Neuronal nitric oxide synthase regulation of calcium cycling in ventricular cardiomyocytes is independent of Cav1.2 channel modulation under basal conditions
Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that—against the currently prevailing view—basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO.
KeywordsCalcium cycling Cav1.2 channel regulation Neuronal nitric oxide synthase Single-channel recordings Ventricular cardiomyocytes Whole cell patch clamp
During the plateau phase of the ventricular action potential, Ca2+ influx through Cav1.2 L-type Ca2+ channels into the cytosol of cardiomyocytes elicits Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR), which finally triggers contraction. This process is called excitation-contraction (EC) coupling. Various regulatory mechanisms control Ca2+ cycling and contractility in ventricular cardiomyocytes. Among those, enhancement of the currents through Cav1.2 channels by the sympathetic nervous system via activation of β-adrenergic receptors during the so-called fight-or-flight response [7, 9, 11] is the most prominent and best established.
Besides upregulation of Cav1.2 activity in response to β-adrenergic signalling, neuronal nitric oxide synthase (nNOS) is also considered a regulator of Cav1.2 and downstream Ca2+ cycling in the heart. The most widely accepted view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels (e.g. [5, 25, 26, 33]). This gives rise to a diminished Ca2+ release from the SR, as reflected by a smaller amplitude of intracellular Ca2+ transients [5, 19, 25], and finally reduced contractility [6, 25, 26]. Evidence for Cav1.2 inhibition by nNOS activity has been derived from studies using nNOS−/− mice and/or pharmacological inhibitors of the enzyme. Ventricular cardiomyocytes derived from nNOS−/− mice showed significantly bigger L-type Ca2+ currents than myocytes from control mouse ventricles [5, 25, 31]. In accordance, pharmacological inhibition of nNOS activity in rodent ventricular cardiomyocytes increased Ca2+ current amplitudes [5, 12, 25, 32], and the application of NO donors reduced Cav1.2 currents [5, 13]. Collectively, these studies suggested that NO, generated by nNOS activity, reduces the currents through Cav1.2 channels in the heart, and this effect was linked with S-nitrosylation of the channel protein [5, 28].
In recent years, we have attempted to reproduce evidence for nNOS- or NO-mediated regulation of Cav1.2 function in mouse ventricular cardiomyocytes, but—much to our surprise—we have failed. A more thorough literature search then revealed evidence from other groups contradicting the established concept of Cav1.2 inhibition by NO via nNOS activity. For example, Barouch and colleagues  reported that cardiomyocytes from nNOS−/− mice exhibit normal Ca2+ currents, and the application of NO donors did not significantly affect the Ca2+ currents in rat ventricular cardiomyocytes  and Cav1.2 channels expressed in HEK293 cells . The apparent inconsistency in the literature concerning nNOS/NO regulation of Cav1.2 channels prompted us to reinvestigate this important issue by using a combination of different methodological approaches.
We find that nNOS/NO activity alters intracellular Ca2+ transients but not through a direct effect on Cav1.2 channels. We conclude from our results that—against the currently prevailing view—basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO regulation of Ca2+ cycling and contractility in myocytes can occur independent of direct effects of NO on Cav1.2 channel function.
Materials and methods
Isolation of ventricular cardiomyocytes
Male C57BL/10ScSnJ mice (15–25 weeks of age) and female Sprague Dawley rats (12–14-week-old) were killed by cervical dislocation. Cardiomyocytes were isolated from the ventricles of their hearts using a Langendorff setup as described in our previous article , and plated on Matrigel (Becton Dickinson)-coated culture dishes. Some myocytes of each preparation were immediately used for intracellular Ca2+ transient measurements, and some for patch clamp recordings (see below). Myocytes from at least 3 preparations (or animals) were used for each individual experiment performed.
Intracellular Ca2+ transient measurements
Intracellular Ca2+ transients were recorded from electrically stimulated ventricular cardiomyocytes, up to 8 h after preparation, using the Ca2+-sensitive fluorescent dye fluo-4 AM (Thermo Fisher Scientific). The respective experimental and analysis procedures are described in our recently published article . The myocytes were bathed in an extracellular solution containing (in mmol/L) 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 5 HEPES, 5 Glucose and pH adjusted to 7.4 with NaOH.
Ca2+ and Ba2+ current recordings
External application of NOS inhibitors and the NO donor SNAP does not affect whole cell currents through L-type Ca2+ channels in mouse ventricular cardiomyocytes. The rundown-corrected current amplitudes are expressed in percentage relative to the respective value at experiment start (= 100%). Decay half-time represents the time period between the current peak and the time point at which the current had decayed to 50%. Values are expressed as means ± SD, and the number of experiments performed (n) is given in brackets. The control (ctl) values were detected immediately before drug application, and the experimental values (drug) were taken 180 s after the start of superfusion with the respective drug. The “wash” values were detected 180 s after the start of drug washout. Ca2+ and Ba2+ indicate the use of bath solution containing 2 mM Ca2+ or 10 mM Ba2+, respectively. The respective concentrations of the drugs used are given in brackets in the leftmost column. No significant difference existed between ctl and drug-treated cells (p values always > 0.2, paired Student’s t test). The only exception (*p = 0.01, ctl versus NPLA Ca2+ current decay half-time) is indicated with an asterisk. For current amplitude value comparisons, t tests were performed on the raw data before normalization
Current amplitude (%, rel. to value at experiment start)
Current decay (decay half-time, ms)
NPLA (1 mM)
100 ± 8 (6)
105 ± 12 (6)
105 ± 11 (5)
8,3 ± 0,9 (6)
9,3 ± 1,0 (6)*
9,1 ± 1,5 (5)
96 ± 4 (5)
98 ± 5 (5)
101 ± 4 (5)
L-NMMA (1 mM)
93 ± 7 (7)
95 ± 9 (7)
97 ± 5 (7)
9,9 ± 2,0 (7)
9,6 ± 2,0 (7)
9,7 ± 1,9 (7)
102 ± 4 (10)
101 ± 4 (10)
100 ± 5 (9)
SNAP (10 μM)
101 ± 18 (8)
106 ± 27 (8)
104 ± 27 (8)
9,3 ± 1,9 (8)
9,6 ± 1,8 (8)
9,8 ± 1,7 (8)
SNAP (500 μM)
108 ± 43 (7)
111 ± 49 (7)
105 ± 19 (5)
9,8 ± 2,0 (7)
9,9 ± 1,8 (7)
8,8 ± 3,2 (5)
Rundown correction procedure
Both Ca2+ and Ba2+ currents in cardiomyocytes, detected with the conventional whole cell patch clamp technique, regularly showed considerable rundown, when recorded over prolonged time periods (up to several min). To correct for this rundown, in each experiment, a single exponential function was fit through all data points under control conditions (for examples see Fig. 2c and d, left). This function was then used to time-dependently correct every actual-measured current amplitude peak value over the whole recording period. This typically resulted in constant current amplitudes until the end of a recording (Fig. 2c and d, right). Only if rundown correction provided a satisfactory result comparable with the experiments shown in Fig. 2c and d, drug superfusion experiments were used for analyses of drug effects. Rundown correction was not necessary in the experiments performed with the perforated patch clamp technique (Fig. 5). In Figs. 3 and 5, as well as in Table 1, relative current amplitude values are given in percentage with respect to the value at experiment start (100%).
Single-channel patch clamp on proteoliposomes
For the proteoliposome experiments, the human cardiac L-type voltage-gated Ca2+ channel α1 subunit long N-terminal (CAC1C_HUMAN isoform 34, Q13936–34, long-NT) variant was used. A pGEM-HJ vector containing the cDNA of the long NT isoform of the α1C subunit of Cav1.2 (α1C,77L) was a gift from N. Dascal (Tel Aviv University, Israel) and N. Soldatov (National Institutes of Health, Baltimore, MD, USA). The cDNA of the long N-terminal isoform of the Cav1.2 was cloned into pcDNA3.1 vector (Invitrogen) and modified to include a HIS6 tag at the N-terminus. More details are available in [9, 20, 30]. The 6X His-tagged protein was expressed in HEK293T cells, then purified by Ni-NTA Agarose beads. Purified proteins were incorporated in artificial liposomes in 1:1000 protein/lipid ratio, following the dehydration/rehydration method as previously described . Single-channel mode of patch clamp technique was used to record openings of the channel. Recording and pipette solutions contained 50 mM NaCl, 100 mM BaCl2, 10 mM HEPES and 2 μM BayK8644 (pH = 7.4). The back-filled microelectrode had an average resistance of 16–17 MΩ. Single-channel currents were filtered at 1 kHz, digitized at 100 kHz and analysed using pClamp software (Molecular Devices). The Cav1.2 channel was determined by the magnitude of the current, changes in open probability of the channel (Po) and sensitivity of the current to the L-type Ca2+ channel antagonist nisoldipine. Current traces from control condition and after 150 μM GSNO (S-nitrosoglutathione) or 100 μM SNP (sodium nitroprusside) as NO donor treatment, or 0.5 μM protein kinase A (PKA, catalytic subunit, reduced 1 mM DTT, 5 mM NEM, substituted 1 mM ATP-disodium salt) were compared relative to control solution within the same patch.
Drug sources and application
S-Nitroso-N-acetyl-DL-penicillamine (SNAP; Sigma, n3398) was used as NO donor in the cardiomyocyte experiments. SNAP was solved in DMSO; the drug-free control bath solutions contained the same amount of DMSO as the experimental solutions. Another S-nitrosothiol compound the S-nitroso-l-glutathione (GSNO, Cayman Chemical, 82,240) was used in the single-channel experiments . Stock solution was made by using DMSO. Since many studies have suggested important role for cell enzymes in GSNO metabolism/NO liberation (γ-glutamyltranspeptidase, superoxide dismutase, glutathione peroxidase, reviewed in ) for our cell-free experimental system, sodium nitroprusside dihydrate (SNP, Sigma 71,778) was used as NO donor.
Data are expressed as means ± SD. Statistical comparisons between drug-free control and drug-treated conditions were made using paired or unpaired (as appropriate, see Figure legends) two-tailed Student’s t tests. A p < 0.05 was considered significant.
Inhibition of nNOS activity increases intracellular Ca2+ transients in ventricular cardiomyocytes
nNOS-derived NO has been shown to regulate Ca2+ cycling and contractility in ventricular cardiomyocytes. NO decreases Ca2+ transients and attenuates contraction (see the “Introduction” section). Here, we first tried to reproduce this well-established nNOS/NO effect in our experimental system. Therefore, we recorded intracellular Ca2+ transients in single ventricular cardiomyocytes and studied the effects of cell-permeable nNOS inhibitors. Figure 1 shows that superfusion with the nNOS inhibitor L-VNIO in a concentration of 100 μM significantly increased the amplitude of Ca2+ transients recorded from mouse ventricular cardiomyocytes (Fig. 1a). A similar effect was obtained by application of 1 mM NPLA, another cell permeable nNOS inhibitor (data not shown). In addition, we performed experiments with ventricular cardiomyocytes derived from rat hearts. As in the mouse myocytes, nNOS inhibition by L-VNIO increased the amplitude of Ca2+ transients in the rat cells (Fig. 1b). Together, these results are consistent with the established inhibitory effect of nNOS/NO activity on Ca2+ cycling in ventricular cardiomyocytes.
nNOS/NO activity does not affect currents through L-type Ca2+ channels in ventricular cardiomyocytes
Next, we tested the effect of another cell-permeable nNOS inhibitor, NPLA. Superfusion of mouse ventricular cardiomyocytes with 1 mM NPLA did not affect the current amplitude (Table 1). As observed with L-VNIO (see above), this was independent of the use of Ca2+ or Ba2+ as charge carrier. Ca2+ current decay was slightly but significantly (p = 0.01, paired Student’s t test) slowed by NPLA (Table 1).
To test whether the lack of considerable effects of L-VNIO and NPLA on the Ca2+ channel properties in ventricular cardiomyocytes was due to the fact that these compounds are selective nNOS inhibitors, we also applied the nonselective cell-permeable NOS inhibitor L-NMMA. Similar to the nNOS inhibitors, superfusion with 1 mM L-NMMA did not affect the Ca2+ channel properties in mouse ventricular cardiomyocytes (Table 1). Together, these results suggest that external application of cell-permeable nNOS and/or NOS inhibitors to ventricular cardiomyocytes does not considerably affect their basal L-type Ca2+ channel properties.
In rat ventricular myocytes, NOS inhibition did not affect the basal Ca2+ current, but in a cAMP-stimulated condition (due to application of the adenylyl cyclase activator forskolin or the phosphodiesterase inhibitor milrinone), the current was significantly augmented by the application of a NOS inhibitor . Here, using a similar experimental strategy, we tested if external L-VNIO (100 μM) application affected the Ca2+ current amplitude in mouse ventricular cardiomyocytes pre-treated with forskolin (5 μM) and milrinone (10 μM) (Suppl. Fig. 1). We found that also in a cAMP-stimulated condition, L-VNIO had no effect on the current amplitude.
As nNOS/NOS inhibition failed to affect L-type Ca2+ channels, we reasoned that direct exposure of cardiomyocytes to NO should be ineffective as well. To test this, we applied the NO donor SNAP in two different concentrations to mouse ventricular cardiomyocytes. Neither superfusion with 10 nor 500 μM SNAP generated any significant effects on Ca2+ current amplitude or current decay kinetics (Table 1). Together with the nNOS/NOS inhibitor studies, these experiments strongly suggest that nNOS/NO activity does not substantially modulate L-type Ca2+ channel properties in ventricular cardiomyocytes.
Human Cav1.2 single-channel currents in artificial membranes are not modulated by NO
nNOS/NO regulation of Ca2+ cycling in ventricular cardiomyocytes is independent of Cav1.2 channel modulation
Among the different existing NOS isoforms, nNOS and endothelial NOS (eNOS) are constitutively expressed in cardiomyocytes, whereas expression of inducible NOS (iNOS) only occurs in the presence of injury and inflammation . Recent consensus is that nNOS is the isoform in cardiomyocytes that plays the predominant role in modulating Ca2+ cycling and contractility [14, 31, 33]. The commonest view is that NO, generated by nNOS activity, reduces the currents through L-type Ca2+ channels [5, 25, 26, 33], which leads to a diminished Ca2+ release from the SR, and finally a reduced contractility [5, 19, 25]. This theory—consistent with a positive correlation between Ca2+ influx and Ca2+ release in cardiomyocytes [3, 8, 10]—however, is not consistent with the findings of several other authors (e.g. [1, 2, 34]; see Introduction).
In the present study, we have carefully reinvestigated potential modulatory effects of nNOS/NO activity on Ca2+ cycling and L-type Ca2+ channels in ventricular cardiomyocytes. Whereas we were able to confirm the well-described inhibitory effect of nNOS/NO on intracellular Ca2+ transients [5, 19, 25], we failed to detect any substantial modulatory effect on basal L-type Ca2+ channel activity. Strikingly, in cells originating from the same cardiomyocyte preparations, superfusion with nNOS inhibitors significantly increased the amplitudes of cytosolic Ca2+ transients, but did not substantially affect the currents through sarcolemmal Ca2+ channels. In addition, Cav1.2 single-channel open probability was unaffected by the presence of NO donors. We therefore propose that, in single isolated ventricular cardiomyocytes, the regulation of Ca2+ cycling by nNOS/NO can occur independent of Cav1.2 channel modulation. This further implies that nNOS/NO regulates Ca2+ cycling by at least one other mechanism than Cav1.2 channel inhibition. Among the various described NOS regulatory mechanisms of cardiac EC coupling (reviewed in [26, 33]), modulation of ryanodine receptor function in the SR membrane and regulation of SR Ca2+ load are obvious candidates .
Potential reasons for inconsistent results in the literature
In contrast to the present study, several authors have demonstrated significant inhibitory effects of nNOS/NO activity on cardiac L-type Ca2+ channels (see the “Introduction” section). On the other hand, there is also evidence from other groups against this concept in line with the work presented herein (e.g. [1, 2, 34]). Although we cannot fully explain the apparent inconsistencies, subsequently potential contributing factors are discussed.
Differences in the methodological approaches used in studies may have contributed to inconsistencies in experimental findings. These may have emerged from differences in animal species, age, and gender, as well as varieties in myocyte isolation procedures, applied patch clamp configurations, pulse protocols, experimental solutions, experimental temperatures or drug application approaches (acute application or pre-incubation). Further, we cannot rule out that small inhibitory effects of nNOS/NO activity on Cav1.2 channels exist, which were below the detection threshold of our methodological approaches, but might have been detected in other laboratories. We have tried to find potential correlations between specifically applied methodological approaches and the gained results. This, however, miserably failed due to incomplete descriptions of the experimental procedures in several of the articles cited herein. One noticeable difference between our study and several papers which, in contrast to us, reported increased Ca2+ current amplitudes following pharmacological inhibition of nNOS activity in rodent ventricular cardiomyocytes [5, 12, 25, 32] was the absence of Na+ in our bath solution. We performed control experiments with a bath solution containing 140 mM Na+ (comparable concentration as used in the named studies), which ruled out that this had actually caused the discrepancy in the results. Thus, irrespective of the absence or presence of external Na+, in our hands, 100 μM L-VNIO failed to increase the Ca2+ currents in mouse ventricular cardiomyocytes (data not shown).
Another factor that may add to the inconsistencies in experimental results in the literature is the actual “β-adrenergic tone” of a cardiomyocyte. Rozmaritsa et al.  reported that the effects of the NO donor SNAP on the Ca2+ currents in human atrial myocytes are dependent on the intracellular cAMP levels. Similarly, in rat ventricular myocytes, NOS inhibition did not affect the basal Ca2+ current, but in a cAMP-stimulated condition, the current was significantly augmented by the application of a NOS inhibitor . In contrast to this study, application of the nNOS inhibitor L-VNIO in our experimental system did not enhance the Ca2+ currents in mouse ventricular cardiomyocytes in a cAMP-stimulated condition (Suppl. Fig. 1). Thus, whereas both Matsumoto and colleagues  and our present work suggest that NOS/nNOS activity does not regulate the basal activity of L-type Ca2+ channels, the situation in case of β-adrenergic stimulation remains controversial. It is possible that a certain “threshold cAMP level” in cardiomyocytes exists, which only when exceeded, allows NOS activity to inhibit Ca2+ currents. Since neither Matsumoto and colleagues  nor we have measured cellular cAMP levels, it is impossible to verify this hypothesis here. Interestingly, some authors (e.g. [5, 25]) have provided evidence for Ca2+ channel inhibition by nNOS activity in cardiomyocytes also in experiments lacking efforts to induce β-adrenergic stimulation. Perhaps, this can occur if isolated myocytes have sufficiently high basal cAMP concentrations. Speculating that nNOS activity inhibits Ca2+ currents in a sufficiently cAMP-stimulated condition, our present results suggest that this does occur indirectly, i.e. via signalling cascades, and not via a direct modulatory effect of NO on the Cav1.2 channel. Consistent with this argument, we report here that the presence of NO donors did not affect human Cav1.2 single-channel open probability in artificial liposomes.
In summary, we cannot unequivocally explain the apparent inconsistencies in experimental results in the literature. Owing to our own findings, we propose that basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS/NO. This interpretation is solidly based on the use of different methodological approaches performed in two independent labs (whole cell patch clamp, perforated patch studies (K. Hilber lab); single channel recordings in liposomes (L.C. Hool lab)). Further, these methods were applied on cardiomyocytes and Cav1.2 channels originating from three different species (mouse, rat and human).
S-Nitrosylation is not a mechanism for significant Cav1.2 post-translational regulation
S-Nitrosylation of cardiac L-type Ca2+ channels was suggested to represent a mechanism for Ca2+ current inhibition by NOS-derived NO [5, 21, 28, 29]. To the best of our knowledge, however, direct evidence for this hypothesis is lacking. Here, we show that the presence of two different NO donors, expected to induce channel S-nitrosylation, did not affect the human Cav1.2 single-channel open probability (Fig. 6). This suggests that S-nitrosylation of Cav1.2 channels does not lead to channel inhibition, which is further supported by our cardiomyocyte experiments. Thus, neither the NO donor SNAP nor the applied NOS inhibitors exerted any substantial effects on the currents trough Ca2+ channels in these cells. Our results are in line with a recent study , which has investigated the molecular basis of the regulation of high voltage-activated Ca2+ channels, heterologously expressed in HEK cells, by S-nitrosylation. The authors found that, in contrast to currents through Cav2.2 (N-type) Ca2+ channels, Cav1.2 currents were hardly affected by the application of the NO donor SNAP. This was attributed to the fact that consensus motifs of S-nitrosylation were much more abundant on Cav2.2 compared with Cav1.2 channels. This study, together with our findings, suggests that S-nitrosylation is not a mechanism for significant Cav1.2 post-translational regulation.
We conclude from our results that—against the currently prevailing view—basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO regulation of Ca2+ cycling and contractility in myocytes can occur independent of Cav1.2 channel modulation.
Open access funding provided by Austrian Science Fund (FWF). We thank J. Uhrinova (Med. Univ. Vienna) for excellent technical assistance.
The conventional whole cell and perforated patch clamp studies, as well as the intracellular Ca2+ transient measurements were performed in the lab of K. Hilber. The single-channel recordings in liposomes were carried out in the lab of L.C. Hool.
J.E., H.K., H.T., L.C.H., K.H. and X.K. conceived or designed the study. J.E., M.C., P.L.S., A.K., B.K.P., H.CS., L.C.H., K.H. and X.K. contributed to acquisition, analysis or interpretation of data for the work. K.H., X.K. and L.C.S. drafted the work, and all authors critically revised it for important intellectual content. All authors approved the final version of the manuscript.
This work was supported by the Austrian Science Fund (FWF) (P30234-B27 to K.H.), and by the National Health and Medical Research Council of Australia (APP1103782 and APP1117366 to L.C.H.).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
All authors declared their informed consent.
The study conforms to the guiding principles of the Declaration of Helsinki, and with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). It further coincides with the rules of the Animal Welfare Committee at the Medical University of Vienna.
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