Phosphodiesterase 4B in the cardiac L-type Ca²⁺ channel complex regulates Ca²⁺ current and protects against ventricular arrhythmias in mice

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (I_Ca,L) is increased only in Pde4b^–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b^–/– and Pde4d^–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b^–/– mice but not in WT mice. These results identify PDE4B in the Ca_V1.2 complex as a critical regulator of I_Ca,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²⁺-induced Ca²⁺ release in cardiomyocytes.

During the cardiac action potential, Ca²⁺ influx through sarcolemmal L-type Ca²⁺ channels (LTCCs) triggers Ca²⁺ release from juxtaposed ryanodine receptor 2 (RyR2) located in the sarcoplasmic reticulum (SR). This allows a rapid and synchronous Ca²⁺ elevation throughout the cell, which activates contraction. During cardiac relaxation, Ca²⁺ is rapidly extruded by the Na⁺/Ca²⁺ exchanger and re-sequestered into the SR by the Ca²⁺-ATPase, SERCA2 (1). This process is highly regulated, in particular, by the sympathetic nervous system. β-Adrenergic receptors (β-ARs) exert strong inotropic and lusitropic effects by increasing intracellular cAMP levels and activating cAMP-dependent PKA. PKA then phosphorylates the key proteins of the excitation-contraction coupling (ECC) process, including LTCC and RyR2 but also phospholamban (PLB), which controls Ca²⁺ reuptake by SERCA2, as well as the myofilament proteins troponin I and myosin binding protein C (1).

The cardiac LTCC consists of the central pore-forming subunit α_1C (Ca_V1.2) and auxiliary β and α₂-δ subunits that modulate its function (2). Upon β-AR stimulation, phosphorylation of Ca_V1.2, the auxiliary β₂ subunit, or the closely associated protein AHNAK by PKA increases channel activity, thus enhancing the L-type Ca²⁺ current (I_Ca,L) (3–5). This regulation involves physical association of PKA with Ca_V1.2 via an A-kinase anchoring protein, AKAP15/18 (6, 7). Similarly, different AKAPs are responsible for the localization of PKA in the immediate vicinity of ECC proteins: the muscle-specific AKAP targets PKA to RyR2 (8, 9), and AKAP18δ localizes PKA to PLB (10).

PKA targeting by AKAPs would not be sufficient to explain hormonal specificity if cAMP could diffuse freely inside cells (11). In addition to discrete production sites, cAMP spreading is restricted by cyclic nucleotides phosphodiesterases (PDEs), the enzymes that degrade cAMP and cGMP into their inactive counterparts, 5′-AMP and 5′-GMP, respectively. Classically, 4 different PDE families (PDE1–PDE4) hydrolyze cAMP in heart: PDE1, which is activated by Ca²⁺-calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4. PDE1 and PDE2 can hydrolyze both cAMP and cGMP, PDE3 preferentially hydrolyzes cAMP, and PDE4 is specific for cAMP (12). A fifth PDE, PDE8A, has been recently shown to modulate cAMP signaling in mouse cardiomyocytes (13). Association of individual PDE families to G_s-coupled receptors enables cardiac cells to generate heterogeneous cAMP signals in response to different hormones and specific control of I_Ca,L (14).

PDE4 is one of the main PDEs expressed in heart. PDE4 becomes predominantly active upon β-AR stimulation and regulates global cAMP levels in cardiac cells (14–19). Particularly, PDE4 is the main PDE modulating I_Ca,L in cardiomyocytes (18, 20). The PDE4 family is encoded by 4 genes (PDE4A–PDE4D), but only PDE4A, PDE4B, and PDE4D are expressed in cardiac tissue (21). These 3 genes give rise to multiple isoforms generated through alternative splicing and by the use of different promoters (22). So far, of these genes, most of our knowledge in the heart concerns the PDE4D gene. Long variants of this gene expressed in the heart include the isoforms PDE4D3, PDE4D5, PDE4D8, and PDE4D9 (23, 24). PDE4D5, PDE4D8, and PDE4D9 were shown to differentially interact with β₁-AR and β₂-AR subtypes in neonatal cardiomyocytes, either directly or by binding to β-arrestin (25–27). The PDE4D3 isoform was found in macromolecular signaling complexes, regulating 2 major players of ECC: KCNQ1/KCNE1 potassium channels (28) and RyR2 (29). In Pde4d^–/– mice, RyR2 is hyperphosphorylated by PKA, leading to abnormal Ca²⁺ release, increased sensitivity to exercise-induced arrhythmias, and development of a late-onset dilated cardiomyopathy (29). In addition, a long PDE4D variant was recently found to associate with SERCA2 in the heart (30).

We believe that until now, the molecular identity of the PDE4 regulating the LTCC was unknown. Because of the prominent role of PDE4D variants in the β-AR cascade, we anticipated that a PDE4D variant might also regulate the LTCC activity. Our initial goal here was to test this hypothesis. We indeed found that PDE4D is part of a Ca_V1.2 signaling complex, but, to our surprise, inactivation of the Pde4d gene had no consequence on basal or β-AR stimulated I_Ca,L, although Ca²⁺ transients and contraction were enhanced. We therefore explored the role of the 2 other cardiac PDE4 isoforms, PDE4A and PDE4B, and found that PDE4B, but not PDE4A, was also associated with the LTCC. In Pde4b^–/– mice, but not in Pde4a^–/– mice, the β-AR response of I_Ca,L was increased, together with an increase in cell contraction and Ca²⁺ transients. In vivo, upon β-AR stimulation, catheter-mediated burst pacing triggered ventricular tachycardia (VT) in Pde4b^–/– mice but not in WT mice. We conclude that not only PDE4D, but also PDE4B, plays a key role during β-AR stimulation of cardiac function. PDE4B, by limiting the amount of Ca²⁺ that enters the cell via the LTCCs, prevents Ca²⁺ overload and arrhythmias.

β-AR stimulation of I_Ca,L and effect of PDE4 inhibition in adult mouse ventricular myocytes. We previously showed that the β-AR stimulation of I_Ca,L is under the control of PDE4 in adult rat ventricular myocytes (18). To determine whether the same was true in mice, I_Ca,L was recorded in adult mouse ventricular myocytes (AMVMs) using the whole-cell patch-clamp technique, and the response of this current to a short application of the nonselective β-AR agonist isoprenaline (Iso) was studied. As shown in Figure 1A, a 15-second stimulation with 100 nM Iso elicited a rapid and transient increase of I_Ca,L amplitude that returned to baseline in less than 10 minutes. Selective PDE4 inhibition with Ro 20-1724 (10 μM) had no effect on basal I_Ca,L but strongly potentiated the stimulation of the current by Iso (Figure 1B). On average, Iso alone increased I_Ca,L maximally by 58.8% ± 3.3% (n = 62; Figure 1C). When PDE4 was inhibited, the stimulatory effect of Iso was prolonged, and the maximal stimulation of I_Ca,L reached 103.3% ± 7.7% (n = 37; Figure 1C; P < 0.001 vs. Iso alone). These results identify PDE4 as an important negative-feedback mechanism of the β-AR regulation of I_Ca,L in AMVMs.

Figure 1

PDE4 modulates β-AR stimulation of I_Ca,L in mouse ventricular myocytes. (A) Typical time course of I_Ca,L amplitude in a cell stimulated by a 15-second pulse of Iso (100 nM). Each square represents the amplitude of I_Ca,L recorded every 8 seconds during a depolarization from –50 to 0 mV. (B) I_Ca,L was further increased when the cell was preincubated with the PDE4 inhibitor Ro 20-1724 (Ro; 10 μM) 2 minutes prior to the 15-second pulse of Iso (100 nM) and maintained throughout the experiment. The individual current traces shown on top of A and B were recorded at the times indicated by the corresponding roman numerals in the graphs below. (C) Mean variation of I_Ca,L after Iso (100 nM, 15 seconds) application alone (black diamonds; n = 62 myocytes) or when PDE4 was inhibited by Ro 20-1724 (gray squares; n = 37 myocytes). All curves represent the mean ± SEM. *P < 0.05 indicates the first of the statistically significant points on the graph. All points that occur after this time point are significant.

Characterization of PDE4 subtypes in mouse heart. To evaluate the contribution of PDE4 to cAMP hydrolysis in the mouse heart, total cAMP hydrolytic activity was measured in ventricle homogenates, and the fraction corresponding to PDE4 was assessed with the specific inhibitor rolipram. As shown in Figure 2A, PDE4 represented about 33% of the total activity. To further dissect the contributions of single PDE4 subtypes to the total PDE4 activity, heart extracts were immunoprecipitated using subtype-specific antibodies against PDE4A, PDE4B, and PDE4D (23), and the PDE activity recovered in the IP pellets was assayed. As shown in Figure 2B, the activities of PDE4A, PDE4B, and PDE4D were comparable in the mouse heart. Western blot experiments indicated that each of these activities results from the expression of long PDE4 variants detected as one major immunoreactive band. The absence of signal in mice deficient for the corresponding gene demonstrates that the antibodies used are specific (Figure 2C).

Figure 2

Characterization of PDE4 activity and subtypes in the adult mouse heart. (A) Heart protein extracts were assayed using 1 μM cAMP as substrate. PDE4 activity is defined as the fraction of total PDE activity inhibited by the specific PDE4 inhibitor rolipram (10 μM). (B) Cardiac extracts were immunoprecipitated with control IgG or using antibodies specific for PDE4A, PDE4B, or PDE4D. The graph shows the PDE activity in the IP pellet using 1 μM cAMP as substrate. Data represent the mean ± SEM of experiments performed at least 3 times. (C) Heart protein extracts from WT mice, Pde4a^–/– mice, and Pde4b^–/– mice were separated on SDS/PAGE and revealed with the corresponding PDE4 subtype-specific antibodies. In the case of PDE4D, cardiac extracts from WT and Pde4d^–/– were first immunoprecipitated with the specific PDE4D antibody and then revealed by Western blot.

PDE4B and PDE4D are part of the L-type calcium channel complex. To determine whether any of these PDE4 associate with the LTCC, we performed IP experiments using mouse heart extracts. The pore-forming subunits of the channel, Ca_V1.2, were immunoprecipitated with an anti-Ca_V1.2 antibody (31). A cAMP-hydrolyzing PDE4 activity was associated with the IP Ca²⁺ channel subunit from WT cardiac tissue and was unaffected in heart extracts from Pde4a^–/– mice. In contrast, this PDE4 activity was significantly decreased in ventricular tissues from Pde4b^–/– and Pde4d^–/– mice (Figure 3A). This suggests that PDE4B and PDE4D are part of a macromolecular complex that includes the LTCC, a finding confirmed by Western blot analysis of Ca_V1.2 IP pellets. Using specific isoform antibodies, both PDE4B (Figure 3B) and PDE4D (Figure 3C) were detected in the cardiac LTCC complex. These interactions were specific, because both enzymes were excluded from IPs using control IgGs (Figure 3, B and C). Furthermore, PDE4B could not be detected in the Ca_V1.2 complex in hearts from Pde4b^–/– mice, and the same was true for PDE4D when the coimmunoprecipitation experiment was performed in hearts from Pde4d^–/– mice. This demonstrates that both PDE4B and PDE4D isoforms associate with Ca_V1.2 in the heart and are part of a macromolecular signaling complex, including the cardiac LTCC.

Figure 3

PDE4B and PDE4D are part of the LTCC complex. (A) Ca_V1.2 was immunoprecipitated from cardiac homogenates of WT mice, Pde4a^–/–, Pde4b^–/–, and Pde4d^–/– mice. The associated PDE4 activity was assessed using rolipram. **P < 0.01. (B) PDE4B was detected in cardiac lysates from WT animals and coimmunoprecipitated with Ca_V1.2. (C) PDE4D was also detected in cardiac lysates from WT animals and was associated with Ca_V1.2. IP IgG, negative control. Data are representative of at least 3 independent experiments.

β-AR stimulation of I_Ca,L and effect of PDE4 inhibition in PDE4-deficient mice. To assess the respective contribution of PDE4A, PDE4B, and PDE4D in the β-AR regulation of I_Ca,L, patch-clamp experiments were performed in AMVMs isolated from mice deficient in the corresponding genes (Pde4a^–/–, Pde4b^–/–, and Pde4d^–/– mice). Cell capacitance and I_Ca,L amplitude at baseline were similar in ventricular myocytes from each genotype. As shown in Figure 4A, I_Ca,L potentiation by Iso (100 nM, 15 seconds) was unchanged in Pde4a^–/– compared to that in WT AMVMs. Unexpectedly, the same was true for myocytes isolated from Pde4d^–/– mice (Figure 4C). In line with these results, the PDE4 inhibitor Ro 20-1724 (10 μM) strongly enhanced the effect of Iso on I_Ca,L in Pde4a^–/– and Pde4d^–/– mice (Figure 4, D and F), and this effect was not different from that obtained in WT mice (compare Figure 4, D and F, with Figure 1C), suggesting that these isoforms do not modulate the β-AR stimulation of I_Ca,L. In contrast, the response of I_Ca,L to a transient β-AR stimulation was significantly enhanced in ventricular myocytes isolated from Pde4b^–/– mice compared with that from WT mice (Figure 4B, maximal I_Ca,L potentiation by Iso was 78.3% ± 3.6% in Pde4b^–/– mice, n = 46; P < 0.001 vs. WT). Moreover, the effect of Ro 20-1724 was severely blunted in Pde4b^–/– myocytes (Figure 4E). These findings indicate that PDE4B is the predominant PDE4 isoform involved in I_Ca,L regulation by short β-AR stimulation in mice. However, since the effect of Ro 20-1724 was not completely abolished in Pde4b^–/– mice, another PDE4 isoform must be involved in the β-AR regulation of I_Ca,L, at least when PDE4B is absent. This is most likely PDE4D, since this isoform was also found to coimmunoprecipitate with the pore-forming subunit of Ca_V1.2 (Figure 3, A and C). The biophysical properties of I_Ca,L were compared in WT and Pde4b^–/– myocytes. As shown in Figure 5A, the current-voltage relationship revealed a similar I_Ca,L density measured in WT and Pde4b^–/– myocytes under basal conditions. I_Ca,L exhibited similar voltage dependence in both genotypes: the half-time for activation and inactivation remained unchanged (Figure 5B). When measured at 0 mV, I_Ca,L amplitude, upon continuous application of Iso at a submaximal concentration (3 nM), was significantly increased in WT AMVMs, from 5.9 ± 0.4 pA/pF to 7.3 ± 0.6 pA/pF (P < 0.05), but the β-AR stimulation induced a greater increase of I_Ca,L amplitude in Pde4b^–/– myocytes than in WT myocytes (Figure 5C). This increase was accompanied by a pronounced leftward shift of the activation curve of I_Ca,L in Pde4b^–/– myocytes, whereas steady-state inactivation was not modified (Figure 5D). As a consequence, under β-AR stimulation, the “window” current between –30 mV and 0 mV was enhanced in myocytes lacking PDE4B.

Figure 4

Pde4b inactivation potentiates the β-AR stimulation of the I_Ca,L in AMVMs. (A–C) Average time course of I_Ca,L amplitude after a brief β-AR stimulation induced by Iso (100 nM, 15 seconds) in WT AMVMs (black squares; n = 62 cells) and PDE4-deficient AMVMs (white squares) (Pde4a^–/–, n = 29 cells; Pde4b^–/–, n = 46 cells; Pde4d^–/–, n = 31 cells). (D–F) Effect of global PDE4 inhibition with 10 μM Ro 20-1724 on I_Ca,L stimulation induced by Iso (100 nM, 15 seconds) in PDE4-deficient AMVMs (Pde4a^–/–, n = 24 cells; Pde4b^–/–, n = 22 cells; Pde4d^–/–, n = 28 cells). Ro 20-1724 was applied 2 minutes prior to the β-AR stimulation and maintained throughout the experiment (gray squares). For comparison, dashed lines indicate the effect of Iso alone on I_Ca,L in the corresponding genotypes. All graphs are average ± SEM. *P < 0.05 indicates the first of the statistically significant points on each graph. All points that occur after those time points are significant.

Figure 5

Pde4b ablation increases LTCC window current upon β-AR stimulation. (A) Mean I_Ca,L density elicited by depolarizing steps from a holding potential of –50 mV, using a double-pulse protocol (inset), was similar in WT (n = 15 cells) and Pde4b^–/– (n = 16 cells) AMVMs under control conditions. (B) Combined activation and steady-state inactivation curves under control conditions illustrate window current of I_Ca,L (half-time for activation [V_{1/2, act}] = –6.1 ± 0.9 mV, k = 5.4 ± 0.1 mV in Pde4b^–/– AMVMs and V_{ν, act} = –5.6 ± 0.7 mV, k = 5.4 ± 0.2 mV in WT [NS]; half-time for inactivation [V_{1/2, inact}] = –21.7 ± 0.8 mV, k = 7.1 ± 0.3 mV in Pde4b^–/– AMVMs and –21.9 ± 0.9 mV, k = 6.4 ± 0.3 mV in WT [NS]. (C) I_Ca,L density recorded during depolarizing pulses from –50 mV was further potentiated in Pde4b^–/– (n = 14) versus WT (n = 16) AMVMs upon sustained β-AR stimulation (3 nM Iso). (D) Increased I_Ca,L window current upon β-AR stimulation in Pde4b^–/– AMVMs. Activation curve of I_Ca,L was more hyperpolarized upon Iso (3 nM) application in Pde4b^–/– AMVMs (V_{1/2, act} = –12.2 ± 1.2 mV, k = 5.3 ± 0.2 mV) than in WT AMVMs (V_{1/2, act} = –7.5 ± 1.3 mV, k = 5.4 ± 0.2 mV, P < 0.05); inactivation was not affected by β-AR stimulation in either genotype (V_{1/2, inact} = –21.7 ± 0.4 mV, k = 6.2 ± 0.4 mV in Pde4b^–/– and V_{1/2, inact} = –21.3 ± 0.7 mV, k = 6.3 ± 0.2 mV in WT).

Colocalization of PDE4B and Ca_V1.2 channels at the Z-line in AMVMs. We next determined the subcellular localization of PDE4B in AMVMs and examined whether PDE4B and Ca_V1.2 colocalize in vivo by using the double-labeling immunofluorescence technique. In agreement with previous studies (7, 32), the staining for Ca_V1.2 obtained with the anti-Ca_V1.2 antibody was observed as a regularly spaced, punctuated pattern, which overlapped with the Z-line marker α-actinin (Figure 6A). Immunolabeling of PDE4B with an antibody that specifically recognizes this isoform (Figure 2C) revealed a strong fluorescent signal throughout the cell and a tight, striated pattern, with the main intensity stripe overlapping with α-actinin (Figure 6B). In AMVMs isolated from Pde4b^–/– hearts, the overall staining intensity for PDE4B was reduced, and, notably, the striation that had overlapped with α-actinin in WT myocytes was absent (Figure 6C). Our interpretation of these results is that Ca_V1.2 and a fraction of PDE4B are colocalized along T-tubule membranes in AMVMs.

Figure 6

Localization of PDE4B and LTCC at the transverse tubules in AMVMs. (A) Confocal images of a WT AMVM double labeled with anti-Ca_V1.2 antibody (CNC1, green) and anti–α-actinin antibody (α-Act, red). (B) Confocal images of a WT AMVM double labeled with anti-PDE4B antibody (green) and anti–α-actinin antibody (red). (C) Confocal images of a Pde4b^–/– AMVM double labeled with anti-PDE4B antibody (green) and anti–α-actinin antibody (red). Scale bar: 20 μm. The graph below each panel indicates the relative fluorescence intensities in the green and red channels measured along a 20-μm distance, as indicated by the yellow line on the enlarged boxed area from the merged images.

β-AR regulation of ECC in AMVMs from Pde4b^–/– and Pde4d^–/– mice. The fact that PDE4B modulates the β-AR stimulation of I_Ca,L and PDE4D regulates RyR2 (29) prompted us to compare the ECC process in WT, Pde4b^–/–, and Pde4d^–/– mice. For this, sarcomere length (SL) and intracellular Ca²⁺ were recorded simultaneously in Fura-2–loaded ventricular myocytes paced at 0.5 Hz. As shown in Figure 7, A and B, under nonstimulated basal conditions, SL shortening and Ca²⁺ transient amplitude were similar in the 3 genetic backgrounds. In WT, Pde4b^–/–, and Pde4d^–/– cells, pulse stimulation with Iso (100 nM, 15 seconds) strongly increased SL shortening and Ca²⁺ transient amplitude. However, these effects were more pronounced in PDE4-deficient myocytes: maximal shortening measured at the peak of Iso stimulation was increased by about 50% in Pde4b^–/– (n = 47) and Pde4d^–/– (n = 47) myocytes compared with that in WT myocytes (n = 51, P < 0.001), whereas Ca²⁺ transient amplitude was increased by about 25% and 28% more in Pde4b^–/– (n = 47) and Pde4d^–/– myocytes (n = 47), respectively, than in WT myocytes (n = 51; P < 0.01). Figure 7C shows the corresponding kinetics of relaxation and decay of the Ca²⁺ transients. In basal conditions, there was no difference in time to half maximal decay (t_1/2off) values of relaxation and Ca²⁺ transient decay among the different genotypes. Iso strongly accelerated relaxation (t_1/2off values were decreased by about 65%) and Ca²⁺ transient decay (t_1/2off values were decreased by about 57%) in WT myocytes (n = 51). In Pde4b^–/– and Pde4d^–/– myocytes (n = 47 each) relaxation was further accelerated (t_1/2off values were decreased by about 81% and 78% of basal value, respectively; P < 0.001 vs. WT), whereas decay kinetics of the Ca²⁺ transients in the presence of Iso were not significantly different from those of WT myocytes (Figure 7C). However, the decay phase of Ca²⁺ transients tended to be accelerated in Pde4b^–/– myocytes compared with WT myocytes (P = 0.059), and the difference was significant compared with Pde4d^–/– myocytes (P < 0.05). As shown in Figure 7, D and E, Pde4b^–/– and Pde4d^–/– myocytes also displayed increased spontaneous Ca²⁺ release (SCR) events upon Iso stimulation. On average, during a 20-second period after the maximal effect of Iso stimulation, 4.5 ± 1.1 SCR events occurred in Pde4b^–/– myocytes (n = 47), and 4.5 ± 0.9 SCR events occurred in Pde4d^–/– myocytes (n = 47), whereas in WT cells, only 1.4 ± 0.5 SCR events were observed (n = 51; P < 0.01). These results show that both PDE4B and PDE4D are required for normal regulation of ECC during β-AR stimulation. To further examine the consequences of PDE4B and PDE4D ablation on SR function, we measured SR Ca²⁺ load by fast application of 10 mM caffeine in basal conditions and at the peak of Iso (100 nM, 15 seconds) stimulation. As shown in the Supplemental Figure 1, A and B (supplemental material available online with this article; doi: 10.1172/JCI44747DS1), SR Ca²⁺ load was not significantly different in the 3 genotypes. However, there was a tendency for a decreased SR Ca²⁺ load in Pde4d^–/– myocytes in basal conditions (–20%). As a consequence, the percentage increase in SR Ca²⁺ load induced by Iso was significantly higher in Pde4d^–/– myocytes versus WT myocytes (P < 0.05, n = 18 each; Supplemental Figure 1C). We thus examined PLB phosphorylation at Ser-16 (PKA site) in isolated myocytes from the 3 genotypes. PLB phosphorylation was not different in the 2 groups under basal conditions. After Iso challenge (100 nM, 15 seconds), PLB phosphorylation was significantly increased in Pde4d^–/– myocytes compared with that in WT myocytes, and a nonsignificant increase was observed in Pde4b^–/– myocytes (Supplemental Figure 1D).

Figure 7

Enhanced ECC and increased arrhythmias upon transient β-AR stimulation in Pde4b^–/– and Pde4d^–/– mice. (A) Representative traces of sarcomere shortening (top) and Ca²⁺ transients (bottom) recorded in electrically paced (0.5 Hz) AMVMs from WT, Pde4b^–/–, and Pde4d^–/– mice. The black traces correspond to control conditions, and the gray traces correspond to the maximal effect produced by a 15-second pulse of Iso (100 nM). (B) Mean data for sarcomere shortening (expressed as the percentage of resting length) and Fura-2 ratio (expressed as the percentage of diastolic ratio) variation measured in control conditions (black) and at the maximum of Iso stimulation (gray) measured in AMVMs from WT (n = 51), Pde4b^–/– (n = 47), and Pde4d^–/– (n = 47) mice. (C) Average t_1/2off values for relaxation (top graph) and Ca²⁺ transient decay (bottom graph) in control conditions (black) and after Iso stimulation (gray) measured in AMVMs from WT (n = 51), Pde4b^–/– (n = 47), and Pde4d^–/– (n = 47) mice. (D) A representative trace of SCR events (arrows) induced by Iso in a Pde4b^–/– AMVM paced at 0.5 Hz. (E) Bar graph representing the average number of SCR events during a 20-second period preceding the application of Iso (black) and an equally long period after the peak of the Iso effect (gray) in WT (n = 51), Pde4b^–/– (n = 47), and Pde4d^–/– (n = 47) AMVMs. Bar graphs represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

ECG, intracardiac recording, and pacing. The arrhythmic behavior of Pde4b^–/– myocytes and the increased susceptibility of Pde4d^–/– mice to exercise-induced cardiac arrhythmias (29) prompted us to evaluate the propensity to ventricular arrhythmias in Pde4b^–/– mice. Six-lead ECGs were recorded in 10 WT and 11 Pde4b^–/– mice (mean age of 17 ± 1 weeks in both groups) in baseline conditions. No significant differences were observed in RR interval (WT, 128 ± 7 ms; Pde4b^–/–, 114 ± 7 ms), P wave (WT, 14 ± 1 ms; Pde4b^–/–, 11 ± 1 ms), PQ interval (WT, 39 ± 1 ms; Pde4b^–/–, 37 ± 1 ms), QRS complex (WT, 12 ± 1 ms; Pde4b^–/–, 11 ± 1 ms), and QTc interval (WT, 47 ± 2 ms; Pde4b^–/–, 50 ± 3 ms) durations. These mice were then used for catheter-mediated ventricular pacing in baseline conditions and after intraperitoneal injections of Iso (0.02 mg/kg and 0.2 mg/kg). Two mice (1 per group) died during the experimental procedure and were excluded from the statistical analysis. In baseline conditions, intracardiac ECG recordings in WT and Pde4b^–/– mice were similar. Pde4b inactivation had no significant effect on the ventricular effective refractory period (VERP) measured at a basic cycle length of 75 ms (WT, 44 ± 2 ms; Pde4b^–/–, 40 ± 2 ms; NS). Iso decreased spontaneous RR interval from 118 ± 5 ms in baseline to 93 ± 4 ms and 90 ± 1 ms at 0.02 mg/kg Iso and 0.2 mg/kg Iso, respectively, in WT mice (P < 0.001 vs. baseline for both doses) and from 109 ± 5 ms to 88 ± 2 ms and 87 ± 1 ms, respectively, in Pde4b^–/– mice (P < 0.001 vs. baseline for both doses; P = NS vs. WT). VERP was reduced to 38 ± 3 ms and 34 ± 3 ms after injection of Iso at, respectively, 0.02 mg/kg and 0.2 mg/kg (P < 0.001 and P < 0.05 vs. baseline) in WT mice and to 34 ± 2 ms and 32 ± 3 ms, respectively, in Pde4b^–/– mice (P < 0.01 and P < 0.05 vs. baseline; P = NS vs. WT). Other ECG parameters were similar in the 2 groups (data not shown). In baseline conditions, burst pacing did not induce arrhythmias either in WT or in Pde4b^–/– mice. However, after pretreatment with Iso, Pde4b^–/– mice responded differently from WT mice. Indeed, burst pacing induced arrhythmias in Pde4b^–/– mice in 1 out of 10 mice at 0.02 mg/kg Iso (P = NS vs. WT) and 4 out of 10 mice at 0.2 mg/kg Iso (P < 0.05 vs. WT; Figure 8, A and B) but not in WT mice. VT was induced in the 4 responding Pde4b^–/– mice. Two of them had sustained VT. Thus, after treatment with Iso, Pde4b gene inactivation favored the occurrence of arrhythmias induced by burst pacing. In contrast, paced extrasystoles induced arrhythmias in both WT and Pde4b^–/– mice (Figure 8C), with a moderately, though not significantly, higher incidence in Pde4b^–/– mice.

Figure 8

Increased susceptibility to VT upon β-AR stimulation in Pde4b^–/– mice. (A) Representative examples of simultaneous lead 1 ECG and intraventricular electrogram recordings obtained in WT and Pde4b^–/– mice under Iso (0.2 mg/kg) after a burst of 30 stimuli at a cycle length (CL) of 55 ms. (B and C) Bar graphs showing the percentage of mice with arrhythmias induced (B) by burst pacing and (C) by 1 to 3 extrastimuli under baseline conditions and after intraperitoneal injections of Iso (0.02 mg/kg and 0.2 mg/kg). PES, paced extrasystole. *P < 0.05.

PDE4 is the predominant PDE family that controls cAMP and LTCC in rodent cardiomyocytes. In the present study, we show that Pde4a, Pde4b, and Pde4d are expressed in mouse heart, but only PDE4B and PDE4D are associated with Ca_V1.2 channels. Whereas β-AR stimulation of I_Ca,L is normal in Pde4a^–/– and Pde4d^–/– myocytes, the β-AR responses of I_Ca,L, Ca²⁺ transients, and contraction are enhanced in myocytes from Pde4b^–/– mice, and this is accompanied by an increased propensity for arrhythmia. β-AR stimulation of Ca²⁺ transients and contraction are also enhanced in myocytes from Pde4d^–/– mice independently of I_Ca,L. For the first time to our knowledge, our results identify PDE4B as a major regulator of I_Ca,L and cardiac function and suggest that PDE4B and PDE4D regulate ECC by different mechanisms.

In mouse ventricular myocytes, pharmacological inhibition of PDE4 does not affect basal I_Ca,L but strongly enhances the effect of β-AR stimulation (Figure 1). A similar regulation of I_Ca,L by PDE4 was found previously in human atrial and rat ventricular myocytes, thus clearly indicating that PDE4 is a major negative regulator of I_Ca,L in the heart (14, 20, 33). Similar to what was reported earlier in the rat (34), PDE4 activity in the mouse ventricle was due to the expression of PDE4A, PDE4B, and PDE4D variants. The migration on SDS/PAGE of these variants corresponds to long forms, and although only one immunoreactive band was detected in each case, this band may correspond to the comigration of several long forms, as demonstrated earlier for PDE4D (23, 24, 29).

Specific variants of PDE4D have been shown to be associated with cardiac ion channels. Indeed, PDE4D3 was found to be coupled to RyR2 (29) and to be included in a macromolecular complex together with the KCNQ1 subunit of a potassium channel regulated by cAMP (28). Here, we found that PDE4D is also associated with the Ca_V1.2 subunit of the LTCC but, surprisingly, does not seem to regulate the channel activity, at least under normal conditions, because inactivation of the Pde4d gene modified neither I_Ca,L nor its β-AR regulation. Since PDE4B was also found in the complex that includes the Ca_V1.2 subunit, and β-AR stimulation of I_Ca,L is upregulated in Pde4b^–/– mice, it is possible that PDE4B is more tightly coupled with the LTCC, hence controlling its phosphorylation, while PDE4D is more remote. This hypothesis is supported by the observation that the PDE4 inhibitor Ro 20-1724 had a small but significant effect on the β-AR response of I_Ca,L in Pde4b^–/– mice (Figure 4E), as if the effect of PDE4D were unmasked in the absence of PDE4B. PDE4D isoforms have been shown previously to interact with β-ARs (25–27). Since β-ARs are part of a signalosome, including the LTCCs (35), it is conceivable that the 2 PDE4 isoforms that immunoprecipitated with Ca_V1.2 are in fact located at different ends of the signalosome, with PDE4D associated with the β-AR and PDE4B associated with the LTCC. Alternatively, PDE4D and PDE4B may control different pools of Ca_V1.2 (36), the latter being responsible for Iso-dependent I_Ca,L regulation.

Since LTCCs are predominantly expressed in the T-tubules (7, 32), PDE4B should be located in the same compartment. Consistent with this idea, immunocytochemical experiments with a specific PDE4B antibody revealed a strong staining at the Z-line in WT myocytes but not in Pde4b^–/– myocytes (Figure 6). This indicates that PDE4B is enriched in the T-tubules, at variance with that found in neonatal rat cardiomyocytes (16). This discrepancy could be due to either interspecies differences or to the higher level of differentiation of adult cardiomyocytes.

I_Ca,L constitutes the trigger for Ca²⁺-induced Ca²⁺ release and cardiac contraction, and its size dictates the amplitude of Ca²⁺ transients and contraction (37, 38). Accordingly, Ca²⁺ transients and contraction were enhanced in Pde4b^–/– myocytes upon β-AR stimulation. This was accompanied by an accelerated relaxation and Ca²⁺ transient decay, although the latter did not quite reach statistical significance (P = 0.059 vs. WT). Accordingly, PLB phosphorylation and SR Ca²⁺ load tended to be increased during Iso stimulation in Pde4b^–/– myocytes, but the differences with WT were not significant (Supplemental Figure 1). In addition, as shown in Supplemental Figure 2, PDE4B was anchored in macromolecular signaling complexes to Ca_V1.2 but not PLB or RyR2, and neither PLB phosphorylation at Ser-16 nor RyR2 phosphorylation at Ser-2808 were increased in Pde4b^–/– hearts compared with that in WT hearts. Altogether, these results suggest that LTCCs constitute the primary target of PDE4B, although Pde4b ablation may not exclusively impact LTCC regulation. Interestingly, the absence of PDE4D also led to increased Ca²⁺ transients and contraction in mouse ventricular myocytes (Figure 7). Since, in these cells I_Ca,L regulation was normal, the augmented ECC in Pde4d^–/– myocytes could be related to PKA hyperphosphorylation of RyR2 during β-AR stimulation and increased open probability of the Ca²⁺ release channel (8, 29). However, increasing RyR activity alone may not be sufficient to explain an increase in Ca²⁺ transient amplitude (38), especially because such phenomenon can result ultimately in a decreased SR Ca²⁺ content (39, 40). PLB mediates part of the positive inotropic effect of β-AR stimulation by increasing SR Ca²⁺ load (41, 42). SR calcium content can by itself determine calcium transient amplitude independently from I_Ca,L amplitude (38). A recent study indicates that PDE4D may also control PLB phosphorylation (30). Consistent with this idea, PLB phosphorylation was increased in Pde4d^–/– myocytes under β-AR stimulation, and Iso more potently increased SR Ca²⁺ load in Pde4d^–/– myocytes than in WT myocytes (Supplemental Figure 1). We propose that in Pde4d^–/– myocytes both SR Ca²⁺ release and SR Ca²⁺ uptake are increased, leading to enhanced Ca²⁺ transients, while trigger activity remains unaltered. In this scheme, the unchanged decay kinetics of the Ca²⁺ transients in Pde4d^–/– myocytes compared to those of WT myocytes under β-AR stimulation could be due to the balancing effect of increased diastolic Ca²⁺ leak and increased Ca²⁺ reuptake by the SR. Altogether, these results provide strong evidence that PDE4B and PDE4D isoforms differentially regulate cardiac ECC.

Alterations in Ca²⁺ handling are linked to cardiac arrhythmias (43). SCR events were increased in Pde4d^–/– cells upon β-AR stimulation. These events were likely due to “leaky” hyperphosphorylated RyR2 channels that promote arrhythmias in mice invalidated for the Pde4d gene (29). Interestingly, inactivation of the Pde4b gene also favored SCR events in isolated cardiomyocytes upon β-AR stimulation, likely by a different mechanism. Therefore, PDE4B deficiency could lead to cardiac dysfunction and arrhythmias. This was further demonstrated in vivo using catheter-based ventricular stimulation that reliably induces ventricular arrhythmias (44). The susceptibility of WT and Pde4b^–/– mice to the development VTs after paced extrasystoles or burst pacing was very low in baseline conditions. Interestingly, under β-AR stress, Pde4b inactivation significantly increased the incidence of arrhythmias induced by burst pacing but not by extrastimuli. In addition, no difference was detected between ECGs recorded from WT and Pde4b^–/– mice. These results are consistent with triggered arrhythmias due to altered Ca²⁺ homeostasis rather than reentries or altered conduction. Arrhythmias triggered by burst pacing during β-AR stimulation could be due to early afterdepolarizations (EADs) or delayed afterdepolarizations (DADs) (43). Interestingly, increased β-AR stimulation also resulted in a larger shift of the activation curve of I_Ca,L in Pde4b^–/– myocytes. As a consequence, a larger window current would occur, promoting Ca²⁺ entry during the action potential. Recovery from inactivation of I_Ca,L within its window may provoke EADs during the action potential plateau (45–47). In addition, increased Ca²⁺ entry in cardiomyocytes could lead to Ca²⁺ overload and increase spontaneous SR Ca²⁺ release, thus promoting the occurrence of DADs. Future studies will be required to distinguish between these 2 possibilities.

In conclusion, our data identify PDE4B as an integral component of the LTCC complex and as the major PDE isoform regulating I_Ca,L during β-AR stimulation. Consequently, PDE4B is crucial for the regulation of cardiac ECC and arrhythmogenesis. Interestingly, we recently reported that PDE4B expression and activity are decreased during cardiac hypertrophy (34). This could lead to increased phosphorylation of LTCC, as reported in failing human ventricular myocytes (48, 49), and therefore provide a mechanistic link to arrhythmias and sudden death in heart failure.

View Supplemental data

The authors wish to thank Agnès Hivonnait (INSERM UMR915, Cardiex facility, Nantes) for technical assistance in ECG and intracardiac recording and pacing, Patrick Lechêne (INSERM U769) for help with data analysis, Pauline Robert and Valérie Domergue-Dupont (animal core facility, University Paris-Sud, IFR141) for handling and genotyping of mice, and Valérie Nicolas (imaging core facility, University Paris-Sud, IFR141) for technical assistance with confocal microscopy. Cardiex facility is supported by the GIS-IBiSA program. This work was supported by Leducq cycAMP grant 06CVD02 (to R. Fischmeister and M. Conti), European Union contract LSHM-CT-2005-018833/EUGeneHeart (to R. Fischmeister), NIH grant HL092788 (to M. Conti), NIH grant R01 HL085372 (to W.A. Catterall), the Fondation Lefoulon-Delalande (to J. Leroy), the Fondation de France (to G. Vandecasteele), and Agence Nationale de la Recherche grant 2010 BLAN 1139 01 (to G. Vandecasteele).

Address correspondence to: Rodolphe Fischmeister or Grégoire Vandecasteele, INSERM UMR-S 769, Université Paris-Sud, Faculté de Pharmacie, 5, Rue J.-B. Clément, F-92296 Châtenay-Malabry Cedex, France. Phone: 33.1.46.83.57.57; Fax 33.1.46.83.54.75; E-mail: rodolphe.fischmeister@inserm.fr (R. Fischmeister), gregoire.vandecasteele@u-psud.fr (G. Vandecasteele).

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

Reference information: J Clin Invest. 2011;121(7):2651–2661. doi:10.1172/JCI44747.

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