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. 2012 Jul 6;423(3):600-5.
doi: 10.1016/j.bbrc.2012.06.024. Epub 2012 Jun 12.

PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes

Misuk Kang  1 , Ka Young Chung
Affiliations

PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes

Misuk Kang et al. Biochem Biophys Res Commun. .

Abstract

Endothelin-1 (ET-1) induces positive inotropy (enhanced contractility) in cardiac muscle, but establishing underlying cellular mechanisms has been controversial in part because of a growing number of signaling pathways and end effectors targeted by ET-1. Here we present evidence that ET-1 induces positive inotropism in ventricular tissue by increasing both systolic Ca2+ and myofilament Ca2+ sensitivity. To examine the roles of PKC-δ and PKC-ε in these acute responses to ET-1, kinase inactive dominant negative PKC (dn-PKC) constructs were expressed in adult rat ventricular myocytes. Yellow fluorescent protein (YFP) was fused to dn-PKC constructs to visualize expression and localization of dn-PKC in living myocytes. Due to an alanine to glutamate mutation in the pseudosubstrate site, dn-PKCs constitutively translocated to anchoring sites and were unaffected by agonist or phorbol ester treatment. Dn-PKC-δ-YFP mainly distributed at Z-lines and at intercalated disks in adult myocytes, whereas dn-PKC-ε-YFP stained the surface sarcolemma, T-tubules/Z-lines and perinuclear region. Myocytes expressing dn-PKC-δ-YFP showed normal systolic Ca2+ and contractile responses to ET-1. In contrast, the entire ensemble of ET-1 responses was blocked in myocytes expressing dn-PKC-ε-YFP including increased Ca2+ transients, enhanced myofilament Ca2+ sensitivity, and positive inotropy. This report provides direct evidence that PKC-ε is activated early and robustly following ET-1 stimulation and thus mediates multiple intracellular changes underlying the acute actions of ET-1 on myocardium.

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Figures

Figure 1
Figure 1. Expression and characterization of dn-PKC-YFPs in myocytes
A. Western blotting of myocyte lysates using anti-GFP antibody. Each lane indicates myocyte lysates prepared 40 hours after adenovirus infection. Total 20 μg lysates were loaded at each lane and the same blot was reprobed with each antibody after stripping for panel B. GFP alone at about 25 kDa clearly showed up in lane 1 and other GFP fused recombinant PKC proteins located at over 100 kDa. B. Western blotting using PKC isoform specific antibodies. C. In vitro kinase assay. GFP alone and fluorescent tagged PKCs were immunoprecipitated with anti-GFP antibody from myocyte lysates and tested for kinase activity using histone H1 as a substrate. Dn-PKC-YFPs show almost no kinase activity compared to wild-type PKC-GFPs. Data represent mean±SD; *P<0.02 versus each isoform of wild-type PKC-GFP. D and E. Representative confocal images of dn-PKC-δ-YFP (D) and dn-PKC-ε-YFP (E) using the 514 nm laser. Arrows and arrowheads indicate the most prominent features of each localization pattern. F and G. Co-localization of dn-PKC-YFPs with Z-line/T-tubular markers in myocytes. F. Myocytes were decorated with mouse monoclonal anti-α-actinin antibody (and an Alexa 568-labeled secondary antibody) as a Z-line marker. Localization of dn-PKC-YFP constructs (Left, green), α-actinin (Middle, red), and merged image (Right) are shown. G. Myocytes were stained with di-8-Anepps, a marker of T-tubular membranes. Localization of dn-PKC-YFP constructs (Left, green), di-8-Anepps (Middle, blue), and merged image (Right) are shown. Bar = 10 μm.
Figure 2
Figure 2. Effects of dn-PKC-YFPs expression on ET-1 induced positive inotropy and Ca2+ signaling
A. Twitch amplitude after 15 minutes treatment of 10 nmol/L ET-1 in control myocytes and myocytes expressing dn-PKC-YFPs. B. Confocal line scan images recorded from an X-rhod-1 loaded myocyte (2 ms/line). Representative images were captured before (left) and 15 min after addition of 10 nmol/L ET-1 (right) (n=10). The Ca2+ signal is shown as a fluorescence ratio (F/F0) with the fluorescence signal (F) being normalized to the signal prior to contraction (F0). C. Ca2+ transient amplitude after ET-1 activation in control myocytes and myocytes expressing dn-PKC-YFPs. Data represents mean±SEM; *P<0.001 compared to control.
Figure 3
Figure 3. Relationships between twitch amplitudes and intracellular Ca2+ amplitudes
The relationship between free Ca2+ and twitch shortening during postrest potentiation was examined to determine myofilament Ca2+ sensitivity. Twitch and Ca2+ transient amplitudes were normalized to their respective maxima. The results are representative of data obtained in five experiments on separate culture preparations.
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