Introduction and Methods The effects of long-term β-adrenergic administration on the expression levels of the cardiac L-type Ca²⁺ channel β₂ subunit, which regulates channel trafficking and function, were characterized in adult rats. Results Systemic administration of isoproterenol (150 mg·kg·h⁻¹) 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 Ca²⁺ transients during action potentials measured in isolated cardiomyocytes (down to 65%). Conclusion These results suggest that cardiac L-type Ca²⁺ 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.

Background: Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca²⁺ 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 Ca²⁺ 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.

Voltage-dependent Ca²⁺ channels and store-operated Ca²⁺ channels (SOCs) are the major routes of Ca²⁺ 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 Ca²⁺ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca²⁺, respectively. Ca²⁺ 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 Ni²⁺ [a blocker of sodium–calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca²⁺ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca²⁺ with a fluorescent Ca²⁺ indicator. These results indicate that influx of Ca²⁺ through SOCs is inhibited by their ROS and Ca²⁺-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca²⁺. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia–reperfusion.

The auxiliary β₄ subunit of the cardiac Ca_v1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the β₄ 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 β₄ subunit in H9c2 cells reduced the expression of IFN-β–stimulated genes. In response to inhibition of the kinase JAK1, the abundances of β₄ subunit mRNA and protein were decreased. β₄ 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 β₄ subunit proteins to nuclei in response to spreading infection. We showed that the β₄ subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter–driven reporter was increased in cells overexpressing the β₄ subunit. Last, overexpressing β₄ 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 Ca²⁺ in these cells. These findings suggest that the β₄ subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection.

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.