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The Na/Ca exchanger encoded by the NCX1 gene plays an important role in calcium homeostasis in cardiac muscle. We previously identified three in vitro signaling pathways that are of major importance in the regulation of Na/Ca exchanger gene expression in neonatal cardiac myocytes, the protein kinase A (PKA) and protein kinase C (PKC) pathways, and intracellular Ca(2+). To determine whether these pathways are important in vivo, we stimulated the PKA and PKC pathways and examined functional expression of the Na/Ca exchanger in adult rat heart. After a 3- and 7-day treatment, norepinephrine (200 microg x kg(-1) x h(-1)), isoproterenol (150 microg x kg(-1) x h(-1)), and phenylephrine (200 microg x kg(-1) x h(-1)) each stimulated a significant increase in NCX1 mRNA levels (35-85%, P < 0.05). Norepinephrine also stimulated a 35% increase in protein abundance (P < 0.05), a 20% decrease in relaxation duration (P < 0.05), and a 25% reduction in the fluorescence decay constant (P < 0.05) after a 7-day treatment. We conclude that a 7-day treatment of alpha- and beta-adrenergic agonists increases the expression of functional Na/Ca exchangers in adult rat heart.

Suberin has been shown to play a role in protection against and response to stress, including defense against soil-borne pathogens. In soybean, oxidized fatty acids are the most predominant monomers contributing to root suberin, with 18-hydroxy-oleic acid being the most abundant. 18-Hydroxy-oleic acid is predicted to be synthesized by members of the CYP86A family of cytochrome P450 enzyme. Six putative CYP86A genes were identified through phylogenetic analysis of the soybean genome. Two of these, CYP86A37 and CYP86A38 show a root-specific gene expression pattern, and were further analyzed to assess their physiological role in suberin deposition using RNAi knockdown in a hairy root system. Soybean hairy roots were found to be nearly identical to soil-grown roots in terms of anatomy, suberin deposition patterns, and suberin chemistry. The RNAi knockdown of CYP86A37 and CYP86A38 yielded hairy root lines with reduced gene expression and a reduction in the oxidized monomers of suberin, most notably 18-hydroxy-oleic acid. Based on this evidence, CYP86A37 and CYP86A38 are predicted to function as fatty acid ω-hydroxylases in vivo.

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To test the hypothesis that mutated beta2-subunits of the L-type calcium channel could serve as a decoy and interdict calcium channel trafficking and function, we engineered a beta2 subunit that contained the beta interaction domain for alpha1c subunit interaction, but lacked N- and C-terminal domains that might be essential for sarcolemmal localization. An adenoviral vector was constructed containing the gene for the beta-interaction domain (BID) fused to green fluorescence protein (GFP), using a vector containing only GFP as control. Freshly plated, dissociated adult rat myocytes were infected and expression and function were assessed at 60 h. Fluorescence microscopy confirmed GFP expression; immunoblot analysis confirmed dose-dependent GFP-BID expression. Mechanical properties of adult rat ventricular myocytes were evaluated using a video edge-detection system. Contractility analysis (optical/video, field stimulation) demonstrated that contracting cells decreased from 60 to 2%. Contractile amplitude (percent shortening) decreases significantly from 5.6 vs. 2.4% with no change in time to peak twitch. Recombinant adenovirus overexpressing mutated beta2 subunits in adult mammalian myocytes can markedly alter excitation-contraction coupling. This paradigm may offer new approaches to understanding and modulating EC coupling.