac specification in vitro with an analysis of gene expression in fetal and adult heart tissue. We discovered that CARDEL (CARdiac DEvelopment Long non-coding RNA; LINC00890; SERTM2) expression coincides with the commitment to the cardiac lineage. CARDEL knockout hPSCs differentiated poorly into cardiac cells, and hPSC-derived cardiomyocytes showed faster beating rates after controlled overexpression of CARDEL during differentiation. Altogether, we provide physiological and molecular evidence that CARDEL expression contributes to sculpting the cardiac program during cell-fate commitment.

Esketamine alleviates hypoxia/reoxygenation injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

OBJECTIVE: This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. METHODS: The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 µM) or TRPV1 inhibitor capsazepine (1 µM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. RESULTS: After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. CONCLUSION: ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes.

In vitro cellular models provide valuable insights into the adaptive biochemical mechanisms triggered by cells to cope with the stress situation induced by hypoxia and reoxygenation cycles. The first biological data generated in studies based on this micrometric life-scale has the potential to provide us a global overview about the main biochemical phenomena presented in some reported preconditioning therapies in life-scale of higher dimensions. Thus, in this study, a cell incubator was designed and manufactured to produce a cellular model of heart hypoxia followed by reoxygenation (HfR) through consecutive repetitions of hypoxia-normoxia gas exchange. Samples of cellular extracts and culture media were obtained from non-proliferative cardiomyocytes (CMs) cultivated under challenging HfR (stressed CMs) and regular cultivation (unstressed CMs) in rounds of four days for each case. Metabolomic based on proton magnetic resonance spectroscopy (1H-MRS) was used as an analytical approach to identify and quantify the metabolomes of these samples, the endo- and exo-metabolome. Despite the stressed CMs presented over 90% higher cellular death rate compared to the unstressed CMs, the metabolic profiles indicates that the surviving cells up-regulate their amino acid metabolism either by active protein degradation or by the consumption of culture media components to increase coenzyme A-dependent metabolic pathways. This cell auto-regulation mechanism could be well characterized in the first two days when the difference smears off under once the metabolomes become similar. The metabolic adaptations of stressed CMs identified the relevance of the cyclic oxidation/reduction reactions of nicotinamide adenine dinucleotide phosphate molecules, NADP+/NADPH, and the increased tricarboxylic acid cycle activity in an environment overloaded with such a powerful antioxidant agent to survive an extreme HfR challenge. Thus, the combination of cellular models based on CMs, investigative methods, such as metabolomic and 1H-MRS, and the instrumental development of hypoxia incubator shown in this work were able to provide the first biochemical evidences behind therapies of gaseous exchanges paving the way to future assays.

In Vitro Modulation of Spontaneous Activity in Embryonic Cardiomyocytes Cultured on Poly(vinyl alcohol)/Bioglass Type 58S Electrospun Scaffolds.

Because of the physiological and cardiac changes associated with cardiovascular disease, tissue engineering can potentially restore the biological functions of cardiac tissue through the fabrication of scaffolds. In the present study, hybrid nanofiber scaffolds of poly (vinyl alcohol) (PVA) and bioglass type 58S (58SiO2-33CaO-9P2O5, Bg) were fabricated, and their effect on the spontaneous activity of chick embryonic cardiomyocytes in vitro was determined. PVA/Bg nanofibers were produced by electrospinning and stabilized by chemical crosslinking with glutaraldehyde. The electrospun scaffolds were analyzed to determine their chemical structure, morphology, and thermal transitions. The crosslinked scaffolds were more stable to degradation in water. A Bg concentration of 25% in the hybrid scaffolds improved thermal stability and decreased degradation in water after PVA crosslinking. Cardiomyocytes showed increased adhesion and contractility in cells seeded on hybrid scaffolds with higher Bg concentrations. In addition, the effect of Ca2+ ions released from the bioglass on the contraction patterns of cultured cardiomyocytes was investigated. The results suggest that the scaffolds with 25% Bg led to a uniform beating frequency that resulted in synchronous contraction patterns.

Alternative polyadenylation and dynamic 3' UTR length is associated with polysome recruitment throughout the cardiomyogenic differentiation of hESCs.

Alternative polyadenylation (APA) increases transcript diversity through the generation of isoforms with varying 3' untranslated region (3' UTR) lengths. As the 3' UTR harbors regulatory element target sites, such as miRNAs or RNA-binding proteins, changes in this region can impact post-transcriptional regulation and translation. Moreover, the APA landscape can change based on the cell type, cell state, or condition. Given that APA events can impact protein expression, investigating translational control is crucial for comprehending the overall cellular regulation process. Revisiting data from polysome profiling followed by RNA sequencing, we investigated the cardiomyogenic differentiation of pluripotent stem cells by identifying the transcripts that show dynamic 3' UTR lengthening or shortening, which are being actively recruited to ribosome complexes. Our findings indicate that dynamic 3' UTR lengthening is not exclusively associated with differential expression during cardiomyogenesis but rather with recruitment to polysomes. We confirm that the differentiated state of cardiomyocytes shows a preference for shorter 3' UTR in comparison to the pluripotent stage although preferences vary during the days of the differentiation process. The most distinct regulatory changes are seen in day 4 of differentiation, which is the mesoderm commitment time point of cardiomyogenesis. After identifying the miRNAs that would target specifically the alternative 3' UTR region of the isoforms, we constructed a gene regulatory network for the cardiomyogenesis process, in which genes related to the cell cycle were identified. Altogether, our work sheds light on the regulation and dynamic 3' UTR changes of polysome-recruited transcripts that take place during the cardiomyogenic differentiation of pluripotent stem cells.

25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

25-hydroxycholesterol (25-HC) plays a role in the regulation of cell survival and immunity. However, the effect of 25-HC on myocardial ischemia/reperfusion (MI/R) injury remains unknown. Our present study aimed to investigate whether 25-HC aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. The overlapping differentially expressed genes (DEGs) in MI/R were identified from the GSE775, GSE45818, GSE58486, and GSE46395 datasets in Gene Expression Omnibus (GEO) database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the database of Annotation, Visualization and Integration Discovery (DAVID). The protein-protein interaction (PPI) network of the overlapping DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These bioinformatics analyses indicated that cholesterol 25-hydroxylase (CH25H) was one of the crucial genes in MI/R injury. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate MI/R injury. Western blot and RT-qPCR analysis demonstrated that CH25H was significantly upregulated in OGD/R-stimulated H9C2 cardiomyocytes. Moreover, knockdown of CH25H inhibited the OGD/R-induced pyroptosis and nod-like receptor protein 3 (NLRP3) inflammasome activation, as demonstrated by cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), RT-qPCR, and western blotting assays. Conversely, 25-HC, which is synthesized by CH25H, promoted activation of NLRP3 inflammasome in OGD/R-stimulated H9C2 cardiomyocytes. In addition, the NLRP3 inhibitor BAY11-7082 attenuated 25-HC-induced H9C2 cell injury and pyroptosis under OGD/R condition. In conclusion, 25-HC could aggravate OGD/R-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cells.

Esketamine alleviates hypoxia/reoxygenation injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration

Abstract Objective This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. Methods The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 μM) or TRPV1 inhibitor capsazepine (1 μM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. Results After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. Conclusion ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

Comparative microRNA profiling of Trypanosoma cruzi infected human cells.

Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, can infect almost any nucleated cell in the mammalian host. Although previous studies have described the transcriptomic changes that occur in host cells during parasite infection, the understanding of the role of post-transcriptional regulation in this process is limited. MicroRNAs, a class of short non-coding RNAs, are key players in regulating gene expression at the post-transcriptional level, and their involvement in the host-T. cruzi interplay is a growing area of research. However, to our knowledge, there are no comparative studies on the microRNA changes that occur in different cell types in response to T. cruzi infection. Methods and results: Here we investigated microRNA changes in epithelial cells, cardiomyocytes and macrophages infected with T. cruzi for 24 hours, using small RNA sequencing followed by careful bioinformatics analysis. We show that, although microRNAs are highly cell type-specific, a signature of three microRNAs -miR-146a, miR-708 and miR-1246, emerges as consistently responsive to T. cruzi infection across representative human cell types. T. cruzi lacks canonical microRNA-induced silencing mechanisms and we confirm that it does not produce any small RNA that mimics known host microRNAs. We found that macrophages show a broad response to parasite infection, while microRNA changes in epithelial and cardiomyocytes are modest. Complementary data indicated that cardiomyocyte response may be greater at early time points of infection. Conclusions: Our findings emphasize the significance of considering microRNA changes at the cellular level and complement previous studies conducted at higher organizational levels, such as heart samples. While miR-146a has been previously implicated in T. cruzi infection, similarly to its involvement in many other immunological responses, miR-1246 and miR-708 are demonstrated here for the first time. Given their expression in multiple cell types, we anticipate our work as a starting point for future investigations into their role in the post-transcriptional regulation of T. cruzi infected cells and their potential as biomarkers for Chagas disease.

Functional defects in hiPSCs-derived cardiomyocytes from patients with a PLEKHM2-mutation associated with dilated cardiomyopathy and left ventricular non-compaction.

Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and ß-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient's iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time.

Morphological changes and Mitochondrial alterations on Cardiomyocytes exposed to Fluoride.

OBJECTIVE: To evaluate the morphological changes of cardiomyocytes exposed to different sodium fluoride (NaF) concentrations, as well as to evaluate the behavior of the mitochondria. METHODS: Rat H9c2 cardiomyocytes were exposed to NaF at concentrations of 0.5 to 5 mmol/L. The morphology and number of mitochondria in these cells were monitored, and the calcium ion (Ca2+) concentration was determined. RESULTS: Morphological changes were evident in the cells treated with different NaF concentrations, and both the number of mitochondria and the Ca2+ concentration decreased in a dose-dependent manner. CONCLUSION: Sodium fluoride induced morphological damage in cardiomyocytes, decreases the Ca2+ concentration and mitochondrial number.

ß-catenin ameliorates myocardial infarction by preventing YAP-associated apoptosis.

OBJECTIVE: To explore whether the effect of ß-catenin on MI and MI-induced cardiomyocyte apoptosis is YAP-dependent. METHODS: The authors established an MI rat model by ligating the anterior descending branch of the left coronary artery, and an MI cell model by treating cardiomyocytes with H2O2. RESULTS: ß-catenin downregulation was observed in MI cardiac tissues and in H2O2-treated cardiomyocytes. Lentiviral-CTNNB1 was administered to MI rats to upregulate ß-catenin expression in MI cardiac tissue. ß-catenin recovery reduced the myocardial infarct area, fibrosis, and apoptotic cell death in MI rats. H2O2 treatment attenuated cell viability and induced cell death in cardiomyocytes, whereas ß-catenin overexpression partially reversed these changes. Moreover, H2O2 treatment caused the deactivation of Yes-Associated Protein (YAP), as detected by increased YAP phosphorylation and reduced the nuclear localization of YAP. Upregulation of ß-catenin expression reactivated YAP in H2O2-treated cardiomyocytes. Reactivation of YAP was achieved by administration of Mitochonic Acid-5 (MA-5) to H2O2-treated cardiomyocytes, and deactivation of YAP by CIL56 treatment in ß-catenin-overexpressing H2O2-treated cardiomyocytes. MA-5 administration increased cell viability and repressed apoptosis in H2O2-treated cardiomyocytes, whereas CIL56 treatment counteracted the effects of ß-catenin overexpression on cell survival and apoptosis. CONCLUSIONS: The present data indicate that ß-catenin and YAP are effective treatment targets for MI, blocking the apoptotic death of cardiomyocytes.

IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes.

A physiological increase in cardiac workload results in adaptive cardiac remodeling, characterized by increased oxidative metabolism and improvements in cardiac performance. Insulin-like growth factor-1 (IGF-1) has been identified as a critical regulator of physiological cardiac growth, but its precise role in cardiometabolic adaptations to physiological stress remains unresolved. Mitochondrial calcium (Ca2+) handling has been proposed to be required for sustaining key mitochondrial dehydrogenase activity and energy production during increased workload conditions, thus ensuring the adaptive cardiac response. We hypothesized that IGF-1 enhances mitochondrial energy production through a Ca2+-dependent mechanism to ensure adaptive cardiomyocyte growth. We found that stimulation with IGF-1 resulted in increased mitochondrial Ca2+ uptake in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes, estimated by fluorescence microscopy and indirectly by a reduction in the pyruvate dehydrogenase phosphorylation. We showed that IGF-1 modulated the expression of mitochondrial Ca2+ uniporter (MCU) complex subunits and increased the mitochondrial membrane potential; consistent with higher MCU-mediated Ca2+ transport. Finally, we showed that IGF-1 improved mitochondrial respiration through a mechanism dependent on MCU-mediated Ca2+ transport. In conclusion, IGF-1-induced mitochondrial Ca2+ uptake is required to boost oxidative metabolism during cardiomyocyte adaptive growth.

β-catenin ameliorates myocardial infarction by preventing YAP-associated apoptosis

Abstract Objective: To explore whether the effect of β-catenin on MI and MI-induced cardiomyocyte apoptosis is YAP-dependent. Methods: The authors established an MI rat model by ligating the anterior descending branch of the left coronary artery, and an MI cell model by treating cardiomyocytes with H2O2. Results: β-catenin downregulation was observed in MI cardiac tissues and in H2O2-treated cardiomyocytes. Lentiviral-CTNNB1 was administered to MI rats to upregulate β-catenin expression in MI cardiac tissue. β-catenin recovery reduced the myocardial infarct area, fibrosis, and apoptotic cell death in MI rats. H2O2 treatment attenuated cell viability and induced cell death in cardiomyocytes, whereas β-catenin overexpression partially reversed these changes. Moreover, H2O2 treatment caused the deactivation of Yes-Associated Protein (YAP), as detected by increased YAP phosphorylation and reduced the nuclear localization of YAP. Upregulation of β-catenin expression reactivated YAP in H2O2-treated cardiomyocytes. Reactivation of YAP was achieved by administration of Mitochonic Acid-5 (MA-5) to H2O2-treated cardiomyocytes, and deactivation of YAP by CIL56 treatment in β-catenin-overexpressing H2O2-treated cardiomyocytes. MA-5 administration increased cell viability and repressed apoptosis in H2O2-treated cardiomyocytes, whereas CIL56 treatment counteracted the effects of β-catenin overexpression on cell survival and apoptosis. Conclusions: The present data indicate that β-catenin and YAP are effective treatment targets for MI, blocking the apoptotic death of cardiomyocytes.

La miocardiopatía hipertrófica induce cambios en la expresión de la triptófano-5-hidroxilasa, el transportador de serotonina y receptores serotoninérgicos / Hypertrophic cardiomyopathy induces changes in the tryptophan-5-hydroxylase, serotonin transporter and serotonergic receptors expressions

Resumen Introducción: Los cardiomiocitos poseen la maquinaria bioquímica capaz de sintetizar, utilizar y recapturar serotonina. Objetivo: Determinar si la miocardiopatía hipertrófica (MCH) induce cambios en la expresión de la triptófano-5-hidroxilasa (TPH) 1 y 2, el transportador de serotonina (SERT) y los receptores serotoninérgicos (RS). Métodos: Estudio transversal de cinco bloques de tejido de corazones con MCH y cinco bloques de corazones de control. Se obtuvieron cinco cortes de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular (SIV) de cada bloque, para determinar la expresión de TPH1 y TPH2, SERT y RS con anticuerpos por inmunofluorescencia. La inmunofluorescencia fue evaluada mediante t de WELCH, con nivel de significación de p < 0.05. Resultados: La PLVI y el SIV de los corazones con MCH mostraron aumento de la expresión de TPH1 y TPH2, así como de los receptores 5-HT2A y 5-HT2B en comparación con los controles (p < 0.01). El receptor 5-HT4 y SERT aumentaron en el SIV de los corazones con MCH (p < 0.01). Conclusiones: Se demostró aumento de las expresiones de TPH, SERT y RS en los cardiomiocitos de los corazones con MCH en comparación con los controles, lo cual podría participar en la fisiopatología de la MCH en los humanos.

Abstract Introduction: Cardiomyocytes have a biochemical machinery with the capacity to synthesize, utilize and reuptake serotonin. Objective: To determine whether hypertrophic cardiomyopathy (HCM) induces changes in the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter (SERT) and serotonergic receptors (SR). Methods: Cross-sectional study of five tissue blocks from hearts with HCM and five controls. Five sections of the left ventricular free wall (LVFW) and interventricular septum (IVS) were obtained from each block to determine the expression of TPH1 and TPH2, SERT and SRs by immunofluorescence with specific antibodies. Immunofluorescence was evaluated by WELCH t-test, with a level of significance of p < 0.05. Results: LVFW and IVS of hearts with HCM showed an increase in the expression of TPH1 and TPH 2 and 5-HT2A and 5-HT2B receptors in comparison with controls (p < 0.01). The 5-HT4 receptor and SERT showed an increase in the IVS of hearts with HCM (p < 0.01). Conclusions: This study demonstrated an increased expression of TPH, SERT and SRs in cardiomyocytes from hearts with HCM in comparison with controls, which could be involved in the pathophysiology of HCM in humans.

Cytotoxicity and Genotoxicity of Azobenzene-Based Polymeric Nanocarriers for Phototriggered Drug Release and Biomedical Applications.

Drug nanoencapsulation increases the availability, pharmacokinetics, and concentration efficiency for therapeutic regimes. Azobenzene light-responsive molecules experience a hydrophobicity change from a polar to an apolar tendency by trans-cis photoisomerization upon UV irradiation. Polymeric photoresponse nanoparticles (PPNPs) based on azobenzene compounds and biopolymers such as chitosan derivatives show prospects of photodelivering drugs into cells with accelerated kinetics, enhancing their therapeutic effect. PPNP biocompatibility studies detect the safe concentrations for their administration and reduce the chance of side effects, improving the effectiveness of a potential treatment. Here, we report on a PPNP biocompatibility evaluation of viability and the first genotoxicity study of azobenzene-based PPNPs. Cell line models from human ventricular cardiomyocytes (RL14), as well as mouse fibroblasts (NIH3T3) as proof of concept, were exposed to different concentrations of azobenzene-based PPNPs and their precursors to evaluate the consequences on mitochondrial metabolism (MTT assay), the number of viable cells (trypan blue exclusion test), and deoxyribonucleic acid (DNA) damage (comet assay). Lethal concentrations of 50 (LC50) of the PPNPs and their precursors were higher than the required drug release and synthesis concentrations. The PPNPs affected the cell membrane at concentrations higher than 2 mg/mL, and lower concentrations exhibited lesser damage to cellular genetic material. An azobenzene derivative functionalized with a biopolymer to assemble PPNPs demonstrated biocompatibility with the evaluated cell lines. The PPNPs encapsulated Nile red and dofetilide separately as model and antiarrhythmic drugs, respectively, and delivered upon UV irradiation, proving the phototriggered drug release concept. Biocompatible PPNPs are a promising technology for fast drug release with high cell interaction opening new opportunities for azobenzene biomedical applications.

Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes.

Introduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery. Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency. Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment. Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.

Different Transcriptomic Response to T. cruzi Infection in hiPSC-Derived Cardiomyocytes From Chagas Disease Patients With and Without Chronic Cardiomyopathy.

Chagas disease is a tropical zoonosis caused by Trypanosoma cruzi. After infection, the host present an acute phase, usually asymptomatic, in which an extensive parasite proliferation and intense innate immune activity occurs, followed by a chronic phase, characterized by low parasitemia and development of specific immunity. Most individuals in the chronic phase remain without symptoms or organ damage, a state called indeterminate IND form. However, 20 to 40% of individuals develop cardiac or gastrointestinal complications at any time in life. Cardiomyocytes have an important role in the development of Chronic Chagas Cardiomyopathy (CCC) due to transcriptional and metabolic alterations that are crucial for the parasite survival and replication. However, it still not clear why some infected individuals progress to a cardiomyopathy phase, while others remain asymptomatic. In this work, we used hiPSCs-derived cardiomyocytes (hiPSC-CM) to investigate patterns of infection, proliferation and transcriptional response in IND and CCC patients. Our data show that T. cruzi infection and proliferation efficiency do not differ significantly in PBMCs and hiPSC-CM from both groups. However, RNA-seq analysis in hiPSC-CM infected for 24 hours showed a significantly different transcriptional response to the parasite in cells from IND or CCC patients. Cardiomyocytes from IND showed significant differences in the expression of genes related to antigen processing and presentation, as well as, immune co-stimulatory molecules. Furthermore, the downregulation of collagen production genes and extracellular matrix components was significantly different in these cells. Cardiomyocytes from CCC, in turn, showed increased expression of mTORC1 pathway and unfolded protein response genes, both associated to increased intracellular ROS production. These data point to a differential pattern of response, determined by baseline genetic differences between groups, which may have an impact on the development of a chronic outcome with or without the presentation of cardiac symptoms.