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The Journal of Physiology logoLink to The Journal of Physiology
. 2002 Jul 26;544(Pt 1):57–69. doi: 10.1113/jphysiol.2002.020552

Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes

L F Santana *, E G Chase *, V S Votaw *, Mark T Nelson *, R Greven *
PMCID: PMC2290559  PMID: 12356880

Abstract

We examined the role of the Ca2+-regulated protein phosphatase calcineurin in controlling Ca2+ signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch-clamp technique. Cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in cells loaded with the fluorescent Ca2+ indicators fluo-4 or fura-2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 nm) or the calcineurin auto-inhibitory peptide (CiP, 100 μM), increased the amplitude and rate of decay of the evoked [Ca2+]i transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 nm) and 100 μM CiP on the [Ca2+]i transient and AP were not additive. Calcineurin inhibition did not modify the K+ currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca2+ current (ICa) and the evoked calcium transient normalized to the ICa. Calcium sparks, which underlie the [Ca2+]i transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co-localize near the T-tubules of ventricular myocytes. We propose that calcineurin and PKA are co-localized to control Ca2+ influx through calcium channels and calcium release through ryanodine receptors.

During excitation-contraction (E-C) coupling, brief openings of voltage-gated Ca2+ channels allow a small amount of Ca2+ to enter the cardiac cytoplasm. This Ca2+ influx causes a local increase in cytoplasmic Ca2+ concentration ([Ca2+]i) that activates nearby sarcoplasmic reticulum (SR) Ca2+ release channels (i.e. ryanodine receptors, RyRs) by the mechanism of calcium-induced calcium release (CICR) (Fabiato, 1983; Nabauer et al. 1989; Niggli & Lederer, 1990). The simultaneous activation of a small number of RyRs allows Ca2+ stored in the lumen of the SR to flow into the cardiac cytoplasm causing a local increase in [Ca2+]i. These local Ca2+ release events, termed ‘Ca2+ sparks’, are considered the elementary Ca2+ release events of E-C coupling (Cheng et al. 1993). Ca2+ sparks could occur spontaneously or be activated by the Ca2+ current (ICa) (López-López et al. 1994, 1995; Cannell et al. 1995). During the action potential (AP), activation of ICa synchronizes the activation of multiple Ca2+ sparks, which sum to produce a large, whole-cell [Ca2+]i transient (Cannell et al. 1994, 1995). As ICa inactivates, the probability of activation of Ca2+ sparks diminishes thereby allowing the SR Ca2+ ATPase and Na+-Ca2+ exchanger to return the [Ca2+]i to resting levels (Balke et al. 1994; López-López et al. 1995).

E-C coupling and Ca2+ sparks are modulated by humoral (e.g. sympathetic stimulation) and hormonal (e.g. angiotensin II) stimulation (Callewaert et al. 1988; Danziger et al. 1990; Kaku et al. 1991; De Mello, 1998; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). The mechanism of action of many of these neurotransmitters or hormones involves changes in the level of phosphorylation of one or more of the proteins involved in controlling Ca2+ in cardiac cells (e.g. Ca2+ channels, phospholamban, RyRs). This suggests that a delicate interplay between protein kinases and phosphatases is critical in determining the function of ventricular myocytes. Indeed, the work by duBell and colleagues examining the effects of protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1) has provided strong evidence that phosphatase activity can modify cardiac E-C coupling by controlling ICa and SR Ca2+ release (duBell et al. 1996, 1997, 2002).

Calcineurin (protein phosphatase 2B, PP2B) is one of the serine/threonine phosphatases expressed in the heart (Rusnak & Mertz, 2000). Calcineurin is of particular interest because Ca2+ and calmodulin regulate its activity. A series of recent papers suggests that calcineurin could modulate E-C coupling in ventricular myocytes. Indeed, Bandyopadhyay et al. (2000) found that inhibition of calcineurin leads to increases in the frequency and amplitude of spontaneous [Ca2+]i oscillations in neonatal rat ventricular myocytes. Similarly, Munch et al. (2002) found that calcineurin depressed the activity of the SR Ca2+ ATPase in human SR vesicles. At present, however, the molecular mechanisms and the functional implications of these findings in adult ventricular myocytes are unclear.

The goal of the present study was to investigate the functional role and cellular mechanisms by which calcineurin could modulate E-C coupling in the heart. To this end we combined epifluorescence and confocal imaging with patch-clamp techniques to measure [Ca2+]i and membrane currents and voltage in adult mouse ventricular myocytes before and after calcineurin inhibition. Our data indicate that calcineurin modulates SR Ca2+ release and ICa in mouse ventricular myocytes. Interestingly, our data show that calcineurin opposes the actions of protein kinase A (PKA) in cardiac myocytes. We also found that calcineurin and PKA co-localize in the T-tubules of mouse ventricular myocytes. Thus, our data suggest that calcineurin and PKA dynamically regulate Ca2+ channels and SR Ca2+ release.

METHODS

Isolation of ventricular myocytes

Animals were handled in strict accordance to the guidelines of the University of Washington Institutional Animal Care and Use Committee. Hearts were obtained from adult (25-30 g) mice (CD-1) killed by an intraperitoneal injection of pentobarbital (100 mg kg−1). Single ventricular myocytes were isolated as previously described (Esposito et al. 2000; Ufret-Vincenty et al. 2001). Dissociated cells were maintained in Dulbecco's MEM at room temperature (25 °C) until used (i.e. 1–6 h after isolation).

Electrophysiology

Ionic currents and action potentials were recorded using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA, USA). Signals were digitized using a Digidata 1320A and stored in a personal computer running the pCLAMP 8 software suite (Axon Instruments). Analysis of the electrical signals was performed using the Clampfit module of pCLAMP 8.

Action potentials were recorded while cells were superfused with a solution (Solution A) with the following constituents (mm): 140 NaCl, 5 KCl, 10 Hepes, 10 glucose, 2 CaCl2, and 1 MgCl2 (pH 7.4). The patch-pipette filling solution used to record AP was composed of (in mm): 110 potassium aspartate, 30 KCl, 10 Hepes, 5 Mg2+-ATP and 10 NaCl (pH 7.3). The 15 mV tip potential produced by this solution was corrected offline. K+ currents were measured using a similar pipette solution, but without NaCl. The external solution used to measure K+ currents contained (in mm): 140 NaCl, 5 KCl, 10 Hepes, 10 glucose, 0.1 CaCl2, 2 MgCl2, 0.01 TTX and 0.01 nifedipine (pH 7.4).

For experiments measuring Ca2+ currents (ICa) the following solutions were used. Cells were superfused with solution A. Once whole-cell voltage clamp was achieved the superfusion solution was changed to one containing (in mm): 140 NaCl, 5 CsCl, 2 CaCl2, 1 MgCl2, 10 Glucose, 10 Hepes, 0.010 TTX. The pipette filling solution used in these experiments contained (in mm) 130 CsCl, 10 TEA-Cl, 5 Mg2+-ATP and 10 Hepes.

In some experiments APs were evoked in ventricular myocytes using an extracellular stimulator (Myopacer, IonOptix Corporation, Milton, MA, USA). Cells were stimulated at a 1.5 excitation threshold through two platinum electrodes at 1 Hz.

Measurements of [Ca2+]i

We measured whole-cell changes in [Ca2+]i in mouse ventricular myocytes using the fluorescent Ca2+ indicator fluo-4. For those experiments that involved the simultaneous measurement of ICa and [Ca2+]i, cells were loaded with the pentapotassium salt of fluo-4 (50 μM) through the patch-pipette on the stage of an Olympus IX-70 inverted microscope (Melville, NY, USA). Fluorescence signals were collected through a 40 × (NA 1.35; Olympus, Melville, NY, USA) lens and detected by an IonOptix photometry system (IonOptix Corp, Milton, MA, USA) coupled to the IX-70. Background-subtracted fluorescence signals were normalized by dividing the fluorescence (F) intensity at each time point by the resting fluorescence (F0).

Imaging of Ca2+ sparks was performed with a BioRad MRC1024 confocal system (Cambridge, MA, USA) coupled to a Nikon TE300 inverted microscope using a Nikon 60 × water immersion lens (NA 1.2). This system was operated using a personal computer running Lasersharp 2000 (v. 4.0) software (BioRad, Cambridge, MA, USA). Images were analysed using custom software written in IDL language (Research Systems, Boulder, CO, USA). Ca2+ sparks were identified using a computer algorithm similar to the one described by Cheng et al. (1999). Calibration of fluorescence signals was performed using the ‘pseudo-ratio’ equation (Cheng et al. 1993):

graphic file with name tjp0544-0057-mu1.jpg

where F is the fluorescence intensity, F0 is the resting fluorescence, Kd is the dissociation constant of fluo (1100 nm) and [Ca2+]i,rest is the resting Ca2+ concentration (150 nm). The rate of decay of [Ca2+]i transients was obtained by fitting the decaying phase of calibrated fluorescence signals with a standard single exponential function.

The amplitude of the [Ca2+]i transient evoked by the application (4 s; using a picospritzer) of a Ca2+- and Na+-free (substituted with N-methyl-d-glucamine) solution containing 20 mm caffeine was used as an indicator of SR Ca2+ content (Santana et al. 1997). To ensure steady-state SR Ca2+ load, cells were subjected to a minimum of 10 preconditioning pulses (1 Hz) before caffeine was applied.

In several experiments cells were injected with the potassium salt of the ratiometric Ca2+ indicator fura-2 (25 μM). Fura-2 fluorescence at 510 nm was measured while cells were excited with 365 (isosbestic point) or 380 nm light. These data are reported as the fluorescence produced by 365 nm excitation divided by fluorescence produced by 380 nm excitation (F365/F380).

Immunofluorescence labelling

For our immunofluorescence studies we used a monoclonal mouse antibody targeting the regulatory subunit of protein kinase A type II (PKA-KII; Chemicon International, Temecula, CA, USA) and a rabbit polyclonal antibody against calcineurin B (Oncogene Research Products, Cambridge, MA, USA). The secondary antibodies were an Alexa Fluor 488-conjugated goat anti-mouse (highly cross-absorbed) obtained from Molecular Probes (Eugene, OR, USA) and a Cy5-conjugated goat anti-rabbit purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Cells were fixed with a cold (-20 °C) solution of 1 % paraformaldehyde in methanol for 10 min. Fixed cells were washed three times for 5 min each in phosphate buffered saline (PBS). Blocking buffer, consisting of 20 % normal goat serum (Jackson ImmunoResearch Laboratories, Inc.) in an antibody dilution buffer (1 % IgG-free, protease-free BSA and 0.1 % Triton X-100 in PBS) was used for 30 min at room temperature to enhance cell permeability and to decrease non-specific binding of the antibodies. Cells were then washed three times in PBS. Myocytes were incubated overnight at 4 °C with the PKA-RII (1:250) and calcineurin (1:250) primary antibodies in antibody dilution buffer. The following day, cells were incubated for 2 h at room temperature with Alexa Fluor 488-conjugated [1:250] and Cy5-conjugated [1:250] secondary antibodies diluted in antibody dilution buffer. Following another wash with PBS, cells were mounted on slides using ProLong Antifade medium (Molecular Probes).

Slides were imaged using the BioRad MRC1024 using the 488 and 567 nm lines of the Kr/Ar laser. Images were collected using a 60 × oil immersion lens (1.4 NA). The fluorescence emitted by Alexa-488 and Cy5 was separated by the appropriate set of filters. The point spread function for this system showed that it has a lateral and axial resolution of 0.33 μm and 0.80 μm, respectively.

Chemicals

H-89, TTX, cyclosporin A and the calcineurin auto-inhibitory peptide were purchased from Calbiochem (La Jolla, CA, USA). Fluo-4, di-8-anepps and fura-2 were purchased from Molecular Probes (Eugene, OR, USA). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Statistics

Data are presented as mean ± standard error of the mean (s.e.m.). Two-sample comparisons were made using Student's t test. Multi-group comparisons were performed using an ANOVA, which, if necessary, was followed by a multi-comparison test (i.e. Tukey's test). A P value of less than 0.05 was used as an indicator of significance. The asterisk (*) symbol is used in the figures to illustrate a significant difference between groups.

RESULTS

Calcineurin modifies the AP and the whole-cell [Ca2+]i transient in mouse ventricular myocytes

To explore the role of calcineurin in the regulation of the AP and Ca2+ signalling a membrane potential and [Ca2+]i were simultaneously measured in mouse ventricular myocytes under control conditions and after the inhibition of calcineurin. The solution in the patch clamp electrode contained 50 μM fluo-4. For some of these experiments the highly specific calcineurin auto-inhibitory peptide (CiP; 100 μM) was also included in the dialyzing pipette solution. APs were evoked by a brief (4 ms) injection of depolarizing current (4-6 nA) at a frequency of 1 Hz.

Figure 1 shows a representative set of [Ca2+]i and AP traces recorded before and after the application of CsA (100 nm) in ventricular myocytes dialysed with or without CiP (100 μM). CsA (100 nm) was used in these experiments for two reasons. First, in vitro, 100 nm CsA maximally inhibits calcineurin activity (Kd= 10 nm) (Swanson et al. 1992). Second, 100 nm CsA is the therapeutic dose used to immunosuppress humans with transplanted organs (de Groot-Kruseman et al. 2001). Note that application of 100 nm CsA increased AP duration at 90 % repolarization (APD90) by about 68 % (control, 116 ± 15 ms, n= 7; CsA, 195 ± 40 ms, n= 10; P < 0.05). Furthermore, the whole-cell [Ca2+]i transient was larger (control, 525 ± 25 nm; CsA, 725 ± 50 nm; P < 0.05) and had a smaller time constant of decay (τdecay) (control, τdecay= 238 ± 15 ms; CsA, τdecay= 176 ± 10 ms; P < 0.05) in CsA-treated cells than in control cells. These data suggest that calcineurin plays a significant role in the control of AP duration and [Ca2+]i in mouse ventricular myocytes. To provide further support to this hypothesis we performed a series of experiments similar to those described above, but involving CiP (100 μM) as an alternative means to inhibit calcineurin. Like CsA-treated cells, ventricular myocytes dialysed with 100 μM CiP had a longer AP (APD90= 192 ± 32 ms; P < 0.05) as well as a larger and faster decaying [Ca2+]i transient (715 ± 47 nm; τdecay= 172 ± 15 ms; P < 0.05) than control cells. It is important to note that the changes in the AP and whole-cell [Ca2+]i transient produced by CiP (100 μM) were similar to those produced by CsA (100 nm).

Figure 1. Calcineurin modulates AP duration and [Ca2+]i in ventricular myocytes.

Figure 1

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A, representative AP and [Ca2+]i records obtained before and after the application of CsA (100 nm) from mouse ventricular myocytes dialysed with or without the calcineurin inhibitory peptide (100 μM; CiP). B, bar plots summarizing the effects of CsA and/or CiP on APD90 duration and the amplitude of the [Ca2+]i transient.

To verify that CsA and CiP modified the AP and whole-cell [Ca2+]i transient of ventricular myocytes through a common mechanism we exposed CiP-dialysed myocytes to 100 nm CsA. We found that the effects of CsA (100 nm) and CiP (100 μM) on the AP and whole-cell [Ca2+]i were not additive; CsA (100 nm) did not produce any further changes in the AP (APD90= 215 ± 36 ms) and whole-cell [Ca2+]i transient (peak [Ca2+]i= 755 ± 33 nm; τdecay= 166 ± 11 ms, P > 0.05) of cells dialysed with CiP (100 μM). Taken together these data provide compelling support for the conclusion that calcineurin modulates both the [Ca2+]i transient and AP duration of mouse ventricular myocytes.

Calcineurin does not alter K+ currents involved in the repolarisation of the mouse ventricle

One possible mechanism by which calcineurin increases AP duration in ventricular myocytes is by decreasing outward K+ currents. We investigated this possibility by measuring ventricular outward K+ currents under control conditions and after the application of 100 nm CsA. Figure 2 illustrates the results of these experiments. Panel A shows two families of K+ current records obtained from a typical cell before and after (≈10 min) it was exposed to CsA (100 nm). K+ currents were evoked by 1200 ms steps to voltages ranging from −50 to 60 mV from the holding potential of −60 mV. Note that outward K+ currents were not affected by CsA (100 nm). Indeed, the current-voltage relationships of the transient (Ito; measured as the difference between the peak current and the current at the end of the 1200 ms voltage pulse) and sustained (Isust; measured as the amplitude of the current at the end of the voltage pulse) components of these currents were unchanged by 100 nm CsA (Fig. 2B). The lack of effect of CsA on outward K+ currents indicates that the increase in AP duration seen during CsA application (see Fig. 2) was most likely the result of an increase in inward current.

Figure 2. Calcineurin does not alter the K+ currents responsible for repolarization of ventricular myocytes.

Figure 2

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A, representative K+ current records obtained from a ventricular myocyte before and after the application of 100 nm CsA. The inset shows the voltage protocol used to evoke these currents. Briefly, cells were depolarized from the holding potential of −60 mV to potentials ranging from −50 to 60 mV for 1200 ms. B, current- voltage relationship of Ito and Isust before and after the application of CsA (100 nm).

The amplitude of whole-cell [Ca2+]i transients and Ca2+ currents is higher in CsA-treated than in control ventricular myocytes

The data described above show that calcineurin inhibition increases the [Ca2+]i transient and AP duration without altering outward K+ currents. These data suggest that the CsA-induced AP prolongation is produced by an increase in inward current. One current that could contribute both to changes in AP duration and [Ca2+]i is ICa (Puglisi & Bers, 2001). Thus, we investigated the effects of CsA (100 nm) on the voltage dependencies of ICa and the [Ca2+]i transient in ventricular myocytes. In these experiments cells were loaded with the free-acid form of fluo-4 (50 μM) via the patch-pipette. [Na+]i was zero in the internal solution to prevent the Na+-Ca2+ exchanger from triggering SR Ca2+ release. In these experiments TTX (10μM) was included in the external solution. ICa and [Ca2+]i transients were evoked by 200 ms voltage steps to potentials ranging from −40 to 60 mV from the holding potential of −50 mV (Fig. 3).

Figure 3. Effects of calcineurin inhibition on E-C coupling.

Figure 3

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A, representative ICa and [Ca2+]i records evoked in a control (black line) and a CsA-treated ventricular myocyte by 200 ms voltage steps to −20 (left), 0 (centre) and +20 (right) mV from the holding potential of −50 mV. B, voltage dependencies of [Ca2+]i and ICa in control and CsA-treated ventricular myocytes. C, this panel shows the voltage dependence of E-C coupling gain (top) and a plot of the dependence of the amplitude of the [Ca2+]i transient (bottom) on ICa in control and CsA-treated myocytes. The lines in the bottom panel represent linear fits to the data. For CsA, slope =−106.63 nm (pA pF−1)−1 with a regression coefficient of 0.94. For control, slope =−48.11 nm (pA pF−1)−1 with a regression coefficient of 0.94.

For this set of experiments we used control cells and cells that had been treated with CsA (100 nm) for 10–30 min. Shown in Fig. 3A are ICa and [Ca2+]i transients that were evoked by depolarizations to −20, 0 and 20 mV from a representative control and CsA-treated ventricular myocyte. Note that the amplitudes of both ICa and the [Ca2+]i transient were larger in the CsA-treated cell than in the control cell at the three voltages shown in this panel. Indeed, our analysis indicated that the amplitudes of ICa and [Ca2+]i transients were larger in CsA-treated cells than in control cells at most voltages examined (Fig. 3B). At 0 mV the amplitude of ICa and [Ca2+]i transients were, respectively, 1.35- and 2.19-fold larger in CsA-treated cells than in control cells (P < 0.01). Interestingly, the peak of ICa was shifted towards more hyperpolarized potentials in CsA-treated cells.

The kinetics of the whole-cell [Ca2+]i transient were also changed by CsA. As discussed above, the [Ca2+]i transient in CsA-treated cells decayed faster than in control cells (at 0 mV, control τdecay= 248 ± 28 ms and CsA τdecay= 172 ± 11 ms, n= 5, P < 0.02). The rate of inactivation of ICa in control and CsA-treated cells was not statistically significant (control, τfast= 7.92 ± 0.74 ms, τslow= 48 ± 1.35 ms; CsA, τfast= 7.12 ± 0.5 ms, τslow= 48.16 ± 1.98; P= 0.25, where τfast and τslow represent the fast and the slow time constants of inactivation, respectively).

We also calculated E-C coupling gain in control and CsA-treated cells (Fig. 3C). E-C coupling gain, defined as the maximum change in [Ca2+]i (Δ[Ca2+]i) divided by the peak ICa density (pA pF−1) density at any given voltage, gives information on the efficacy of ICa to trigger SR Ca2+ release. Figure 3C shows that E-C coupling gain was larger (P < 0.05) in CsA-treated cells than in control cells. A plot of the relationship between [Ca2+]i and ICa in control and CsA-treated cells is also shown in Fig. 3C. Together these data suggest that inhibition of calcineurin increases the amplitude of the evoked [Ca2+]i transient by increasing ICa and the amount of Ca2+ released by a given ICa.

Calcineurin controls Ca2+ spark activity

An elevation in SR calcium load would be predicted to increase the probability and amplitude of the elementary SR Ca2+ release event, the Ca2+ spark. This increase in Ca2+ spark probability would reflect an increase in RyR open probability in response to an elevated SR Ca2+ load (Lukyanenko et al. 2001). Recent work has suggested that changes in SR Ca2+ content could significantly affect Ca2+ spark frequency and amplitude in cardiac ventricular myocytes (Lukyanenko et al. 1996, 2001; Santana et al. 1997). An elevation in spark amplitude could reflect an elevation in the calcium driving force due to increased SR Ca2+ (Lukyanenko et al. 2001). Figure 4 shows representative line-scan images of spontaneous Ca2+ sparks in control and CsA-treated cells. We found that in the presence of 100 nm CsA (10.2 ± 0.97 sparks s−1 (100 μm)−1, n= 239), the rate of spontaneous Ca2+ sparks was 3-fold higher than in control cells (3.4 ± 0.51 sparks s−1 (100 μm)−1, n= 69; P < 0.01). In a separate set of experiments involving the ratiometric fluorescent Ca2+ indicator fura-2, we found that CsA (100 nm) did not change resting, spatially-averaged (i.e. whole-cell) [Ca2+]i (control F365/F380= 0.78 ± 0.04 vs. CsA F365/F380= 0.82 ± 0.06, n= 5, P > 0.05, data not shown). These data suggest that the increase in Ca2+ spark rate induced by 100 nm CsA was produced by an increase in RyR activity, which could result from an elevation in SR Ca2+ load. Consistent with this hypothesis, we found that CsA produced a small, but significant increase in Ca2+ spark amplitude (277 ± 8 nm, control, vs. 297 ± 7 nm, CsA; P < 0.05).

Figure 4. Calcineurin modulates Ca2+ sparks.

Figure 4

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A, representative line-scan images from control (top) and CsA-treated (100 nm; bottom) ventricular myocytes, respectively. The traces on the right side of these images represent the time-course of fluorescence along the region indicated by the line. B, bar plot of the mean ±s.e.m. of the rate of spontaneous Ca2+ sparks in control and 100 nm CsA-treated ventricular myocytes. C, surface plot of an averaged Ca2+ spark from control and CsA-treated ventricular myocytes. D, bar plot of the mean ±s.e.m. of the amplitude of spontaneous Ca2+ sparks in control and CsA-treated ventricular myocytes. *P < 0.05.

Inhibition of calcineurin increases SR Ca2+ load

The increase in Ca2+ spark frequency and amplitude in response to CsA (100 nm) suggests that SR Ca2+ load is greater during calcineurin inhibition. We therefore investigated whether SR Ca2+ load is greater in the presence of CsA (100 nm). The amplitude of a global [Ca2+]i transient induced by 20 mm caffeine was used as an indicator of SR Ca2+ content. In these experiments, cells were field stimulated at a frequency of 1 Hz for at least 10 s before caffeine (20 mm) was applied. This protocol ensured steady-state SR Ca2+ loading at the time of caffeine application. As Fig. 5 shows SR Ca2+ load was nearly 25 % greater in cells exposed to 100 nm CsA (10-15 min) than in control cells (1600 ± 150, CsA, vs. 1200 ± 150 nm, control, n= 5; P < 0.05). These data and Ca2+ spark data suggest that inhibition of calcineurin increases whole-cell [Ca2+]i transients, at least in part, through elevation of the SR Ca2+ content.

Figure 5. Calcineurin increases SR Ca2+ load.

Figure 5

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A, Two records of caffeine-induced [Ca2+]i transients before and after the application of CsA (100 nm) from a typical ventricular myocyte stimulated at 1 Hz with extracellular platinum electrodes. Caffeine was applied after attaining steady-state conditions. B, bar plot of the mean ±s.e.m. of the amplitude of the caffeine-induced Ca2+ transient under control conditions and after the application of CsA (100 nm). *P < 0.05.

Dynamic control of E-C coupling by calcineurin and protein kinase A

One possible mechanism by which calcineurin could affect ICa and SR function in the heart is by opposing the effects of an active protein kinase. Thus, inhibition of calcineurin by CsA (100 nm) could lead to an enhanced level of phosphorylation of E-C coupling proteins. Indeed, many of the effects of calcineurin inhibition resemble the activation of protein kinase A (PKA) on cardiac myocytes (i.e. enhanced ICa and [Ca2+]i transients, increased Ca2+ spark frequency, etc.). Therefore, we investigated the possibility that calcineurin inhibition by CsA increases the [Ca2+]i transients because it allows increased PKA-mediated phosphorylation. First, we examined whether the actions of CsA (100 nm) on the AP-evoked [Ca2+]i transient depended on the activity of PKA. During the entire duration of these experiments cells were subjected to field stimulation at a frequency of 1 Hz to evoke an AP and a [Ca2+]i transient. After a steady-state was achieved the specific PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 μM) (Chijiwa et al. 1990) was introduced into the bath. Once we had established that H-89 had produced its maximal effect (usually after 4–5 min under our experimental conditions), cells were then exposed to a solution containing H-89 (1 μM) and CsA (1 μM). Figure 6 summarizes the results of these experiments. Note that in mouse ventricular myocytes H-89 (1 μM) decreased the amplitude of the [Ca2+]i transient by about 35 % (control, 525 ± 25 nm; H-89, 341 ± 18 nm; n= 5, P < 0.01), thus suggesting PKA is active in these cells even under control conditions. More importantly, we found that in the presence of 1 μM H-89, even 1 μM CsA did not modify the amplitude of the AP-evoked [Ca2+]i transient (H-89, 341 ± 18 nm; H-89 + CsA, 331 ± 19 nm; n= 5, P > 0.05). These data clearly suggest that inhibition of calcineurin increases the amplitude of the AP-evoked [Ca2+]i via a PKA-dependent pathway.

Figure 6. Functional coupling between calcineurin and PKA.

Figure 6

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A, three [Ca2+]i transients recorded from a typical ventricular myocyte under control conditions, after the application of H-89 (1 μM) and after the addition of H-89 and CsA (1 μM). The plot on the right of this panel summarizes the results obtained from a population of cells. B, [Ca2+]i transients recorded from a control and CsA-treated (10-15 min) ventricular myocyte in the presence of varied concentrations (0 (control), 0.1, 1 and 10 μM) of forskolin. The bar plot on the right of this panel shows the mean ±s.e.m. of the amplitude of the evoked [Ca2+]i transient in control (□) and CsA-treated (Inline graphic) cells in the presence of 0.1, 1 and 10 μM forskolin. *P < 0.05.

One testable prediction of this hypothesis is that PKA activators such as forskolin would have larger effects on the [Ca2+]i transient in cells with their calcineurin inhibited than in control cells. To investigate this we examined the effects of 0.1, 1 and 10 μM forskolin on the AP-evoked [Ca2+]i transient in control cells and in cells that had been treated with 1 μM CsA, to inhibit calcineurin (Fig. 6B). As predicted, we found that 0.1, 1 and 10 μM forskolin produced incrementally larger increases in the amplitude of the AP-evoked [Ca2+]i transients in cells exposed to CsA (1 μM) than in control cells (P < 0.03). Thus, taken together these data suggest that calcineurin and PKA oppose each other to regulate the amplitude of ICa and the whole-cell [Ca2+]i transient.

Co-localization of the regulatory subunit of protein kinase A and calcineurin in the T-tubules of mouse ventricular myocytes

The data presented above suggest that PKA and calcineurin share common targets: the Ca2+ channel and the SR. Therefore, PKA and calcineurin should be localized near one another, the Ca2+ channel and the SR. To investigate this possibility, we used immunofluorescence techniques to determine the location of calcineurin and PKA in ventricular myocytes. Figure 7A shows a set of confocal immunofluorescence images from a ventricular myocyte labelled with antibodies against calcineurin (left) and the regulatory subunit of PKA (RII, centre). The secondary antibodies used in these experiments were linked to Cy5 and Alexa-488 for calcineurin and PKA-RII, respectively, to minimize emission overlap. The image on the right of Fig. 7A shows the overlay of the calcineurin and PKA-RII images. Note that calcineurin and PKA-RII fluorescence occurs in transverse lines of high intensity in the cell. The image resulting from the merging of the calcineurin (red) and PKA-RII (green) images suggests that these proteins are co-localized (yellow) in ventricular myocytes. To provide further support to this conclusion we measured the fluorescence intensity along the cell shown in Fig. 7A. The longitudinal fluorescence profile of calcineurin and PKA-RII in the region of the cell marked by the dotted line is shown in Fig. 7B. Note that the fluorescence profile of PKA and calcineurin are in register, which suggests that these proteins are found in close proximity to one another (i.e. their lateral separation must be lower than 0.33 μm, the resolution of our confocal microscope) in mouse ventricular myocytes. Interestingly, the distance between peak fluorescence intensities was, respectively, 1.92 ± 0.23 and 1.84 ± 0.35 μm for calcineurin and PKA, a value that is similar to the distance between T-tubules in ventricular myocytes. To investigate this issue further we exposed mouse ventricular myocytes to the voltage-sensitive dye di-8-anepps, which partitions into the outer leaflet of the plasma membrane and fluoresces depending on the voltage across the membrane. Di-8-anepps has been used by other investigators to identify T-tubular membranes in ventricular myocytes (Shacklock et al. 1995). A confocal image of a di-8-anepps-loaded myocyte is shown in Fig. 7C. Note that di-8-anepps (red) produces periodic lines of fluorescence. The distance between the peak fluorescence intensities of these lines is 1.87 ± 0.19 μm, a value that is similar to the ones observed in the calcineurin and PKA-RII images. Taken together these data suggest that both calcineurin and PKA-RII are localized in the T-tubules of ventricular myocytes.

Figure 7. Co-localization of calcineurin and the regulatory subunit of PKA in ventricular myocytes.

Figure 7

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A, confocal images of a ventricular myocyte showing the fluorescence pattern for antibodies targeted against calcineurin (left), the regulatory subunit of PKA (centre) and the overlay of these two images. The bottom row shows an expanded view of the region outlined by the square in the top row images. The arrows point at transverse lines (i.e. Z-lines) of high fluorescence intensity in this cell. B, spatial profile of fluorescence obtained from the calcineurin and PKA images shown in panel A. Fluorescence intensity (measured in arbitrary units; A.U.) was measured along the dotted lines shown in the images in panel A. C, confocal image of a ventricular myocyte loaded with the fluorescent indicator di-8-anepps. The plot below shows the fluorescence intensity along the dotted line in the image above.

DISCUSSION

In this study, we provide evidence that calcineurin and PKA have opposing effects on ICa and the [Ca2+]i transient. Immunofluorescence imaging of calcineurin and PKA suggests that this functional coupling is facilitated by co-localization of these two proteins near the T-tubules of ventricular myocytes, where they modulate Ca2+ channels and SR function. Our results are consistent with the concept that calcineurin and PKA dynamically control calcium entry and release by regulating the level of PKA-mediated phosphorylation of at least two targets: the Ca2+ channel and phospholamban (Fig. 8). Inhibition of calcineurin would lead to PKA-mediated enhancement of the Ca2+ current and to dis-inhibition of the SR Ca2+ ATPase due to phosphorylation of phospholamban. The latter effect would elevate SR calcium load, which would elevate Ca2+ spark frequency and amplitude as well as the amplitude of the whole-cell [Ca2+]i transient. These results also suggest that calcineurin serves as a negative feedback element to limit the actions of PKA on Ca2+ channels and the SR. Specifically, an elevation of Ca2+ influx and SR calcium release would increase the activity of calcineurin to moderate the effects of sympathetic stimulation (PKA) on calcium currents and release. The degree of modulation of ICa and SR Ca2+ release would be determined by local cAMP and Ca2+ levels, which would respectively determine the activities of PKA and calcineurin.

Figure 8. Proposed actions of calcineurin and PKA on phospholamban and Ca2+ channels in mouse ventricular myocytes.

Figure 8

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Upon activation PKA phosphorylates phospholamban (PLB) and Ca2+ channels. Phosphorylation of Ca2+ channels increases ICa. Phosphorylation of PLB elicits an increase in SR Ca2+ ATPase activity, which increases SR Ca2+ load. An increase in SR Ca2+ load then increases the frequency and amplitude of Ca2+ sparks, which could then lead to an increase in the activity of calcineurin (CaN). Activation of calcineurin de-phosphorylates Ca2+ channels and PLB, which reduces ICa and SR Ca2+ uptake and thereby opposes the actions of PKA.

Mechanisms by which calcineurin and PKA modulate E-C coupling in ventricular myocytes

A recent series of papers provides insights into the mechanisms by which calcineurin could control [Ca2+]i in ventricular myocytes (Kim et al. 1998; Park et al. 1999; Munch et al. 2002). Munch et al. (2002) recently found that calcineurin-mediated de-phosphorylation of phospholamban leads to reduced SR Ca2+ ATPase activity. Drago et al. (1998) found that phospholamban and SR Ca2+ are co-localized in the T-tubules of ventricular myocytes. The immunofluorescence data shown here, together with the data presented in the recent studies cited above, provide structural support for this hypothesis. Similarly, our data suggest that PKA and calcineurin are localized near the T-tubules of ventricular myocytes. PKA, and possibly calcineurin, are targeted to this region of ventricular cells by their association to A kinase anchoring protein 100 (AKAP100) (Yang et al. 1998). Thus, the Z-lines of ventricular myocytes could be viewed as a specialized region for the control of Ca2+ signalling by local changes in PKA and calcineurin activity.

The work by Munch et al. (2002) also suggests that the increases in the rate of decay of the [Ca2+]i transient and increased SR Ca2+ load we observed during calcineurin inhibition could be due to higher Ca2+ uptake by the SR Ca2+ ATPase activity due to sustained phospholamban phosphorylation. An increase in SR Ca2+ load would increase SR Ca2+ release through an elevation of opening probability (Po) and single channel conductance of RyRs (Lukyanenko et al. 1996). However, we cannot rule out the possibility that calcineurin could also control SR Ca2+ release by controlling the phosphorylation state of RyRs. Indeed, a recent study found that calcineurin could directly interact with cardiac RyRs (Bandyopadhyay et al. 2000). Interestingly, the authors of this study proposed that calcineurin is linked to cardiac RyRs via FK506 binding protein (FKBP) 12.6. This observation is consistent with our data showing that calcineurin is found in the T-tubules of ventricular myocytes, where RyRs are located (Franzini-Armstrong et al. 1999). Although these results suggest that calcineurin could control RyR phosphorylation in heart, it is controversial whether phosphorylation could modify the function of RyRs in ventricular myocytes (Li et al. 2002). Planar lipid bilayer work by Valdivia et al. (1995) and Marx's research group (Marx et al. 2000; Antos et al. 2001) have provided compelling evidence that PKA phosphorylation of RyRs can produce significant changes in the function of this channel. However, it appears that PKA-induced changes in RyR channel function in planar lipid bilayers do not necessarily translate to alterations of its function in intact cardiac myocytes (Li et al. 2002). In this context it should be noted that Eisner and colleagues recently examined the effects of altering RyR sensitivity on E-C coupling (Eisner et al. 2000; Trafford et al. 2000). They found that modulation of RyR Ca2+ sensitivity alone did not have sustained effects on the [Ca2+]i transient. This occurs because the increase in Ca2+ release produced by an increase in the sensitivity of RyRs to Ca2+ causes an increase in Na+-Ca2+ exchanger-mediated Ca2+ extrusion. Furthermore, there is reduced Ca2+ influx via ICa due to greater Ca2+-dependent inactivation of this current. The combination of lower Ca2+ influx (i.e. faster rate of inactivation of ICa) and larger extrusion (i.e. increased outward Na+-Ca2+ exchanger current) would thereby lead to a reduced SR Ca2+ load and reduced Ca2+ release during subsequent beats, even when the RyRs are more sensitive to activating Ca2+. The studies by the Eisner group (Eisner et al. 2000; Trafford et al. 2000) therefore suggest that while RyR phosphorylation could be increased during calcineurin inhibition, the sustained changes in [Ca2+]i observed in our studies could be due to increased SR Ca2+ ATPase activity. Such an increase in SR Ca2+ ATPase activity would facilitate elevated SR Ca2+ levels. This high SR Ca2+ load conspires with a larger ICa and increased RyR sensitivity to produce a long-lasting increase in E-C coupling.

Mechanism of CsA-induced AP prolongation

Under physiological conditions calcineurin could also modulate Ca2+ dynamics in the heart by controlling AP duration. The duration of the cardiac AP is set by the time-course and amplitude of inward and outward currents in cardiac myocytes. AP prolongation can occur when potassium channel currents are decreased and/or when inward currents are increased, as classic presentations on AP biophysics would require (Hille, 2001). Our data suggest that calcineurin inhibition increases the inward ICa current. Using a recent mathematical model of the rabbit AP (Puglisi & Bers, 2001) we found that an increase in the amplitude of ICa similar to the one reported here during calcineurin inhibition - approximately 35 % - could also lead to a significant increase in AP duration. Thus, use of the Puglisi-Bers model (Puglisi & Bers, 2001) supports our conclusion that the increase in AP duration we report is produced by an increase in ICa.

Comparison with other studies

Recently another group of investigators examined the role of calcineurin in rat ventricular myocytes (duBell et al. 1998). They found that application of CsA (10 μM) did not produce any significant effects on the AP-evoked [Ca2+]i transient in rat ventricular myocytes. There are several explanations for these apparently conflicting results. First, calcineurin activity could be higher under our experimental conditions than under those used by duBell et al. (1998). Second, calcineurin may have different targets in rat and mouse ventricular myocytes. Third, because calcineurin inhibition can only modify the [Ca2+]i transients when PKA activity is high, it is possible that a low basal PKA activity in the rat prevented calcineurin inhibition from having any effects on E-C coupling. Indeed, recent results obtained from rat ventricular myocytes suggest that these cells have relatively low basal PKA activity (Santana et al. 1998). Any of these scenarios could underlie the differences between our results and those of duBell et al. (1998).

Conclusions

In conclusion, our results indicate that inhibition of calcineurin increases the whole-cell [Ca2+]i transient by increasing PKA-mediated phosphorylation of Ca2+ channels and phospholamban. The long-lasting effects of calcineurin-inhibition on E-C coupling are due to an increase in SR Ca2+ ATPase activity produced by phospholamban phosphorylation, which elevates SR Ca2+. Furthermore, our results support the concept that the opposing actions of co-localized PKA and calcineurin modulate the activity of Ca2+ channels and RyRs, and that calcineurin provides a negative feedback element to this process. PKA-induced increases in ICa and SR Ca2+ release are limited by subsequent Ca2+-induced activation of calcineurin.

Acknowledgments

This work was supported by grants HL70556-02 (L.F.S.) and U54-NS34905-02 (L.F.S. and M.T.N.). The authors would like to thank Dr Carmen Ufret-Vincenty for critically reading earlier versions of this manuscript and Mrs Marla Feinstein for technical help on immunofluorescence studies.

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