Published online before print October 2, 2003, doi: 10.1161/01.RES.0000099244.01926.56
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Neuropeptide Y Is an Essential In Vivo Developmental Regulator of Cardiac ICa,L
From the Department of Pharmacology (L.P., A.B., J.Q., V.O.R., S.F.S., R.B.R.) and Center for Molecular Therapeutics (S.F.S., R.B.R.), Columbia University, New York, NY; and the Department of Biochemistry and Howard Hughes Medical Institute (R.D.P.), University of Washington, Seattle, Wash.
Correspondence to Richard B. Robinson, PhD, Columbia University, Department of Pharmacology, 630 W 168th St, New York, NY 10032. E-mail rbr1{at}columbia.edu
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Abstract |
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Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (ICa,L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in ICa,L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy-/-) and matched controls (Npy+/+). Whole-cell voltage clamp demonstrates ICa,L density increases postnatally in Npy+/+ (by 56%), but is unchanged in Npy-/-. Both ICa,L density and action potential duration are significantly greater in adult Npy+/+ than Npy-/- myocytes, whereas ICa,L density is equivalent in neonatal Npy+/+ and Npy-/- myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in ICa,L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy+/+ and Npy-/-, indicating NPY does not influence the developmental change in ICa,L voltage-dependence. Immunoblot analyses and measurements of maximally activated ICa,L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy+/+ and Npy-/- cannot be attributed to altered Ca2+ channel
1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in ICa,L density in cardiac myocytes results from regulation ICa,L properties by NPY.
Key Words: neuropeptide Y • development • Ca2+ channel • innervation • ventricle
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Introduction |
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The mammalian cardiac L-type Ca2+ current (ICa,L) increases in density during postnatal development. Cell culture studies suggest sympathetic innervation may contribute, because in vitro innervation of neonatal rat ventricular myocytes by sympathetic neurons increases ICa,L density.1,2 However, the nature of the in vitro neural signal has been debated, and the extent to which neural signaling contributes to normal maturation of ICa,L during development in vivo is unknown.
Some data3 suggest the neural signal regulating ICa,L maturation is the neurotransmitter norepinephrine (NE). Our own studies suggest the relevant neural signal is neuropeptide Y (NPY), a 36-amino acid peptide present in and released from sympathetic nerve terminals. NPY fully mimics the effect of innervation in cell culture, and sustained exposure of innervated neonatal myocytes to an NPY antagonist fully prevents the effect of innervation in vitro.2 However, evidence is lacking for the relevance of NPY to normal postnatal maturation of ICa,L density in vivo. Therefore, we have taken advantage of a transgenic mouse containing a targeted deletion of the NPY gene4 to determine the effect of NPY deficiency during normal development on ICa,L properties in adult ventricular myocytes. The results demonstrate the essential role of NPY as an in vivo developmental regulator of ICa,L in the postnatal mammalian heart and provide insights into the mechanism of ICa,L modulation by NPY during maturation.
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Materials and Methods |
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Animals were maintained and experiments conducted in accordance with the US NIH Guide for the Care and Use of Laboratory Animals (publication No. 85-23). NPY-deficient (Npy-/-) mice in a 129SvEv background4 were bred at Columbia University and strain-matched wild-type (Npy+/+) mice were obtained from Taconic Laboratories (Germantown, NY).
Isolation of Cardiac Myocytes
Single ventricular cells were isolated from adult (8- to 10-week-old male mice) and newborn (3- to 4-day-old mice of either sex) hearts using a Langendorff perfusion of enzyme containing solution. A mixture of collagenase and trypsin (adult) or collagenase and protease (newborn) was used in the initial perfusion (see expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org). Isolated cells were maintained in enzyme-free solution at 4°C and used within 8 hours of isolation. Single cells were prepared from 44 adult (21 Npy+/+, 23 Npy-/-) and 6 neonatal (3 of each type) animals.
Electrophysiology
Only clearly striated rod-shaped adult and spindle-shaped newborn cells were studied. Cells were superfused with 35°C Tyrode solution containing (in mmol/L) 140 NaCl, 5.4 KCl, 1.0 CaCl2, 1.0 MgCl2, 5.0 HEPES, and 10 dextrose (pH 7.4). Whole-cell patch clamp recordings used 2 to 3 M
borosilicate glass pipettes (Sutter Instrument), pCLAMP 7 software, DigiData 1200 interface, and Axopatch 1D or 200B amplifier (Axon Instruments). To record action potentials, pipettes contained (in mmol/L) 130 aspartic acid, 146 KOH, 10 NaCl, 2.0 CaCl2, 1.0 MgCl2, 5.0 EGTA, 10 HEPES, and 2.0 MgATP (pH 7.2). For ICa,L recordings, pipettes contained (in mmol/L) 80 aspartic acid, 10 NaCl, 70 CsOH, 40 CsCl, 2.0 MgCl2, 10 EGTA, 10 HEPES, 2.0 ATP Na2, and 0.1 GTP Na2 (pH 7.2).
ICa,L was recorded in Cs-containing bath solution: (in mmol/L) 135 NaCl, 10 CsCl, 1 CaCl2, 1 MgCl2, 5 HEPES, 10 dextrose, and 0.01 tetrodotoxin (pH 7.4). In control experiments, ICa,L was recorded as a single voltage step every 4 seconds to assess rundown and eliminate possible changes in density and inactivation kinetics from facilitation (see Anderson5). No significant difference in rundown rate was seen between adult Npy-/- and Npy+/+ myocytes: after 4 minutes, peak amplitude was (as percent initial) 91±6 (n=8) and 90±7 (n=7), respectively. In I-V experiments, current stability was usually checked for 2 minutes before beginning the protocol. In cells from newborn mice, rundown was faster (peak amplitude 85±10% after 2 minutes in Npy-/-, n=3), and the I-V protocol completed within 1 minute of membrane rupture.
I-V curves were generated as nifedipine-sensitive (10 µmol/L) difference currents using 200-ms steps from -30 to +60 mV (holding potential -40 mV), and activation relations calculated as previously described.6 To insure that larger current amplitudes in adult myocytes did not cause loss of voltage control and error in the activation relation, we performed regression analysis of current amplitude versus midpoint and slope of activation; no significant correlation was observed. Steady-state inactivation and recovery from inactivation were measured in Na-free solution as previously described,6 with minor differences in voltage commands (see Figures). Na-free solution also was used to measure the response to forskolin and BayK 8644 from a -60-mV holding potential. To measure facilitation, ICa,L was generated by 100-ms voltage steps from -40 to 20 mV every 2 seconds. After 60 seconds, the protocol was paused for 60 seconds, then resumed and peak current of consecutive traces measured.
Transient outward current (Ito) was recorded with solutions and protocols as described by Dun et al,7 except the test voltage duration was 3 seconds, and defined as the peak 4-aminopyridine (4-AP, 3 mmol/L)–sensitive current.
Electrophoresis and Immunoblotting
Light sarcolemmal (LS) and heavy membrane (HM) fractions were isolated from ventricles of Npy+/+ and Npy-/- hearts according to Anborgh et al,8 with modification. Proteins were resolved by SDS-PAGE, and immunoblot analysis was performed with antibodies to the cardiac type 1C Ca2+ channel, caveolin-3, the Na/K-ATPase 1 subunit, and cardiac types V/VI adenylyl cyclase (AC). See online data supplement for details.
All samples were run at 2-fold different levels of protein loading, which were optimized in preliminary experiments to be well within the dynamic range of the ECL detection for each protein (to allow for differences in protein abundance and/or antibody sensitivity). ECL is not a linear detection system: under the conditions used in these experiments, a 2-fold difference in protein loading corresponded to 
2- to 3-fold difference in immunoreactivity (necessitating a separate analysis of results obtained at different protein loadings).
Data Analysis
Activation and inactivation data were fit to a Boltzmann function and recovery from inactivation data fit to a biexponential function (Origin 6, Microcal). All data are presented as mean±SE. Statistical significance was determined by ANOVA or Student’s t test as appropriate and defined as P<0.05.
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Results |
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Basic Characterization of Npy-/- and Npy+/+ Myocytes
As previously reported,4 Npy-/- mice were of normal body weight, with no significant difference in weight of adult ventricles (107.6±5.9 mg in Npy+/+ versus 95.7±2.8 mg in Npy-/-). Myocyte size did not differ for either adult [capacitance, 126±3.9 pF (n=74) for Npy+/+; 136±3.8 pF (n=68) for Npy-/-] or newborn [23.7±4.1 pF (n=8) and 24.7±2.2 pF (n=6), respectively] animals.
Action potential characteristics were studied in adult myocytes (Figure 1). Neither action potential amplitude nor resting potential differed significantly between Npy+/+ and Npy-/- myocytes (amplitude, 125.9±2.8 versus 123.4±2.8 mV; resting potential, -67.2±1.1 versus -64.9±0.9 mV) (P>0.05; n=17 and 15, respectively). Action potential duration (APD) was measured at times ranging from 30% to 90% repolarization. ANOVA indicated APD was significantly greater in Npy+/+ than Npy-/- myocytes.
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Because Ito is a major repolarizing current in mouse ventricle, we asked if the reduced APD in Npy-/- myocytes resulted from enhanced Ito. However, Ito magnitude was not significantly greater in Npy-/-. Peak current at +60 mV was 26.6±4.5 and 23.3±3.8 pA/pF in Npy+/+ and Npy-/-, respectively (n=11 and 8; P=0.596).
ICa,L Activation and Inactivation
In Figure 2, typical traces of the nifedipine-sensitive current and mean I-V curves for adult Npy+/+ and Npy-/- cells are shown. Peak ICa,L density in adult cells was maximal at 0 mV in both preparations; however, current density was significantly smaller in Npy-/- than Npy+/+ cells (ANOVA, two curves differ significantly; P<0.001). At 0 mV Npy-/- current density was 36% smaller (9.64±0.77 pA/pF, n=9, versus 15.05±1.28 pA/pF, n=10). Unlike in adult, ICa,L from Npy-/- and Npy+/+ newborn cells had statistically equivalent I-V relations (Figure 3), with maximum at +10 mV. Maximal peak-current density was 9.19±1.26 pA/pF (n=8) and 9.91±1.04 pA/pF (n=6) for newborn Npy-/- and Npy+/+ cells, respectively. Thus, there is no prenatal effect of NPY deficiency on cardiac ICa,L. Both peak current density and the I-V relation were equivalent in newborn ventricle cells from Npy-/- and Npy+/+ animals. Further, in the absence of NPY exposure in vivo, there is no postnatal increase in ICa,L density, as the adult peak current density in the Npy-/- animals is equivalent to that in either newborn animal (Figure 4A).
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The reversal potential, determined from extrapolation of the linear portion of the positive limb of the I-V curves, did not differ between groups and was used to construct activation relations (Figure 4B). The two newborn curves were the most positive and essentially superimposable, with midpoints (V1/2) of -2.0±2.0 mV (n=8) and -2.9±1.5 mV (n=6) in Npy-/- and Npy+/+, respectively. The adult activation curves were significantly more negative (V1/2: -11.1±1.9 mV, n=10, for adult Npy+/+, P=0.005 relative to newborn Npy+/+; -9.2±1.0 mV, n=9, for adult Npy-/-, P=0.004 relative to newborn Npy-/-). The adult V1/2 values did not differ from each other. Slope factors of the activation curves for adult Npy+/+ and Npy-/- cells also did not differ significantly (4.9±0.2 and 5.1±0.2 mV, respectively), but were significantly smaller than values for newborn cells (6.2±0.2 and 6.5±0.4 mV for Npy+/+ and Npy-/-, respectively; P<0.005 for both adult versus newborn comparisons), suggesting an age-dependent steepening of the activation relation independent of postnatal NPY exposure.
The reduced current density in adult Npy-/- could result from a difference in voltage dependence of inactivation or recovery from inactivation, such that a smaller fraction of channels were available during the voltage protocol in Npy-/- than Npy+/+ cells. Therefore, we determined the steady-state inactivation relation. We found no significant difference in ICa,L availability between adult Npy+/+ and Npy-/- cells (Figure 5). Threshold for inactivation was about -40 mV for both groups. V1/2 values were -25.3±0.8 mV (n=8) and -26.7±1.3 mV (n=6), respectively; slope factors were 4.7±0.2 and 5.2±0.5 mV, respectively. Thus, the reduced peak current in Npy-/- cells does not result from a shift in inactivation relation.
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The reduced peak current in Npy-/- cells also could arise from slower recovery from inactivation. Figure 6 illustrates this is not the case, and that recovery from inactivation is faster in Npy-/-. Mean time dependence of recovery of ICa,L from inactivation is illustrated, with the initial portion expanded in the inset. The time course of recovery is best described by a biexponential function. Although both the fast and slow time constants (
) are statistically equivalent in both groups (fast , 47.9±9.2 ms, n=10, and 49.4±5.2 ms, n=8 for Npy-/- and Npy+/+, respectively; slow , 470.3±144.3 and 355.2±66.2 ms, respectively), the fraction of current ascribed to the fast component was significantly greater in Npy-/- (0.85±0.03 versus 0.68±0.07, P=0.033). In some cases, at interpulse intervals of 1.0 to 1.6 seconds, ICa,L produced by the second (test) pulse was greater than control current, which may be a result of facilitation. This was seen in 1 of 8 Npy+/+ and 3 of 10 Npy-/- cells. We further explored this phenomenon in conditions (see Materials and Methods) similar to those in the recovery experiments. In both groups the amplitude of the current induced by 0.5 Hz stimulation after a 60-second period of rest increased, and reached a maximum at the 4th test pulse (not shown); the increase was modest for both preparations but significantly (P=0.011) greater in Npy-/- cells (8.5±1.4%, n=7) than Npy+/+ (3.5±1.1%, n=8). Both the faster recovery from inactivation and greater facilitation in Npy-/- myocytes, if also present at physiological resting potentials, would tend to mitigate the difference in peak current density at the rapid heart rates typical of mouse.
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Calcium Channel 1C Subunit Protein Expression and Maximal Responsiveness
Given that the reduced peak current density of adult Npy-/- myocytes cannot be explained by a difference in steady-state inactivation or recovery from inactivation, we used immunoblot analysis to determine whether L-type Ca2+ channel 1C subunit protein expression differs between the two preparations. To optimize immunodetection of the membrane-associated Ca2+ channel protein, we used a differential centrifugation protocol to separate LS membranes from other cellular membranes. The LS fraction contains only a minor amount of total membrane protein and is highly enriched in Na+/K+-ATPase, cardiac type V/VI AC isoforms, and caveolin-3 (plasma membrane and lipid raft markers), relative to the HM fraction (Figure 7A); unexpectedly, protein recovery in the LS from Npy+/+ is significantly higher than from Npy-/-. Protein recovery in the HM from Npy+/+ and Npy-/- does not differ. Figure 7B shows that Ca2+ channel 1C subunit immunoreactivity is detected as a 200-kDa band, which corresponds to mobility of the full-length protein,9 in both LS and HM fractions. An additional minor 160-kDa immunoreactive species (which is consistent with a C-terminal truncation of the pore-forming Ca2+ channel 1C subunit) was detected with the anti-1C subunit antibody (directed against the N-terminus) only in HM. Both 200- and 160-kDa bands are epitope specific and are blocked by competing antigen peptide. Figures 7C and 7D show that 1C immunoreactivity (expressed per mg protein) is similar in LS membranes from Npy+/+ and Npy-/-; 1C immunoreactivity in HM fractions (which contain the bulk of membrane protein) is actually 30% higher in Npy-/- ventricles, compared with the Npy+/+. These results indicate that the smaller current density in Npy-/- hearts does not result from a gross decrease in total L-type Ca2+ channel 1C subunit expression. Rather, the reduced yield of total LS protein, in the context of higher levels of HM fraction 1C subunit immunoreactivity in Npy-/- ventricles (relative to Npy+/+ controls), could suggest a role for NPY in calcium channel targeting to a functionally important membrane pool.
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To further investigate whether there is a difference in total available functional channel density, we conducted experiments to compare maximally activated current density in myocytes from Npy+/+ and Npy-/- animals. We reasoned that if there is a difference in basal channel activity rather than density of functional channel protein, then a protocol designed to maximally stimulate protein kinase A (PKA)–dependent phosphorylation might eliminate the difference between Npy-/- and Npy+/+ cells. Forskolin is a direct activator of AC (which is expressed at equivalent levels in Npy-/- and Npy+/+ ventricles; Figure 7) and has been used successfully in previous developmental studies of ICa,L in rabbit myocytes.10 Therefore, we exposed adult cells to forskolin (10 µmol/L) to maximally stimulate AC during repetitive imposition of a voltage step to 0 mV, and determined the full I-V relation before and 2 to 3 minutes after start of forskolin exposure. For these experiments ICa,L was defined as peak minus steady-state current, rather than the nifedipine-sensitive current. There is a strong and reversible response to forskolin (Figure 8) and the magnitude of that response is significantly greater in Npy-/- than Npy+/+ myocytes (P=0.025). In the absence of forskolin, the measured ICa,L density statistically differs between Npy-/- and Npy+/+ cells (P=0.015), but after exposure to forskolin, current density no longer statistically differs between the two groups. A similar result was obtained using the L-type channel agonist BayK 8644 (Figure 8C).
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Discussion |
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This is the first study to provide direct evidence that NPY exerts an effect on maturation of any cardiac ion channel during postnatal development in vivo. Our main finding is that ICa,L recorded in ventricular myocytes from NPY-deficient mice has a reduced density compared with the current from Npy+/+ animals. Consistent with this finding, APD was significantly shorter in NPY-/- myocytes, and this was not due to an increase in Ito. These results demonstrate that NPY is an obligatory developmental factor controlling the properties of L-type Ca2+ channels in the heart and confirms our previous results using an in vitro cell culture system.2
Additional information on the role of NPY in developmental regulation of L-type Ca2+ current was provided in the newborn animal experiments. The existence of ICa,L in cultured ventricular myocytes was previously demonstrated for fetal or postnatal stages of mouse development,11–13 but this study provides the first data comparing ICa,L in freshly isolated postnatal newborn and adult mouse cardiac myocytes. In agreement with data from other mammalian species,14–16 the results with Npy+/+ cells show a developmental increase in ICa,L density. However, Npy-/- and Npy+/+ newborn myocytes had ICa,L of identical density and voltage-dependence, indicating the divergence of ICa,L in Npy-/- and Npy+/+ ventricles is established postnatally, along with maturation of sympathetic innervation of the heart. Further, it is striking that in the absence of NPY, there is no postnatal increase in current density, suggesting that NPY is the sole contributor to this developmental change in the mouse. It also is striking that the difference in current density, with adult Npy-/- being 36% reduced from that of Npy+/+, is remarkably similar to the difference observed in neonatal rat ventricle cultures as a result of sympathetic innervation (39%) or NPY (34%).2
Not all developmental changes in ICa,L are NPY-dependent. NPY-independent factors appear responsible for the age-dependent shift in voltage-dependence of ICa,L activation (seen in both the voltage corresponding to the peak of the I-V relation and in the calculated activation relations), because an equivalent negative developmental shift occurred in both Npy-/- and Npy+/+ cells. This shift was not parallel: the steepness of the activation curves constructed in both adult cell preparations was significantly greater than that of either newborn cell preparation. For reasons detailed in the Materials and Methods section, we do not believe this negative shift in activation results from loss of voltage control when clamping the larger adult cells with their correspondingly greater ICa,L. Further, the shift is equivalent in Npy-/- and Npy+/+ cells despite current amplitude being significantly greater in Npy+/+ myocytes. However, because these are calculated activation relations, the results should be confirmed by direct determination of the activation relation from tail current measurements.
Although the present results argue that the postnatal increase in L-type current density is entirely NPY-dependent, others have suggested a contribution of adrenergic agonists.3 Cell culture studies using rat heart cells confirm that exposure to NE exerts transcriptional effects on the 1C subunit of the channel, increases message and protein levels, and increases current density.3 Additional experiments in vivo demonstrate that sustained exposure to elevated NE levels in the adult animal also affects these parameters.17 However, it has not been demonstrated that exposure of innervated myocytes in culture to adrenergic receptor blockers prevents the effect of sympathetic innervation. Further, the effect of innervation in vitro is localized, affecting only physically innervated myocytes and not adjacent ones.1 We previously demonstrated that effects of innervation in vitro on the Na+ channel, which are mediated by NE, are global and can be replicated by nerve-conditioned medium as well as by physical innervation.18 In contrast, other effects of innervation in vitro that are mediated by NPY are not replicated by nerve-conditioned medium,19,20 confirming the localized nature of this signaling pathway in cell culture. Thus, although NE clearly exerts transcriptional effects on the L-type Ca2+ channel, there is no direct evidence that this contributes to the increased ICa,L density resulting from either sympathetic innervation in vitro or normal development. In addition, given the known function of NPY as a negative feedback inhibitor of NE release, it would be expected that sympathetic activity might be greater in the Npy-/- animals. Thus, if NE exerts an important developmental influence on ICa,L one might predict current density would have actually been greater in the Npy-/- myocytes.
Liu et al21 reported an approximate 2-fold increase in 1C subunit protein levels in rat heart homogenates between birth and adult, whereas Haase et al22 reported a decline in 1C protein levels postnatally in rat heart. We are not aware of comparable immunoblot studies in mouse heart. Although we did not conduct immunoblot studies in neonatal hearts from Npy+/+ or Npy-/-, our immunoblot analyses of adult tissue failed to identify a gross difference in 1C subunit expression that could account for the NPY-dependent increase in calcium current density in mouse heart. However, the bulk of the 1C subunit immunoreactivity (including the truncated form of the 1C subunit) was recovered in a HM fraction that contained relatively low levels of surface membrane protein markers, and which exhibited elevated (rather than reduced) levels of 1C subunit immunoreactivity in the Npy-/- hearts. This raises the possibility that 1C subunits in the HM might be intracellular channels, and that a functionally important pool of 1C subunits in the LS membrane fraction is reduced in Npy-/- hearts (with lower protein recovery in the LS fraction, relative to Npy+/+). However, we cannot rule out several alternative interpretations of the results. (1) It is possible that an NPY-induced change in cellular cytoarchitecture alters the efficiency of LS membrane recovery (ie, that the difference in LS protein recovery is not due to a bone fide difference in the total amount of cell surface LS membrane in the tissue). (2) Because any biochemical approach that compares L-type calcium channel expression (or the expression of any other low abundance channel/signaling protein) must be performed on some type of enriched membrane fraction, some level of uncertainty related to the efficiency of channel protein recovery from the starting tissue sample is unavoidable. (3) Finally, NPY-dependent changes in ICa,L might be due to an alteration in the expression of other Ca2+ channel isoforms or subunits. In this regard, studies in other systems predict that an increase in 1D expression could contribute to the negative shift in activation observed developmentally,23 whereas an increase in ß2-subunit expression could contribute to increased ICa,L density.24
The functional studies with forskolin and BayK 8644, in conjunction with the immunoblotting results, strongly suggest that the difference in current density between Npy+/+ and Npy-/- myocytes might be attributable to differences in posttranslational modifications or cellular factors that regulate channel activity, rather than differences in actual expression of channel protein. Consistent with this hypothesis, in the presence of maximal stimulation by forskolin or BayK 8644, current density no longer differs between the two preparations. A similar differential effect of forskolin in ventricle myocytes was observed in a developmental study of ICa,L in rabbit.10 The present results suggest that functionally relevant absolute protein levels are similar in the two preparations, but that under basal conditions the L-type Ca2+ channels of Npy+/+ and Npy-/- myocytes possess distinct properties. Whereas the forskolin data are compatible with a difference in basal activation by the adenylyl cyclase pathway, the results with BayK 8644 argue that the difference in current density more likely arises from differences in the biophysical properties of the channel. Additional experiments, including single channel analysis, are needed to identify the precise biophysical characteristics influenced by NPY. Further, elucidation of the relevant signaling cascade that NPY influences will require studies with selective activators or inhibitors. In this regard, it may be relevant that exposure of neonatal mouse cardiac myocytes to NPY results in activation of a pathway that includes the Y5 NPY receptor subtype, a pertussis toxin sensitive guanine nucleotide regulatory protein and activation of mitogen-activated protein kinase.25 Whether the same cascade is involved in long-term regulation of ICa,L density by NPY remains to be determined.
The effects of NPY in this preparation are not necessarily restricted to direct action on myocardial cell NPY receptors, as there may be indirect and compensatory actions in the developing animal in vivo. Because the magnitude of the NPY-dependent increase in ICa,L density observed in this study is comparable to that reported during incubation of neonatal myocytes with NPY in cell culture, such secondary NPY actions may not be relevant to regulation of cardiac L-type Ca2+ channels. However, secondary and/or compensatory effects, as well as additional direct effects not studied, could affect the physiological impact of reduced Ca2+ current density. For example, NPY has been reported to exert additional long-term effects on myocardial cells, including increased ß-adrenergic receptor density26 and transient outward current density,27 and Npy-/- animals also would be expected to have reduced presynaptic feedback inhibition of autonomic activity. At least some of these effects could act to reduce the physiological impact of reduced ICa,L density on Ca2+ influx during an action potential in vivo. Although we did not observe a significant difference in Ito in this study, we only measured peak 4-AP sensitive current. There are known to be multiple components of transient outward current in mouse ventricle with complex kinetics,28 and a more careful analysis of the individual components is required to fully resolve this question. In addition, the faster recovery from inactivation in Npy-/- myocytes also might mitigate any difference in ICa,L density, although the extent of this difference at a normal resting potential remains to be determined. Similarly, the difference in APD and therefore its influence on repolarization time course and activation of other currents is likely to be highly dependent on intrinsic heart rate. Thus, the extent of the differences in APD and ICa,L in the intact animal, whether there are additional differences in other aspects of Ca2+ homeostasis and the effect of any such differences on cardiac output, remain to be determined.
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Acknowledgments |
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Acknowledgments
This work was supported by NIH program project grant HL-28958 and a grant-in-aid from the Heritage Affiliate of the American Heart Association.
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Footnotes |
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Original received February 20, 2003; revision received September 22, 2003; accepted September 24, 2003.
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References |
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