Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II

Abstract

beta(1)-adrenergic receptor (beta(1)AR) stimulation activates the classic cAMP/protein kinase A (PKA) pathway to regulate vital cellular processes from the change of gene expression to the control of metabolism, muscle contraction, and cell apoptosis. Here we show that sustained beta(1)AR stimulation promotes cardiac myocyte apoptosis by activation of Ca(2+)/calmodulin kinase II (CaMKII), independently of PKA signaling. beta(1)AR-induced apoptosis is resistant to inhibition of PKA by a specific peptide inhibitor, PKI14-22, or an inactive cAMP analogue, Rp-8-CPT-cAMPS. In contrast, the beta(1)AR proapoptotic effect is associated with non-PKA-dependent increases in intracellular Ca(2+) and CaMKII activity. Blocking the L-type Ca(2+) channel, buffering intracellular Ca(2+), or inhibiting CaMKII activity fully protects cardiac myocytes against beta(1)AR-induced apoptosis, and overexpressing a cardiac CaMKII isoform, CaMKII-deltaC, markedly exaggerates the beta(1)AR apoptotic effect. These findings indicate that CaMKII constitutes a novel PKA-independent linkage of beta(1)AR stimulation to cardiomyocyte apoptosis that has been implicated in the overall process of chronic heart failure.

Figure 1

Inhibition of the cAMP/PKA signaling pathway did not protect myocytes from β₁AR-induced apoptosis. Cardiac myocytes from β₁β₂ DKO mice were infected with either Adv-β₁AR or Adv–β-gal at an MOI of 100. (a) Typical micrographs of TUNEL staining of myocytes. Treatment with 1 μM ISO for 24 hours increased the number of apoptotic cells (arrows); the βAR antagonist propranolol (10 μM), but not the PKA inhibitor PKI (5 μM), prevented the β₁AR apoptotic effect. (b) ISO-induced phosphorylation of PLB at Ser16 (P-PLB-Ser¹⁶) in the absence (Ctr) or presence of Rp-cAMP (100 μM) or PKI (5 μM). Similar results were obtained in four other experiments. Pretreatment periods of 1 hour (shown) and 6 hours (not shown) of cells with the PKA inhibitors were equally effective in blocking PKA-dependent PLB phosphorylation in response to ISO treatment (1 μM for 10 minutes). (c–e) Effects of PKA inhibitors on ISO-induced increase in TUNEL staining (c), Hoechst staining (d), or DNA fragmentation as assayed by cell death ELISA (e). Data are presented as mean ± SE (n = 4–8 independent experiments in 5,000–6,000 cells from 10–20 hearts for each group). *P < 0.01 vs. ISO-untreated cells or those pretreated with propranolol. Prop, propranolol.

Figure 2

Effects of PKA and CaMKII inhibitors on β₁AR-mediated increase in TUNEL-positive cells in β₂AR KO (a) or WT (b) mouse cardiac myocytes. β₁ARs in β₂AR KO myocytes were stimulated with 1 μM ISO, and β₁ARs in WT cells were stimulated with 1 μM ISO plus the β₂AR blocker ICI 118,551 (0.5 μM). *P < 0.01 vs. ISO-untreated cells or those pretreated with AIP (10 μM) or KN93 (0.5 μM) (n = 6 for each group). Rp, Rp-cAMP.

Figure 3

G_βγ or G_i signaling is not involved in β₁AR-induced cardiomyocyte apoptosis. Neither inhibition of G_βγ signaling by adenoviral expression of βARK-ct nor disruption of G_i signaling by pretreatment of cells with PTX (1 μg/ml for 3 hours) altered ISO-induced (1 μM) DNA fragmentation assayed by cell death ELISA in β₁β₂ DKO cells infected by Adv-β₁AR. *P < 0.01 vs. ISO-untreated myocytes. n = 6–7 independent experiments for each group.

Figure 4

PKA-independent increase in intracellular Ca²⁺ is essential for the β₁AR apoptotic effect. After β₁β₂ DKO myocytes were infected by Adv-β₁AR, cells were incubated with designated reagents for 1 hour, then ISO (1 μM) was added and cells were incubated for another 3–6 hours (a, b, d, and e) or 24 hours (c). (a) Prolonged β₁AR stimulation elevated basal intracellular free Ca²⁺ in unpaced cardiac myocytes. This effect was blocked by the L-type Ca²⁺ channel antagonist nifedipine (1 μM), but not the PKA inhibitor PKI (5 μM). *P < 0.01 vs. ISO-untreated groups and those pretreated by nifedipine (n = 20–35 cells from six hearts). (b) Intracellular Ca²⁺ transients were measured in a subset of cells electrically paced at 0.5 Hz for at least 10 minutes in the absence (n = 29 cells from four hearts) and presence (n = 22 cells from four hearts) of sustained β₁AR stimulation by ISO. *P < 0.05 vs. ISO-untreated myocytes. (c) Effects of nifedipine, EGTA-AM (1 μM), or the SR ATPase inhibitor thapsigargin (1 μM) on β₁AR-induced increase in TUNEL-positive cells. *P < 0.01 vs. ISO-untreated myocytes and those pretreated with EGTA-AM, nifedipine, or thapsigargin (n = 4–8). (d) Representative confocal linescan images of caffeine-elicited SR Ca²⁺ release in ISO-treated (1 μM, 3 hours, bottom) and untreated cells (top). The x axis shows the time courses for caffeine treatment, and the y axis shows the spatial profiles of Ca²⁺ transients along a scan line inside the cell. (e) Average amplitude of caffeine-elicited Ca²⁺ transients in ISO-treated or untreated group. *P < 0.01 vs. ISO-untreated myocytes. n = 25–30 cells from six hearts in each group. Nif, nifedipine; TG, thapsigargin.

Figure 5

Role of CaMKII and calcineurin in the β₁AR-mediated apoptotic effect. After β₁β₂ DKO myocytes were infected by Adv-β₁AR, cells were pretreated with the CaMKII inhibitors AIP (10 μM) or KN93 (0.5 μM) or the inactive KN93 analogue KN92 (2 μM); with the calcineurin inhibitors FK506 (10 μM) or cyclosporin A (5 μM); with the PKA inhibitor PKI (5 μM); or with the Ca²⁺ channel blocker nifedipine (1 μM), all for 1 hour (except 3 hours for AIP) prior to administration of 1 μM ISO. Apoptosis was assessed after incubation for another 24 hours. (a) AIP or KN93 fully protected cardiomyocytes against ISO-elicited apoptosis. Arrows indicate TUNEL-positive nuclei. (b) The ISO-induced increase in TUNEL-positive cells was prevented by AIP or KN93, but not the inactive KN93 analogue KN92 or the calcineurin inhibitors FK506 or cyclosporin A. n = 4–8. *P < 0.01 versus ISO-untreated groups or those treated with KN93 or AIP. (c) ISO-induced DNA laddering in the absence (control) or presence of KN93, PKI, or the βAR antagonist propranolol. Similar results were obtained in four other experiments. CyA, cyclosporin A.

Figure 6

Temporal and pharmacological profiles of CaMKII activation in response to β₁AR stimulation. (a) In Adv-β₁AR–infected β₁β₂ DKO myocytes, β₁AR stimulation (1 μM ISO for 6 hours) increased CaMKII autophosphorylation. This effect was blocked by KN93 (5 μM) but not by the PKA inhibitor PKI (5 μM). Similar results were obtained in three other experiments. (b) Time course of ISO-induced increase in CaMKII activity assayed by ³²P incorporation into a specific peptide substrate of the kinase (see Methods; n = 4 for each data point). (c) Pharmacological profile of CaMKII activation (n = 4–6). *P < 0.01 vs. cells in the absence of ISO or those in the presence of KN93, AIP, or nifedipine. P-CaMKII, autophosphorylated CaMKII.

Figure 7

Overexpression of CaMKII-δC exaggerates β₁AR-induced myocyte apoptosis. (a) Confocal imaging of HA immunofluorescence in typical β₂AR KO myocytes expressing either β-gal (I), or HA-tagged CaMKII-δC (II, cell surface scan; III, cell nucleus level scan), or HA-tagged CaMKII-δB (IV). (b) Expression of HA-tagged CaMKII-δC assayed by Western blot with an antibody reacting with HA. (c) Dose responses of β₁AR-induced increase in apoptotic cells in β₂AR KO myocytes infected by Adv–CaMKII-δC (with or without KN93) or Adv–β-gal (n = 6 for each data point).