Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET.

Calcium (Ca2+) and 3',5'-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after β-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under β-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation

Ca²⁺ transient amplitudes in ISO and forskolin treated cardiomyocytes.

<p>Cells were loaded with Fura2-AM, paced at 1 Hz and treated with 100 nM ISO or 10 μM forskolin with subsequent applications of PDE inhibitors rolipram 10 μM, 8-MMX 30 μM and IBMX 100 μM. Shown are baseline Ca²⁺ amplitudes (black bars) and systolic Ca²⁺ transient amplitudes (grey bars) measured in paced <b>(A)</b> and resting <b>(B)</b> cells. Means ± SEM, *—p<0.05; **—p<0.01; ***—p<0.001 with ANOVA (compared to control, second value after / as compared to previous stimulation) followed by the Gasser-Greenhouse correction. n = 7 for ISO and n = 9 for forskolin cells in A, and n = 4 and 5 for ISO and forskolin cells in B, respectively (all isolated from at least 3 mice for each condition). Effects of ISO and forskolin in A are significantly different (p = 0.03 by one-way ANOVA).</p

Schematic diagram highlighting Ca²⁺ and cAMP changes observed in this study under different experimental conditions (basal, ISO and forskolin stimulated cardiomyocytes with and without pacing).

<p>Pacing leads to an increase in Ca²⁺ levels which is further augmented by forskolin>ISO via PKA-dependent phosphorylation of Ca²⁺ handling proteins. However, pacing has little effect on cAMP levels, apart from the case when it is combined with forskolin stimulation, together both lead to PDE1 activation. Increase of PDE1 activity affects presumably a discrete subcellular microdomain which constitutes a small percentage of the whole cellular AMP content and can be therefore revealed in the cytosol only by the use of a PDE1 inhibitor. Forskolin and ISO generate quantitatively comparable but differently shaped amounts of cAMP which may come from ISO-induced dissociation of PDE4D8 from the β₁-adrenergic receptor. This mechanism regulates local second messenger pool at the receptor and allows more rapid increase of cAMP in the cytosol, as compared to forskolin stimulation.</p

cAMP dynamics in adult mouse ventricular Epac1-camps expressing cardiomyocytes upon treatment with cAMP elevating agents and PDE4 inhibition.

<p><b>(A)</b> Representative calcium traces in Fura2-AM loaded Epac1-camps transgenic cardiomyocytes under resting conditions (left) and upon electric field stimulation at 1 Hz (right). <b>(B)</b> Non-normalized FRET ratios do not differ between resting and paced Epac1-camps cardiomyocytes under basal and stimulated conditions (isoproterenol—ISO, 100 nM—plus 3-isobutyl-1-methylxathin—IBMX, 100 μM). <b>(C)</b> Representative FRET traces from Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or <b>(E)</b> with the direct AC activator forskolin (10 μM) leading to an increase of cAMP visualized as a decrease in the FRET ratio. Inhibition of PDE4 by rolipram (Roli, 10 μM) strongly enhances this effect, whereas the unselective PDE inhibitor IBMX (100 μM) has only little additional effect. <b>(D and F)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes. Cells were paced at 1 Hz. Values are means ± SEM; from n = 6 cells isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p

cAMP dynamics in adult mouse cardiomyocytes upon preincubation with IBMX and after low concentration of ISO.

<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes preincubated with 3-isobutyl-1-methylxanthin (IBMX, 100 μM) and then stimulated with non-saturating concentrations of the β-AR agonist isoproterenol (ISO, 1nM). The AC activator forskolin (10 μM) was used to reach the maximal FRET response. <b>(B)</b> Representative control traces (n = 4 and 8 for unpaced and paced, respectively) showing FRET response to IBMX response over the whole time-course involved in these experiments. Representative FRET responses (n = 7 each) to 1 nM ISO applied along (without IBMX prestimulation), followed by forskolin plus IBMX <b>(D)</b> Quantification of experiments from A and C shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with IBMX and ISO. Cells were paced at 1 Hz. Values are means ± SEM, n = 8 cells for each A graph and n = 7 cells for each C graph isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p

cAMP dynamics in adult mouse cardiomyocytes upon treatment with cAMP elevating agents and PDE1 inhibition.

<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or with the direct AC activator forskolin (10 μM) <b>(C)</b>. Subsequent application of the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX, 30 μM) enhances the cAMP stimulatory effect of ISO and forskolin. Stimulation with the unselective PDE inhibitor 3-isobutyl-1-methylxanthin (IBMX, 100 μM) leads to a further increase of cAMP. <b>(B and D)</b> Quantification of experiments shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with ISO. Forskolin stimulated cardiomyocytes show significant differences in PDE1 contribution to total PDE inhibition which is significantly higher in paced cardiomyocytes as compared to resting cells. Pretreatment of resting cardiomyocytes with calcium elevating reagents such as thapsigargin (100 nM) and calcium ionophore A23187 (10 μM) mimics the effect of field stimulation. Cells were paced at 1 Hz. Values are means ± SEM; n = 6–10 cells isolated from 3 hearts per condition; *—significant difference at p<0.05 by one-way ANOVA; n.s.- not significant.</p

Subsarcolemmal cAMP dynamics in adult mouse ventricular cardiomyocytes transgenically expressing pmEpac1 biosensor.

<p><b>(A)</b> Representative FRET traces from pmEpac1 cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) and subsequently by the PDE4 inhibitor rolipram (Roli, 10 μM) followed by the unselective PDE inhibitor IBMX (100 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167974#pone.0167974.g001" target="_blank">Fig 1C and 1D</a>. <b>(C)</b> Representative FRET traces from pmEpac1 cardiomyocytes stimulated with ISO (100 nM) and subsequently by the PDE1 inhibitor 8-MMX (30 μM) followed by the unselective PDE inhibitor IBMX (100 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167974#pone.0167974.g003" target="_blank">Fig 3A and 3B</a>. <b>(B and D)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes treated with ISO, rolipram or 8-MMX. Cells were paced at 1 Hz. Values are means ± SEM; from n = 12 and n = 11 cells (unpaced and paced, respectively) isolated from 3 hearts per condition in B and n = 9 cell from 2 hearts each in D; n.s.—not significant by one-way ANOVA.</p