Cardiac Myocyte Signaling Responses

To assess the suitability of the adult mouse cardiac myocytes for signaling studies, we measured ligand-induced changes in the accumulation of cyclic adenosine monophosphate (cAMP) in the phosphorylation of a panel of signaling proteins and in excitation-contraction coupling. For all experiments, myocytes were cultured overnight (approximately 18 hours) before treatment with ligands.

Ligand induced changes in the accumulation of cAMP. To measure ligand-induced changes in cAMP, three sentinel ligands were used: (i) isoproterenol, a nonselective b-adrenergic receptor ( b-AR) agonist that activates the Gs subtype of G proteins and adenylate cyclase; (ii) forskolin, a direct activator of adenylate cyclase; and (iii) carbachol, a muscarinic receptor agonist that activates the Gi subtype of G proteins and inhibits adenylate cyclase. Cyclic adenosine monophosphate concentrations were measured by enzyme-linked immunosorbent assay (ELISA) . Isoproterenol induced a rapid (30 seconds), seven-fold increase in cAMP that declined to a steady state plateau, about 60% of the peak, by three minutes (Fig. 2A). Forskolin, a direct activator of adenylate cyclase, caused a gradual increase in cAMP for the entire twenty-minute time course (Fig. 2B). Finally, carbachol inhibited increases in cAMP induced by either isoproterenol or forskolin (Fig. 2C and 2D). These observations are consistent with the known effects of these ligands on the accumulation of cAMP in cardiac myocytes from other species and whole heart.

Fig. 2. Accumulation of cAMP in myocytes treated with sentinel ligands. (A, B) Myocytes were cultured for 18 hr (overnight) and then treated for 0, 1, 3, 8, or 20 min with (A) 1 mM isoproterenol or (B) 100 mM forskolin. After treatment, myocytes were lysed and the amounts of cAMP were determined by ELISA (protocol number PP00000015). (C, D) To assess inhibition of cAMP accumulation, myocytes were cultured overnight and then pretreated for 3 min with 10 mM carbachol and treated either (C) for 1 min with 1 mM isoproterenol or (D) for 10 min with 100 mM forskolin. After treatment, myocytes were lysed and the amounts of cAMP were determined by ELISA (protocol number PP00000015). Each graph shows mean ± S.E.M. n = 3, with each measurement made in duplicate.

Ligand-induced changes in the phosphorylation of signaling proteins. To measure ligand-induced changes in the phosphorylation of signaling proteins, five sentinel ligands were used: (i) phenylephrine (an a1-AR agonist) and (ii) angiotensin II, which are both hypertrophic agonists that activate Gq-coupled receptors; (iii) insulin and (iv) insulin-like growth factor 1, which are both hypertrophic agonists that activate the PI3-kinase signaling pathway; and (v) isoproterenol, a b-AR agonist. Protein phosphorylation was monitored by Western blot analysis using two phosphoprotein antibody mixtures . One antibody mixture contained phosphospecific antibodies to Akt (Ser 473), p38 MAP kinase (Thr 180/Tyr 182), and S6 ribosomal protein (Ser 235/236), and the second antibody mixture contained phosphospecific antibodies to ERK1/2 (Thr 202/Tyr 204), p70 S6 kinase (Thr 421/Ser 424), and phospholamban (Ser 16) (Fig. 3A). Phenylephrine significantly increased the phosphorylation of ERK and p38. Insulin and insulin-like growth factor-1 (IGF-1) rapidly increased the phosphorylation of Akt, p70 S6 kinase, and S6 ribosomal protein, all downstream targets of PI3-kinase. Isoproterenol greatly increased phosphorylation of phospholamban (about 40-fold) and also increased the phosphorylation of ERK, p38 MAP kinase, and S6 ribosomal protein (Fig. 3B-F). These results confirm previous reports for the ligand-induced changes in the phosphorylation of signaling proteins, for example, the increased phosphorylation of ERK in response to phenylephrine(18) and the increased phosphorylation of Akt with insulin in mouse heart in vivo(19).

Fig. 3. Phosphorylation of signaling proteins in myocytes treated with sentinel ligands. Myocytes were cultured for 18 hr (overnight) and then treated for 0, 1, 3, 15, 60, or 240 min with five sentinel ligands: 30 mM phenylephrine (with 2 mM timolol, a b-adrenergic receptor antagonist); 100 nM angiotensin II; 100 nM insulin; 10 nM insulin-like growth factor-1; or 1 mM isoproterenol. After treatment, myocytes were lysed with SDS-PAGE sample buffer (PP00000132 and PS00000437) and the cell extracts were examined by Western immunoblotting (PP00000007) with two mixtures of phosphospecific antibodies. (A) Western blots were performed using two antibody mixtures. Antibody mixture A contained phosphospecific antibodies to Akt (Ser 473), p38 MAP kinase (p38 MAPK; Thr 180/Tyr 182), and S6 ribosomal protein (ribosomal S6; Ser 235/236), and antibody mixture B contained phosphospecific antibodies to p70 S6 kinase (p70S6K; Thr 421/ Ser 424), ERK 1 and 2 (ERK1/2; Thr 202/Tyr 204), and phospholamban (PLB; Ser16; pentamer, dimer, and single PLB bands are observed). The specific bands are identified to the right of each blot. The band densities for each phosphoprotein were measured (PP00000007), normalized to the band density for Rho-GDI, and the fold change was calculated relative to the 0 time point (= fold change defined as 1). (B-F) Each graph shows the fold change in phosphorylation for each of the five sentinel ligands at 0 (red), 1 (light blue), 3 (orange), 15 (green), 60 (purple), and 240 (blue) min, sequentially. For all bar graphs, data are mean ± S.E.M. n = 6 for three independent preparations from both the Cell Preparation and Analysis Laboratory and the Laboratory for the Development of Signaling Assays (LDSA), except for results with phenylephrine/timolol treated cells, n = 3, which were prepared in the LDSA. NR = no response; * P < 0.05 (t-test vs. 0 min).

Ligand-induced changes in myocyte excitation-contraction coupling (contraction and calcium transients). To assess excitation-contraction coupling in cultured adult mouse cardiac myocytes, we measured contractile responses and calcium transients in response to electrical stimulation and isoproterenol . Myocytes were cultured overnight and then loaded with the calcium indicator fura-2. Measurements of contraction (percent shortening) and calcium transients on individual myocytes were made with electrical stimulation (1 Hz, 20 V, 4 ms) before and after treatment with isoproterenol (Fig. 4A-D). Isoproterenol increased the extent of myocyte shortening (Fig. 4E; basal 3.79 ± 0.92 mm; isoproterenol 9.11 ± 0.59 mm; mean ± SEM; n = 7 for each; P < 0.05), the amplitude of the calcium transient (Fig. 4F; 171 ± 15%; mean ± SEM; n = 7; P < 0.05), and the rate of decline of the calcium transient (Fig. 4G; basal 0.185 ± 0.010 sec; isoproterenol 0.159 ± 0.004 sec; mean ± SEM; n = 7 for each; P < 0.05). The contractile and calcium responses to electrical stimulation in cultured adult mouse myocytes indicate intact excitation-contraction coupling mechanisms. In addition, increased myocyte shortening and calcium transients in response to isoproterenol correlated with the increase in phospholamban phosphorylation observed by Western blot analysis (8,15,20,21).

Fig. 4. Calcium transients and contraction in myocytes treated with isoproterenol. Myocytes were cultured for 18 hr (overnight) and then loaded with the calcium indicator fura-2AM at 25 °C in HEPES buffer. For all measurements, myocytes were electrically paced (1 Hz, with 20 V pulse amplitude and 4 ms pulse duration), and basal and ligand stimulated changes in length and calcium were recorded. (A, B) Contraction in a representative myocyte, measured as length change, is shown before and 5 min after treatment with 1 mM isoproterenol. The y-axis shows myocyte length in m and the x-axis shows time in seconds. (C, D) Calcium transients are shown in a single myocyte before and 5 min after treatment with 1 mM isoproterenol. The y-axis shows fura-2 emission ratios (360 nm/380 nm) and the x-axis shows time in seconds. (E) Average myocyte shortening, (F) calcium transient amplitude (plotted as fold increase), and (G) calcium re-uptake time constant were measured before and 5 min after isoproterenol (1 mM). Data are mean ± SEM; n = 7 myocytes from two cultures; * P < 0.05.