Tatiana M. Vinogradova, Didier X.P. Brochet, Syevda Sirenko, Yue Li, Harold Spurgeon, Edward G. Lakatta

Transcription

1 Sarcoplasmic Reticulum Ca 2 Pumping Kinetics Regulates Timing of Local Ca 2 Releases and Spontaneous Beating Rate of Rabbit Sinoatrial Node Pacemaker Cells Tatiana M. Vinogradova, Didier X.P. Brochet, Syevda Sirenko, Yue Li, Harold Spurgeon, Edward G. Lakatta Rationale: Sinoatrial node cells (SANCs) generate local, subsarcolemmal Ca 2 releases (LCRs) from sarcoplasmic reticulum (SR) during late diastolic depolarization. LCRs activate an inward Na -Ca 2 exchange current (I NCX ), which accelerates diastolic depolarization rate, prompting the next action potential (AP). The LCR period, ie, a delay between AP-induced Ca 2 transient and LCR appearance, defines the time of late diastolic depolarization I NCX activation. Mechanisms that control the LCR period, however, are still unidentified. Objective: To determine dependence of the LCR period on SR Ca 2 refilling kinetics and establish links between regulation of SR Ca 2 replenishment, LCR period, and spontaneous cycle length. Methods and Results: Spontaneous APs and SR luminal or cytosolic Ca 2 were recorded using perforated patch and confocal microscopy, respectively. Time to 90% replenishment of SR Ca 2 following AP-induced Ca 2 transient was highly correlated with the time to 90% decay of cytosolic Ca 2 transient (T-90 C ). Local SR Ca 2 depletions mirror their cytosolic counterparts, LCRs, and occur following SR Ca 2 refilling. Inhibition of SR Ca 2 pump by cyclopiazonic acid dose-dependently suppressed spontaneous SANCs firing up to 50%. Cyclopiazonic acid and graded changes in phospholamban phosphorylation produced by -adrenergic receptor stimulation, phosphodiesterase or protein kinase A inhibition shifted T-90 C and proportionally shifted the LCR period and spontaneous cycle length (R ). Conclusions: The LCR period, a critical determinant of the spontaneous SANC cycle length, is defined by the rate of SR Ca 2 replenishment, which is critically dependent on SR pumping rate, Ca 2 available for pumping, supplied by L-type Ca 2 channel, and ryanodine receptor Ca 2 release flux, each of which is modulated by camp-mediated protein kinase A dependent phosphorylation. (Circ Res. 2010;107: ) Key Words: sinoatrial nodal pacemaker cells sarcoplasmic reticulum Ca 2 pumping -adrenergic receptor signaling The sinoatrial (SA) node is the primary physiological pacemaker of the heart. Sinoatrial node cells (SANCs) are able to generate spontaneous action potentials (AP) because of the gradual, spontaneous depolarization of the membrane, ie, diastolic depolarization (DD). 1 Multiple mechanisms are involved in the generation of DD, including numerous ionic currents, 1 and the most recently discovered mechanism, local subsarcolemmal Ca 2 releases (LCRs) from ryanodine receptors (RyRs). 2 4 Similar to ventricular myocytes, SANC cycles Ca 2 via sarcoplasmic reticulum (SR) equipped with SR Ca 2 pumps (SERCAs) and release channels, RyR, and extrudes Ca 2 from the cell via Na - Ca 2 exchanger. 2,5,6 LCRs appear during late DD, before the AP upstroke, 2 4 and do not require a change in the membrane potential, but occur spontaneously: they persist during acute switch to the voltage clamp and are present in permeabilized SANCs. 7 During each spontaneous cycle, Ca 2 influx through L-type Ca 2 channels, triggered by the AP upstroke, produces a global Ca 2 transient, causing a global SR Ca 2 depletion and RyR inactivation. When the SR Ca 2 content is replenished by SERCA, which constantly pumps Ca 2 back into the SR, and RyRs recover from inactivation, LCRs begin to appear. The restitution time, ie, the time from the APtriggered global Ca 2 transient to the onset of LCRs during DD is the LCR period. LCR occurrence activates an inward Na -Ca 2 exchange current (I NCX ), which produces an exponential rise of the late DD and is a determinant of the time at which the next rapid AP upstroke will occur. 7 9 The LCR period, therefore, is a regulator of the spontaneous SANC Original received March 17, 2010; revision received June 25, 2010; accepted July 12, In June 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days. From the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore, Md. Correspondence to Edward G. Lakatta, MD, Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr, Baltimore, MD LakattaE@grc.nia.nih.gov 2010 American Heart Association, Inc. Circulation Research is available at DOI: /CIRCRESAHA

2 768 Circulation Research September 17, 2010 Non-standard Abbreviations and Acronyms AP AR CPA DD IBMX I NCX ISO LCD LCR PDE PKA PLB RyR SA SANC SERCA SR action potential adrenergic receptor cyclopiazonic acid diastolic depolarization 3-isobutyl-1-methylxanthine Na -Ca 2 exchange current isoproterenol subsarcolemmal local Ca 2 release generated local SR Ca 2 depletion subsarcolemmal local Ca 2 release phosphodiesterase protein kinase A phospholamban ryanodine receptor sinoatrial sinoatrial nodal cell sarco-/endoplasmic reticulum Ca 2 /ATPase sarcoplasmic reticulum beating rate. 7 9 Although it has been assumed that the LCR period depends, at least in part, on the rate at which SR is refilled with Ca 2, an intrinsic link between the former and latter has never been demonstrated. The first goal of the present study is to test the hypothesis that SR Ca 2 refilling is a crucial determinant of both the LCR period and the spontaneous SANC beating rate. The velocity of Ca 2 pumping into SR by SERCA is modulated by phospholamban (PLB) phosphorylation at the protein kinase A (PKA)-dependent Ser16 site. 10 Although a prior study has noted a close correlation between graded changes in PLB phosphorylation and the LCR period, 11 the mechanisms accountable for this close relationship have never been revealed. The second goal of the present study is to define mechanisms accountable for the close link between PLB phosphorylation and the LCR period in SANCs. Methods An expanded Methods section, detailing SA node cell isolation and electrophysiological recordings, confocal imaging of SR Ca 2 depletions, cytosolic Ca 2 transients and LCRs, 2,7,11 13 cell permeabilization, 7,9 and Western blotting, 9,11 is available in the Online Data Supplement at Statistical Analysis Data are presented as means SEM. The statistical significance of effects was evaluated by Student s t test or ANOVA where appropriate. A value of P 0.05 was considered statistically significant. Results To determine the role of SERCA pumping in spontaneous SANC firing at 35 C, we used a specific and reversible SERCA inhibitor, cyclopiazonic acid (CPA). 14 CPA decreased the spontaneous SANC beating rate in a dosedependent manner (EC 50, 1.2 mol/l) to a maximal suppression of % (Figure 1A and 1B); all effects were reversed after CPA washout. The suppression in the beating rate was attributable to a marked decrease in the DD rate (Figure 1A) from 59 3 to21 3 mv/sec (n 4, P 0.01), supporting the idea that SR refilling plays an essential role in the control of the basal pacemaker function. To clarify specific mechanisms involved in the CPAinduced suppression of DD and spontaneous firing of intact SANCs, we studied how CPA affects Ca 2 cycling, specifically, subsarcolemmal LCRs. Representative images and average data in Figure 1C and 1D show that at 4 minutes of superfusion with 3 mol/l CPA, there was a marked decrease in LCR size as well as a reduction of LCR number during each spontaneous cycle (Figure 1C and 1D). CPA also markedly increased the LCR period, and the increase in the LCR period was highly correlated with a prolongation of the Figure 1. Inhibition of SERCA by CPA suppresses LCRs and SANC beating rate. A, APs recorded in a representative rabbit SANC before and during superfusion with 3 mol/l CPA. B, The relative dose-dependent decrease in SANC firing rate in response to different CPA concentrations. C, Confocal line-scan images of a representative SANC before and following exposure to 3 mol/l CPA; LCRs are indicated by arrowheads. The inset below the top image illustrates how the LCR period is defined, ie, as the time from the prior AP-induced Ca 2 transient to the onset of LCR. D, In 6 SANCs, CPA (3 mol/l) markedly decreases number of LCRs per spontaneous cycle, as well as LCR size, measured as full width at half maximum (FWHM). *P 0.05.

3 Vinogradova et al SR Ca Pumping Controls Cardiac Pacemaker Function 769 Figure 2. CPA decreases the number and size of LCRs and the SR Ca 2 content in permeabilized rabbit SANCs. A, Confocal line-scan images of a representative saponin-permeabilized SANC bathed in 100 nmol/l free [Ca 2 ] before and following exposure to 3 mol/l CPA. B, Left, The average frequency of LCRs (normalized per 1 second and 100 m). B, Right, LCR size, measured as full width at half maximum (FWHM) in skinned SANCs in control conditions (4 SANCs; 160 LCR) and after 3 minutes of superfusion with 3 mol/l CPA (4 SANCs; 46 LCR). C, Effect of a rapid application of caffeine to a representative permeabilized SANC in the absence (top) and presence (bottom) of 3 mol/l CPA. D, Average effect of CPA on the initial rapid component of the caffeine-induced SR Ca 2 release, indexed by F/F 0 ;n 9 SANCs in a control group; n 7 SANCs following 3 minutes of superfusion with 3 mol/l CPA. *P spontaneous cycle length (Online Figure I, D), suggesting that changes in LCR characteristics could be a major mechanism of CPA-induced decrease in spontaneous SANC firing. To define direct effects of CPA on LCRs in the absence of regularly occurring AP-induced Ca 2 transients, SANCs were permeabilized with saponin and bathed at 100 nmol/l cytosolic free [Ca 2 ]. Similar to its effect in intact SANCs, a 4-minute superfusion with CPA reduced the LCR frequency and size in permeabilized SANCs (Figure 2A and 2B); this was due, at least in part, to a substantial reduction in the SR Ca 2 content, assessed by a rapid spritz of 20 mmol/l caffeine directly onto the skinned SANCs (Figure 2C and 2D). Next, we determined how the increase in the LCR period produced by CPA in intact SANCs is related to CPA-induced suppression of SERCA function. In rabbit ventricular myocytes the rate of [Ca 2 ] i decline during AP-induced Ca 2 transient is highly dependent on Ca 2 pumping into SR, 15,16 and either the time to 90% decay of Ca 2 transient (T-90 C )or monoexponential [Ca 2 ] i decline constant ( ) are convenient measures to characterize the kinetics of the SR Ca 2 pumping and SR Ca 2 refilling. 15,16 Figure 3A illustrates CPA-induced time-dependent prolongation of the decay of AP-induced Ca 2 transient, as reflected in the increase of either the monoexponential decline constant,, or time to 90% decay of the cytosolic Ca 2 transient, T-90 c (Figure 3A and 3B). Note Figure 3. Effect of CPA to increase the LCR period is linked to a prolongation of the decay of AP-induced Ca 2 transient. A, Confocal line-scan images and Ca 2 waveforms of a representative spontaneously beating SANC before, during, and after superfusion with 3 mol/l CPA. In this SANC, LCRs were inhibited after a 5-minute CPA superfusion. B, Relationship between average changes in the monoexponential decline constant, (y 0.7x 45.5 ms, R ), and time to 90% decay of the AP-induced Ca 2 transient, T-90 C (y 0.7x 10.9 ms, R ), and average changes in the LCR period in a representative SANC (A) before, during, and after superfusion with 3 mol/l CPA. C, The average CPAinduced increase of the LCR period in B is linked to the average increase in the spontaneous cycle length (y 1.0x 53.2 ms, R ).

4 770 Circulation Research September 17, 2010 Figure 4. Refilling of SR Ca 2 after AP-induced SR Ca 2 depletion in SANCs is faithfully reported by the decay of cytosolic Ca 2.A,Confocal line-scan image of SR luminal Ca 2 (top) and corresponding Ca 2 waveform (bottom) in a representative spontaneously beating SANC visualized using lowaffinity Ca 2 indicator Fluo-5N. Bottom, Demonstration of how LCD period and AP-induced global Ca 2 depletions, T-90 SR, are defined. B, Confocal linescan image (top) and Ca 2 waveform (bottom) illustrate changes in cytosolic Ca 2 in a representative SANC. The inset below the bottom image illustrates how the LCR period and decay of AP-induced Ca 2 transient, T-90 C, are defined. C, Histograms of the SR Ca 2 - refilling times, T-90 SR (14 SANCs), and decay of Ca 2 transient, T-90 C (9 SANCs), overlap, indicating that T-90 C truthfully reproduce T-90 SR. D, Histograms LCDs periods (82 LCDs from 28 SANCs) and LCR periods (92 LCRs from 9 SANCs) overlap, indicating that LCR period truthfully reproduce LCD periods. that changes in T-90 c were paralleled by changes in, suggesting that either parameter faithfully indexes the Ca 2 transient decay. The CPA-induced time-dependent increase in the relaxation time of the Ca 2 transient was accompanied by a concomitant increase in the LCR period. There was a strong correlation between the time-dependent prolongation in SR Ca 2 refilling and prolongation of the LCR period, suggesting that Ca 2 refilling of SR following AP-induced Ca 2 release is a key determinant of the LCR period (Figure 3B). The time-dependent CPA-induced increase in LCR period predicted the time-dependent increase in the spontaneous cycle length (R ), and both parameters recovered after washout (Figure 3C). On average, after a 4 minute superfusion with 3 mol/l CPA (n 5), there was 51% slowing of the decay of the AP-induced Ca 2 transient, T-90 c (from to ms, P 0.01); 30% decrease in its amplitude (from to F/F 0, P 0.05), accompanied by 53% increase in the LCR period (from to ms, P 0.02). To confirm that Ca 2 refilling of SR is indeed a determinant of the occurrence of subsequent LCRs, luminal SR Ca 2 in SANCs was visualized using Fluo-5N, a low-affinity Ca 2 indicator. 12 The SR refilling times after AP-induced Ca 2 transient were indexed by 90% refilling of SR Ca 2 store (T-90 SR ) (Figure 4A). LCR-generated local SR Ca 2 depletions (LCDs) were detected as spatially restricted darkenings, clearly seen after the Ca 2 image was normalized (F/F 0 ) and processed using customized software, 12 which permitted detection of LCDs as apparent dips among background noise (Figure 4A). To confirm that the relaxation time of global AP-induced Ca 2 transient at 90%, T-90 c, reflects the SR refilling rate, T-90 SR, and that the LCR period is similar to the period of LCD, we compared histograms of T-90 SR and T-90 c (Figure 4C), as well as histograms of LCD periods and LCR periods (Figure 4D). That histograms of T-90 SR and T-90 c overlap indicates that relaxation time of global AP-induced Ca 2 transient, T-90 c, reflects the SR refilling time, T-90 SR. Histograms of LCD periods and LCR periods also overlap (Figure 4D), indicating that the LCR period reflects the LCD period. In ventricular myocytes, -adrenergic receptor ( -AR) stimulation increases camp-mediated, PKA-dependent PLB phosphorylation, leading to relief of inhibition of SERCA and to a decrease in the SR refilling time. 10,16 Prior studies have demonstrated that spontaneous beating of rabbit SANCs is critically dependent on camp-mediated, PKA-dependent phosphorylation, particularly, PLB phosphorylation at PKAdependent Ser16 site. 9,11 However, the relationship between changes in PLB phosphorylation during -AR stimulation in SANCs, changes in SR refilling time and LCD period were not directly measured. Figure 5 shows that -AR stimulation with isoproterenol (ISO) (0.1 mol/l) decreases SR refilling times and shifts the histogram of T-90 SR to the left (Figure 5C). The decrease in the SR refilling time is linked to the -AR stimulation induced increase in PLB phosphorylation and consequent acceleration of the SERCA Ca 2 pumping rate. The decrease in T-90 SR is accompanied by a decrease in LCD period and a shift of LCD period histogram to the left (Figure 5B versus 5C). The decrease in the LCD period is accompanied by a decrease in the spontaneous cycle length (Figure 5D), which confirms that the refilling time is indeed the key factor that controls the LCD period and spontaneous SANC beating rate. Because of high Fluo-5N bleaching, it is not possible to measure refilling times, T-90 SR, and LCD periods in the same SANCs before and after -AR stimulation. However, because the relationship of T-90 C and LCR period is similar to that of T-90 SR and LCD period (Figure 4), we can use T-90 C and LCR period readouts to index effects of -AR stimulation in the same SANCs. Online Figure II shows that -AR stimulation decreases both T-90 C (from to

5 Vinogradova et al SR Ca Pumping Controls Cardiac Pacemaker Function 771 Figure 5. -AR stimulation induced reduction in the spontaneous AP cycle length is linked to acceleration of SR Ca 2 refilling. A, Confocal line-scan image of SR luminal Ca 2 (middle) and SR luminal Ca 2 waveform from the whole image (top) and from the area inside a dashed line (bottom) during -AR stimulation visualized using lowaffinity Ca 2 indicator Fluo-5N in a representative spontaneously beating SANC. B and C, Histograms of the SR Ca 2 -refilling times, T-90 SR, and LCD periods (82 LCD) in subset of control SANCs and in another subset of SANCs, after superfusion with 0.1 mol/l ISO (62 LCDs), respectively. D, Reduction in the spontaneous cycle length during -AR stimulation is linked to the reduction in the LCD period. ms, n 6) and LCR period (from to ms), shifting their histograms to the left. The decrease in the LCR period is accompanied by the decrease in the spontaneous cycle length (Online Figure II). -AR stimulation with ISO also increases the average number of LCRs per each spontaneous cycle (from to ; P 0.01); LCR size (from to m) and LCR amplitude (from to F/F 0, P 0.02). Like -AR stimulation, phosphodiesterase (PDE) inhibition markedly increases the level of camp and consequentially increases camp-mediated PKA-dependent phosphorylation in conjunction with its effect to accelerate spontaneous firing of rabbit SANCs. 11 Similar to -AR stimulation, an increase in PKA-dependent PLB phosphorylation by PDE inhibition would be expected to relieve SERCA from PLB inhibition, accelerate pumping Ca 2 into SR and its refilling decreasing the time of the decay of AP-induced Ca 2 transient, T-90 C. Indeed, suppression of PDE activity by either a broad-spectrum PDE inhibitor, 3-isobutyl-1- methylxanthine (IBMX), or PDE3 inhibitor, milrinone, markedly decreases T-90 C and concomitantly decreases the LCR period, shifting their histograms to the left (for IBMX, Figure 6; for milrinone, Online Figure III, respectively). The reduction in the LCR period produced by either IBMX or milrinone is paralleled by a reduction in the spontaneous cycle length (Figure 6D; and Online Figure III, D, respectively). Both IBMX and milrinone markedly increase the number of LCRs per cycle, LCR size and amplitude (Online Figure IV). The suppression of PKA-dependent phosphorylation in SANCs by a specific PKA inhibitor peptide, PKI, is likely Figure 6. Suppression of PDE activity with IBMX increases SR Ca 2 refilling rate and reduces T-90 C and LCR period. A, Confocal line-scan images of a representative SANC before and during exposure to 100 mol/l IBMX; LCRs are indicated by arrowheads. B, Histograms of the decay of AP-induced Ca 2 transient, T-90 C, before and during superfusion with IBMX. C, Histograms of the LCR period in control (46 LCRs from 5 SANCs) and after superfusion with IBMX (124 LCRs from 5 SANCs). D, IBMX-induced decrease in the spontaneous cycle length is linked to the decrease in the LCR period.

6 772 Circulation Research September 17, 2010 Figure 7. Suppression of PKAdependent phosphorylation with PKI increases SR Ca2ⴙ refilling time and prolongs the LCR period. A, Confocal line-scan images of a representative SANC before and following exposure to 5 mol/l PKI; LCRs are indicated by arrowheads. B, Histograms of the decay of the Ca2 transient, T-90C, before and during superfusion with PKI. C, Histograms of the LCR period in control (n 5 cells, 52 LCRs) and during superfusion with PKI (n 5 cells, 18 LCRs). D, The PKI-induced increase in the spontaneous cycle length is linked to the increase in the LCR period. mediated by a reduction in phosphorylation of multiple proteins that regulate SANC Ca2 balance, including PLB, RyR, L-type Ca2 channels, and probably others.9 In SANCs, PKI markedly prolongs a decay of AP-induced Ca2 transient, T-90C, shifting its histogram and that of LCR periods, to longer times (Figure 7). This increase in the LCR period is accompanied by an increase in the spontaneous SANC cycle length (Figure 7D). PKI also substantially decreases the number of LCRs per each spontaneous cycle (from to ; n 4, P 0.05), LCR amplitude (from to F/F0; n 5, P 0.05), and LCR size (from to m, n 4, P 0.05). Figure 8B illustrates the continuum between changes in the PLB phosphorylation produced by -AR stimulation, PDE inhibition, or PKI and changes in T-90C. Thus, the rate at which the SR reloads with Ca2 is dependent on the level of PLB phosphorylation: an increase or decrease in PLB phosphorylation is linked to proportional inverse changes in SR refilling times (Figure 8B). Furthermore, the effects of PLB phosphorylation on T-90C are reflected in the changes of the LCR period (Figure 8C), ie, the acceleration of SR refilling produced by either -AR stimulation or PDE inhibition decreases, whereas PKI increases, the LCR period. Note that the increase in the SR refilling time and LCR period produced by direct inhibition of SERCA by CPA, which does not change PLB phosphorylation, lies along the same function that depicts the effects of maneuvers that change PLB phosphorylation (Figure 8C). Finally, relative changes in the LCR period, effected by perturbations that alter SR pumping rate in Figure 8C, are tightly linked to the relative changes in the spontaneous cycle length (R2 0.98), and the function describing this conforms to the line of identity (Figure 8D). Figure 8. Graded changes in PLB phosphorylation are paralleled by proportional changes in SR Ca2ⴙ refilling time, T-90C, and LCR period, which are highly correlated with concurrent changes in the spontaneous cycle length. A, Representative Western blots of PLB phosphorylated at serine 16 site and total PLB in rabbit SANCs in the basal state and following milrinone (50 mol/l), IBMX (100 mol/l), -AR stimulation (0.1 mol/l [ISO1], or 1 mol/l [ISO2] isoproterenol), and PKI (10 mol/l). B, Graded changes in PLB phosphorylation by -AR stimulation (0.1 mol/l ISO, n 8), a broad-spectrum PDE inhibitor (100 mol/l IBMX, n 8), specific PDE-3 inhibitor (50 mol/l milrinone, n 8), or by specific PKA inhibitor peptide (10 mol/l PKI, n 8) are linked to inverse changes in T-90C. C, Changes in T-90C produced by either changes in PLB phosphorylation or by CPA are paralleled by concomitant changes in the LCR period. D, Changes in the spontaneous cycle length are tightly linked to the concurrent changes in the LCR period.

7 Vinogradova et al SR Ca Pumping Controls Cardiac Pacemaker Function 773 Discussion The first novel finding of the present study is that the restitution time for LCRs, the LCR period, and the spontaneous SANC cycle length, are both determined by the speed at which SR is refilled with Ca 2. The counterpart of cytosolic LCRs, ie, LCDs, visualized with a low-affinity Ca 2 indicator Fluo-5, appear only when SR is replenished with Ca 2 (Figure 4). A crucial role of SR Ca 2 cycling and, particularly, SERCA function for normal spontaneous pacemaker firing (Figure 1) is confirmed by suppression of SERCA function and SR Ca 2 reuptake with CPA, a specific Ca 2 -ATPase inhibitor, 14,17 which dramatically, in a time- and dosedependent manner, decreases the spontaneous beating rate of rabbit SANCs by up to 50% (Figure 1B). The second novel finding of the present study is that the CPA-induced increase in the spontaneous cycle length (reduction in the beating rate) results from the prolongation of the LCR period, caused by a substantial increase in the time of SR refilling with Ca 2 (Figure 3 and Online Figure I). The increase in the LCR period and decrease in LCR number and size produced by CPA postpones the occurrence of LCR-activated, I NCX and reduces its amplitude leading to decrease in the slope of DD (Figure 1A) and, as a result, prolongation of the spontaneous cycle length (Online Figure I). In skinned SANCs, the suppression of SERCA function by CPA decreases the SR Ca 2 load (Figure 2D) and markedly suppresses LCRs (Figure 2B) indicating that SR Ca 2 load is a crucial determinant of LCR characteristics. Similar to intact SANCs, CPA also markedly increases a time interval between spontaneous LCRs in skinned SANCs (Figure 2A). When SR Ca 2 pumping rate is inhibited by CPA, LCR number and size both in intact (Figure 1D) and permeabilized SANCs are markedly decreased (Figure 2B). The decay rate of the AP-induced transient increase in cytosolic [Ca 2 ] reflects the activities of both the SR Ca 2 - ATPase and the Na -Ca 2 exchanger, and their contribution is species-dependent. 15,16 In rabbit ventricular myocytes, 70% of Ca 2 released in cytosol during AP-induced Ca 2 transient is actively transported back into SR by SR Ca 2 - ATPase, and 30% is extruded from the cell via Na -Ca 2 exchanger. 15,16 Thus, the dominance of SERCA over Na -Ca 2 exchanger in rabbit ventricular myocytes is 2 to 3-fold. 15,16 In the present study, in rabbit SANCs, inhibition of SERCA function by CPA increased the time of the decay of AP-induced Ca 2 transient, T-90 C,by 2-fold, which is consistent with data in rabbit ventricular myocytes. 15,16 In SANCs, the subsystems of ionic channels and Ca 2 cycling work together to guarantee the beating rate stability in a given steady state. 18,19 There are multiple interactions between these 2 subsystems: surface membrane proteins not only control changes in membrane potential but also directly or indirectly regulate intracellular Ca 2 cycling; and vice versa, intracellular Ca 2 cycling proteins also regulate membrane potential via Ca 2 modulation of surface membrane electrogenic molecules. The present results show how manipulations that specifically target the Ca 2 cycling subsystem, ie, suppression of SERCA function by CPA, affect the entire coupled system and lead to decrease in the spontaneous beating of SANCs. Although a moderate CPA-induced reduction of guinea pig isolated SA node or SANC beating rate has been noted before, 6 neither the full range of this effect nor the mechanisms of CPA-induced suppression of the SANC beating rate had been studied. The experimentally observed CPA-induced decrease in the SANC spontaneous beating rate via modulation of the Ca 2 -ATPase pumping rate in the present study is faithfully simulated by a novel numeric model of SANCs. 20 Consistent with experimental results, these model simulations predict 40% suppression of the SANC beating rate when Ca 2 -ATPase pumping rate is decreased to approximately one-third of its basal value. 20 The speed at which Ca 2 is pumped back in the SR is modulated by PLB, which, in its unphosphorylated state, binds to SERCA and inhibits its function. 10,21 In ventricular myocytes, phosphorylation of PLB by camp-mediated, PKA-dependent activation relieves SERCA inhibition, leading to acceleration of Ca 2 uptake by SR and an increase in the speed of cardiac muscle relaxation. 10,16 Although it has been demonstrated that the positive chronotropic effect of -AR stimulation is critically dependent on its effect to accelerate SR Ca 2 cycling, 3,18,19,22 24 shifting the LCR period and LCR-activated I NCX current to earlier times during DD, 24 the mechanisms responsible for these changes have never been demonstrated. The third novel finding of the present study, which directly monitors changes in the SR Ca 2 in SANCs, is that an increase in PKA-dependent phosphorylation by -AR stimulation, indexed by PLB phosphorylation (Figure 8A), decreases the SR refilling time, T-90 SR, and consequently shortens the counterpart of LCR period, the LCD period, producing a decrease in the spontaneous cycle length (Figure 5D). The effects of -AR stimulation to decrease the LCD period (Figure 5C) are attributable, in part at least, to the increase in the SERCA pumping rate, which is relieved by PLB phosphorylation. As noted, PKA-dependent effects on sarcolemmal molecules are also involved, because these effects regulate Ca 2 that is provided for the SR for pumping. The important role of PLB phosphorylation in cardiac pacemaker function has also been confirmed in genetically manipulated mice (see the Online Data Supplement). A comparison of the time of SR Ca 2 refilling, T-90 SR, and the decay time of AP-induced Ca 2 transient, T-90 C, demonstrates that the latter faithfully reproduce the dynamics of SR Ca 2 refilling and can be used to index it (Figure 4). This observation has also been confirmed during -AR stimulation, ie, changes in cytosolic Ca 2, T-90 C, and LCR period (Online Figure II) are fully consistent with the data in Figure 5, which illustrates changes in the SR Ca 2 refilling time, T-90 SR, and LCD period. Inhibition of PKA-dependent phosphorylation by PKI decreases PLB phosphorylation (Figure 8) and markedly increases the SR refilling time, indexed by T-90 C (Figure 7). The decrease in PLB phosphorylation, produced by PKI, inhibits SERCA function and SR Ca 2 uptake, prolonging SR Ca 2 refilling time and increasing both the LCR period and spontaneous cycle length.

8 774 Circulation Research September 17, 2010 In the basal state spontaneous SANC firing is highly regulated by constitutive adenylyl cyclase activity 25,26 and constitutive PDE activity. 11 The concurrent activation of both camp production and degradation mechanisms permits rapid responses to signals that change Ca 2. When PDE activity is inhibited either by broad-spectrum PDE inhibitor, IBMX, or by PDE3 inhibitor, milrinone, a substantial decrease in the SR refilling time, indexed by T-90 C, accompanied by a decrease in the LCR period is observed (Figure 6 and Online Figure III, respectively). However, PKA-dependent phosphorylation affects several major targets involved in the regulation of Ca 2 cycling and spontaneous SANC beating rate: L-type Ca 2 current, 11,16,24 PLB, 9,11,16 and RyR. 9,27,28 Because these proteins operate in concert and are all critically involved in the regulation of Ca 2 cycling in SANCs, it is a challenging task to estimate individual contributions of each of these players. PKA-dependent phosphorylation of RyR might modulate RyR Ca 2 release characteristics and, similar to ventricular myocytes, 27,28 contribute to SR Ca 2 replenishment and Ca 2 cycling in SANCs. An increase in camp-mediated PKA-dependent phosphorylation produced by -AR stimulation or PDE inhibition markedly increases L-type Ca 2 current (I Ca,L ) amplitude in SANCs by 75% and 45% respectively. 11,24 An increase in Ca 2 influx through I Ca,L increases the amount of Ca 2 available for pumping into SR, contributing to increase of SR Ca 2 load. 11,16,24 As a result, LCR amplitude and spatial width are markedly increased and LCR-activated inward current via Na -Ca 2 exchanger is substantially augmented, 11,24 leading to an increase in the spontaneous SANC beating rate. The present study demonstrates a key role of SR Ca 2 replenishment, controlled by SERCA function and its regulation by PLB phosphorylation, to modulate the SERCA pumping rate and SR Ca 2 load to control the LCR period. To delineate the relative contributions of the SR Ca 2 pump and ionic channels to the positive chronotropic effect of PDE inhibition, experimentally measured amplifications of ionic currents produced by PDE inhibition (ie, I Ca,L increased by 45% 11 and I K increased by 12% 11 ) have been introduced in our new numeric model. 20 The model faithfully simulated the experimentally observed 50% increase in the spontaneous SANC beating rate when Ca 2 -ATPase pumping rate was increased by 4-fold. 20 However, in the absence of changes in the SR Ca 2 -ATPase pumping rate, changes in membrane currents alone produced only a modest 13% increase in the spontaneous SANC beating rate, supporting the interpretation of the present experimental results and confirming a pivotal role of Ca 2 -ATPase pumping rate and SR refilling time in the modulation of the LCR period and spontaneous SANC beating rate. In summary, present study shows, for the first time, that the SR Ca 2 refilling time is a key parameter in regulation of the LCR period and the spontaneous SANC cycle length. Gradations in SR Ca 2 refilling time via PKAdependent phosphorylation, indexed by PLB phosphorylation at Ser16 site or direct SERCA inhibition by CPA, are closely linked to gradations in the LCR period and cycle length across the wide physiological range of the SANC beating rate. Sources of Funding This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging. None. Disclosures References 1. Irisawa H, Brown HF, Giles W. Cardiac pacemaking in the sinoatrial node. Physiol Rev. 1993;73: Bogdanov KY, Vinogradova TM, Lakatta EG. Sinoatrial nodal cell ryanodine receptor and Na Ca2 exchanger: molecular partners in pacemaker regulation. Circ Res. 2001;88: Wu Y, Gao Z, Chen B, Koval OM, Singh MV, Guan X, Hund TJ, Kutschke W, Sarma S, Grumbach IM, Wehrens XH, Mohler PJ, Song LS, Anderson ME. Calmodulin kinase II is required for fight or flight sinoatrial node physiology. Proc Natl Acad Sci U S A. 2009;106: Ju YK, Allen DG. The distribution of calcium in toad cardiac pacemaker cells during spontaneous firing. Pflugers Arch. 2000;441: Lyashkov AE, Juhaszova M, Dobrzynski H, Vinogradova TM, Maltsev VA, Juhasz O, Spurgeon HA, Sollott SJ, Lakatta EG. Calcium cycling protein density and functional importance to automaticity of isolated sinoatrial nodal cells are independent of cell size. Circ Res. 2007;100: Sanders L, Rakovic S, Lowe M, Mattick PA, Terrar DA. Fundamental importance of Na -Ca2 exchange for the pacemaking mechanism in guinea-pig sino-atrial node. J Physiol. 2006;571: Vinogradova TM, Zhou YY, Maltsev V, Lyashkov A, Stern M, Lakatta EG. Rhythmic ryanodine receptor Ca2 releases during diastolic depolarization of sinoatrial pacemaker cells do not require membrane depolarization. Circ Res. 2004;94: Bogdanov KY, Maltsev VA, Vinogradova TM, Lyashkov AE, Spurgeon HA, Stern MD, Lakatta EG. Membrane potential fluctuations resulting from submembrane Ca2 releases in rabbit sinoatrial nodal cells impart an exponential phase to the late diastolic depolarization that controls their chronotropic state. Circ Res. 2006;99: Vinogradova TM, Lyashkov AE, Zhu W, Ruknudin AM, Sirenko S, Yang D, Deo S, Barlow M, Johnson S, Caffrey JL, Zhou YY, Xiao RP, Cheng H, Stern MD, Maltsev VA, Lakatta EG. High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2 store oscillations and spontaneous beating of cardiac pacemaker cells. Circ Res. 2006;98: MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. 2003;4: Vinogradova TM, Sirenko S, Lyashkov AE, Younes A, Li Y, Zhu W, Yang D, Ruknudin A, Spurgeon HA, Lakatta EG. Constitutive phosphodiesterase activity restricts spontaneous beating rate of cardiac pacemaker cells by suppressing local Ca2 releases. Circ Res. 2008;102: Brochet DX, Yang D, Di Maio A, Lederer WJ, Franzini-Armstrong C, Cheng H. Ca2 blinks: rapid nanoscopic store calcium signaling. Proc Natl Acad Sci U S A. 2005;102: Perreault CL, Mulieri LA, Alpert NR, Ransil BJ, Allen PD, Morgan JP. Cellular basis of negative inotropic effect of 2,3-butanedione monoxime in human myocardium. Am J Physiol. 1992;263:H503 H Goeger D, Rieley R, Dorner J, Cole R. Cyclopiazonic acid inhibition of the Ca2-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles. Biochem Pharmac. 1988;37: Bassani JW, Bassani RA, Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol. 1994;476: Bers DM. Competition among Na/Ca exchanger, sarcolemmal Ca-pump and SR Ca-pump during relaxation and at rest. In: Bers DM. Excitation- Contraction Coupling and Cardiac Contractile Force. Boston, Mass: Kluwer; 2001: Nelson EJ, Li CC, Bangalore R, Benson T, Kass RS, Hinkle PM. Inhibition of L-type calcium-channel activity by thapsigargin and 2,5- t-butylhydroquinone, but not by cyclopiazonic acid. Biochem J. 1994; 302: Lakatta EG, Maltsev V, Vinogradova TM. A coupled SYSTEM of intracellular Ca2 clocks and surface membrane voltage clocks controls the

9 Vinogradova et al SR Ca Pumping Controls Cardiac Pacemaker Function 775 timekeeping mechanism of the heart s pacemaker. Circ Res. 2010;106: Vinogradova TM, Lakatta EG. Regulation of basal and reserve cardiac pacemaker function by interactions of camp-mediated PKA-dependent Ca2 cycling with surface membrane channels. J Mol Cell Cardiol. 2009;47: Maltsev VA, Lakatta EG. Synergism of coupled subsarcolemmal Ca2 clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model. Am J Physiol. 2009;296:H594 H Colyer J, Wang JH. Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban. J Biol Chem. 1991;266: Ju YK, Allen DG. How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2 release in amphibian pacemaker cells. J Physiol. 1999;516: Rigg L, Heath BM, Cui Y, Terrar DA. Localization and functional significance of ryanodine receptors during beta-adrenoceptor stimulation in the guinea-pig sino-atrial node. Cardiovasc Res. 2000;48: Vinogradova TM, Bogdanov KY, Lakatta EG. -Adrenergic stimulation modulates ryanodine receptor Ca2 release during diastolic depolarization to accelerate pacemaker activity in rabbit sinoatrial nodal cells. Circ Res. 2002;90: Mattick P, Parrington J, Odia E, Simpson A, Collins T, Terrar D. Ca2 stimulated adenylyl cyclase isoform AC1 is preferentially expressed in guinea-pig sino-atrial node cells and modulates the If pacemaker current. J Physiol. 2007;582: Younes A, Lyashkov AE, Graham D, Sheydina A, Volkova MV, Mitsak M, Vinogradova TM, Lukyanenko YO, Li Y, Ruknudin AM, Boheler KR, van Eyk J, Lakatta EG. Ca(2 ) -stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells. J Biol Chem. 2008;283: Blayney LM, Lai FA. A mechanism of ryanodine receptor modulation by FKBP12/12.6, protein kinase A, and K201. Cardiovasc Res. 2010;85: Ginsburg KS, Bers DM Modulation of excitation contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca2 load and Ca2 current trigger. J Physiol. 2004;556: Novelty and Significance What Is Known? For more than 60 years, a prevailing view has been that the physiological timekeeping mechanism of the heart s pacemaker resides primarily in an ensemble of surface membrane ion transport proteins. More recently, the view that the sarcoplasmic reticulum (SR) of sinoatrial nodal cells (SANCs) functions as a Ca 2 clock by generating rhythmic local Ca 2 releases (LCRs) beneath the cell membrane has emerged as an important player in pacemaker timing. Thus, the timekeeping mechanism of the heart s pacemaker cells is regulated by a robust, coupled-clock system involving surface membrane and intracellular oscillators. What New Information Does This Article Contribute? By directly measuring local SR Ca 2 depletion and refilling time, the rate at which the SR refills with Ca 2 following the prior AP-triggered Ca 2 release is shown to regulate the LCR period. The Ca 2 refilling time of the SR can be inferred from the decay kinetics of the cytosolic Ca 2 transient to predict changes in LCR period and spontaneous AP cycle length in response to -adrenergic receptor stimulation ( -ARs). The present study presents the first direct evidence that the LCR period (ie, the restitution time for LCR occurrence following a prior AP, or the Ca 2 clock s ticking speed ) is determined by the speed at which SR refills with Ca 2. Changes in the LCR period (eg, in response to G protein coupled receptor stimulation) induce changes in the timing of LCR-activated inward Na -Ca 2 current, leading to changes in the spontaneous depolarization of SANCs, resulting in variations of the spontaneous AP cycle length. The findings of the present study should motivate further studies of how the integrated response of Ca 2 -handling proteins, including L-type Ca 2 channels, ryanodine receptors, and SR Ca 2 pumps, contribute to the SR Ca 2 refilling time and, consequently, the rate of pacemaker firing. Overall, the present work elucidates the molecular interactions that determine the complex coupled-clock systems that robustly regulate basal SANC AP firing rate and yet are sufficiently flexible to respond to neurotransmitter input to shift the clock s ticking speed. These findings could help in the development of a rational design of biological pacemakers that can be translated into therapies for patients with heart rate and rhythm disorders.