IP3-mediated Ca2+ release regulates atrial Ca2+ transients and pacemaker function by stimulation of adenylyl cyclases

Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias. NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.


INTRODUCTION
Ca 2 þ handling in the heart is vital to normal physiological function and regulation of excitation-contraction coupling (1,2). Ca 2 þ signaling in cardiomyocytes is tightly regulated, for example, by protein kinase A (PKA) and Ca 2 þ /calmodulin-dependent protein kinase II (CaMKII), or by Ca 2 þ -mobilizing elements such as inositol trisphosphate (IP 3 ), cADPribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), as well as by Ca 2 þ itself (1)(2)(3). The atrial and ventricular chambers of the heart have very different functions, and therefore it is not surprising that there are many differences between atrial and ventricular myocytes in excitation-contraction coupling and in the handling of Ca 2 þ ions by different intracellular compartments. One characteristic feature of atrial myocytes is the relative abundance of receptors for inositol trisphosphate (IP 3 ) compared with ventricular myocytes (3). IP 3 is a Ca 2 þ -mobilizing second messenger (4) that acts to open IP 3 receptors (IP 3 R), located on the sarcoplasmic reticulum (SR) of cardiomyocytes (3,5). IP 3 is positively inotropic in atrial (3) and ventricular (6) preparations and is positively chronotropic in the sinoatrial node (SAN) (7,8). IP 3 is synthesized upon stimulation of phospholipase C (PLC) commonly, but not exclusively, by G-protein coupled receptors associated with G q (9). In cardiac myocytes, endothelin-1 (ET-1), angiotensin II (Ang-II), and phenylephrine (PE) all increase intracellular IP 3 level (10) via their actions at the G q -coupled ET-A, Ang-II, and a-adrenergic receptors, respectively.
Early functional studies revealed a much greater effect of IP 3 -associated stimuli on the contractility of atrial preparations than upon their ventricular counterparts (11), and expression of IP 3 R type 2 (IP 3 R2) is now known to be at least six times greater in atrial myocytes (3). IP 3 R expression is significantly increased during atrial fibrillation in both human patients (12) and animal models (13). Inhibiting G q -coupled Ang-II receptors has been shown to prevent the early remodeling associated with rapid atrial pacing (14), whereas in healthy atrial myocytes, G q -associated signaling causes an IP 3 R-dependent increase in the Ca 2 þ spark rate of quiescent myocytes and amplitude of the stimulated Ca 2 þ transient (15), effects matched on direct application of IP 3 [Ca 2 þ sparks (3,16), Ca 2 þ transients (3)]. Interestingly, even in healthy cells, IP 3 -dependent stimulation can be associated with the generation of spontaneous diastolic Ca 2 þ events (3,5).
Cardiac function is also controlled by pathways involving production of cAMP (1,17). These pathways are commonly thought to be distinct and separate from the previously mentioned IP 3 -dependent mechanisms, although evidence supports a cAMP-mediated regulation of the sensitivity of IP 3 Rs to Ca 2 þ (18,19). Here, we consider a possible novel link between cAMP-dependent mechanisms and IP 3 -mediated Ca 2 þ release from the SR, which arises from the existence and location of the Ca 2 þ -stimulated isoforms of adenylyl cyclase, AC1 and AC8, which have been shown to be selectively expressed in SAN (20,21) and atria (22), whereas in contrast, ventricular myocytes predominantly express AC5 and AC6 (23), which lack Ca 2 þ sensitivity. Interestingly, AC1 and AC8, like IP 3 R, have been shown to be expressed close to the surface of SA node and atrial myocytes, although the relative positions of these adenylyl cyclases and IP 3 Rs have not been previously explored (20).
Previous experiments also provided evidence for a role for Ca 2 þ -stimulated adenylyl cyclases in regulating Ca 2 þ transients and L-type Ca 2 þ currents in atrial myocytes (22). In particular, loading atrial myocytes with the Ca 2 þ chelator, BAPTA, was shown to cause a substantial reduction of the amplitude of L-type Ca 2 þ currents in atrial myocytes, but BAPTA was without effect when cytosolic cAMP levels were maintained at 200 μM by application from a patch pipette. These observations were interpreted as evidence for ongoing enhancement of Ltype Ca 2 þ currents, which was dependent on basal activity of Ca 2 þ -stimulated adenylyl cyclases in atrial myocytes. This interpretation was further supported by the observations that the amplitudes of L-type Ca 2 þ currents were also reduced when adenylyl cyclases were inhibited by MDL12-330A [cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride] (22).
In view of the functional importance of Ca 2 þ -stimulated adenylyl cyclases in regulating atrial and SAN function, and the likelihood that IP 3 Rs may be located close to AC1 and AC8, the hypothesis to be tested is that actions of IP 3 in atria and SAN depend on IP 3 -mediated Ca 2 þ release from the SR leading to activation of the adenylyl cyclases, AC1 and AC8.

METHODS
All animal experiments were performed in accordance with the United Kingdom Home Office Guide on the Operation of Animal (Scientific Procedures) Act of 1986. All experimental protocols (Schedule 1) were approved by the University of Oxford, Procedures Establishment License (PEL) Number XEC303F12.

Atrial Myocyte Isolation
Male Dunkin Hartley guinea pigs (350-550 g, Envigo, UK) were housed and maintained in a 12-h light/dark cycle with ad libitum access to standard diet and sterilized water. Guinea pigs were culled by cervical dislocation in accordance with Home Office Guidance on the Animals (Scientific Procedures) Act (1986). Atrial myocytes were isolated following the method of Collins et al. (2011) (29) and stored at 4 C in a high potassium medium containing (in mmol/L): KCl 70, MgCl 2 5, K þ glutamine 5, taurine 20, EGTA 0.1, succinic acid 5, KH 2 PO 4 20, HEPES 5, glucose 10 at pH 7.2 with KOH. Healthy atrial myocytes were identified on the basis of morphology.

Immunocytochemistry
Immunocytochemistry staining and analysis was carried out using the method of Collins and Terrar (2012) (22). AC1 (sc25743) and AC8 (sc32128) primary antibodies were purchased commercially (Santa Cruz Biotechnology, Santa Cruz, CA) and used at a dilution of 1:200. Specificity of sc25743 and sc32128 for AC1 and AC8 was confirmed by Western blot [methods and data previously published in Mattick et al. (20)]. IP 3 R monoclonal primary antibodies (IP 3 R1 KM1112, IP 3 R2 KM1083, IP 3 R3 KM1082) were a kind gift from Professor Katsuhiko Mikoshiba (30) and used at a dilution of 1:1,000. The specificity of antibodies KM1112, KM1083, and KM1082 has been previously verified using Western blot by Sugiyama et al. (31) as well as in previous publications (30). Use of these IP 3 R antibodies has been extensively covered in previous studies (32)(33)(34)(35). All primary antibody staining was carried out overnight at 4 C. Secondary antibody labeling was carried out using either Alexa Fluor 488 or 555 conjugated secondary antibodies (Invitrogen, UK), raised against the appropriate species, for 60 min at room temperature at a dilution of 1:400. Observations were carried out using a Zeiss LSM 510 confocal microscope (Â40 or Â63 oil objectives). For detection of Alexa Fluor 488, fluorescence excitation was at 488 nm with emission collected at 505-530 nm. An excitation filter of 543 nm and an emission filter at >560 nm were used to detect Alexa Fluor 555. The two channels were imaged sequentially. Control cells where the primary or secondary antibody was to be excluded were incubated with 5% donkey serum alone without addition of the relevant antibody. To quantify the relationship between the red and green signals that were imaged during double labeling experiments, we carried out a pixel-by-pixel colocalization analysis on whole cells in ImageJ [using the plugin "Just Another Colocalization Plugin" (36)]. The analysis assessed, pixel by pixel across the whole image, the correlation between intensity values of the two dyes viewed as grayscale images and used produced Pearson's coefficient, which is between À1 (total exclusion of the signals) and þ 1 (complete colocalization of the signals).
For photorelease experiments, isolated atrial myocytes were incubated for 60 min at room temperature with 0.5 mmol/L membrane-permeant caged IP 3 (caged-IP 3 /PM) and 0.025% pluronic F127 (Enzo Life Sciences, UK). Fluo-5F-AM (3 mmol/L) was added for the last 10 min of incubation. DMSO concentrations were 0.5% during IP3/PM loading and 0.75% during IP 3 /PM þ Fluo-5F. Cells were visualized using a Zeiss Axiovert 200 with attached Nipkow spinning disk confocal unit (CSU-10, Yokogawa Electric Corporation, Japan). Excitation light, transmitted through the CSU-10, was provided by a 488-nm diode laser (Vortran Laser Technology Inc., Sacramento, CA). Emitted light was passed through the CSU-10 and collected by an iXON897 EM-CCD camera (Oxford Instruments, UK) at 60 frames per second with 2 Â 2 binning (pixel size = 0.66667 mm 2 ). UV uncaging was carried out using 3Â rapid flashes of a Xenon arc lamp (Rapp Optoelectronics, Germany), delivered through the objective lens. To avoid dye bleaching, the cells were not continually exposed to 488 nm light. Instead, a video of 8-10 s of calcium transients was recorded at a number of timepoints immediately before photorelease (denoted 0 s) and at the indicated times after UV exposure. Calcium transients were measured using regions of interests (ROIs) in Andor iQ software (v 1.7) to record average whole cell fluorescence. For inhibitor work, each aliquot of IP 3 /PM (3-4 experiments) was first used for a control experiment and inhibitor data were excluded if control cells did not respond. Cells were also excluded if, upon analysis, control (pre-photorelease) data exhibited alternans, missed beats, or were otherwise unstable. Adenylyl cyclases were inhibited using the nonselective adenylyl cyclase inhibitor MDL 12-330 A (3 mmol/ L) (37), whereas for inhibition of PKA, we used H89 (1 mmol/L). 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mmol/L) and N x -nitro-L-arginine methyl ester (L-NAME; 100 mmol/L) were used to inhibit soluble guanylyl cyclase and nitric oxide synthase, respectively. All inhibitors were sourced from Tocris Bioscience (UK), except L-NAME which was sourced from Sigma-Aldrich, and applied for at least 10 min, of which at least 5 min were stimulated at 1 Hz. Calcium transient time courses were analyzed in ClampFit (v 10.4) and rise and decay curve-fitting were carried out by fitting single exponentials using Prism (v 8).

Murine Atrial Studies
Adult male CD1 mice (CD-1 IGS 30-35 g, Charles River, UK) were housed maintained in a 12-h light/dark cycle with ad libitum access to standard diet and sterilized water. Mice were culled by cervical dislocation in accordance with Home Office Guidance on the Animals (Scientific Procedures) Act (1986). The heart was rapidly excised and washed in heparincontaining PSS. The ventricles were dissected away under a microscope, and the area adjacent to the sinoatrial node was cleared of connective tissue. The spontaneously beating atrial preparation was mounted in a 37 C organ bath containing oxygenated PSS and connected to a force transducer (MLT0201 series, ADInstruments, UK) to visualize contractions. Resting tension was set between 0.2 and 0.3 g, the tension signal was low-pass filtered at 20 Hz, and beating rate was calculated from the time interval between contractions. After stabilization (variation in average rate of a 10-s sample of no more than 2 beats/min over a 10-min period), cumulative concentrations of PE were added to the bath (range 0.1-30 mmol/L) in the presence of metoprolol (1 mmol/L, applied 30 min before PE) to ensure specificity to a-adrenergic effects. Preparations were excluded if stabilized beating rate under control conditions (PSS only) was less than 300 beats/ min or if preparations were not rhythmic. In addition to the inhibitors listed previously, IP 3 receptors were inhibited using 2-aminoethyl diphenylborinate (2-APB) (2.5 mmol/L, Merck, UK) (37). AC1 was inhibited using the AC1 selective inhibitor ST034307 (1 mmol/L, Tocris, UK) (38), whereas U73122 (5 mmol/L, Tocris, UK) was used to inhibit IP 3 production by PLC. Inhibitors were added after stabilization of the preparations and applied for either 30 min (2-APB, ODQ, L-NAME, U73122, and ST034307) or 60 min (MDL, H89) before PE additions. PE dose-response curves were started only after tissue had reached a stable response, where any occurred.

Statistics
For all single-cell data, t tests or ANOVA were used as appropriate with Dunnett's or Tukey's post hoc test to compare groups to a single control or to all other groups, respectively, as required. Experimenters were not blinded to the conditions being analyzed. Log(concentration)-response curves, used to estimate EC 50 s and maximum responses, were calculated using Prism v8.4.0 software (GraphPad, CA), by fitting an agonist-response curve with a fixed slope to normalized response data. Normalized data were used to compare responses as it was expected some inhibitors used would significantly affect the control beating rate or Ca 2 þ transient amplitude. Fitted values were compared using ANOVA with Dunnett's or Tukey's post hoc test. For analysis of Ca 2 þ transient rise and decay times at 0 s and 120 s after IP 3 photorelease, Ca 2 þ data were analyzed using pClamp v10 (Molecular Devices, CA) to generate times corresponding to 10%-90% and 10%-50% rise time and 90%-10%, 90%-75%, 90%-50%, and half-width decay time. Decay phases of transients were also fitted using one phase decay least squares regression (Prism v8.4.0). Data are presented as means ± SE of recorded values, other than doseresponse curve maximum, which is given as means ± SE of best-fit value, and EC 50 , which is presented as best-fit value with 95% confidence interval.

Type 2 IP 3 Receptors are Colocalized with AC8 in Cardiac Atrial Myocytes
In agreement with published literature (3), type 2 IP 3 receptors (IP 3 R2) were visualized in a punctate pattern at the cell periphery, consistent with a position on junctional SR (Fig. 1B). The vast majority of IP 3 R expression in cardiac atrial myocytes is thought to be IP 3 R2, with IP 3 R1 and IP 3 R3 as minimal components (3). Consistent with this notion, staining for IP 3 R1 (Fig. 1A) and IP 3 R3 (Fig. 1C) receptors did not demonstrate a distinct subcellular pattern and may represent at least some nonspecific labeling. Negative controls for immunocytochemistry (application of secondary antibody only) are shown in Fig. 1D.
Similar to previously published work in guinea pig SAN (20) and atrial myocytes (20,22) and murine SAN (21), immunolocalization of AC8 indicated a band at or just beneath the sarcolemma. Pixel-by-pixel analysis revealed substantial colocalization between AC8 and IP 3 R2 in isolated guinea pig atrial myocytes, Pearson's overlap coefficient R = 0.81 ± 0.02 (n = 14 cells), representative cell shown in Fig. 1, E-G.
AC1 staining was localized to a band which was consistently nearby but predominantly on the intracellular side of IP 3 R2 staining and signals were not substantially overlapping (R = 0.48 ± 0.05, n = 5, representative cell shown in Fig.  1, H-J). The pattern of AC1 and AC8 expression observed matched that for both AC1 and AC8 described previously in SAN cells (20).
The Effect of IP 3 on Cellular Ca 2 þ Transients Requires Functional Adenylyl Cyclases and PKA IP 3 is not cell permeant and is broken down rapidly within cells. In addition, as activation of a-ARs (e.g., using PE) may result in signaling via alternative pathways including activation of PKC via diacylglycerol (DAG) (39), for our experiments, we used a cell-permeant, caged version of the compound (IP 3 /PM) to provide cell stimulation specifically via this second messenger from an exogenous source. This IP 3 compound crosses the cell membrane, is de-esterified by constitutive esterase activity and trapped, and finally can be activated by "uncaging" through brief exposure to UV light. Exposure of cells to UV light alone under the conditions of this experiment did not affect calcium transient amplitude (Fig. 2B) or shape.
Guinea pig atrial myocytes exhibited the classical "Ushaped" activation pattern (40) of atrial myocyte Ca 2 þ transients ( Fig. 2A, i and ii). Photorelease of IP 3 in isolated cardiac atrial myocytes led to a gradual increase in stimulated Ca 2 þ transient amplitude (e.g., 31 ± 6% increase 60 s after photorelease, n = 16, Fig. 2, B and C. This response was completely abolished in the presence of either the adenylyl cyclase inhibitor MDL (3 mmol/L, n = 6, Fig. 2, B and D) or PKA inhibitor H89 (1 mmol/L, n = 9, Fig. 2, B and E), e.g., change in Ca 2 þ transient at 60 s after photorelease of À9 ± 2% in the presence of MDL and À16 ± 8% in the presence of H89. The control IP 3 response was significantly greater than that of MDL or H89 at all measured timepoints after IP 3 photorelease (P < 0.0002 for all comparisons except 30 s after photorelease in H89 where P = 0.0235, ANOVA with Tukey's test for multiple comparisons), whereas the responses seen in the presence of MDL or H89 were not significantly different from one another, or time controls, throughout all timepoints (P > 0.85 for all comparisons).
As explained in the previous paragraph, our simple hypothesis is that the effects of photoreleased IP 3 to increase CaT amplitude results from Ca 2 þ release from the SR which then activates Ca 2 þ -stimulated adenylyl cyclases that are located nearby. However, a more complex hypothesis deserves consideration since PE responses in cat atrial myocytes have been reported to be dependent on nitric oxidemodulated soluble guanylyl cyclase activity by a mechanism that involves stimulation of NO synthase (eNOS) by IP 3mediated Ca 2 þ release from the SR (41). We therefore carried out IP 3 photorelease in the presence of either 10 mmol/L ODQ to inhibit soluble guanylyl cyclase, or 100 mmol/L L-NAME to inhibit nitric oxide synthase (Fig. 2, B, F, and G). There was no change in the response to IP 3 photorelease in the presence of ODQ (P > 0.56 for all timepoints, ANOVA, n = 10, Fig.  2, B and F) or L-NAME (P > 0.45 for all timepoints, ANOVA, n = 4, Fig. 2, B and G); under both conditions, Ca 2 þ transient amplitude increased significantly over time, beginning rapidly after photorelease of IP 3 and was not significantly different to control at any timepoint (Fig. 2B). It therefore appears that under the conditions of our experiments, there is little or no contribution of the nitric oxide-stimulated guanylyl cyclase pathway to the effects of photoreleased IP 3 .
Further analysis of calcium transient characteristics at the 0 and 120 s timepoints (Fig. 2, H-M and Supplemental Fig. S1; all Supplemental material is available at https://doi.org/ 10.6084/m9.figshare.12333305) showed that photorelease of IP 3 led to a significant increase in maximum upstroke velocity without affecting 10%-90% rise time (Fig. 2J) and a significant reduction in time constant of decay (Fig. 2I) as well as time to 90% and 50% recovery (Fig. 2, L and M). These changes were no longer seen in the presence of MDL. In the presence of H89, photorelease of IP 3 had no significant effect on maximum upstroke velocity or time to 50% recovery, however time to 90% recovery was significantly reduced, though to a lesser extent than the effects seen after exposure to IP 3 under control conditions (control; P < 0.0001; H89; P = 0.04).
Separate experiments were performed using external application of PE (10 mmol/L) to determine the effect of inhibition of adenylyl cyclases or PKA where alternative signaling pathways (e.g., PKC, DAG) may be involved (Supplemental Fig. S2). PE caused a 39 ± 10% increase in calcium transient amplitude (n = 6, P = 0.0006 PE condition vs. paired PSS control). As expected, PE no longer caused a significant increase in calcium transient amplitude when applied during inhibition of IP 3 Rs or a 1 -adrenergic receptors using 2-APB or prazosin, respectively (P = 0.961 for 2-APB and P = 0.998 prazosin condition vs. drug þ PE condition, two-way ANOVA). In a separate set of experiments, PE caused a 35 ± 9% increase in peak-stimulated Ca 2 þ transient amplitude (n = 8). In the presence of adenylyl cyclase inhibitor MDL (10 mmol/L, n = 5) or PKA inhibitor H89 (1 mmol/L, n = 4), 10mmol/L PE was no longer able to significantly enhance calcium transient amplitude (P = 0.689 for MDL and P = 0.137 for H89 condition vs. drug þ PE condition, two-way ANOVA, Supplemental Fig. S2). This is consistent with the findings of the Blatter group working in cat atrial myocytes (41).
In assessing the importance of the above observations, it is necessary to take into account the influence of ongoing activity of adenylyl cyclases in the absence of stimulation by IP 3 -mediated Ca 2 þ release from the SR [described in Refs. (20) and (22)], as well as possible effects of cAMP on the IP 3evoked Ca 2 þ release (18,42), and these points are considered in more detail in the DISCUSSION. Both MDL and H89 alone were previously shown (22) to reduce the amplitude and slow the time course of CaTs [MDL reduced CaT amplitude by 48 ± 8% (P < 0.001, n = 7), increased the time to peak by 45 ± 5% (P < 0.001, n = 7) and increased the time to 50% decay by 37 ± 13% (P < 0.05, n = 7), whereas H89 reduced CaT amplitude by 37 ± 5% (P < 0.01, n = 6), increased the time to peak by 19 ± 3% (P < 0.001, n = 6) and increased the time to 50% decay by 20 ± 6% (P < 0.05, n = 6)]. However, although CaT amplitude was reduced by both MDL and H89 because of ongoing adenylyl cyclase activity under resting conditions, there was still a substantial remaining CaT (greater than 50% peak amplitude), and if there were important effects of IP 3 -stimulated Ca 2 þ release from the SR that did not depend on adenylyl cyclases or downstream PKA effects then these would still be expected to operate and lead to observable increases in CaT amplitude under these conditions. The complete lack of any detectable increase in CaT amplitude following photorelease of IP 3 in the presence of MDL or H89 therefore provides a clear indication for the requirement of adenylyl cyclase activity and downstream PKA to bring about the effects of IP 3 in the absence of drugs. In a similar way, although some reduction of IP 3 -evoked Ca 2 þ release in the presence of MDL and PKA, as suggested from the work of Colin Taylor (18), cannot be excluded, the Ca 2 þ sensitivity of IP 3 Rs to IP 3 would not be expected to be reduced to zero in the presence of MDL or H89, and therefore again the complete lack of detectable effects of photoreleased IP 3 in the presence of MDL or H89 are attributable to the essential requirement of adenylyl cyclase activity and downstream PKA actions for the effects of IP 3 in the absence of drugs.

The Positive Chronotropic Effect of PE on the Sinoatrial Node Also Requires Functional Adenylyl Cyclases
It has been established that endogenous generation, or exogenous administration, of IP 3 in the SAN leads to an increase in spontaneous beating rate, accompanied by an increase in Ca 2 þ transient amplitude (7), whereas cAMP from Ca 2 þ -stimulated adenylyl cyclases has been shown to modulate murine heart rate (21) and, specifically, I(f) in these cells (20). Spontaneously beating atrial tissue preparations can also provide a measure of sinoatrial node activity through measurement of beating rate. IP 3 R2 (7) and AC8 (21) expression have previously been demonstrated in murine sinoatrial node, with that of AC8 highly similar to data presented in Fig. 1 and that of IP 3 R2 including both peripheral staining as seen in atrial myocytes (e.g., Fig. 1) and separate bands on non-junctional SR. Our own observations (20) demonstrate that AC1 and AC8 appear to show similar distribution within SAN cells as atrial cells. Dose-response curves to PE in the concentration range 0.1-30 mmol/L were carried out on spontaneously beating isolated murine right atria in the presence of 1 mmol/L metoprolol to ensure no confounding action of b-adrenergic receptors and fit with Log(agonist) versus response curves (three-parameter model) by nonlinear regression using a least squares method (Prism 8). Preparations were allowed to reach a stable beating rate in PSS and cumulative addition of PE then took place after either 30 min of metoprolol exposure (used as a time-control for the effect of other inhibitors) or exposure to metoprolol plus named inhibitor. Under these conditions, the positive chronotropic response to PE fits a standard agonist dose-response curve with an EC 50 of 0.91 mmol/L [95% confidence interval (CI) 0.68-1.21] and a maximum rate increase of 15.1 ± 0.2% (n = 10, Fig. 3A).
In the presence of 1 mmol/L MDL to inhibit adenylyl cyclase activity, we observed a 34.5 ± 6.4% reduction in beating rate in the absence of further intervention (Fig. 3B, P < 0.0001, one-way ANOVA with Dunnett's correction vs. timecontrol, n = 5). Under these conditions, bath application of cumulative doses of PE no longer led to an increase in beating rate (maximum rate change 0.7 ± 0.2%, n = 5, Fig. 3A) without significant effect on EC 50 (0.85 mmol/L, 95% CI 0.08-7.63). Application of 1 mmol/L ST034307, a selective inhibitor of AC1 (8), led to a significant reduction in the effect of PE (P < 0.0001 vs. PE, maximum response from dose-response curve fit 7.0 ± 0.3%, n = 8, Fig. 3A) and a significant shift in EC 50 (to 5.60 mmol/L, 95% CI 3.73-9.64, P = 0.0163 vs. response to PE, ANOVA with Dunnett's correction) without a significant change in initial beating rate (Fig. 3B, P = 0.9998). Application of 1 mmol/L H89 to inhibit PKA also led to a significant reduction in beating rate (by 13.7 ± 3.2%, P = 0.0014 vs. time-control, n = 5, Fig. 3B) and completely abolished the effect of PE (largest measured rate change, at 30 mM PE, À1.9 ± 5.2%, n = 5, Fig. 3A). In the presence of H89, it was no longer possible to accurately fit a dose-response curve using the same model, it is therefore excluded from the statistical analyses based on curve fitting.
In agreement with the IP 3 photorelease data, neither L-NAME (100 mmol/L, n = 6), nor ODQ (30 mmol/L, n = 5) had a significant effect upon spontaneous beating rate under control conditions, the maximum response to PE or EC 50 (Fig. 3,  A and B).

DISCUSSION
The present observations indicate the need for an important extension to the proposed signaling pathways underlying the well-recognized actions of IP 3 in atria and SAN and provide evidence for an alternative to the previous hypotheses that Ca 2 þ released from the SR via IP 3 receptors may increase the amplitude of Ca 2 þ transients as a direct result of priming nearby ryanodine receptors (RyRs) (3) or by activating of eNOS located in caveolae in the surface membrane (41). This study represents the first measurements that link direct cellular stimulation with IP 3 in atrial myocytes to downstream actions via the generation of cAMP and activation of PKA. Our work is consistent with the hypothesis that interaction of IP 3 -mediated Ca 2 þ release with the cAMP system is essential for the positive inotropic and chronotropic effects of this compound in the cardiac atria and sinoatrial node, and that this is physiologically important in the response of these tissues to a-adrenoceptor stimulation. The generation of cAMP by adenylyl cyclases in this newly proposed IP 3 -dependent pathway will itself have functional effects in SAN by increasing the activation of I(f) (20), and subsequent stimulation of PKA will have well-established downstream effects on phospholamban/SERCA (45), L-type Ca 2 þ channels (22), and perhaps RyR (46) in both SAN and atria. Structural studies using immunostaining methods (Fig. 1), which initially led us to investigate this intriguing possibility within our preparations, highlight the Ca 2 þstimulated isoforms AC8 and AC1 as probable candidates for this interaction.
Before considering subtypes of adenylyl cyclase in more detail, our proposal of a direct involvement of Ca 2 þ -stimulated adenylyl cyclases in the actions of IP 3 in atria and SA node should be set in the broader context of interactions between IP 3 and cAMP pathways. A particularly interesting possibility is that cAMP influences IP 3 -evoked Ca 2 þ release sensitivity to Ca 2 þ (42). It has been shown that IP 3 Rs, including IP 3 R2, can be phosphorylated by PKA enhancing IP 3 -evoked Ca 2 þ release (18,42). In addition, high concentrations of cAMP can cause PKA-independent modulation of IP 3 Rs (18,42). We cannot exclude the possibility that such mechanisms also operate in atria and SA node under the conditions of our experiments, but as set out in the RESULTS, we do not believe that such a possibility invalidates our use of MDL and H89 to investigate the requirement of adenylyl cyclases for the actions of IP 3 in these tissues. We argue that substantial (greater than 50% peak amplitude) CaTs remain in the presence of MDL and H89, and even if the IP 3 -evoked Ca 2 þ release were to be reduced in the presence of MDL or H89, this IP 3 -evoked Ca 2 þ release would not be expected to be reduced to zero. The suggestion that IP 3 receptors remain functional in the absence of cAMP or phosphorylation is based on extensive published work on regulation of IP 3 R2 receptors, and in all cases, it has been observed that these receptors continue to be activated by IP 3 in the absence of cAMP or phosphorylation by PKA (18,42,47,48). In the case of phosphorylation of IP 3 R2, the extent of enhancement of Ca 2 þ release has been observed to vary with IP 3 concentration, and at 1 mM [see Fig. 2C in (47)], there was no detectable Figure 3. The positive chronotropic effect of PE requires function of adenylyl cyclases and a proposed scheme for regulation of intracellular calcium via IP 3 signaling. A: dose-response curves to show the change in beating rate on cumulative addition of PE to spontaneously beating murine right atrial preparations under control conditions (n = 10) and in the presence of either 2-APB (IP 3 R inhibitor, 2.5 mmol/L, n = 7), MDL (AC inhibitor, 1 mmol/L, n = 5), ST034307 (AC1 inhibitor, 1 mmol/L, n = 8), H89 (PKA inhibitor, 1 mmol/L, n = 5), U73122 (PLC inhibitor, 5 mmol/L, n = 5), L-NAME (NOS inhibitor, 100 mmol/L, n = 6), or ODQ (sGC inhibitor, 30 mmol/L, n = 5). Dose-response curves (solid lines) were fit with log(agonist) versus response (three-parameter model) using GraphPad Prism 8. *significant reduction in maximum response of the fitted curve by ANOVA in comparison to PE. B: comparison of beating rate change in spontaneously beating murine atrial preparations from stable beating in PSS on addition of the inhibitors used in A, prior to stimulation by PE. Control indicates addition of b-blockade only as a time control (1 mmol/L metoprolol, n = 10). Other conditions are b-blockade plus indicated inhibitor. *P < 0.001 compared to control (one-way ANOVA with Dunnett's post hoc correction). Data plotted as means ± SE. C: scheme indicates potential mechanisms by which activation of a1 adrenergic receptors (a1-AR) by PE may lead to increased atrial cytoplasmic calcium transients (indicated by [Ca 2 þ ] i ) and sinoatrial node beating rate based on published observations in addition to our present results. Activation of a1-AR leads to elevated IP 3 resulting from cleavage of PIP 2 to DAG and IP 3 by PLC. IP 3 activation of IP 3 R2 results in release of Ca 2 þ from the SR, which subsequently leads to activation of Ca 2 þsensitive adenylyl cyclases (AC8 or AC1) and activation of PKA by cAMP, or direct effects of cAMP on the funny current I(f). In the proposed scheme, AC8 is placed in the sarcolemma, but it remains to be established whether there is an additional location in the junctional SR and whether nearby AC1 may also be activated by IP 3 -mediated Ca 2 þ release. Image created with BioRender. DAG, diacylglycerol; IP 3 , inositol trisphosphate; IP 3 R2, inositol trisphosphate receptor type 2; PE, phenylephrine; PSS, physiological salt solution; SR, sarcoplasmic reticulum; 2-APB, 2-aminoethoxydiphenyl borate.
change in the sensitivity of IP 3 R2 to IP 3 following PKA effects. Since no previous work is consistent with the proposal that IP 3 receptors are unresponsive to IP 3 in the absence of cAMP or without phosphorylation, we argue that our observation of a complete suppression of the effects of IP 3 photorelease on CaTs in the presence of MDL and H89 provides convincing evidence supporting the proposal that, at least in atria, IP 3 -evoked Ca 2 þ release from the SR activates Ca 2 þ -sensitive adenylyl cyclases and downstream PKA to bring about an enhancement of CaT amplitude. Similar arguments can be advanced for suppression of PE effects on beating rate in SA node, but in this case, additional complications that need to be taken into account are direct effects of cAMP (inhibited by MDL but not H89) and possible roles of DAG that are activated via a receptors in addition to IP 3 release, and future experiments will be necessary to further test these possibilities.
Ten mammalian adenylyl cyclase isoforms have been discovered, nine membrane bound and one soluble form. Of these, three are Ca 2 þ sensitive; AC1 is CaM dependently Ca 2 þ stimulated (49) with an EC 50 for Ca 2 þ of 75 nmol/L (50), AC8 is CaM dependently stimulated (51, 52) with a Ka for Ca 2 þ activation of $0.5 mmol/L (53) and AC5 is CaM-independently inhibited (54,55). The majority of previous studies on AC1 and AC8 pertain to roles in the brain, where these enzymes have been implicated in a range of processes including spatial memory formation (56)(57)(58), neurodevelopment (59), responses to inflammatory pain (60), and opioid dependence (61). AC1 may also have a role in podocytes of the glomerulus of the kidney (62). Our immunocytochemistry data demonstrate that AC8 is found in close proximity to IP 3 Rs in cardiac atrial myocytes, whereas AC1 is found in a band just intracellular to these receptors. AC8, therefore, is ideally positioned to transduce local changes in Ca 2 þ into the cAMP-dependent and PKA-dependent effects detailed in this paper, namely, the modulation of cellular Ca 2 þ transients in response to IP 3 . Given the known position of IP 3 Rs on the junctional SR (3,5), it is not possible for our staining to distinguish whether AC8 is located on the SR itself or on the surface membrane, situated less than 20 nm away (63,64). Sucrose-based fractionation of isolated SAN myocytes has indicated that AC1 and AC8 activity is most associated with fractions also containing caveolin-3 (65). In other cell types, AC8 has been localized to caveolae (66), and disruption of lipid rafts has been seen to abolish the stimulation of this cyclase by Ca 2 þ (53). Taken together, this evidence is consistent with a surface membrane distribution of this enzyme. Although it seems most likely that Ca 2 þ released via IP 3 Rs activates colocalized AC8, the selective AC1 inhibitor ST034307 (38) significantly attenuated the response of whole atria beating rate to PE (Fig. 3A), raising the possibility that this Ca 2 þ could also activate nearby AC1.
The schematic shown in Fig. 3C provides an illustration of the cellular pathway, supported by the data presented in this paper, which may result following activation of the IP 3 signaling cascade. Increased generation of cAMP via Ca 2 þ activation of AC8 or AC1 may lead to activation of PKA and multiple downstream actions that result in increases in cellular Ca 2 þ transient amplitude or beating rate at the sinoatrial node. AC1 and AC8 expression has previously been demonstrated in the SAN and these AC isoforms have been implicated in the regulation of SAN pacemaker activity (20,21). Moen et al. (2019) (21) recently demonstrated that overexpression of AC8 in SAN cells results in an increase in heart rate and decrease in heart rate variability. Furthermore, work from the Terrar group has previously demonstrated the involvement of Ca 2 þ -sensitive ACs in the regulation of I(f) in SAN myocytes (20) as well as L-type Ca 2 þ current in atrial myocytes (22). Taken together, these previous studies implicate both LTCC and HCN4 as potential targets for regulation via IP 3 signaling in the context of the data presented in Figs. 2 and 3. The involvement of other targets for PKA phosphorylation however, including ryanodine receptors (RyRs) (46), phospholamban (PLB) (45), and the Na þ /Ca þ exchanger (NCX) (67,68) cannot yet be discounted. It is possible that the activity of PKA augments regulation by PKC, which has also been well documented at these same target sites and is similarly activated via stimulation of G q -coupled receptors (24,(69)(70)(71)(72). The use of caged-IP 3 in this study however rather than stimulation of G q -coupled receptors (Fig. 2) demonstrates that the effects on cellular Ca 2 þ observed in the present study can occur via the effects of IP 3 signaling specifically and are independent of activation of DAG.
Under the conditions of our study, inhibition of ACs or PKA significantly reduced baseline beating rate in right atrial preparations (Fig. 3B). This is consistent with published data from our group (20,22) and others (73,74). Indeed, it has previously been shown that heart rate in AC8 overexpressing mice is significantly higher than in their wild-type counterparts (75). The sinoatrial node has a constitutive level of cAMP, which is significantly greater than that of the ventricle in the absence of adrenergic stimulation (26). How much of this activity is attributable specifically to Ca 2 þ -stimulated ACs is not discernable from our experiments, as selective inhibitors are not currently available for all ACs, although 1 μmol/L ST034307 did not affect baseline rate (Fig. 3). The diastolic cell Ca 2 þ concentration in SAN myocytes, $225 nmol/L (76), is considerably higher than that in ventricular cells. Given that AC1 and AC8 proteins have not been shown to be expressed in ventricular tissue (20), it seems possible that differences in the expression of Ca 2 þ -stimulated AC isoforms could contribute to the differences between resting SA nodal and ventricular cAMP concentration as previously reported (26). Indeed, cAMP synthesis activity is high in SAN myocyte lysates in 1 mmol/L Ca 2 þ but almost abolished in Ca 2 þ -free solution (65), suggesting Ca 2 þ -stimulated cAMP production may be the dominant mechanism in these cells at rest.
Our data indicate that high constitutive cAMP production in the sinoatrial node cannot be attributed to background IP 3 R activity. Background activity under the conditions of our experiments appears to be negligible as neither 2-APB, nor U73122 had a significant effect on the spontaneous beating rate of intact right atrial preparations when applied in our organ bath setup. This is in contrast to published work from Ju et al. (2011) (7) and Kapoor et al. (2015) (8), who both report significant reductions in spontaneous Ca 2 þ transient firing rate in response to 2-APB measured using Ca 2 þ fluorophores in dissected nodal tissue and isolated sinoatrial node myocytes, respectively.
In atrial myocytes isolated from cat, the effects of PE to enhance I CaL have been reported to occur through inhibition of phosphodiesterase downstream of PI-3K-mediated eNOS activation (41). Although we agree that cAMP and PKA are central to the response of atrial myocytes and the sinoatrial node to PE, and IP 3 , we did not find evidence that nitric oxide or soluble guanylyl cyclase activity were required under the conditions of our experiments.
It has previously been hypothesized that Ca 2 þ release through IP 3 Rs acts to enhance atrial myocyte Ca 2 þ transients by increasing the local Ca 2 þ concentration around RyRs and thereby enhancing RyR response to the opening of LTCC (3,16). Although this has been directly observed in an IP 3 R overexpression model (77), our data investigating the effect of IP 3 photorelease in primary isolated myocytes is consistent with the notion that the functional effects of IP 3 Rmediated Ca 2 þ release on RyRs occur downstream of intermediary signals. In particular, it would be expected that recruitment of local RyRs in response to photoreleased IP 3 would remain after inhibition of adenylyl cyclases or PKA. We observed no change in Ca 2 þ transient amplitude over time in response to direct application of IP 3 in the presence of MDL or H89. It remains possible that RyR sensitization resulting from localized Ca 2 þ release via IP 3 Rs is essential in the period before our measurements take place, or that augmented RyR release could itself contribute to recruitment of adenylyl cyclase and PKA signaling.
The results of this study provide a novel mechanism by which a ubiquitous second messenger pathway contributes to physiological signaling in the heart. The presented hypothesis, however, may also provide interest in the context of cardiac pathology. For instance, Mougenot et al. (2019) (78) have recently demonstrated that overexpression of AC8 accelerates age-related cardiac dysfunction through increased hypertrophy and interstitial fibrosis in transgenic mice. Even in healthy atrial myocytes, IP 3 R signaling can generate arrhythmogenic Ca 2 þ waves (3,5). The IP 3 pathway may contribute to spontaneous generation of action potentials in pulmonary vein sleeve cells (79,80), one of the main sites for AF initiation (81), whereas IP 3 R is known to be upregulated in atrial myocytes from patients with chronic AF (12) and from animal models of atrial fibrillation (AF) (13). In fact, atrial IP 3 R expression appears to correlate with markers of atrial dysfunction regardless of diagnosed disease state (12). In contrast, RyR expression becomes downregulated in chronic AF (82) and the organization of RyR clusters is disrupted (83). The relative time course of these expression changes during disease development is unknown. The position and character of the signaling domains presented in this paper remain to be determined in the context of the remodeling associated with AF, though the evidence presented in this paragraph highlights the importance of understanding the cellular mechanisms of the IP 3 pathway and role of Ca 2 þ -sensitive ACs in both healthy and diseased cardiomyocytes.
The present paper provides novel information on the signaling pathways responsible for physiological responses to IP 3 , demonstrating a crucial requirement for cAMP and PKA. We have focused on Ca 2 þ -stimulated ACs as an effector of this interaction. Our new observations provide an added level of complexity to Ca 2 þ modulation in the atria and sinoatrial node and raise questions concerning the importance of these interacting signaling pathways in atrial fibrillation and related pathology.

DISCLOSURE
No conflicts of interest, financial or otherwise, are declared by the authors.