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Circulation Research. 2007;101:1274-1282
Published online before print November 1, 2007, doi: 10.1161/CIRCRESAHA.107.158428
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(Circulation Research. 2007;101:1274.)
© 2007 American Heart Association, Inc.

Cellular Biology

Altered Heart Rate and Sinoatrial Node Function in Mice Lacking the cAMP Regulator Phosphoinositide 3-Kinase-{gamma}

Robert A. Rose, M. Golam Kabir, Peter H. Backx

From the Departments of Physiology and Medicine (R.A.R., P.H.B.), Division of Cardiology at the University Health Network (R.A.R., P.H.B.), and Heart and Stroke/Richard Lewar Centre of Excellence (R.A.R., M.G.K., P.H.B.), University of Toronto, Ontario, Canada.

Correspondence to Dr Peter H. Backx, University of Toronto, Heart & Stroke/ Richard Lewar Centre, Room 68, Fitzgerald Building, 150 College St, Toronto, Ontario, Canada M5S 3E2. E-mail p.backx{at}utoronto.ca


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Ablation of the enzyme phosphoinositide 3-kinase (PI3K){gamma} (PI3K{gamma}–/–) in mice increases cardiac contractility by elevating intracellular cAMP and enhancing sarcoplasmic reticulum Ca2+ handling. Because cAMP is a critical determinant of heart rate, we investigated whether heart rate is altered in mice lacking PI3K{gamma}. Heart rate was similar in anesthetized PI3K{gamma}–/– and wild-type (PI3K{gamma}+/+) mice. However, IP injection of atropine (1 mg/kg), propranolol (1 mg/kg), or both drugs in combination unmasked elevated heart rates in PI3K{gamma}–/– mice, suggesting altered sinoatrial node (SAN) function. Indeed, spontaneous action potential frequency was {approx}35% greater in SAN myocytes isolated from PI3K{gamma}–/– mice compared with PI3K{gamma}+/+ mice. These differences in action potential frequency were abolished by intracellular dialysis with the cAMP/protein kinase A antagonist Rp-cAMP but were unaffected by treatment with ryanodine to inhibit sarcoplasmic reticulum Ca2+ release. Voltage-clamp experiments demonstrated that elevated action potential frequencies in PI3K{gamma}–/– SAN myocytes were more strongly associated with cAMP-dependent increases in L-type Ca2+ current (ICa,L) than elevated hyperpolarization-activated current (If). In contrast, ICa,L was not increased in working atrial myocytes, suggesting distinct subcellular regulation of L-type Ca2+ channels by PI3K{gamma} in the SAN compared with the working myocardium. In summary, PI3K{gamma} regulates heart rate by the cAMP-dependent modulation of SAN function. The effects of PI3K{gamma} ablation in the SAN are unique from those in the working myocardium.


Key Words: ion channels • electrophysiology • action potentials • arrhythmia


*    Introduction
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 up arrowAbstract
 *Introduction
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Genetic knockout of phosphoinositide 3-kinase (PI3K){gamma} (PI3K{gamma}–/–) in mice results in reduced phosphodiesterase activity, increased intracellular cAMP levels, and enhanced ventricular contractility.1–5 We have previously demonstrated that increased contractility in ventricular myocytes isolated from PI3K{gamma}–/– mice is attributable to elevated sarcoplasmic reticulum (SR) Ca2+ levels and increased SR Ca2+ release without changes in L-type Ca2+ current (ICa,L).4,6 These results establish that the regulation of cAMP by PI3K{gamma} in ventricular myocytes occurs in a subcellular compartment containing the SR, but not L-type Ca2+ channels.7

Heart rate is determined by the electrical properties of specialized myocytes located in the sinoatrial node (SAN).8,9 The spontaneous electrical activity of SAN myocytes results from slow diastolic depolarizations that increase the membrane potential toward the threshold for eliciting action potentials (APs). The activity of several currents involved in the regulation of spontaneous AP firing in SAN myocytes are modulated by cAMP, including the hyperpolarization-activated current (If), ICa,L, a delayed rectifier K+ current (IKr) and a Na+– Ca2+ exchange current (INCX) that is driven by SR Ca2+ release.8–14 Because PI3K{gamma} has emerged as an important regulator of cAMP in the myocardium, and because cAMP is a critical modulator of SAN pacemaker cell activity, we sought to evaluate the role of PI3K{gamma} in heart rate regulation using transgenic mice lacking PI3K{gamma}. Our results indicate that PI3K{gamma} modulates the intrinsic activity of the SAN in a cAMP-dependent fashion and that the effects of PI3K{gamma} ablation in the SAN are unique compared with the working myocardium of the atria and ventricles. Some of these data have been presented in abstract form.15


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
 down arrowReferences
 
Animals
In the present study, male wild-type (PI3K{gamma}+/+) and PI3K{gamma}–/–16 littermate mice aged 10 to 14 weeks were used. All experimental procedures were in accordance with the regulations of The Canadian Council on Animal Care and were approved by the University of Toronto animal care facility.

In Vivo Heart Rate Measurements
Heart rate was measured in anesthetized mice using a pressure catheter (Millar Instruments) inserted into the aorta via the carotid artery, as described in the online data supplement at http://circres.ahajournals.org.

Isolation of Mouse SAN and Right Atrial Myocytes
The procedures for isolating single pacemaker myocytes from the SAN, as well as working right atrial myocytes from the mouse have been described17–19 and are available in the online data supplement. Single SAN and right atrial myocytes were used for patch-clamp studies using standard solutions and electrophysiological protocols, which are detailed in the online data supplement.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Consistent with previous reports,1–3 no differences in heart rate were observed between PI3K{gamma}–/– and PI3K{gamma}+/+ mice in control conditions (Figure 1). To assess whether the intrinsic electrical properties of the SAN differed between PI3K{gamma}–/– and PI3K{gamma}+/+ mice, heart rate was also measured following selective blockade of the autonomic nervous system. Figure 1A shows that administration of atropine (1 mg/kg) to block parasympathetic nervous system activity caused heart rate to increase (P<0.05) from 544.1±22.2 to 589.9±17.5 beats per minute (bpm) over 20 minutes in PI3K{gamma}+/+ mice and from 526.4±17.3 to 635.4±14.3 bpm over shorter periods ({approx}5 minutes) in PI3K{gamma}–/– mice. After atropine treatment, heart rate was higher (P<0.05) in PI3K{gamma}–/– than PI3K{gamma}+/+ mice. Treatment with propranolol (1 mg/kg; Figure 1B) to block sympathetic nervous system activity reduced (P<0.05) heart rate in PI3K{gamma}+/+ (511.2±9.8 to 343.3±16.5 bpm) and PI3K{gamma}–/– (523.4±15.5 to 406.9±13.0 bpm) mice. Following propranolol treatment, heart rate was greater (P<0.05) in PI3K{gamma}–/– than PI3K{gamma}+/+ mice. Combined treatment with atropine and propranolol (Figure 1C) produced heart rate changes that were comparable to the effects of propranolol alone.


Figure 1
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  		Figure 1. Effects of PI3K{gamma} ablation on heart rate in anesthetized mice under basal conditions and following nervous system blockade. Effect of atropine (1 mg/kg) (A), propranolol (1 mg/kg) (B), and a combination of atropine and propranolol (C) on heart rate in PI3K{gamma}+/+ and PI3K{gamma}–/– mice. Drugs were give by IP injection at 0 minutes. Following all 3 pharmacological interventions, heart rate was significantly greater (*) in PI3K{gamma}–/– mice than wild-type controls (n=6 to 7 mice for each test group).

Because the results above suggest that PI3K{gamma} modulates the intrinsic pacemaker properties of the SAN, we directly assessed the electrical properties of isolated SAN myocytes. Spontaneous APs could be continuously recorded for at least 20 minutes and, as summarized in Figure 2, occurred at a higher frequency (P<0.05) in PI3K{gamma}–/– myocytes (173±8 bpm) than PI3K{gamma}+/+ myocytes (129±6 bpm). The relative differences in SAN myocyte firing rate between PI3K{gamma}–/– and PI3K{gamma}+/+ were similar to the heart rate differences observed in mice following autonomic blockade, although the absolute rates differed because of temperature differences between the studies. These changes in AP frequency were associated with increases (P<0.05) in the slope of the diastolic depolarization (DD) from 25.1±1.5 mV/sec in PI3K{gamma}+/+ myocytes to 41.7±1.4 mV/sec in PI3K{gamma}–/– myocytes, without changes (P=0.62) in the maximum diastolic potential between genotypes (supplemental Table I). Because the modulation of ventricular cardiomyocyte contractility by PI3K{gamma} depends on cAMP,2,4 we examined the effects of the cAMP/protein kinase (PKA) antagonist adenosine-3',5' cyclic phosphorothioate (Rp-cAMP; 1x10–4 mol/L)20 on SAN myocytes. Eight minutes of treatment with Rp-cAMP had no effect (P=0.69) on spontaneous AP frequency in PI3K{gamma}+/+ SAN myocytes but reduced (P<0.05) firing frequency from 170±6 to 140±4 bpm and the DD slope from 48.9±3.5 to 22.2±1.2 mV/S in PI3K{gamma}–/– SAN myocytes, without affecting the maximum diastolic potential (Figure 2 and supplemental Table I). Firing frequencies were identical between the groups after Rp-cAMP.


Figure 2
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  		Figure 2. Effects of PI3K{gamma} ablation on spontaneous AP frequency in isolated SAN myocytes. A and B, Representative recordings of spontaneous APs in control conditions and following application of the cAMP/PKA antagonist Rp-cAMP (1x10–4 mol/L) in PI3K{gamma}+/+ and PI3K{gamma}–/– myocytes. Recordings were made using the whole-cell configuration of the patch-clamp technique, and Rp-cAMP was included in the recording pipette, so that it could enter the cells by diffusion. C, Summary data demonstrating that spontaneous AP frequency was elevated in PI3K{gamma}–/– myocytes in a cAMP/PKA-dependent fashion. *P<0.05 between genotypes under the same conditions. {dagger}P<0.05 for Rp-cAMP vs control within genotypes; n values in parentheses. Measurements were performed at room temperature (22°C to 23°C).

To explore the ionic basis for the changes in AP-firing frequency, we measured ICa,L and If because these currents alter the spontaneous firing rate of SAN myocytes in a cAMP-dependent manner by influencing the DD slope. Figure 3 shows representative ICa,L recordings, originating from CaV1.2 and CaV1.3 channels, measured with voltage-clamp protocols designed to minimize T-type Ca2+ currents.21 These recordings were done in the presence of tetrodotoxin (3x10–5 mol/L) or QX-314 (3x10–2 mol/L) to block voltage-gated Na+ channels. ICa,L current densities were larger (P<0.05), and the peak of the current–voltage relationship (I-V) curve was shifted toward negative potentials in PI3K{gamma}–/– versus PI3K{gamma}+/+ SAN myocytes. Note that these I-V curves peak at more negative membrane potentials than ventricular myocytes because of the functional expression of the CaV1.3 channel isoform.21 To better quantify the differences in ICa,L between the groups, steady-state conductance analysis was performed, which revealed that the maximum conductance (Gmax) was elevated (P<0.05) in PI3K{gamma}–/– (93.5±7.5 pS/pF) compared with PI3K{gamma}+/+ (72.7±7.9 pS/pF) SAN myocytes (Figure 3C). Furthermore, the voltage required for 50% channel activation (V1/2) was shifted (P<0.05) leftward from –30.6±1.7 mV for PI3K{gamma}+/+ SAN myocytes to –38.5±2.0 mV in PI3K{gamma}–/– SAN myocytes (Figure 3C). Despite clear differences in channel activation, no differences (P=0.89) in the time constants for ICa,L inactivation (ie, {tau}fast and {tau}slow) were observed between genotypes (supplemental Figure I). These changes in channel activation are expected to increase depolarizing currents leading to increased DD slopes and spontaneous firing frequencies.


Figure 3
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  		Figure 3. Effects of PI3K{gamma} ablation on ICa,L in SAN myocytes. A, Representative current recordings of SAN ICa,L in myocytes from PI3K{gamma}+/+ and PI3K{gamma}–/– mice. B, Summary I-V curves demonstrating that SAN ICa,L current density was elevated in PI3K{gamma}–/– mice (n=26 myocytes) compared with wild-type controls (n=21 myocytes). *P<0.05 at the given membrane potential. C, Activation curves for ICa,L conductance in SAN myocytes demonstrating that Gmax was elevated and V1/2 of activation was left shifted in PI3K{gamma}–/– SAN myocytes compared with wild-type SAN myocytes (see text for details). D, Representative ICa,L recordings (250-ms voltage-clamp step to –10 mV) in SAN myocytes from PI3K{gamma}+/+ mice and PI3K{gamma}–/– mice before and after application of Rp-cAMP (1x10–4 mol/L). E, Summary data for the effects of Rp-cAMP on peak ICa,L. *P<0.05 between genotypes under the same conditions, {dagger}P<0.05 for Rp-cAMP vs control within the same genotype (n values in parentheses).

Because the increase in Gmax and the shift in V1/2 for ICa,L in PI3K{gamma}–/– SAN myocytes are reminiscent of changes observed following activation of cAMP/PKA in the myocardium, we examined the effects of the cAMP/PKA antagonist Rp-cAMP (1x10–4 mol/L) on ICa,L measured at –10 mV, a voltage at which the conductance is maximal. As summarized in Figure 3D and 3E, Rp-cAMP had no effect (P=0.30) on ICa,L in PI3K{gamma}+/+ SAN myocytes but reduced (P<0.05) peak ICa,L in PI3K{gamma}–/– to levels not different from PI3K{gamma}+/+ myocytes in the presence or absence of Rp-cAMP. Similarly, Rp-cAMP had no effect (P=0.12) on Gmax or V1/2 of activation for ICa,L in PI3K{gamma}+/+ SAN myocytes but reduced (P<0.05) Gmax and shifted V1/2 in PI3K{gamma}–/– SAN myocytes to values that were not different (P=0.48) from PI3K{gamma}+/+ (supplemental Figure II).

The cAMP-dependent ICa,L elevations in PI3K{gamma}–/– SAN myocytes contrasts with the lack of effect of PI3K{gamma} ablation on ICa,L in ventricular myocytes.4 Because ICa,L is only produced by CaV1.2 channels in ventricular myocytes, but is generated by CaV1.2 and CaV1.3 channels in SAN myocytes, we considered the possibility that ICa,L elevations in PI3K{gamma}–/– SAN myocytes originate from cAMP-dependent regulation of CaV1.3 channels. To test this possibility, we took advantage of the fact that, like SAN myocytes, working atrial myocytes also express CaV1.2 and CaV1.3 L-type Ca2+ channels.21–23 As summarized in Figure 4 (and supplemental Figure I), ICa,L in right atrial myocytes did not differ (P=0.85) between PI3K{gamma}+/+ and PI3K{gamma}–/–, suggesting that increased ICa,L in PI3K{gamma}–/– SAN myocytes is unlikely to result from distinct regulation of CaV1.3 L-type Ca2+ channels by PI3K{gamma}.


Figure 4
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  		Figure 4. Effects of PI3K{gamma} ablation on ICa,L in right atrial working myocytes. A, Representative ICa,L recordings in right atrial myocytes from wild-type and PI3K{gamma}–/– mice. B and C, Summary I-V and activation curves showing that ICa,L current density, Gmax, and activation kinetics in right atrial myocytes were not significantly different between genotypes (n=17 PI3K{gamma}+/+ myocytes and n=19 PI3K{gamma}–/– myocytes).

Next, we recorded If. Our data illustrate that If current density was higher (P<0.05) in PI3K{gamma}–/– SAN myocytes, compared with PI3K{gamma}+/+ myocytes, at membrane potentials less than or equal to –80 mV (Figure 5). These elevated current densities were accompanied by a shift (P<0.05) in the V1/2 for channel activation (–96.4±1.0 mV for PI3K{gamma}+/+ myocytes versus –88.5±0.9 mV for PI3K{gamma}–/– myocytes) with no change (P=0.14) in slope factor (18.5±0.9 for PI3K{gamma}–/– versus 19.0±0.8 for PI3K{gamma}+/+). These differences in If are anticipated to increase depolarizing currents, leading to a higher DD slopes and firing rates in PI3K{gamma}–/– myocytes.


Figure 5
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  		Figure 5. Properties of If in SAN myocytes from PI3K{gamma}–/– mice. A, Representative If recordings from wild-type and knockout myocytes. B, Summary I-V curves (measured at the time point indicated by the open circles in (A) demonstrating that If is increased in PI3K{gamma}–/– mice (n=19 myocytes) compared with PI3K{gamma}+/+ mice (n=22 myocytes). *P<0.05 at the given membrane potential. C, If activation curves in wild-type and PI3K{gamma}–/– mice, which were measured at the points indicated by black circles in A. V1/2 was significantly shifted to the right in PI3K{gamma}–/– mice (n=14 myocytes) compared with PI3K{gamma}+/+ mice (n=15 myocytes).

To determine whether these If differences resulted from altered cAMP/PKA signaling between the groups, the effects of Rp-cAMP (1x10–4 mol/L) were examined (Figure 6A and 6B). In these studies, the control current levels were measured 30 seconds after rupturing the cell membrane, and the effects of Rp-cAMP were measured after 8 minutes of dialysis (see Materials and Methods). Rp-cAMP increased (P<0.05) If (at –120 mV) slightly from –10.2±0.8 to –11.9±0.9 pA/pF in PI3K{gamma}+/+ SAN myocytes while reducing (P<0.05) If from –18.4±0.8 to –15.7±1.2 pA/pF in PI3K{gamma}–/– SAN myocytes. The ability of Rp-cAMP to increase If in PI3K{gamma}+/+ SAN myocytes is not unexpected because, whereas Rp-cAMP can block PKA activation, it also directly activates hyperpolarization-activated, cyclic nucleotide–gated (HCN) channels.24,25 Thus, to dissect the contributions of PKA to the enhanced If in PI3K{gamma}–/–, myocytes were dialyzed with a 14-22-amide protein kinase inhibitor (PKI14–22) (1x10–5 mol/L), which is the inhibitor peptide for the PKA catalytic subunit.26 PKI14–22 had no effect on If in PI3K{gamma}+/+ SAN myocytes, but caused If reductions in PI3K{gamma}–/– SAN myocytes similar to those seen with Rp-cAMP (Figure 6C and 6D).


Figure 6
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  		Figure 6. Effects of Rp-cAMP and PKI14–22 on If in SAN myocytes from wild-type and PI3K{gamma}–/– mice. A, Representative If recordings (2-second voltage-clamp step to –120 mV) in myocytes from PI3K{gamma}+/+ mice and PI3K{gamma}–/– mice before and after treatment with Rp-cAMP (1x10–4 mol/L). B, Summary data for the effects of Rp-cAMP on If. C, Representative If recordings before and after treatment with PKI14–22. D, Summary data for the effects of PKI14–22 of If. *P<0.05 between genotypes under the same conditions, {dagger}P<0.05 for Rp-cAMP vs control within the same genotype (n values in parentheses).

The results above establish that increased spontaneous firing rates of SAN myocytes in PI3K{gamma}–/– mice are related to elevated cAMP/PKA signaling. To explore cAMP signaling further, we investigated the effects of isoproterenol (ISO). As expected, application of ISO (1x10–6 mol/L) increased (P<0.05) AP frequency from 118±9 to 154±6 bpm in PI3K{gamma}+/+ myocytes (Figure 7A and 7B), in association with increases (P<0.05) in DD slope, AP overshoot, and AP duration measured at 50% repolarization (APD50) (supplemental Table I). In PI3K{gamma}–/– myocytes, ISO application increased (P<0.05) AP frequency from 183±6 to 207±6 bpm with relatively similar changes in AP profile, as seen in PI3K{gamma}+/+ SAN myocytes (Figure 7B and supplemental Table I), establishing that cAMP-dependent signaling is not saturated under baseline conditions in PI3K{gamma}–/– SAN myocytes. Indeed, both ICa,L and If were increased (P<0.05) by β-adrenergic receptor stimulation with ISO in PI3K{gamma}–/– and PI3K{gamma}+/+ SAN myocytes (Figure 7C through 7F).


Figure 7
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  		Figure 7. Effects of ISO on spontaneous AP firing, ICa,L, and If in SAN myocytes from wild-type and PI3K{gamma}–/– mice. A, Representative recordings of spontaneous APs in control conditions and following the application of the β-adrenergic receptor agonist ISO (1x10–6 mol/L) for 5 minutes in PI3K{gamma}+/+ and PI3K{gamma}–/– SAN myocytes. Summary data are illustrated in B. C, Representative ICa,L recordings during a 250-ms voltage-clamp step to –10 mV in control conditions and after superfusion with ISO with summary data shown in D. E, Representative If recordings during a 2-second voltage-clamp step to –120 mV before and after superfusion with ISO. Summary data for If are shown in F. *P<0.05 between genotypes under the same conditions. {dagger}P<0.05 for ISO vs control within genotypes (n values in parentheses).

SAN pacemaker activity has been shown to be modulated by SR Ca2+ loading and release,14,27,28 which are both regulated by cAMP/PKA and markedly increased in PI3K{gamma}–/– ventricular myocytes.4,5 Therefore, we tested the effects of the SR Ca2+ release blocker ryanodine (1x10–5 mol/L) on spontaneous AP frequency. Representative AP recordings in control conditions and after treatment with ryanodine (minimum 8 minutes; Figure 8) show firing frequency was reduced (P<0.05) by approximately the same percentage in PI3K{gamma}–/– SAN myocytes (166±11 to 131±8 bpm) as in PI3K{gamma}+/+ SAN myocytes (135±5 to 105±6 bpm). Ryanodine also reduced (P<0.05) DD slope and increased (P<0.05) APD50 (supplemental Table I). Thus, AP-firing frequencies remained elevated (P<0.05) in PI3K{gamma}–/– SAN myocytes after inhibition of SR Ca2+ release, suggesting that differences in SR function are not responsible for the higher firing rates in PI3K{gamma}–/– SAN myocytes.


Figure 8
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  		Figure 8. Effects of ryanodine on spontaneous AP firing in SAN myocytes from wild-type and PI3K{gamma}–/– mice. A and B, Representative recordings of spontaneous APs in control conditions and following the application of ryanodine (1x10–5 mol/L). C, Summary data demonstrating that AP-firing frequency was reduced in both genotypes following treatment with ryanodine. *P<0.05 between genotypes under the same conditions, {dagger}P<0.05 for ryanodine vs control within genotypes (n values in parentheses).

Heart rate is also potently regulated by parasympathetic nervous system-mediated stimulation of M2 muscarinic receptors in the SAN, which activate hyperpolarizing acetylcholine-sensitive K+ currents (IKACh) via Gβ{gamma} subunits.29 Because heart rate (Figure 1) differed between PI3K{gamma}+/+ and PI3K{gamma}–/– mice after autonomic blockade, we also examined IKACh in SAN myocytes. IKACh evoked by carbachol (1x10–5 mol/L)17 did not differ between PI3K{gamma}+/+ and PI3K{gamma}–/– SAN myocytes (supplemental Figure III), indicating that the properties of IKACh are not directly affected by PI3K{gamma}.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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Although our studies demonstrate that PI3K{gamma} negatively regulates the spontaneous firing rate of isolated SAN myocytes, heart rate was not different between wild-type and PI3K{gamma}–/– mice under basal conditions. This is not unexpected because heart rate in vivo is regulated by the autonomic nervous system.30,31 Indeed, blockade of the sympathetic or parasympathetic arms of the autonomic nervous system unmasked elevated heart rates in PI3K{gamma}–/– mice with the relative heart rate differences after combined propranolol and atropine treatment being similar to the relative differences in spontaneous firing rate observed in isolated SAN myocytes. Interestingly, whereas both propranolol and atropine treatment affected heart rate, sympathetic blockade had a relatively larger effect in agreement with previous mouse studies.32–34 Furthermore, the larger heart rate changes with atropine in PI3K{gamma}–/– (compared with PI3K{gamma}+/+) mice suggest that elevated parasympathetic activity is used by PI3K{gamma}–/– mice to suppress higher intrinsic AP rates of their SANs, even though IKACh was not directly affected by PI3K{gamma}.

Consistent with previous reports showing that PI3K{gamma} is a negative regulator of cAMP in the ventricular myocardium,1–3 we found that the cAMP/PKA blocker Rp-cAMP reduced AP firing frequency in PI3K{gamma}–/– SAN myocytes to levels indistinguishable from PI3K{gamma}+/+ SAN myocytes in the absence or presence of Rp-cAMP. The differences in AP firing observed between PI3K{gamma}+/+ and PI3K{gamma}–/– SAN myocytes, as well as the changes induced by Rp-cAMP, correlated tightly with changes in the slope of the DD. Under physiological conditions, several channels whose activities are increased by β-adrenergic receptor-mediated elevations in cAMP/PKA signaling provide depolarizing current during the diastolic period.

ICa,L is an attractive candidate to explain the differences in spontaneous AP firing between PI3K{gamma}+/+ and PI3K{gamma}–/– SAN myocytes and the response to Rp-cAMP in PI3K{gamma}–/– SAN myocytes, for several reasons. First, ICa,L in SAN myocytes is generated by 2 distinct L-type Ca2+ channel {alpha}1 subunits: CaV1.2 and CaV1.3. Whereas CaV1.2 channels contribute primarily to the AP upstroke, CaV1.3 channels activate at more negative membrane potentials that correspond to the late phase of the DD in SAN myocytes.21,22 Second, ICa,L was increased in PI3K{gamma}–/– SAN myocytes because of an increased conductance (Gmax) and a leftward shift in V1/2 for channel activation, changes that mimic the alterations seen with β-adrenergic receptor–mediated elevations in cAMP/PKA signaling. The leftward shift in V1/2 preferentially increases ICa,L at potentials corresponding to the DD. Third, the effects of Rp-cAMP on ICa,L closely mirrored the effects on spontaneous firing rates. Specifically, the elevation in ICa,L density that was observed in PI3K{gamma}–/– SAN myocytes was eliminated by Rp-cAMP. On the other hand, Rp-cAMP had no effect on ICa,L in PI3K{gamma}+/+ SAN myocytes. Thus, our findings suggest that cAMP-dependent regulation of ICa,L is a major determinant of the firing rate of the mouse SAN, as concluded previously.19,21 Consistent with this suggestion, ICa,L is a prototype for cAMP/PKA-dependent phosphorylation,35,36 and ICa,L blockers are routinely used in the effective treatment of atrial tachyarrhythmias.37 It could be hypothesized that the negative shift in ICa,L activation properties (ie, V1/2) between genotypes results from an increased role for Cav1.3 channels in PI3K{gamma}–/– SAN myocytes. However, this seems unlikely because V1/2 (as well as Gmax) was identical between genotypes after Rp-cAMP addition.

Another potential contributor to the elevated AP-firing rates in PI3K{gamma}–/– SAN myocytes is the "pacemaker current" If, even though the role of If in cardiac pacemaking has been challenged.9 Careful analysis of our AP recordings reveal that the maximum diastolic potential in mouse SAN myocytes is approximately –60 mV for both genotypes. Because our If I-V curves revealed that the If density was not statistically different between PI3K{gamma}+/+ and PI3K{gamma}–/– SAN myocytes at potentials above –80 mV, it could be concluded that If does not contribute to the rate differences between these genotypes. However, our If activation curve measurements reveal that voltages of approximately –60 mV are sufficiently positive to activate If in both genotypes, consistent with previous studies showing that If contributes to AP firing in mouse SAN.18,38 More importantly, because V1/2 is significantly shifted to more positive potentials and because the maximal If densities are increased, the degree of If activation is greater in PI3K{gamma}–/– SAN myocytes at these diastolic voltages, suggesting that If differences can contribute to higher firing rates in PI3K{gamma}–/– SAN myocytes. It is also conceivable that differences in If channel gating attributable to hysteresis, a property demonstrated in HCN channels,39,40 which was not assessed in our studies, could increase the impact of If on SAN firing rates in the 2 genotypes.

Our results show that Rp-cAMP reduced If in PI3K{gamma}–/– SAN myocytes but slightly increased If in wild-type myocytes. Moreover, If remained elevated in PI3K{gamma}–/– SAN myocytes after Rp-cAMP treatment. These complex results can be understood by recognizing that Rp-cAMP can affect If in 2 ways. First, like cAMP and other cAMP analogs, Rp-cAMP can increase If by directly binding to HCN channels via a cyclic nucleotide binding domain.10,25 On the other hand, If is also regulated by PKA-dependent phosphorylation,41,42 consistent with the presence of several consensus PKA phosphorylation sites in HCN4 channels, the major HCN homolog in mouse SAN.38,43 Thus, in PI3K{gamma}+/+ SAN myocytes Rp-cAMP addition is expected to enhance If by direct binding to the channels. By contrast, in PI3K{gamma}–/– myocytes Rp-cAMP treatment is expected to inhibit PKA, thereby reducing If while simultaneously adding to the elevated cAMP pool, which results in elevations of If above those seen in wild-type SAN. Consistent with this interpretation, PKA inhibition with PKI14–22 had no affect on If in PI3K{gamma}+/+ SAN myocytes but reduced If in PI3K{gamma}–/– SAN to levels still exceeding wild-type mice, because of persistent cAMP elevations in PI3K{gamma}–/– myocytes. These data suggest that elevated cAMP in PI3K{gamma}–/– mice increases If both by direct binding and by PKA-mediated effects. Furthermore, because Rp-cAMP in PI3K{gamma}–/– mice did not reduce If to wild-type levels but did normalize firing rates, it appears that If differences make relatively minor contributions to the elevated AP-firing rates in PI3K{gamma}–/– SAN.

Previous work from our laboratory demonstrated that ventricular myocytes from PI3K{gamma}–/– mice have large increases in SR Ca2+ cycling.7 Because SR Ca2+ cycling has been shown to be an important modulator of spontaneous firing rates of SAN myocytes, we tested the effects of ryanodine on these myocytes at doses that block SR Ca2+ release.27 Ryanodine reduced AP-firing frequency by {approx}20% in both genotypes, confirming a role for the SR in pacemaker function in the mouse SAN. Importantly, after ryanodine treatment, AP firing continued in both genotypes and remained significantly elevated in PI3K{gamma}–/– SAN myocytes, although a greater beat-to-beat variability in AP-firing pattern was observed in the wild-type mice as described previously.44 These data, together with our voltage-clamp results, support the conclusion that the elevated SAN firing rate of PI3K{gamma} knockout mice is primarily driven by increases in sarcolemmal currents, such as ICa,L and possibly If, although contributions from changes in SR Ca2+ cycling cannot be fully ruled out.

Increased ICa,L in PI3K{gamma}–/– SAN myocytes is clearly distinct from the absence of differences in ICa,L in PI3K{gamma}–/– ventricular myocytes.7 In the current study, we also observed no differences in ICa,L density or activation kinetics between wild-type and PI3K{gamma}–/– right atrial myocytes. Importantly, CaV1.3 channels are functional in working mouse atrial myocytes,23 as well as SAN myocytes.21,22 Therefore, if PI3K{gamma} were selectively regulating CaV1.3 channels, alterations in ICa,L activation kinetics would be expected for the working right atrial myocytes in PI3K{gamma}–/– mice, which was not observed. These results suggest that selective cAMP-dependent modulation of CaV1.3 versus CaV1.2 channels does not underlie the elevated ICa,L in PI3K{gamma}–/– SAN myocytes. Rather, our data support the conclusion that PI3K{gamma} is critical for the baseline suppression of cAMP levels in intracellular microdomains containing L-type Ca2+ channels in SAN myocytes but not in working atrial or ventricular myocytes. The basis for these differences between myocytes from different regions is unclear, but tight spatiotemporal regulation of cAMP in microdomains of cells appears to involve macromolecular complexes containing many players, including phosphodiesterases and A kinase anchoring proteins.45–47 It is possible that PI3K{gamma} is differentially integrated into such macromolecular complexes in a regional-dependent manner in the heart.

Significance
SAN dysfunction is a major burden that progressively increases with age and in disease states. For example, bradyarrhythmias associated with sick sinus syndrome account for a large proportion of sudden deaths during heart failure.30 The cause of SAN dysfunction in heart failure is unclear, but heart disease is associated with impaired β-adrenergic/cAMP/PKA signaling, as well as altered function of L-type Ca2+ channels in the SAN.30,48 In the present study, we show that PI3K{gamma} profoundly modulates SAN function and heart rate and that this modulation is cAMP dependent. Previous studies have shown that PI3K{gamma} expression is strongly increased in disease49; therefore, it is possible that PI3K{gamma} may contribute to the onset and maintenance of bradyarrhythmias in heart disease, as well as to SAN dysfunction more generally.


*    Acknowledgments
 
We thank Dr Benoit-Gilles Kerfant for helpful discussions.

Sources of Funding

This work was supported by Canadian Institutes for Health Research funding (to P.H.B.). P.H.B. is a career investigator with the Heart and Stroke Foundation of Ontario. R.A.R. is the recipient of Postdoctoral fellowships from the Heart and Stroke Foundation of Canada, The Alberta Heritage Foundation for Medical Research, and the Canadian Institutes of Health Research Tailored Advanced Collaborative Training in Cardiovascular Science (TACTICS) program.

Disclosures

None.


*    Footnotes
 
Original received June 21, 2007; revision received October 10, 2007; accepted October 19, 2007.


*    References
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