Linkage of β₁-adrenergic stimulation to apoptotic heart cell death through protein kinase A–independent activation of Ca²⁺/calmodulin kinase II

β₁-adrenergic receptor (β₁AR) 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 β₁AR stimulation promotes cardiac myocyte apoptosis by activation of Ca²⁺/calmodulin kinase II (CaMKII), independently of PKA signaling. β₁AR-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 β₁AR proapoptotic effect is associated with non–PKA-dependent increases in intracellular Ca²⁺ and CaMKII activity. Blocking the L-type Ca²⁺ channel, buffering intracellular Ca²⁺, or inhibiting CaMKII activity fully protects cardiac myocytes against β₁AR-induced apoptosis, and overexpressing a cardiac CaMKII isoform, CaMKII-δC, markedly exaggerates the β₁AR apoptotic effect. These findings indicate that CaMKII constitutes a novel PKA-independent linkage of β₁AR stimulation to cardiomyocyte apoptosis that has been implicated in the overall process of chronic heart failure.

Stimulation of β-adrenergic receptor (βAR), a prototypical G protein–coupled receptor, is broadly involved in metabolic regulation, growth control, muscle contraction, cell survival, and cell death. In the heart, βAR stimulation by catecholamines serves as the most powerful regulatory mechanism to enhance myocardial performance in response to stress or exercise by activating the classic stimulatory pathway comprising the G protein G_s, adenylyl cyclase, cAMP, and protein kinase A (PKA) (1, 2). However, sustained activation of β₁AR, the predominant βAR subtype expressed in the heart, also induces cardiac myocyte apoptosis (3–6). Apoptotic heart cell death has been implicated in the overall process of myocardial remodeling and the transition from cardiac hypertrophy to chronic heart failure (7–10), an illness afflicting more than five million Americans, with a 5-year mortality greater than 80% (11). However, the mechanism underlying the β₁AR apoptotic effect remains poorly understood.

Previous studies suggested that β₁AR-induced cardiac myocyte apoptosis was mediated by the cAMP/PKA pathway (12, 13), the only known intracellular mechanism underlying β₁AR-elicited cellular responses (1, 2). However, transgenic overexpression of type V (14) or type VI (15) adenylyl cyclase in mouse hearts does not cause cell death, although it markedly augments basal PKA activity (14) and cardiac contractility (14, 15). More ironically, in cultured cardiac myocytes or in vivo, selective β₂AR subtype stimulation elicits a profound cardiac protective effect (4, 5, 16), in spite of overtly enhanced cAMP formation (16–18).

The goal of the present study is to determine the mechanism of β₁AR-induced apoptosis. In addition to pharmacological approaches used in WT mouse cardiac myocytes, we created genetically “pure” β₁AR experimental settings using adult cardiac myocytes from β₂AR KO mice (19) or by adenovirus-mediated gene transfer (20) of the mouse β₁AR in myocytes from β₁β₂ double knockout (DKO) mice (21). These approaches enabled us to avoid the complicated interactions between the coexisting β₁AR and β₂AR subtypes that exert opposing effects on cardiac cell survival and cell death (4, 5, 16). In the present study, we demonstrate that β₁AR-induced cardiac myocyte apoptosis is independent of cAMP and PKA signaling, but requires a novel signaling pathway involving Ca²⁺ and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII).

Sustained β₁AR stimulation delivers a potent apoptotic signal in cardiac myocytes. In β₁β₂ DKO cells infected with an adenoviral vector encoding β₁AR (Adv-β₁AR) at an MOI of 100 for 24 hours, the β₁AR density was 550 ± 46 fmol/mg protein (n = 4), which was approximately 16-fold greater than the receptor density in WT cells (33.5 ± 1.5 fmol/mg protein, n = 3). Stimulation of the expressed β₁ARs by ISO (1 μM) for 24 hours caused cardiomyocyte death by increased apoptosis (Figure 1a). β₁AR stimulation led to a threefold augmentation in cells positive for either TUNEL (Figure 1, a and c) or Hoechst staining (Figure 1d). Apoptotic nuclei appeared blue by TUNEL staining, with a varying degree of chromatin condensation and fragmentation, as indicated by arrows (Figure 1a). Concomitantly, there was a marked increase in DNA fragmentation assayed by cell death ELISA (Figure 1e) and DNA laddering (see Figure 5c) in myocytes subjected to ISO treatment. Propranolol (10 μM), a βAR antagonist, protected myocytes from ISO-induced apoptosis (Figure 1, a, c, and e, and Figure 5c), indicating that the ISO effect is mediated by receptor activation. The earliest significant apoptotic effect occurred after 8 hours of ISO treatment, as evidenced by a 2.2-fold increase in the percentage of TUNEL-positive cells (n = 3, P < 0.05). Similarly, β₁AR stimulation by ISO markedly increased apoptotic cell death in β₂AR KO myocytes (Figure 2a). Furthermore, selective β₁AR stimulation by ISO (1 μM) in the presence of a β₂AR blocker, ICI 118,551 (0.5 μM), clearly increased WT myocyte apoptosis (Figure 2b). These results validate the relevance of our data in DKO cells expressing β₁AR using adenoviral gene transfer. Thus, sustained β₁AR stimulation delivers a potent apoptotic signal, in agreement with previous reports (3–6). Since a similar maximal apoptotic effect occurred in all three experimental systems examined, the receptor density appears not to be a rate-limiting factor in transducing β₁AR apoptotic signal in cardiac myocytes.

β₁AR-induced apoptosis is independent of cAMP/PKA signaling. To determine the potential role of cAMP/PKA in β₁AR-mediated myocyte apoptosis, we first used H-89, a widely used PKA inhibitor, and found that H-89 at a high concentration (20 μM) protected cardiac myocytes against β₁AR-induced apoptosis. TUNEL-positive cells were reduced from 11.4% ± 1.1% to 3.3% ± 0.8% by H-89 (n = 4, P < 0.01), consistent with previous reports (12). However, the interpretation of experiments using H-89 is complicated by the recent finding that H-89 is also a potent βAR blocker (24). We therefore inhibited this pathway using a highly specific, membrane-permeable peptide PKA inhibitor, PKI (25), and an inactive cAMP analogue, Rp-cAMP (26). Pretreatment of myocytes with either Rp-cAMP (100 μM) or PKI (5 μM) completely abolished PKA-dependent phosphorylation of PLB (Figure 1b), a key cardiac PKA target protein that regulates the SR Ca²⁺ pump activity (27), thus validating the effectiveness of these PKA inhibitors in abrogating cAMP/PKA signaling. Surprisingly, neither PKA inhibitor at the same respective concentrations had any significant effect on β₁AR-mediated apoptosis (Figure 1, a and c–e, and Figure 5c).

The inability of PKA inhibitors to protect cardiac myocytes against β₁AR-induced apoptosis was confirmed in the β₂AR KO model, in which the density and functionality of native β₁ARs remain unaltered (19), and in WT mouse myocytes subjected to ISO in the presence of β₂AR blockade. Stimulation of the native β₁AR in adult β₂AR KO or WT cardiac myocytes by ISO similarly increased TUNEL-positive cells even in the presence of PKI or Rp-cAMP (Figure 2). Thus, while the cAMP/PKA signaling pathway has been thought to be the sole mechanism responsible for β₁-adrenergic responses, β₁AR apoptotic signal transduction essentially bypasses this pathway.

Role of G_βγ or G_i signaling. To identify the molecular mediator for the non–PKA-dependent β₁AR apoptotic effect, we tested several candidates, including free G_βγ released from heterotrimeric G_s proteins or G proteins other than G_s (e.g., G_i proteins). The possible involvement of G_βγ or G_i signaling in β₁AR-mediated apoptosis was examined by adenoviral gene transfer of the C-terminal domain of βAR kinase (βARK-ct) to inhibit G_βγ signaling (28) and by pretreatment of cells with PTX to disrupt G_i signaling (29). Neither βARK-ct nor PTX significantly affected β₁AR-mediated apoptotic DNA fragmentation (Figure 3). In contrast, both interventions effectively blocked the G_βγ- and G_i-mediated β₂AR antiapoptotic effect under similar experimental conditions (5) and prevented G_βγ-mediated activation of PI3K (30). These results rule out the possibility that G_βγ or G_i signaling is responsible for β₁AR-mediated cardiomyocyte apoptosis.

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.

Essential role of Ca²⁺ entry and intracellular Ca²⁺ in β₁AR apoptotic signaling. It has been shown that altered Ca²⁺ signaling promotes apoptosis in a variety of cell types (31). We next explored the possibility that Ca²⁺, instead of cAMP, acts as the second messenger to transmit the β₁AR apoptotic signal in cardiac myocytes. In Adv-β₁AR–infected DKO myocytes, ISO treatment for 3–6 hours (just prior to manifestation of β₁AR-induced apoptosis) significantly elevated intracellular free Ca²⁺ as measured with the Ca²⁺-sensitive fluorescent probe indo-1. As shown in Figure 4a, sustained β₁AR stimulation increased basal cytosolic Ca²⁺ from 122.8 ± 5.8 (n = 32 cells from six hearts) to 308.7 ± 9.7 nM (n = 29 cells from six hearts). This Ca²⁺ response remained largely intact in the presence of PKI (5 μM) but was abolished by nifedipine (1 μM), an L-type Ca²⁺ channel antagonist, indicating an elevation in intracellular Ca²⁺ mediated by L-type Ca²⁺ currents (I_Ca) (Figure 4a). Furthermore, we measured phasic intracellular Ca²⁺ transients in cultured, paced (0.5 Hz) cardiac myocytes in the presence and absence of prolonged β₁AR stimulation by ISO. β₁AR stimulation significantly increased both diastolic and systolic Ca²⁺, with the diastolic effect most prominent, in paced cardiac myocytes (Figure 4b). Thus, β₁AR stimulation increases cytosolic Ca²⁺ in both contracting and noncontracting cardiac myocytes. In order to determine whether the altered Ca²⁺ homeostasis is causally linked to β₁AR-induced apoptosis, we inhibited Ca²⁺ entry by nifedipine or buffered intracellular Ca²⁺ by incubating cells with EGTA-AM. Both treatments rescued cardiac myocytes from β₁AR-induced apoptosis (Figure 4c).

In addition, we measured SR Ca²⁺ load, as indexed by the amplitude of Ca²⁺ transients generated in response to a bolus administration of caffeine (20 μM). We found that sustained β₁AR stimulation significantly elevated the caffeine-releasable SR Ca²⁺ content, by 38% (Figure 4, d and e). Confocal imaging further revealed that the caffeine-induced Ca²⁺ release was spatially uniform (at the optical resolution), regardless of β₁AR stimulation (Figure 4d). Paralyzing SR Ca²⁺ recycling by inhibiting the SR Ca²⁺ pump with thapsigargin also protected myocytes from β₁AR-induced apoptosis (Figure 4c). Together, these results indicate that the enhanced L-type channel Ca²⁺ influx, the subsequent increases in cytosolic Ca²⁺, and the SR Ca²⁺ overload are all required for β₁AR-induced myocyte apoptosis.

β₁AR-induced apoptosis requires PKA-independent activation of CaMKII. To further delineate the specific pathway transducing Ca²⁺-mediated β₁AR apoptotic signaling, we examined possible roles of Ca²⁺-dependent protein phosphatases and kinases, particularly calcineurin and CaMKII. It remains controversial whether activation of calcineurin participates in Ca²⁺-induced cardiac myocyte apoptosis (32, 33), whereas its apoptotic effect is well established in other cell types (34). The present data show that inhibition of calcineurin by cyclosporin A (5 μM) or FK506 (10 μM) failed to block β₁AR-induced apoptotic heart cell death, suggesting that calcineurin does not play an essential role in β₁AR apoptotic signaling (Figure 5b). In sharp contrast, a highly specific membrane-permeable peptide inhibitor of CaMKII, autocamtide-2–related inhibitory peptide (AIP, 10 μM) (35), fully protected myocytes from β₁AR-induced apoptosis, as evidenced by the typical micrographs (Figure 5a) and the average results of TUNEL staining (Figure 5b). Similar protective effects were observed with another specific CaMKII inhibitor, KN93 (0.5 μM), but not its inactive analogue, KN92 (2 μM) (Figure 5, a and b). DNA laddering assays confirmed that β₁AR-induced DNA fragmentation was resistant to the PKA inhibitor PKI, but suppressed by the CaMKII inhibitor KN93 (Figure 5c). Moreover, in either β₂AR KO or WT mouse cardiac myocytes, inhibition of CaMKII by AIP or KN93 similarly prevented β₁AR-mediated apoptosis (Figure 2). Norepinephrine (at 1 μM), a physiological catecholamine, in the presence of an α₁AR blocker, prazosin (1 μM), similarly promoted apoptosis in WT cells, an effect that was also abolished by KN93 or AIP (data not shown). These results provide the first demonstration that activation of CaMKII, rather than PKA, is required for β₁AR-induced apoptosis in the heart.

The pivotal role of CaMKII in β₁AR-mediated apoptotic signaling was reinforced by data on the pharmacological profile of β₁AR-induced CaMKII activation. In Adv-β₁AR–infected DKO cells, ISO stimulation for 6 hours increased the level of autophosphorylated CaMKII, an active form of the kinase (36), without altering the abundance of the kinase protein (Figure 6a). Notably, the increase in CaMKII activity was PKI-resistant, but was abolished by KN93 and AIP (Figure 6, a–c) at the same respective concentrations that blocked the β₁AR-mediated apoptotic effect. Since CaMKII activity was elevated prior to the manifestation of cardiomyocyte apoptosis, we conclude that CaMKII activation constitutes an early β₁AR-elicited event to signal cell apoptosis.

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.

Overexpression of CaMKII-δC exaggerates β₁AR-induced apoptosis. Based on the aforementioned results, an increase in CaMKII abundance should enhance the β₁AR apoptotic effect in cardiac myocytes. To test this hypothesis, we expressed HA-tagged CaMKII-δC, a predominant cardiac cytoplasmic CaMKII isoform (37), or CaMKII-δB, a nuclear CaMKII isoform (37), in β₂AR KO myocytes. Confocal immunocytochemical analysis confirmed that the expressed CaMKII-δC was distributed in cytoplasm and the surface membranes, including the transverse tubules (Figure 7a, II and III), but not in the nuclei (Figure 7a, III), whereas CaMKII-δB was concentrated in the nuclei (Figure 7a, IV). Expression of HA-tagged CaMKII-δC was also confirmed by Western blotting using a monoclonal antibody reacting with HA (Figure 7b). Remarkably, overexpression of CaMKII-δC shifted the dose-response curve of ISO-induced apoptosis leftward by nearly an order of magnitude (EC₅₀, 1.1 nM and 9.0 nM for myocytes infected by Adv–CaMKII-δC and Adv–β-gal, respectively) and increased the maximal apoptotic effect by 50%, whereas it exerted no appreciable apoptotic effect in the absence of β₁AR stimulation by ISO (Figure 7c). Again, inhibition of CaMKII by KN93 in cardiac myocytes overexpressing CaMKII-δC abolished the β₁AR apoptotic effect over a wide range of agonist concentrations (Figure 7c). By contrast, overexpression of CaMKII-δB neither enhanced nor reduced β₁AR-induced myocyte apoptosis (data not shown), indicating that CaMKII-δC but not CaMKII-δB is involved in β₁AR apoptotic signaling. The fact that increased CaMKII-δC abundance exaggerates the β₁AR apoptotic effect without altering basal cell apoptosis substantiates the conclusion that CaMKII mediates the β₁AR apoptotic signal.

β₁AR apoptotic signaling pathway. The prevalent theory of β₁AR signal transduction is that the cAMP/PKA pathway is solely responsible for β₁AR-mediated cellular responses. A close inspection of studies to date, however, reveals no convincing evidence to validate that this is also the case for β₁AR-evoked apoptotic signal in the heart. Using two genetically defined β₁AR systems, we have provided the first documentation that sustained β₁AR stimulation delivers a powerful cardiac apoptotic signal via a CaMKII-dependent, rather than a PKA-dependent, mechanism. This conclusion is based on several lines of evidence. First, inhibition of PKA by a specific peptide inhibitor, PKI, or an inactive cAMP analogue, Rp-cAMP, does not affect β₁AR-induced myocyte apoptosis under conditions in which PKA-mediated protein phosphorylation is completely blocked. Second, sustained β₁AR stimulation elicits PKA-independent augmentation of intracellular Ca²⁺ as well as SR Ca²⁺ load in both resting and electrically paced myocytes, and CaMKII is activated in a time-dependent fashion. More importantly, either blocking I_Ca, buffering intracellular Ca²⁺, or inhibiting CaMKII activity fully protects cardiac myocytes against β₁AR-induced apoptosis. The essential role of CaMKII in β₁AR apoptotic signaling is also corroborated by the fact that overexpression of a cardiac isoform of CaMKII, CaMKII-δC, markedly enhances β₁AR-induced apoptosis. Similarly, in cultured adult rat cardiac myocytes, β₁AR-mediated apoptosis is blocked by nifedipine or CaMKII inhibition with KN93, but not by PKA inhibition with PKI (data not shown). Thus, we conclude that the β₁AR-evoked apoptotic signal is delivered by a PKA-independent, CaMKII-mediated signaling pathway. Nevertheless, this should not be taken as evidence that basal CaMKII activation (such as in the beating heart) alone is sufficient to induce apoptosis. Our previous studies have shown that 2.0-Hz electrical pacing is able to augment CaMKII activation, as shown by a twofold increase in CaMKII-dependent phosphorylation of PLB at Thr¹⁷, and that the effect of pacing on PLB-Thr¹⁷ phosphorylation is synergistically enhanced to sixfold when combined with β₁AR stimulation in freshly isolated rat cardiac myocytes (38), indicating that β₁AR stimulation and electrical pacing exert a synergistic effect on CaMKII activation in cardiac myocytes.

Although unexpected, the present finding that the β₁AR apoptotic effect is PKA-independent is supported by previous observations that transgenic overexpression of adenylyl cyclase and the resultant elevation of intracellular cAMP in mouse hearts are dissociated from myocyte apoptosis (14, 15), and that β₂AR stimulation exhibits a profound antiapoptotic effect despite elevated intracellular cAMP formation in cardiac myocytes (3–5, 16). Together, the present and previous studies indicate that an increase in cAMP/PKA signaling does not necessarily cause heart cell apoptosis. This perhaps reflects distinct compartmentation of intracellular cAMP under different circumstances (2, 26, 39–42). In contrast, the linkage of sustained β₁AR stimulation to cardiac myocyte apoptosis by the multifunctional protein kinase, CaMKII, is in general agreement with recent findings that, in naive cell lines, numerous insulting factors including UV light, TNF-α (43), and protein phosphatase inhibitors (44) can activate CaMKII, which then contributes to the insult-induced apoptosis.

Distinct signaling modes underlie sustained versus acute β₁AR stimulation. Given that both Ca²⁺ and cAMP serve as the second messenger, β₁AR signal transduction bifurcates into two pathways mediated by CaMKII and PKA, respectively. Interestingly, the two pathways are called upon in tandem to fulfill distinctly different functional roles. Acute β₁AR stimulation rapidly activates the cAMP/PKA pathway, with the peak response within 1 minute (45), whereas prolonged β₁AR stimulation causes desensitization of cAMP/PKA signaling within 30 minutes (1, 24). This fast cAMP/PKA response is crucial to sympathetic control over the heart rate and myocardial contraction, allowing the heart to increase its output within seconds in response to a “fight-or-flight” situation. In contrast, the newly identified CaMKII signaling is evoked gradually and appears to contribute little to acute β₁AR contractile response, but is essential to the β₁AR cardiac apoptotic effect.

The slow kinetics of CaMKII activation may reflect a cumulative increase in intracellular Ca²⁺ due to a small but persistent Ca²⁺ entry through L-type channels. This interpretation is consistent with the facts that CaMKII activation is blocked by the L-type channel antagonist niphedipine and that CaMKII-δC is enriched beneath cell surface membranes, thus permitting an intimate interplay between CaMKII and the L-type channel (23). Alternatively, the gradual elevation of CaMKII activity may reflect that multiple steps, e.g., upregulation of intermediate signaling components, are involved in transducing receptor signal to the kinase activation.

Regardless of the exact mechanism, the time-dependent switch of signaling modes during enduring receptor stimulation may represent a new paradigm of G protein–coupled receptor signal transduction. In this regard, it has recently been shown that β₂AR stimulation induces a switch from cAMP/PKA signaling to a G_i-dependent MAPK signaling pathway (46), and that these two events are causally linked, i.e., activation of cAMP/PKA is a prerequisite for the receptor coupling to G_i proteins and the subsequent activation of the MAPK pathway (46). In the case for sustained β₁AR stimulation, however, the gradual activation of CaMKII is independent of PKA signaling (Figure 6). Both examples show a time-dependent homologous regulation of G protein–coupled receptor signaling.

Regarding possible mechanisms underlying the non–PKA-dependent increase in intracellular Ca²⁺, it has been proposed that persistent β₁AR stimulation increases I_Ca via a direct coupling of G_sα to the Ca²⁺ channel (47). More recently, it has been shown that cardiac-specific overexpression of G_sα similarly augments I_Ca amplitude in a PKA-independent manner (48). Both lines of evidence support the idea that PKA-independent cross-talk between G_sα and the L-type Ca²⁺ channel may contribute to β₁AR-induced increase in intracellular Ca²⁺ and subsequent activation of CaMKII.

Pathophysiological relevance of β₁AR apoptotic signaling. Increasing evidence indicates that prolonged β₁AR stimulation exerts a cardiotoxic effect that often outweighs the short-term gain in cardiac contractile support. Both in vivo and in vitro studies have shown that enhanced β₁AR signaling by either selective receptor stimulation or receptor overexpression increases proapoptotic protein levels and promotes cardiac apoptosis (3–6). Since cardiac myocytes are terminally differentiated cells, preventing the loss of irreplaceable cells is critical for the maintenance of normal cardiac function. These studies explain, at least in part, the poor prognosis of heart failure patients with tonically elevated plasma norepinephrine, an endogenous β₁AR agonist (49), as well as the beneficial effects of βAR blockers, particularly β₁AR blockers in chronic heart failure (50). In light of the present findings, an increase in intracellular Ca²⁺ and subsequent activation of CaMKII, rather than the long-suspected cAMP/PKA cascade, are particularly cardiotoxic in terms of cardiac myocyte apoptosis. Thus, unraveling the new pathway and the novel signaling mode of β₁AR signal transduction hold promise for identifying key therapeutic targets and new strategies for minimizing detrimental effects of β₁AR stimulation in the failing heart.

We thank E.G. Lakatta, M. Crow, M. Mattson, A. Chelsey, N.M. Soldatov, and D.R. Abernethy for valuable discussions, and B. Ziman for excellent technical support. This work was supported by the NIH Intramural Research Program(K. Chakir, H. Cheng, and R.-P. Xiao) and by Major State Basic Research Development Program (grant no. 973) of China (R.-P. Xiao).

See the related Commentary beginning on page 597.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: β-adrenergic receptor (βAR); protein kinase A (PKA); Ca²⁺/calmodulin-dependent protein kinase II (CaMKII); isoproterenol (ISO); pertussis toxin (PTX); Rp-8-CPT-cAMPS (Rp-cAMP); protein kinase A inhibitor 14-22 (PKI); autocamtide-2–related inhibitory peptide (AIP); sarcoplasmic reticulum (SR); hemagglutinin (HA); phospholamban (PLB).

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