In cardiac muscle cells, both adrenergic agonists and antagonists induce reactive oxygen species from NOX2 but mutually attenuate each other's effects.

In cardiac muscle cells adrenergic agonists stimulate the generation of reactive oxygen species, followed by redox signaling. We postulated that the antagonists would attenuate such reactive oxygen species generation by the agonists. H9c2 cardiac myoblasts, neonatal rat cardiac myocytes, and HEK293 cells expressing ß1/ß2 adrenoceptors were stimulated with several agonists and antagonists. All the agonists and antagonists independently generated reactive oxygen species; but its generation was minimum whenever an agonists was added together with an antagonist. We monitored the Ca++ signaling in the treated cells and obtained similar results. In all treatment sets, superoxide and H2O2 were generated in the mitochondria and the cytosol respectively. NOX2 inhibitor gp91ds-tat blocked reactive oxygen species generation by both the agonists and the antagonists. The level of p47phox subunit of NOX2 rapidly increased upon treatment, and it translocated to the plasma membrane, confirming NOX2 activation. Inhibitor studies showed that the activation of NOX2 involves ERK, PI3K, and tyrosine kinases. Recombinant promoter-reporter assays showed that reactive oxygen species generated by both the agonists and antagonists modulated downstream gene expression. Mice injected with the ß-adrenergic agonist isoproterenol and fed with the antagonist metoprolol showed a robust induction of p47phox in the heart. We conclude that both the agonism and antagonism of adrenoceptors initiate redox signaling but when added together, they mutually counteract each other's effects. Our study thus highlights the importance of reactive oxygen species in adrenoceptor agonism and antagonism with relevance to the therapeutic use of the ß blockers.

Transcriptomic Validation of the Protective Effects of Aqueous Bark Extract of Terminalia arjuna (Roxb.) on Isoproterenol-Induced Cardiac Hypertrophy in Rats.

Aqueous extract of the bark of Terminalia arjuna (TA) is used by a large population in the Indian subcontinent for treating various cardiovascular conditions. Animal experiments have shown its anti-atherogenic, anti-hypertensive, and anti-inflammatory effects. It has several bioactive ingredients with hemodynamic, ROS scavenging, and anti-inflammatory properties. Earlier we have done limited proteomic and transcriptomic analysis to show its efficacy in ameliorating cardiac hypertrophy induced by isoproterenol (ISO) in rats. In the present study we have used high-throughput sequencing of the mRNA from control and treated rat heart to further establish its efficacy. ISO (5 mg/kg/day s.c.) was administered in male adult rats for 14 days to induce cardiac hypertrophy. Standardized aqueous extract TA bark extract was administered orally. Total RNA were isolated from control, ISO, ISO + TA, and TA treated rat hearts and subjected to high throughput sequence analysis. The modulations of the transcript levels were then subjected to bio-informatics analyses using established software. Treatment with ISO downregulated 1,129 genes and upregulated 204 others. Pre-treatment with the TA bark extracts markedly restored that expression pattern with only 97 genes upregulated and 85 genes downregulated. The TA alone group had only 88 upregulated and 26 downregulated genes. The overall profile of expression in ISO + TA and TA alone groups closely matched with the control group. The genes that were modulated included those involved in metabolism, activation of receptors and cell signaling, and cardiovascular and other diseases. Networks associated with those genes included those involved in angiogenesis, extracellular matrix organization, integrin binding, inflammation, drug metabolism, redox metabolism, oxidative phosphorylation, and organization of myofibril. Overlaying of the networks in ISO and ISO_TA group showed that those activated in ISO group were mostly absent in ISO_TA and TA group, suggesting a global effect of the TA extracts. This study for the first time reveals that TA partially or completely restores the gene regulatory network perturbed by ISO treatment in rat heart; signifying its efficacy in checking ISO-induced cardiac hypertrophy.

Apocynin prevents isoproterenol-induced cardiac hypertrophy in rat.

Oxidative stress is implicated in the pathogenesis of a plethora of cardiovascular diseases including interstitial fibrosis, contractile dysfunction, ischemia-reperfusion injury, and cardiac remodeling. However, antioxidant therapies targeting oxidative stress in the progression of those diseases have largely been unsuccessful. The current study evaluated the effects of a NADPH oxidase inhibitor, apocynin (Apo), on the production of reactive oxygen species and the development of pathological cardiac hypertrophy under sustained ß-adrenergic stimulation in male Wistar rats. As evident from the HW/BW ratio, HW/TL ratio, echocardiography, and histopathology, hypertrophic responses induced by isoproterenol (Iso; 5 mg/Kg body weight, subcutaneous) were blocked by Apo (10 mg/Kg body weight, intraperitoneal). Iso treatment increased the transcript levels of cybb and p22-phox, the two subunits of Nox. Iso treatment also caused a decrease in reduced glutathione level that was restored by Apo. Increase in mRNA levels of a number of markers of hypertrophy, viz., ANP, BNP, ß-MHC, and ACTA-1 by Iso was either partially or completely prevented by Apo. Activation of key signaling kinases such as PKA, Erk, and Akt by Iso was also prevented by Apo treatment. Our study thus provided hemodynamic, biochemical, and molecular evidences supporting the therapeutic value of Apo in ameliorating adrenergic stress-induced cardiac hypertrophy.

Activation of adrenergic receptor in H9c2 cardiac myoblasts co-stimulates Nox2 and the derived ROS mediate the downstream responses.

In recent years, NADPH oxidases (Noxes) have emerged as an important player in cardiovascular pathophysiology. Despite the growing evidences on the role of specific Nox isoforms, mechanisms of their activation, targets of reactive oxygen species (ROS) generated, and their downstream effects are poorly understood as yet. In this study, we treated H9c2 cardiac myoblasts with norepinephrine (NE, 2 µM), inducing ROS generation that was inhibited by Nox2-specific peptide inhibitor gp91ds-tat. Organelle-specific hydrogen peroxide-sensitive probe HyPer showed that the site of ROS generation is primarily in the cytosol, to some extent in the endoplasmic reticulum (ER) but not the mitochondria. Modulation of mRNAs of marker genes of cardiac hypertrophy i.e. induction in ANP and ß-MHC, and reduction in α-MHC by NE treatment was prevented by specific inhibition of Nox2 by gp91ds-tat. Induction of ANP and ß-MHC at the protein level were also attenuated by the inhibition of Nox2. Induction of c-Jun and FosB, the two members of the transcription factor family AP-1, were also blocked by the inhibition of Nox2 by gp91ds-tat. Induction of promoter-reporter constructs harboring multiple AP-1 elements and the upstream of FosB and ANP genes by NE were also blocked by the inhibition of Nox2 by gp91ds-tat and a dominant negative mutant of p22phox, a constituent of Nox2 that prevents its activation. This study for the first time establishes the significant role of Nox2 in mediating the NE-induced pathological adrenergic signaling in cardiac myoblasts.

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress.

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.