Effects of increased systolic Ca²⁺ and β-adrenergic stimulation on Ca²⁺ transient decline in NOS1 knockout cardiac myocytes

Abstract

We have previously shown that the main factor responsible for the faster [Ca²⁺]_i decline rate with β-adrenergic (β-AR) stimulation is the phosphorylation of phospholamban (PLB) rather than the increase in systolic Ca²⁺ levels. The purpose of this study was to correlate the extent of augmentation of PLB Serine¹⁶ phosphorylation to the rate of [Ca²⁺]_i decline. Thus, ventricular myocytes were isolated from neuronal nitric oxide synthase knockout (NOS1^−/−) mice, which we observed had lower basal PLB Serine¹⁶ phosphorylation levels, but equal levels during β-AR stimulation. Ca²⁺ transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca²⁺ ([Ca²⁺]_o) and a non-specific β-AR agonist isoproterenol (ISO, 1μM) with 1mM [Ca²⁺]_o. This allowed us to get matched Ca²⁺ transient amplitudes in the same myocyte. Similar to our previous work, Ca²⁺ transient decline was significantly faster with ISO compared to 3mM [Ca²⁺]_o, even with matched Ca²⁺ transient amplitudes. Interestingly, when we compared the effects of ISO on Ca²⁺ transient decline between NOS1^−/− and WT myocytes, ISO had a larger effect in NOS1^−/− myocytes, which resulted in a greater percent decrease in the Ca²⁺ transient RT₅₀. We believe this is due to a greater augmentation of PLB Serine¹⁶ phosphorylation in these myocytes. Thus, our results suggest that not only the amount but the extent of augmentation of PLB Serine¹⁶ phosphorylation are the major determinants for the Ca²⁺ decline rate. Furthermore, our data suggest that the molecular mechanisms of Ca²⁺ transient decline is normal in NOS1^−/− myocytes and that the slow basal Ca²⁺ transient decline is predominantly due to decreased PLB phosphorylation.

INTRODUCTION

An enhanced lusitropic response is a hallmark of β-adrenergic (β-AR) stimulation ¹. This is, in part, due to the faster [Ca²⁺]_i uptake into the sarcoplasmic reticulum (SR) by the SR Ca²⁺ ATPase (SERCA). SERCA’s function is inhibited by its regulatory protein, phospholamban (PLB) ². The PLB-mediated inhibition of SERCA is relieved by its dissociation, which is caused by an increase in systolic Ca²⁺ levels and/or phosphorylation of PLB ³. During β-AR stimulation, not only is PLB phosphorylated at Serine¹⁶ by the cAMP-dependent protein kinase (PKA) but there are higher systolic Ca²⁺ levels as well. In our previous study ⁴, we set out to determine the major factor in the faster [Ca²⁺]_i decline during β-AR stimulation (i.e., the high systolic levels of [Ca²⁺]_i or PLB phosphorylation). We demonstrated that the faster [Ca²⁺]_i decline rate with β-AR stimulation was due to the phosphorylation of PLB, specifically at Serine¹⁶, instead of the increase in systolic Ca²⁺ levels.

Cardiac relaxation is altered in many cardiomyopathies such as heart failure ^5–7. The slowed relaxation can be attributed to changes in myofilament kinetics and/or slowed [Ca²⁺]_i decline. Fully understanding each aspect of muscle relaxation will be critical for successful treatment of these diseases. Thus, the purpose of this study was to correlate the extent of augmentation of PLB Serine¹⁶ phosphorylation to the rate of [Ca²⁺]_i decline. Previous studies have shown that neuronal nitric oxide synthase (NOS1) signaling is able to modulate basal PLB phosphorylation ^{8, 9}. Namely, basal PLB phosphorylation at Serine¹⁶ is decreased in NOS1 knockout (NOS1^−/−) compared to wildtype (WT) myocytes ^{8, 9.} However, during β-AR stimulation, PLB Serine¹⁶ phosphorylation levels are similar in NOS1^−/− and WT myocytes ^{8, 9}. Since NOS1^−/− myocytes have lower basal PLB Serine¹⁶ phosphorylation but equal phosphorylation levels with β-AR stimulation, the extent of the increase of PLB Serine¹⁶ phosphorylation is greater in NOS1^−/− compared to WT myocytes. We hypothesize that not only the amount but the extent of augmentation of PLB Serine¹⁶ phosphorylation are the key determinants of the Ca²⁺ transient decline rate. We expect a greater effect of ISO that should result in a more prominent effect on Ca²⁺ transient decline in NOS1^−/− vs WT myocytes.

METHODS AND MATERIALS

Cardiomyocyte isolation

Ventricular myocytes were isolated from NOS1^−/− and C57BL/6 (WT) (Jackson Laboratories, Bar Harbor, Maine) as previously described ⁸. Briefly, the heart was cannulated and hung on a Langendorff apparatus. It was then perfused with Ca²⁺ free tyrode solution for 4 min. The solution was then switched to tyrode solution containing Liberase Blendzyme II (0.077 mg/ml) (Roche Applied Science, Indianapolis, IN). After 3–5 min, the heart was taken down, the ventricles minced, and myocytes were dissociated by trituration. Subsequently the myocytes were filtered through mesh, centrifuged, and resuspended in Tyrode solution containing 200 μmol/L Ca²⁺. Myocytes were used within 6 hours of isolation. All the animal protocols and procedures were performed in accordance with National Institutes of Health guidelines and approved by the Institutional Laboratory Animal Care and Use Committee at The Ohio State University.

Measurement of Myocyte Ca²⁺ transients

Ca²⁺ transient measurements were performed as previously described ¹⁰. Briefly, myocytes were loaded at room temperature with Fluo-4 AM (10 μmol/L, Molecular Probes, Eugene, OR) for 30 min, and then another 30 min were allowed for intracellular de-esterification. The solution for de-esterification was Tyrode solution containing 200 μmol/L Ca²⁺. The instrumentation used for cell fluorescence measurements was a Cairn Research Limited (Faversham, UK) epifluorescence system. [Ca²⁺]_i was measured by Fluo-4 epifluorescence with excitation at 480±20 nm and emission at 535±25 nm. The illumination field was restricted to collect the emission of a single cell. Data were expressed as ΔF/F₀, where F is the fluorescence intensity and F₀ is the intensity at rest. Myocytes were stimulated at 1 Hz via platinum electrodes connected to a Grass Telefactor S48 stimulator (West Warwick, RI).

Western blot for PLB Serine¹⁶ phosphorylation

Whole hearts were perfused with the various experimental solutions (3mM [Ca²⁺]_o or ISO) for 3 min using a Langendorff apparatus, homogenized, and analyzed via western blot as previously described ¹¹. Membranes were probed using a custom antibody to PLB (Zymed, San Francisco, CA), Serine16 phosphospecific antibody (Badrilla, Leeds, UK) and normalized to calsequestrin (ABR, Golden, CO). Data expressed as Serine¹⁶ phosphorylation normalized to total PLB.

Solutions and drugs

Normal Tyrode (NT) solution consisted of (in mmol/L): 140 NaCl, 4 KCl, 1 MgCl₂, 1 CaCl₂, 10 glucose, 5 HEPES, pH 7.4 adjusted with NaOH or HCl. Isoproterenol (ISO, 1 μmol/L, a non-selective β-AR agonist) was prepared fresh each day. All chemicals were from Sigma (St. Louis, MO).

Experimental protocol

Our experimental protocol consisted of the myocyte first being superfused with control solution (NT with 1 mM [Ca²⁺]) until steady-state was reached, the myocyte was then superfused with 3 mM [Ca²⁺] NT solution, which resulted in increased systolic Ca²⁺ levels. After reaching steady-state, the solution was then switched back to control solution (1mM [Ca²⁺] NT) resulting in the washout of 3 mM [Ca²⁺]_o and the Ca²⁺ transient amplitude returning back to basal levels (~ 3 minutes). The myocyte was then superfused with 1μM ISO (with 1mM [Ca²⁺]_o) which increased myocyte systolic Ca²⁺ levels. Experiments were performed at room temperature.

Statistics

Data were presented as mean±SEM. Differences between groups were evaluated for statistical significance (P < 0.05) by ANOVA for multiple groups or paired/unpaired Student’s t tests for two groups.

RESULTS

Ca²⁺ transient kinetics experimental protocol

Shown in Figure 1A is a representative experiment showing Ca²⁺ transients over time recorded in a NOS1^−/− myocyte. The maximal Ca²⁺ transient amplitude for each treatment for NOS1^−/− myocytes and adapted data for WT myocytes ⁴ is shown in Figure 1B. 3 mM [Ca²⁺]_o and ISO caused a significant increase in maximal Ca²⁺ transient amplitude compared to 1 mM [Ca²⁺]_o in WT and NOS1^−/− myocytes. However, NOS1^−/− myocytes had significantly lower maximal Ca²⁺ transient amplitudes at 1 mM [Ca²⁺]_o, 3 mM [Ca²⁺]_o, and ISO compared to WT myocytes. Shown in Figure 1C is the Ca²⁺ transient decline rate measured as time to 50% relaxation (RT₅₀) with 1 mM [Ca²⁺]_o, 3 mM [Ca²⁺]_o, and ISO. 3 mM [Ca²⁺]_o and ISO resulted in a faster RT₅₀ compared to 1 mM [Ca²⁺]_o in WT and NOS1^−/− myocytes. However, NOS1^−/− myocytes had significantly slower RT₅₀ at 1 mM and 3 mM [Ca²⁺]_o. Interestingly, RT₅₀ with ISO is similar between WT and NOS1^−/− myocytes. Thus, Ca²⁺ transient decline was slower in NOS1^−/− myocytes, but normalized with β-AR stimulation.

Figure 1.

Experimental Protocol. A) Representative time plot of the experimental protocol. B) Summary data (mean±sem) of maximum Ca²⁺ transient amplitude with 1 mM [Ca²⁺]_o, 3mM [Ca²⁺]_o, or ISO from WT (clear bar) and NOS1^−/− (black bar) myocytes. C) Summary data (mean±sem) of Ca²⁺ transient decline measured as the RT₅₀. a P<0.05 vs corresponding WT, b P<0.05 vs 1 mM [Ca²⁺]_o, c P<0.05 vs 1mM and 3mM [Ca²⁺]_o.

Since we observed differences in Ca²⁺ transient decline in NOS1^−/− myocytes with 3mM [Ca²⁺]_o and ISO treatments, we further analyzed Ca²⁺ transient decline in NOS1^−/− myocytes. We first examined PLB phosphorylation. We measured PLB Serine16 phosphorylation under similar experimental conditions (i.e., perfusion with 3 mM [Ca²⁺]_o or ISO for 3 min, which is a similar time point in which we matched the Ca²⁺ transient amplitudes). Shown in Figure 2A, WT and NOS1^−/− myocytes perfused with ISO had significantly increased Serine16 phosphorylation compared with myocytes perfused with 3 mM [Ca²⁺]_o. However, NOS1^−/− myocytes perfused with 3 mM [Ca²⁺]_o, had decreased Serine16 phosphorylation levels but similar phosphorylation levels with ISO when compared to WT myocytes. Further analysis reveals that ISO produces a much larger increase in PLB Serine¹⁶ phosphorylation in NOS1^−/− myocytes compared to WT myocytes (Figure 2B). Thus, the extent of the increase of PLB Serine¹⁶ phosphorylation with ISO was greater in NOS1^−/− vs WT myocytes.

Figure 2.

Effects of ISO and 3mM [Ca²⁺]_o on WT and NOS1^−/− PLB Serine¹⁶ phosphorylation and Ca²⁺ transient decline in NOS1^−/− myocytes. A) Summary data (mean±sem) of PLB Serine¹⁶ phosphorylation with ISO or 3mM [Ca²⁺]_o (A.U.- arbitrary units). B) Summary data (mean±sem) of the ISO mediated increase in PLB Serine¹⁶ phosphorylation expressed as the % of 3mM [Ca²⁺]_o. Summary data (mean±sem) of Ca²⁺ transient amplitude (left) and decline (right) in NOS1^−/− myocytes which had a higher maximal Ca²⁺ transient amplitude to ISO (C) or 3 mM [Ca²⁺]_o (D). *P<0.05) vs corresponding WT or 3 mM [Ca²⁺]_o. E) Individual values of peak [Ca²⁺]_i versus RT₅₀ with Ca²⁺ (black, r²=0.70) and ISO (gray, r²=0.007).

Although the maximum Ca²⁺ transient amplitudes with 3mM [Ca²⁺]_o and ISO were not statistically different, the faster Ca²⁺ transient decline rates with ISO may have resulted from the slightly higher peak systolic Ca²⁺ levels. Therefore, we further investigated the Ca²⁺ transient decline by grouping the NOS1^−/− myocytes that had a higher maximal response to ISO together (n=18) and the myocytes that had a higher maximal response to 3mM [Ca²⁺]_o together (n=5). When grouping the data, the myocytes that had a higher maximum peak systolic Ca²⁺ with ISO also had a faster RT₅₀ (Figure 2C). In this case, it is unknown if the faster Ca²⁺ transient decline with ISO is due to higher systolic Ca²⁺ levels or PLB Serine¹⁶ phosphorylation. However, when comparing the group that had a higher maximum peak systolic Ca²⁺ with 3mM [Ca²⁺]_o (Figure 2D), ISO still resulted in a faster Ca²⁺ transient decline RT₅₀. We observed the same phenomenon in WT myocytes ⁴. We further analyzed this relationship between maximum peak systolic Ca²⁺ levels and RT₅₀. We plotted the maximum peak systolic Ca²⁺ levels with 1mM [Ca²⁺]_o, 3mM [Ca²⁺]_o, and ISO against their respective RT₅₀ (Figure 2E). The higher peak systolic Ca²⁺ in the 1mM and 3mM [Ca²⁺]_o group correlated with a faster RT₅₀ (slope of −41.3±4.3). However, in the ISO group, there was no correlation between peak systolic Ca²⁺ and RT₅₀ (slope of −1.5±4.3). We observed the same phenomenon in WT myocytes ⁴. These data suggest that with ISO at 1 mM [Ca²⁺]_o, unlike in the absence of ISO, the rate of Ca²⁺ transient decline is not dependent on peak Ca²⁺ transient amplitude. These data suggest that during β-AR stimulation PLB Serine¹⁶ phosphorylation is the major factor responsible for the faster Ca²⁺ transient decline rate. Furthermore, these data suggest that the molecular mechanisms for Ca²⁺ transient decline are normal in NOS1^−/− myocytes.

Ca²⁺ Transient kinetics with matched amplitudes

For accurate comparisons of Ca²⁺ transient decline rates, it has been shown that one must match the Ca²⁺ transient amplitudes ¹². By using our experimental protocol, we were able to match Ca²⁺ transient amplitude levels between the 3mM [Ca²⁺]_o and ISO groups and reduce the variability of Ca²⁺ handling between cells exposed to 3mM [Ca²⁺]_o and ISO by superfusing each myocyte with both solutions. Representative matched individual Ca²⁺ transient traces are shown in Figure 3A and the matched peak values of 3mM [Ca²⁺]_o and ISO are shown in Figure 3B.

Figure 3.

Effects of 3 mM [Ca²⁺]_i and ISO on Ca²⁺ transient decline with matched Ca²⁺ transient amplitudes in NOS1^−/− myocytes. A) Representative trace of matched [Ca²⁺]_i amplitudes with 3 mM [Ca²⁺]_o (black) or 10⁻⁶ M ISO (gray). B) Summary data (mean±sem) of matched Ca²⁺ transient amplitude with 3mM [Ca²⁺]_o (black) or ISO (gray). C) Summary data (mean±sem) of Ca²⁺ transient decline with 3mM [Ca²⁺]_o (black) or ISO (gray). (D) Summary data (mean±sem) of Ca²⁺ transient decline time intervals with 3mM [Ca²⁺]_o or ISO. * P<0.05 vs corresponding 3 mM [Ca²⁺]_o.

Using the matched Ca²⁺ transient amplitude data, we examined the effects of 3mM [Ca²⁺]_o and ISO on Ca²⁺ transient decline by analyzing the time it takes the Ca²⁺ transient to decline by 25% (RT₂₅), 50% (RT₅₀), 75% (RT₇₅) and 90% (RT₉₀) from its peak amplitude. The Ca²⁺ decline with ISO at each time point was significantly faster compared to 3mM [Ca²⁺]_o (Figure 3C). Our previous study ⁴ showed that the faster effect of ISO occurred only in the first 50% of the decline in WT myocytes. This was determined by dividing the declining Ca²⁺ transient into intervals: RT_50-25, RT_75-50, and RT_90-75. For example, we subtracted the RT₂₅ from the RT₅₀ to get the RT_50-25 interval, reflecting the time of the Ca²⁺ transient to decline from 25% from the peak amplitude to the 50% point. Data are shown in Figure 3D. The RT_50-25 and RT_75-50 intervals were significantly different between 3mM [Ca²⁺]_o and ISO. Thus, these data suggest that faster Ca²⁺ transient decline with ISO compared to 3mM [Ca²⁺]_o occurs in the first 75% in NOS1^−/− myocytes.

Different effect of ISO on Ca²⁺ transient decline in NOS1^−/− vs WT myocytes

The interval data observed in the NOS1^−/− myocytes are somewhat different than what was observed in WT myocytes (i.e., first 75% vs 50% of the decline) ⁴. Hence, we wanted to further investigate if there were any more differences in the effects of ISO on Ca²⁺ transient decline between WT and NOS1^−/− myocytes. This was done by examining the effects of ISO on the decline rate (i.e., RT₂₅, RT₅₀, RT₇₅, and RT₉₀) of matched Ca²⁺ transients as a percent change of the decline with 3mM [Ca²⁺]_o. For example, at the RT₂₅, ISO in WT myocytes resulted in 18±1% faster decline compared to 3mM [Ca²⁺]_o, but in NOS1^{−/ −} myocytes, ISO had a 26±3% faster decline (P<0.05 vs WT). ISO also had a faster decline at the RT₅₀ in NOS1^−/− myocytes, but not at the RT₇₅ or the RT₉₀ (Figure 4A). We performed the same analysis investigating the effects of ISO in WT and NOS1^−/− myocytes on our relaxation time intervals. Shown in Figure 4B, ISO had a significantly greater effect at the RT_50-25 interval in NOS1^−/− vs WT myocytes. This pronounced effect of ISO on Ca²⁺ transient decline also resulted in a greater effect in reducing the Ca²⁺ transient RT₅₀ when inspecting the maximal responses to ISO (Figure 4C). That is, ISO resulted in a 48±2% faster Ca²⁺ transient RT₅₀ (compared to the RT₅₀ with 1mM [Ca²⁺]_o) in NOS1^{−/ −} myocytes, but only a 39±2% in WT myocytes (P<0.05 vs NOS1^−/−). There was no difference in the effects of 3mM [Ca²⁺]_o on Ca²⁺ decline between NOS1^−/− and WT myocytes. Thus, these data suggest that ISO has a more prominent effect to augment Ca²⁺ transient decline in NOS1^−/− myocytes.

Figure 4.

Effect of ISO on Ca²⁺ transient decline in NOS1^−/− vs WT myocytes. A) Summary data (mean±sem) of the effects of ISO on Ca²⁺ transient decline normalized to 3mM [Ca²⁺]_o in WT (clear bars) and NOS1^−/− (black bars) myocytes. B) Summary data (mean±sem) of the effects of ISO on Ca²⁺ transient decline time intervals normalized to 3mM [Ca²⁺]_o in WT (clear bars) and NOS1^−/− (black bars) myocytes. C) Summary data (mean±sem) of the effects of maximum 3mM [Ca²⁺]_o (left) and ISO (right) on Ca²⁺ transient RT₅₀ normalized to 1mM [Ca²⁺]_o in WT (clear bars) and NOS1^−/− (black bars) myocytes. * P<0.05 vs corresponding WT.

DISCUSSION

The lowering of [Ca²⁺]_i is the initiating event that permits relaxation. The majority of the decline of [Ca²⁺]_i is due to Ca²⁺ resequestation into the SR via the SERCA/PLB complex and extrusion from the cell by the Na⁺/Ca²⁺ exchanger (NCX). In murine myocytes, the bulk (>95%) of the [Ca²⁺]_i is resequestered back into the SR via SERCA ¹³, which is reversibly inhibited by PLB ³. Ca²⁺ binding to SERCA results in the dissociation of PLB from SERCA to relieve this inhibition. PLB is also a key phosphoprotein in the heart, which can be phosphorylated on Serine¹⁶ through PKA or on Threonine¹⁷ through Ca²⁺/calmodulin-dependent protein kinase. Phosphorylation at either site also results in the dissociation of PLB from SERCA. Thus, increasing [Ca²⁺]_i or PLB phosphorylation results in greater Ca²⁺ resequestation into the SR and faster Ca²⁺ decline.

Ca²⁺ decline during β-AR stimulation

Stimulation of the β-AR receptor results in increased myocyte contraction and faster relaxation ¹. In terms of Ca²⁺ handling, this will lead to an increase in peak systolic Ca²⁺ levels and faster decline. The higher systolic Ca²⁺ levels during β-AR stimulation should result in PLB dissociation and greater SR Ca²⁺ uptake. Furthermore, β-AR stimulation results in the phosphorylation of PLB at Serine^16,14, which will also dissociate PLB from SERCA. We have previously shown that in WT myocytes the major factor for the faster Ca²⁺ decline during β-AR stimulation is PLB Serine¹⁶ phosphorylation.

In addition to the greater Ca²⁺ uptake into the SR with β-AR stimulation, greater Ca²⁺ extrusion from the cell could also enhance the Ca²⁺ transient decline rate ¹⁵. Thus, increased NCX, which removes Ca²⁺ from the cytosol throughout the action potential (besides phase 0), could result in faster Ca²⁺ transient decline. However, we believe that NCX does not play a role in the effects of β-AR stimulation on Ca²⁺ transient decline. Studies have found that β-AR stimulation does not affect NCX function ¹⁶. In addition, NCX plays a minor role in the Ca²⁺ decline rates in mouse myocytes (<5%), so an effect, if any, would be very minor at best. Further, using the NCX knockout mouse, there was no difference in Ca²⁺ transient decline rates with β-AR stimulation in WT compared to knockout myocytes ¹⁷.

Besides greater Ca²⁺ uptake or extrusion, β-AR stimulation also decreases myofilament Ca²⁺ sensitivity via TnI phosphorylation. It is widely accepted that TnI phosphorylation during β-AR stimulation accelerates relaxation ^{18, 19.} A previous study ²⁰ has shown that in unloaded myocyte experiments, there is little effect of TnI phosphorylation on myocyte relengthening. However, under loaded conditions (e.g. trabeculae), TnI phosphorylation does play a significant role in relaxation. Thus, under our experimental conditions, (i.e. unloaded myocyte) we believe that TnI does not play a role. Hence, with our previous work ⁴ showing that the major mechanism for the faster Ca²⁺ transient decline is PLB Serine¹⁶ phosphorylation, we wanted to extend this observation and correlate the extent of augmentation of PLB Serine¹⁶ phosphorylation to the rate of [Ca²⁺]_i decline.

NOS1 signaling in myocytes

Nitric oxide produced via NOS1 is an important cardiac signaling molecule ²¹. Many studies have shown that NOS1 is a key modulator of cardiac myocyte function. By using cardiac myocyte NOS1 overexpressing mice ²², NOS1^−/− mice ^{8, 23–26}, and specific NOS1 inhibitors ^{8, 26}, studies found that NOS1 signaling results in enhanced inotropy, lusitropy and augments the functional response to β-AR stimulation. In terms of Ca²⁺ handling, NOS1 signaling will accelerate Ca²⁺ transient decline under basal conditions and increase basal and β-AR stimulated systolic Ca²⁺ levels. Hence, NOS1^−/− myocytes have slowed basal Ca²⁺ transient decline and depressed basal systolic Ca²⁺ levels. A molecular component of NOS1 regulation of myocyte contraction occurs via modulation of basal PLB Serine¹⁶ phosphorylation levels. We⁸ and others^{9, 27} have shown that WT myocytes with acute NOS1 inhibition or NOS1^−/− myocytes have reduced basal levels of PLB Serine¹⁶ phosphorylation, while myocyte-specific NOS1 overexpression results in increased basal PLB Serine¹⁶ phosphorylation. Interestingly, studies ^{8, 9} have shown that NOS1^−/− myocytes have similar PLB Serine¹⁶ phosphorylation levels and Ca²⁺ transient decline rates during β-AR stimulation. Since NOS1^−/− myocytes have a lower basal PLB Serine¹⁶ phosphorylation but equal phosphorylation levels with ISO, the extent of the increase of PLB Serine¹⁶ phosphorylation is greater in NOS1^−/− compared to WT myocytes. Our current results examining PLB Serine¹⁶ phosphorylation (Fig 2A and 2B) are consistent with these previous studies, in which we show that ISO resulted in a 692±107% increase in PLB Serine¹⁶ phosphorylation in NOS1^−/− myocytes but only a 306±51% increase in WT myocytes. Thus, NOS1^−/− myocytes represent a straightforward model system to investigate the role of PLB Serine¹⁶ phosphorylation augmentation in modulating the Ca²⁺ transient decline.

Ca²⁺ transient decline in NOS1^−/− and WT myocytes with high extracellular Ca²⁺ vs ISO

We investigated Ca²⁺ transient decline kinetics with ISO and 3mM [Ca²⁺]_o in NOS1^−/− myocytes and compared them to WT data ⁴. As shown in Figure 1C, WT had significantly faster Ca²⁺ transient RT₅₀ during 1mM [Ca²⁺]_o and 3mM [Ca²⁺]_o compared to NOS1^−/− myocytes. The slowed Ca²⁺ decline during 1mM [Ca²⁺]_o and 3mM [Ca²⁺]_o is consistent with lower basal PLB Serine¹⁶ phosphorylation levels and agrees with previous work ^{8, 9}. Moreover, the Ca²⁺ transient RT₅₀ with ISO is similar between WT and NOS1^−/− myocytes, which suggest that β-AR stimulation normalizes the Ca²⁺ transient RT₅₀ in NOS1^−/− myocytes via similar PLB Serine¹⁶ phosphorylation levels between WT and NOS1^−/− myocytes and agrees with previous work ⁴. Nevertheless, the faster RT50 with ISO (compared to 3 mM [Ca²⁺]_o) in the NOS1^−/− myocytes may be due to the higher (although not significant) systolic Ca²⁺ levels. However, if we group the NOS1^−/− myocytes that had a larger Ca²⁺ transient amplitude with 3mM [Ca²⁺]_o (Figure 2D), the Ca²⁺ transient decline with ISO was still faster than 3mM [Ca²⁺]_o. Thus, this highlights the importance of PLB Serine¹⁶ phosphorylation in modulating the rate of Ca²⁺ decline.

In addition to PLB Serine¹⁶ phosphorylation, an increase in diastolic SR Ca²⁺ leak via increased ryanodine receptor (RyR2) activity could contribute to slowed basal Ca²⁺ transient decline. Indeed, Gonzalez et al ²⁸ previously demonstrated that NOS1^−/− myocytes have increased basal diastolic SR Ca²⁺ leak via increased RyR2 activity and slowed basal [Ca²⁺]_i decline. Thus, there is a balance shift towards increased SR Ca²⁺ release that slows the Ca²⁺ decline. However, we observed decreased RyR2 activity (and diastolic SR Ca²⁺ leak) ²⁹ and decreased basal PLB Serine¹⁶ phosphorylation in NOS1^−/− myocytes (8 and Figure 2A). Thus, our data suggests, that the balance is shifted towards a decrease in SR Ca²⁺ uptake that is responsible for the slowed basal [Ca²⁺]_i decline. During β-AR stimulation, RyR2 activity is increased ^{30, 31}. However, there is no difference in RT₅₀ between WT and NOS1^−/− myocytes (in which PLB Serine¹⁶ phosphorylation is the same). Furthermore, our previous work ⁴ showed that increased RyR activity with β-AR stimulation contributed to a faster rate of [Ca²⁺]_i rise, but had no effect on [Ca²⁺]_i decline. Therefore, during β-AR stimulation, the balance is shifted to greater SR Ca²⁺ uptake via PLB Serine¹⁶ phosphorylation that accelerates Ca²⁺ decline. Thus, if RyR2 activity was the major reason for the slowed Ca²⁺ transient decline, then we should still have observed slowed Ca²⁺ transient decline with ISO in the NOS1^−/− myocytes vs WT.

We also examined the relationship between maximum systolic Ca²⁺ levels and the Ca²⁺ transient RT₅₀ (Figure 2E). These data show that in the absence of ISO as systolic Ca²⁺ levels increased there was a direct relationship for a faster Ca²⁺ transient decline. However, this is not the case during β-AR stimulation. That is, the ISO group (at 1 mM [Ca²⁺]_o) was not dependent upon systolic Ca²⁺ levels. We did not measure this relationship with ISO at 3 mM [Ca²⁺]_o and cannot establish that the same phenomenon will occur. The ISO at 1 mM [Ca²⁺]_o data are similar to what we observed in WT myocytes ⁴. Thus, we suggest that the molecular mechanisms for the Ca²⁺ transient decline are not different between NOS1^−/− and WT myocytes but just slower under basal conditions due to decreased PLB Serine¹⁶ phosphorylation.

As suggested in previous studies ^{4, 12}, to properly analyze Ca²⁺ transient decline between groups, one should use matched Ca²⁺ transient amplitudes. Thus, we investigated the RT₂₅, RT₅₀, RT₇₅, RT₉₀ and the time intervals (RT_50-25 and RT_75-50) of the Ca²⁺ transient decline with matched Ca²⁺ transient amplitudes (Figure 3). ISO resulted in a faster decline at all time points and a faster decline at the RT_50-25 and RT_75-50 intervals. This suggests that ISO has its greatest effect in increasing Ca²⁺ decline during the initial 75%. The effects of PLB Serine¹⁶ phosphorylation are observed in the initial 75% of the decline in NOS1^−/− myocytes because, we believe that the increase in systolic Ca²⁺ with 3 mM [Ca²⁺]_o (or ISO) will not dissociate all the PLB from SERCA. However, phosphorylation of PLB Serine16 results in the dissociation of more PLB from SERCA (compared to 3 mM [Ca²⁺]_o) resulting in a faster decline during the initial 50% in WT and 75% in NOS1^−/− Ca²⁺ transient decline.

Ca²⁺ transient decline in NOS1^−/− vs WT myocytes

Our data suggest that ISO has a more pronounced effect on Ca²⁺ transient decline in NOS1^−/− myocytes compared to WT myocytes. This effect of ISO also resulted in a greater decline in RT₅₀ in NOS1^−/− vs WT myocytes (Figure 4C). We believe this greater effect of ISO in NOS1^−/− myocytes is due to the lower basal PLB Serine¹⁶ phosphorylation. Thus, there is a greater quantitative increase in PLB Serine¹⁶ phosphorylation with ISO that results in a greater effect on Ca²⁺ transient decline. Furthermore, although not significant, there was a trend that 3mM [Ca²⁺]_o had a greater effect on RT₅₀ in NOS1^−/− myocytes (Figure 4C). NOS1^−/− myocytes also had a steeper slope in the peak systolic Ca²⁺ levels with 1mM and 3mM [Ca²⁺]_o and their respective RT₅₀ (Figure 2) compared to WT myocytes ⁴. We believe that this may be due to the increased SERCA/PLB ratio in the NOS1^−/− myocytes ^{24, 32}. These data highlight the major role of PLB phosphorylation in modulating Ca²⁺ transient decline. Such that there is a compensatory change in SERCA/PLB ratio in NOS1^−/− myocytes, but basal Ca²⁺ transient decline is still slower (compared to WT myocytes), which we believe is due to decreased basal PLB Serine¹⁶ phosphorylation.

In conclusion, our data suggest that the molecular mechanisms for the decline of [Ca²⁺]_i is similar between NOS1^−/− and WT myocytes and emphasizes that not only the amount but the extent of augmentation of PLB Serine¹⁶ phosphorylation are key determinants for the Ca²⁺ transient decline rate.

• Molecular mechanisms of [Ca²⁺]_i decline are similar in NOS1^−/− and WT myocytes

• β-AR stimulation resulted in greater effect on [Ca^2+]_i decline in NOS1^−/− myocytes

• The amount of PLB phosphorylation is major determinant for the [Ca²⁺]_i decline rate

• Extent of augmentation of PLB phosphorylation also is a major determinant