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Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavβ4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavβ4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavβ4. Finally, we found that overexpression of Cavβ4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavβ4 subunit expression.

Exercise plays an important role in cardiac health and enhances the transport of glucose in cardiac muscle by increasing the glucose transporter-4 (GLUT4) content at the cell membrane. The GLUT4 gene is a target of myocyte enhancer transcription factor 2A (MEF2A). Several transcription factors are regulated by microRNAs (miRs), small non-coding RNAs that control gene expression at the posttranscriptional level. In this study we tested the hypothesis that exercise regulates the expression of miR-223 and that MEF2A is a direct target of miR-223. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that GLUT4 gene expression and protein abundance increased by 30 and 23%, respectively, in the microsomal fraction immediately after exercise, and had returned to control levels after 18 h. In contrast, the increase in GLUT4 in the membrane fraction was delayed. Exercise also increased the protein abundance of transcription factors involved in GLUT4 expression. Immediately after exercise, the protein abundance of MEF2A, nuclear respiratory factor 1 (NRF1), and forkhead box O1 (FOXO1) increased by 18, 30, and 40%, respectively. qRT-PCR experiments showed that miR-223-3p and miR-223-5p expression decreased immediately after exercise by 60 and 30%, respectively, and luciferase assays indicated that MEF2A is a target of the 5p strand of miR-223. Overexpression of miR-223-5p in H9c2 cells decreased the protein abundance of MEF2A. Our results suggest that the exercise-induced increase in GLUT4 content in cardiac muscle is partly due to the posttranscriptional increase in MEF2A protein abundance caused by the decrease in miR-223-5p expression. The exercise-induced decrease in miR-223-3p expression likely contributes to the increases in NRF1 and FOXO1 abundance and GLUT4 content., (© 2023. Springer Nature Limited.)

Dengue virus (DENV) infection elevates intracellular Ca 2+ concentration ([Ca 2+ ] i ), but it is unknown whether Ca 2+ and calmodulin (CaM) are involved in DENV infection. We conducted immunofluorescence and western blot experiments and measured [Ca 2+ ] i examining the effects of DENV infection and drugs that alter Ca 2+ /CaM functions on CaM translocation, DENV2 infection, protein expression, virus-inducible STAT2 protein abundance, and CREB phosphorylation in H9c2 cells. DENV infection increased CaM expression, its nuclear translocation and NS3 and E viral proteins expression and colocalization in a manner that could be blocked by the ryanodine receptor antagonist dantrolene. DENV infection also increased CREB phosphorylation, an effect inhibited by either dantrolene or the CaM inhibitor W7. Dantrolene substantially hindered infection as assessed by focus assays in Vero cells. These results suggest that Ca 2+ and CaM play an important role in DENV infection of cardiac cells and that dantrolene may protect against severe DENV cardiac morbidity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca 2+ elevations produced by ischemia-reperfusion, protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca 2+ entry (SOCE) STIM1 and Orai1., Results: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Immunofluorescence analyses revealed that diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N -acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Confocal microscopy revealed that diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFκB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expression., Conclusions: Our results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Voltage-dependent Ca 2+ channels and store-operated Ca 2+ channels (SOCs) are the major routes of Ca 2+ entry into mammalian cells. Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca 2+ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca 2+ , respectively. Ca 2+ store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate (5-HD; a selective mitochondrial ATP-sensitive K + channel blocker), an intracellular mitochondrial energizing solution, or Ni 2+ [a blocker of sodium-calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca 2+ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca 2+ with a fluorescent Ca 2+ indicator. These results indicate that influx of Ca 2+ through SOCs is inhibited by their ROS and Ca 2+ -dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca 2+ . We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia-reperfusion., (Copyright © 2020 Sampieri, Fuentes, Carrillo, Hernández, García and Sánchez.)

The auxiliary β 4 subunit of the cardiac Ca v 1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the β 4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-β (IFN-β)] and of the IFN-β-related genes Ddx58 , Ifitm3 , Irf7 , Stat2 , Ifih1 , and Mx1 , as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the β 4 subunit in H9c2 cells reduced the expression of IFN-β-stimulated genes. In response to inhibition of the kinase JAK1, the abundances of β 4 subunit mRNA and protein were decreased. β 4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-β, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of β 4 subunit proteins to nuclei in response to spreading infection. We showed that the β 4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter-driven reporter was increased in cells overexpressing the β 4 subunit. Last, overexpressing β 4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca 2+ in these cells. These findings suggest that the β 4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Introduction and Methods: The effects of diazoxide on cardiac hypertrophy and miR-132 expression were characterized in adult rats and in cardiomyocytes. Diazoxide effects on reactive oxygen species (ROS) production and on the cAMP-response element binding (CREB) transcription factor's abundance in cardiomyocytes were also analyzed. ROS measurements used a fluorescent dye. Western blot analysis and quantitative Reverse Transcription Polymerase Chain Reaction were used to measure phosphorylated form of CREB (pCREB) abundance and miR-132 expression, respectively., Results: Isoproterenol (ISO) induced cardiac hypertrophy, an effect that was mitigated by diazoxide. The rate of ROS production, CREB phosphorylation, and miR-132 expression increased after the addition of ISO. H2O2 increased pCREB abundance and miR-132 expression; upregulation of miR-132 was blocked by the specific inhibitor of CREB transcription, 666-15. Consistent with a role of ROS on miR-132 expression, diazoxide prevented the increase in ROS production, miR-132 expression, and pCREB abundance produced by ISO. Phosphorylation of CREB by ISO was prevented by U0126, an inhibitor of mitogen-activated protein kinase., Conclusions: Our data first demonstrate that diazoxide mitigates hypertrophy by preventing an increase in miR-132 expression. The mechanism likely involves less ROS production leading to less phosphorylation of CREB. Our data further show that ROS enhance miR-132 transcription, and that ISO effects are probably mediated by the mitogen-activated protein kinase pathway.

Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aims: To characterize the effects of long-term β-adrenergic receptor stimulation on Rem protein and mRNA expression in rat heart and possible involvement of miR-132., Methods: Adult rats were treated with isoproterenol (ISO, 150 µg.kg.h(-1)) for 2 d and Rem, miR-132, and α1c (the principal subunit of Cav1.2 channels) were measured at protein and mRNA levels with western blot and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiments, respectively. Ca(2+) currents and intracellular Ca(2+) signals were evaluated in isolated cardiomyocytes., Results: Systemic administration of ISO led to decreases in Rem protein and mRNA levels (down to 49%). Furthermore, levels of the microRNAs (miRs) miR-132 and miR-214 were upregulated 5- and 9-fold, respectively. Transfection of miR-132, but not miR-214, into HEK293 cells reduced the expression of a luciferase reporter gene controlled by a conserved 3´-untranslated region (UTR) of Rem by half. Chronic ISO administration also led to a 25% decrease in the amplitude of peak L-type Ca(2+) currents, a 40% decrease in α1c subunit protein abundance at the membrane level, and a 60% decrease in expression of α1c channel subunit mRNA., Conclusions: These results suggest that Rem expression is down-regulated posttranscriptionally by miR-132 in response to long-term activation of β-adrenergic signaling, but this down-regulation does not produce a larger Ca(2+) influx through Cav1.2 channels.

Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K(+) channel openers such as diazoxide, provides protection against ischemia in cardiac muscle, skeletal muscle, and other tissues. Effects on Ca(2+) homeostasis during the late phase of PPC have been described in cardiomyocytes, but no information is available regarding intracellular Ca(2+) changes in skeletal muscle fibers during late PPC. Intracellular Ca(2+) signals were measured in single fibers of adult mouse skeletal muscle, with fluorescent probes, 48 h after the administration of diazoxide. Parvalbumin levels in the myofibers were quantitated by Western blot. Diazoxide induction of late PPC was confirmed by partial protection of muscles from peroxide-induced damage. Late PPC was associated with a significant decrease in the duration of Ca(2+) signals during single twitches and tetanus with no changes in peak values. This effect was prevented by the reactive oxygen species (ROS) scavenger tiron. Late PPC was accompanied by a 30% increase in parvalbumin levels, and this effect was also blocked by tiron. Our data show, for the first time, a role of parvalbumin in late PPC in skeletal muscle.

Introduction and Methods: The effects of long-term β-adrenergic administration on the expression levels of the cardiac L-type Ca channel β2 subunit, which regulates channel trafficking and function, were characterized in adult rats., Results: Systemic administration of isoproterenol (150 mg·kg·h) for 2 d led to a 50% increase in the ventricular wet weight-to-body weight ratio (mg/g) and of more than two-fold in the expression of actin protein. In contrast, β2 subunit protein levels decreased (down to 49%), while mRNA levels remained unchanged. Furthermore, levels of microRNAs (miRs), including miR-21 and miR-132, were upregulated (7.2 and 7.9 fold, respectively). Transfection of these miRs into HEK293 cells attenuated expression of a luciferase reporter gene controlled by a conserved 3'-untranslated region (UTR) of the β2 subunit (down to 67% and 56%, respectively). Systemic administration of isoproterenol also led to briefer intracellular Ca transients during action potentials measured in isolated cardiomyocytes (down to 65%)., Conclusion: These results suggest that cardiac L-type Ca channel β2 subunit protein expression may be downregulated by miRs in response to long-term activation of β-adrenergic signaling, possibly as an adaptive response in cardiac hypertrophy and sustained β-adrenergic states.

Up to 30% of liver transplants will develop graft cirrhosis within 5 years after liver transplantation (LT) due to recurrent HCV-infection forwarding accelerated graft damage. Genetic variants of cytokines involved in the immune response may contribute to the degree of graft inflammation, fibrosis progression, and antiviral therapy outcome. The aim of our study was to analyze biochemical and histological inflammation extent based on protocol liver biopsies and to evaluate the role of genetic variants of IL-28b in HCV-related graft disease and antiviral treatment response. 183 patients, who underwent liver transplantation for HCV-induced liver disease, were genotyped for IL-28b (rs8099917, G ≥ T) by TaqMan Genotyping Assay. 56 of 159 patients have been successfully treated with interferon-based antiviral therapy. 605 protocol liver biopsies performed 0.5 to 10 and more than 10 years after transplantation were evaluated according to Desmet and Scheuer classification of inflammation and fibrosis. Prevalence of IL-28b-genotypes was correlated with histological severity of graft damage, levels of aminotransferases, occurrence of acute cellular rejection, pre-treatment viremia, and antiviral therapy outcome. Significant association of IL-28b-genotype distribution was observed to the median grade of inflammation (p < 0.001), mean levels of aminotransferases (ALT: p = 0.001, AST: p = 0.003), median pre-treatment viremia level within 1 year after LT (p = 0.046) and interferon-based antiviral therapy failure (p < 0.001). Among successfully treated patients, G-allele was significantly less frequent, and the genotype GG was not present at all. No differences were observed regarding acute cellular rejection (p = 0.798) and fibrosis stages (p = 0.586). IL-28b polymorphism seems to influence the degree of graft inflammation at biochemical and histological levels. G-allele might serve as a marker for graft inflammation and as a predictor for unfavorable antiviral therapy outcome in HCV-re-infected LT-population., (Copyright © 2011 American Association for the Study of Liver Diseases.)

The expression of two different transcripts for Ca(V)1.2 in rat tissues mirrors that which has previously been described for human tissue, in that expression of transcripts expressing exon 1a is predominant only in heart, whereas expression of transcripts expressing exon 1b is greater in smooth muscle rich tissues such as aorta and intestine. Transcripts expressing exon 1b also predominate in brain and in diaphragm. Western blots indicate that the N-terminus coded for by exon 1b is present in much of the protein in all these tissues except heart. The promoter just upstream of exon 1b has been cloned, sequenced and utilized to drive expression of luciferase in smooth muscle A7r5 cells, cardiac HL-1 cells, skeletal muscle L6 cells and neuronal PC12 cells. The nucleotide sequence of the promoter exhibits 80% identity with the equivalent promoter previously identified in humans and 94% identity with the sequence of the equivalent region of the mouse genome. Evidence in favor of still another promoter upstream of exon 2 has been uncovered.