H-89 decreases the gain of excitation-contraction coupling and attenuates calcium sparks in the absence of beta-adrenergic stimulation

Randi J. Parks, Susan E. Howlett

    Research output: Contribution to journalArticlepeer-review

    Abstract

    This study used the selective protein kinase A (PKA) inhibitor H-89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) to determine the role of basal PKA activity in modulating cardiac excitation-contraction coupling in the absence of β-adrenergic stimulation. Basal intracellular cyclic AMP (cAMP) levels measured in isolated murine ventricular myocytes with an enzyme immunoassay were increased upon adenylyl cyclase activation (forskolin; 1 and 10 μM) or phosphodiesterase inhibition (3-isobutyl-1-methylxanthine, IBMX; 300 μM). Forskolin and IBMX also caused concentration-dependent increases in peak Ca2 transients (fura-2) and cell shortening (edge-detector) measured simultaneously in field-stimulated myocytes (37 °C). Similar effects were seen upon application of dibutyryl cAMP. In voltage-clamped myocytes, H-89 (2 μM) decreased basal Ca2 transients, contractions and underlying Ca2 currents. H-89 also decreased diastolic Ca2 and the gain of excitation-contraction coupling (Ca2 release/Ca2 current), especially at negative membrane potentials. This was independent of alterations in sarcoplasmic reticulum (SR) Ca2 loading, as SR stores were unchanged by PKA inhibition. H-89 also decreased the frequency, amplitude and width of spontaneous Ca2 sparks measured in quiescent myocytes (loaded with fluo-4), but increased time-to-peak. Thus, H-89 suppressed SR Ca2 release by decreasing Ca2 current and by reducing the gain of excitation-contraction coupling, in part by decreasing the size of individual Ca2 release units. These data suggest that basal PKA activity enhances SR Ca2 release in the absence of ß-adrenergic stimulation. This may depress contractile function in models such as aging, where the cAMP/PKA pathway is altered due to low basal cAMP levels. © 2012 Elsevier B.V. All rights reserved.
    Original languageEnglish
    Pages (from-to)163-172
    Number of pages9
    JournalEuropean journal of pharmacology
    Volume691
    Issue number1-3
    DOIs
    Publication statusPublished - 15 Sep 2012

    Keywords

    • Calcium
    • Cardiac myocyte
    • Cyclic AMP
    • Excitation-contraction coupling
    • H-89
    • Sarcoplasmic reticulum

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