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In utero exposure to nicotine abolishes the postnatal response of the cardiac sodium current to isoproterenol in newborn rabbit atrium

      Background

      In utero exposure to tobacco smoke is associated with sudden infant death syndrome (SIDS) and cardiac arrhythmias in newborns. The arrhythmogenic mechanisms seem linked to alterations of the cardiac sodium current (INa). We previously reported that in utero exposure to nicotine delays the postnatal development of the heart sinoatrial node in rabbits and altered expression of the sodium channels NaV1.5 and NaV1.1 in the atrium surrounding it. These channels react differently to sympathetic stimulation.

      Objective

      The purpose of this study was to test whether nicotine altered the response of INa to stimulation by the β-adrenoreceptor agonist isoproterenol in atrial myocytes. Our hypothesis is that changes in the sympathetic response of sinoatrial node peripheral cells may create a substrate for arrhythmia.

      Methods

      Using the patch-clamp technique we measured the effect of nicotine on the response of INa to adrenergic stimulation in isolated cardiomyocytes.

      Results

      Isoproterenol increased INa by 50% in newborn sham rabbits but had no effect in newborn rabbits exposed to nicotine in utero. Our data also show that nicotine increases the late sodium current, an effect that may promote QT prolongation.

      Conclusion

      We provide the first evidence linking fetal exposure to nicotine to long-term alterations of INa response to isoproterenol. These changes may impair INa adaptation to sympathetic tone and prevent awakening from sleep apnea, thus leading to arrhythmias that could potentially be involved in SIDS. Our data also raise concerns about the use of nicotine replacement therapies for pregnant women.

      Keywords

      Introduction

      Sudden infant death syndrome (SIDS) is the leading cause of death in the first year of life. In utero exposure to tobacco smoke remains the highest risk factor in 85% of cases.
      • Haglund B.
      • Cnattingius S.
      Cigarette smoking as a risk factor for sudden infant death syndrome: a population-based study.
      Therefore, it seems logical to prescribe nicotine replacement therapy to pregnant women who wish to quit smoking. However, of all the toxic compounds found in smoke, only nicotine is associated with cardiac arrhythmias in newborns.
      • Chow F.A.
      • Seidler F.J.
      • McCook E.C.
      • Slotkin T.A.
      Adolescent nicotine exposure alters cardiac autonomic responsiveness: beta-adrenergic and m2-muscarinic receptors and their linkage to adenylyl cyclase.
      • D'Alessandro A.
      • Boeckelmann I.
      • Hammwhoner M.
      • Goette A.
      Nicotine, cigarette smoking and cardiac arrhythmia: an overview.
      This raises concerns regarding nicotine replacement therapy during pregnancy and suggests that nicotine altered ion channels involved in cardiac function.
      Evidence links SIDS to failed coordination of the cardiovascular and respiratory systems during postnatal development of the heart causing failure to awake from sleep apnea and sudden death.
      • Tuladhar R.
      • Harding R.
      • Michael A.T.
      • Horne R.S.
      Comparison of postnatal development of heart rate responses to trigeminal stimulation in sleeping preterm and term infants.
      Although multiple causes of sleep apnea have been proposed, the final step in SIDS is cardiac arrhythmia.
      Changes in cardiac conduction, cellular excitability, and prolongation of action potential duration (APD)
      • Schwartz P.J.
      • Priori S.G.
      • Dumaine R.
      • et al.
      A molecular link between the sudden infant death syndrome and the long-QT syndrome.
      are common occurrences in SIDS.
      • Biet M.
      • Morin N.
      • Lessard-Beaudoin M.
      • et al.
      Prolongation of action potential duration and QT interval during epilepsy linked to increased contribution of neuronal sodium channels to cardiac late Na+ current: a potential mechanism for sudden death in epilepsy.
      These changes suggest that INa is involved because it is responsible for conduction and modulates APD.
      • Biet M.
      • Morin N.
      • Lessard-Beaudoin M.
      • et al.
      Prolongation of action potential duration and QT interval during epilepsy linked to increased contribution of neuronal sodium channels to cardiac late Na+ current: a potential mechanism for sudden death in epilepsy.
      • Dumaine R.
      • Antzelevitch C.
      Molecular mechanisms underlying the long QT syndrome.
      Moreover, the sustained (late) component of INa modulates the slope of diastolic depolarization in pacemaker cells, thereby influencing heart rhythm.
      • Huang X.
      • Ma A.Q.
      • Yang P.
      • Du Y.
      • Xi Y.T.
      • Geng T.
      [Expression and function of voltage-gated Na+ channel isoforms in rat sinoatrial node].
      • Lei M.
      • Jones S.A.
      • Liu J.
      • et al.
      Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking.
      Therefore, we tested whether in utero exposure to nicotine altered INa activity in a way that could explain these electrophysiological changes associated with SIDS.
      During postnatal development, the sinoatrial node (SAN) gradually shrinks, and the surrounding tissues are replaced by transitional atrial cells.
      • Boyett M.R.
      • Honjo H.
      • Kodama I.
      The sinoatrial node, a heterogeneous pacemaker structure.
      This developmental process is associated with increased expression of neuronal sodium channels (nNaV) in the central regions of the SAN but enhanced expression of the cardiac isoform NaV1.5 in peripheral cells.
      • Lei M.
      • Jones S.A.
      • Liu J.
      • et al.
      Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking.
      Because half-maximal inactivation of NaV1.5 channels occurs at voltages more hyperpolarized (−15 to −25 mV) than nNaVs,
      • Biet M.
      • Barajas-Martinez H.
      • Ton A.T.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      About half of the late sodium current in cardiac myocytes from dog ventricle is due to non-cardiac-type Na(+) channels.
      • Haufe V.
      • Cordeiro J.M.
      • Zimmer T.
      • et al.
      Contribution of neuronal sodium channels to the cardiac fast sodium current I(Na) is greater in dog heart Purkinje fibers than in ventricles.
      NaV1.5 channels are completely inactivated at the maximum diastolic potential of SAN cells (−60 mV)
      • Schram G.
      • Pourrier M.
      • Melnyk P.
      • Nattel S.
      Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function.
      while remaining available at the resting potential of atrial myocytes (Vr ∼ −70 mV) and in the ventricle (Vr ∼ −90 mV).
      • Dumaine R.
      • Cordeiro J.M.
      Comparison of K+ currents in cardiac Purkinje cells isolated from rabbit and dog.
      • Cordeiro J.M.
      • Zeina T.
      • Goodrow R.
      • et al.
      Regional variation of the inwardly rectifying potassium current in the canine heart and the contributions to differences in action potential repolarization.
      Therefore, depolarized atrial cells in the close periphery of the SAN are less excitable during postnatal development, thus creating a layer of cardiomyocytes that electrically isolate the pacemaker center from the surrounding atrium. On the other hand nNaVs continue to remain active within the SAN, thereby contributing to diastolic depolarization. INa is responsible for propagation of the electrical impulse. This maturation process is essential to guiding this impulse through conduction pathways from the SAN to the ventricle, preventing the SAN from exciting peripheral atrial cells and meeting cardiac demand as the infant is developing.
      • Lei M.
      • Jones S.A.
      • Liu J.
      • et al.
      Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking.
      • Maier S.K.
      • Westenbroek R.E.
      • Yamanushi T.T.
      • et al.
      An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node.
      We previously reported that nicotine decreased postnatal expression of nNaVs (especially NaV1.1) in the SAN but increased expression of the cardiac isoform NaV1.5.
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      The reduced contribution of nNaVs to diastolic depolarization within the SAN may slow sinus rhythm,
      • Lei M.
      • Jones S.A.
      • Liu J.
      • et al.
      Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking.
      and enhanced expression of NaV1.5 in the atrium may change excitability and conduction. Sympathetic tone is a strong modulator of conduction and excitability. Interestingly, β-adrenergic receptors have opposite effects on NaV1.5 and nNaVs, and changes in their activity during development may influence the cardiac response to adrenergic stimulus. The effect of nicotine on the sympathetic response of INa is unknown.
      • Murray K.T.
      • Hu N.N.
      • Daw J.R.
      • et al.
      Functional effects of protein kinase C activation on the human cardiac Na+ channel.
      • Li M.
      • West J.W.
      • Numann R.
      • Murphy B.J.
      • Scheuer T.
      • Catterall W.A.
      Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase.
      • Gershon E.
      • Weigl L.
      • Lotan I.
      • Schreibmayer W.
      • Dascal N.
      Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes.
      • Smith R.D.
      • Goldin A.L.
      Functional analysis of the rat I sodium channel in xenopus oocytes.
      We and others demonstrated that protein kinase A (PKA) activation, after isoproterenol stimulation, increases INa by recruiting NaV1.5 channels to the sarcolemma in native myocytes and HEK cells.
      • Murray K.T.
      • Hu N.N.
      • Daw J.R.
      • et al.
      Functional effects of protein kinase C activation on the human cardiac Na+ channel.
      • Li M.
      • West J.W.
      • Numann R.
      • Murphy B.J.
      • Scheuer T.
      • Catterall W.A.
      Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase.
      • Gershon E.
      • Weigl L.
      • Lotan I.
      • Schreibmayer W.
      • Dascal N.
      Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes.
      • Smith R.D.
      • Goldin A.L.
      Functional analysis of the rat I sodium channel in xenopus oocytes.
      In contrast, stimulation of protein kinase C (PKC) favors internalization of NaV1.5
      • Murray K.T.
      • Hu N.N.
      • Daw J.R.
      • et al.
      Functional effects of protein kinase C activation on the human cardiac Na+ channel.
      • Qu Y.H.
      • Rogers J.C.
      • Tanada T.N.
      • Scheuer T.
      • Catterall W.A.
      Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C.
      and decreases the activity of nNaVs. Because PKA and PKC differentially modulate each isoform, we tested the hypothesis that postnatal changes in the balance of sodium channels expressed in the atrium alter INa response to the β-adrenergic receptor agonist isoproterenol and create a substrate for arrhythmias.

      Material and methods

      All animal procedures, care, and maintenance were approved by the ethics review board of the Faculty of Medicine and Health Sciences of the Université de Sherbrooke and follow the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

      Animal model

      New Zealand female rabbits (Charles River, Quebec, Canada) were mated and implanted subcutaneously with 2 osmotic pumps (Alzet, Cupertino, CA) each containing 2 mL of nicotine solution (Sigma-Aldrich, Canada) after 14 days of gestation. Sham does were exposed to saline solution instead of nicotine. Serum concentration of cotinine (nicotine metabolite) was measured every 2 days (Supplemental Methods).

      Cardiomyocyte dissociation

      Twenty-six to 30 days after birth, rabbits of either sex were deeply anesthetized, and their hearts were quickly excised and perfused with a collagenase II solution (see Supplemental Methods for details).

      Electrophysiology

      Myocytes were placed in a chamber mounted on the stage of an inverted microscope and superfused with a low-sodium solution cocktail containing K+ and Ca2+ blockers as previously described
      • Biet M.
      • Barajas-Martinez H.
      • Ton A.T.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      About half of the late sodium current in cardiac myocytes from dog ventricle is due to non-cardiac-type Na(+) channels.
      (see Supplemental Methods for details). INa was measured at room temperature (22°C) in the whole-cell configuration of the patch-clamp technique.

      Data analysis

      Data acquisition and analysis were performed using the pCLAMP program suite V10 (Axon Instruments, Molecular Devices, CA) and ORIGIN 8 (Microcal Software, MA) software, respectively. Activation and inactivation data were fitted to a Boltzmann distribution function. Conductance (GNa) was calculated from the ratio INa/(Vm – ENa). GNa,Max was obtained from slope of the linear portion of the current–voltage (I/V) relationship. Window current was calculated using the Hodgkin–Huxley formalism
      • Hodgkin A.L.
      • Huxley A.F.
      The dual effect of membrane potential on sodium conductance in the giant axon of Loligo.
      (detailed explanation in Supplemental Methods).

      Statistical analysis

      Data are expressed as mean ± SEM. Significance was established by analysis of variance (with repetition) for unpaired data. An F test was used to assess differences between fit to data. Data were significantly different for P <.05.

      Results

      Using subcutaneous osmotic pumps in gestating rabbit does, we showed that a nicotine plasma concentration similar to that found in light smokers (100 and 150 ng/mL) can be maintained.
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      Survival plot shows that 5 of 20 newborn rabbits exposed to nicotine died suddenly within the first 5 days of life compared to none in untreated animals (Figure 1). Because cotinine plasma level remains elevated up to 25 days after birth
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      and we wanted to test for long-term alterations of INa, we performed our measurements 30 days postnatal.
      Figure thumbnail gr1
      Figure 1Kaplan–Meier plot (percent of survival) for 60 kittens exposed to nicotine in utero (Nico). No sudden death occurred in the nonexposed kittens (Sham; n = 60).
      During postnatal development, the periphery of the SAN is replaced by less excitable atrial cardiomyocytes, a process called resorptive degeneration.
      • Ottaviani G.
      • Matturri L.
      • Rossi L.
      • James T.N.
      Crib death: further support for the concept of fatal cardiac electrical instability as the final common pathway.
      • Matturri L.
      • Ottaviani G.
      • Lavezzi A.M.
      • Turconi P.
      • Cazzullo A.
      • Rossi L.
      Expression of apoptosis and proliferating cell nuclear antigen (PCNA) in the cardiac conduction system of crib death (SIDS).
      We previously reported that nicotine modulates expression of neuronal and cardiac sodium channels during this process
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      and may influence INa activity. To complement those data, we tested how nicotine altered INa response to adrenergic stimulus in the right atrium close to the SAN. INa maximum amplitude in cardiomyocytes from nicotine-exposed kittens was 48% ± 9% higher vs sham (Figures 2A and 2C). INa was maximum at −40 mV (Figure 2C) and −30 mV (Figure 2B) in nicotine-exposed and sham myocytes, respectively. In sham myocytes, superfusion with 10 μmol/L isoproterenol increased INa maximum amplitude by 91% ± 9% (Figures 2A and 2B). Analysis of the I/V relationship in sham myocytes showed INa activation threshold occurred at a voltage of –60 mV in control and –70 mV (Figure 2B) with isoproterenol, thus indicating that cells are excitable at voltages closer to the resting membrane potential during adrenergic stimulation. Maximum INa amplitude voltage was similarly shifted from –30 mV in control to –45 mV with isoproterenol. Analysis of the maximum conductance (Figure 2E) obtained from the I/V curves revealed that isoproterenol did not change GMax density, with values of 2.9 ± 0.2 pS/pF and 3.1 ± 0.2 pS/pF in control and isoproterenol-exposed myocytes, respectively. Because GMax represents the maximum number of channels available, this result suggests the increase in INa amplitude in sham myocytes is in part due to alterations in the gating of the NaVs. This was confirmed by analysis of the activation curve (Figure 2F), which showed that isoproterenol shifted the voltage at which INa reached 50% of its maximum value (V0.5) from −37.5 ± 0.2 mV in control to −51.7 ± 0.5 mV.
      Figure thumbnail gr2
      Figure 2In utero exposure to nicotine abolished the effect of β–adrenergic stimulation on the cardiac sodium current (INa). A: Representative sodium current recordings (INa) in cardiomyocytes isolated from the right atrium of sham and kittens exposed to nicotine in utero in control (Ctrl) and after perfusion with isoproterenol (+Iso). INa was elicited by 30-ms test pulses from a holding potential of –120 mV (inset). B, C: Current–voltage (I/V) relationship. Peak INa was normalized to the capacitance of their respective cells and plotted as current density (pA/pF) for each test potential. Number of cells: sham (n = 8), sham Iso (n = 9), nicotine (n = 8), nicotine + Iso (n = 6). Cells from 3 animals were tested for each condition (minimum 2 cells per animal). D: Maximum current density (from B and C) (**P <.01, t test on paired values). E: Isoproterenol did not increase the maximum conductance (GNa,Max) of INa. GNa was calculated as the ratio INa/(Vm-ENa), where the denominator represents the driving force for the current, and ENa and Vm are the reversal potential for the current (from B and C) and the test voltage, respectively. The ratio GNa/(GNa,Max) therefore represents the fraction of channels activated at each voltage. F, G: Isoproterenol (open symbols) shifted the-mid activation potential (V0.5) of sham myocytes from −37.5 ± 0.2 mV to −49.5 ± 0.9 mV (P <.05, paired t test) but did not in nicotine-exposed (filled symbols) animals (−53.4 ± 0.3 mV and −46.3 ± 0.4 mV). Difference between control values in sham and nicotine were significant (P <.01, 2-way analysis of variance). Data are given as mean ± SEM.
      In nicotine-exposed cells (Figures 2D and 2E), isoproterenol had no effect on INa amplitude or conductance, nor did it change its activation threshold (Figure 2C) or maximum amplitude voltage (Figure 2D), which were similar to the one observed in stimulated sham myocytes. As a result, INa remained 24% smaller compared to sham values during the adrenergic stimulation (Figure 2D). This loss of sensitivity to isoproterenol was confirmed by the absence of variation in the mid-activation potential of INa (Figure 2G). Therefore, nicotine abolished the increase in cardiac cell excitability after adrenergic stimulation.
      We next measured the influence of nicotine on the availability of the channels using a standard inactivation protocol (Figure 3A). The voltages with 50% of INa available were −81.3 ± 0.2 mV and −85.3 ± 0.1 mV in sham and nicotine-exposed myocytes, respectively (P <.05, F test). Isoproterenol shifted mid-voltage of inactivation to −85.3 ± 0.2 mV in sham (P <.05) but had no effect on nicotine-exposed myocytes (V1/2: −84.1 ± 0.1 mV). There was no difference between the sham and nicotine-exposed myocytes after application of isoproterenol (Figure 3B).
      Figure thumbnail gr3
      Figure 3Nicotine blunts the effect of isoproterenol (10 μM) on sodium channel availability. A: Representative current recordings during a test pulse to −10 mV preceded by conditioning pulses from −140 mV to +40 mV in increments of 5 mV from a holding potential of –120 mV (inset). B: Inactivation curves (availability) in sham (n = 7), +Iso (n = 8), nicotine (n = 8), nicotine + Iso (n = 7) conditions were obtained by plotting the ratio of INa to its maximum value against the conditioning pulse voltage. Data were fitted to a Boltzmann distribution function. Isoproterenol significantly shifted mid-inactivation potential (V0.5) from −81.3 ± 0.2 mV to −85.3 ± 0.2 mV (P <.05, f test) in sham cardiomyocytes but had no effect in nicotine-exposed animals (−85.3 ± 0.1 mV and −84.1 ± 0.1 mV). Data are given as mean ± SEM. Cells from 3 animals were tested for each condition (minimum 2 cells per animal).
      Steady-state activation curves determine the probability of opening of the channels at various voltages, whereas steady-state inactivation represents the fraction of the population available at a given potential. The overlap between the 2 curves thus creates a voltage range at which Na+ channels transit spontaneously between the open and inactivated states and generate the so-called window current (IW) (Figure 4). In sham animals, isoproterenol shifted this overlap toward more negative potentials, resulting in a higher probability of opening at voltages close to the resting membrane potential of the cell (Figure 4A).
      Figure thumbnail gr4
      Figure 4Exposure to nicotine blunts the effect of isoproterenol (Iso) on the sodium window current. A, B: Activation ( and ) and inactivation () of INa in the range of voltage at which each curve overlaps. C, D: Window currents calculated from the overlap of the activation and inactivation curves using the standard Hodgkin and Huxley formalism (see ). Ctrl = control.
      The overlap was similar in control nicotine-exposed cells and sham myocytes exposed to isoproterenol (Figures 4A and 4B), suggesting that nicotine-exposed cells are hyperexcitable in control conditions. However, as opposed to sham animals, isoproterenol shifted the voltage overlap toward more positive potentials, away from the normal resting membrane potential, in nicotine-exposed myocytes.
      IW was calculated from the product of the fraction of channels available, their opening probability, and GNa,max using the Hodgkin–Huxley model. The model predicted that in sham myocytes, isoproterenol will increase IW by 332% and shift its peak value from –35 to –47 mV (Figure 4C), suggesting more excitable atrial cells. Nicotine exposure alone increased IW by 325% to the level observed in sham myocytes exposed to isoproterenol. As opposed to its effect on sham myocytes, isoproterenol reduced IW by 54% and depolarized its maximum amplitude voltage in nicotine-exposed cells (Figure 4D), thus predicting that isoproterenol reduces excitability in nicotine-exposed myocytes.
      To test the model, we measured INa after fast inactivation was completed and IW dominated the residual Na+ current (Figure 5A). In agreement, isoproterenol shifted the I/V relationship of this late current (INaL) toward more negative potentials and increased its amplitude by 260% ± 15% in sham myocytes (Figures 5B and 5D).
      Figure thumbnail gr5
      Figure 5Modulation of the late sodium current (INaL) by isoproterenol (Iso) reflects the dependence on voltage predicted by the window current (). A: Representative INa recordings from a cardiomyocyte exposed to nicotine in utero after a series of voltage pulses in 5-mV increments from a holding potential of −120 mV (protocol in inset). Arrow indicates measurement of the late current (INaL) once fast inactivation of the channels is completed. B, C: Current–voltage relationship of INaL in cardiomyocytes isolated from sham (n = 9) and nicotine-exposed kittens, respectively (n = 6) in control (Ctrl) and after perfusion with Iso (10 μmol/L). Nicotine abolished the response to Iso. D: Maximum late Na+ current density INaL,max. Statistical significance: *P <.05, **P <.01, ***P <.001 (*vs sham control); †P < 0.05 (vs sham Iso). Analysis of variance from 4–6 animals.
      Nicotine by itself increased INaL maximum amplitude by 175% ± 15% (2.5-fold) and hyperpolarized the peak I/V relationship by 15 mV vs sham myocytes (Figures 5C and 5D). Isoproterenol had no significant effect on INaL in nicotine-exposed myocytes (Figure 6C). As a result, the amplitude of INaL was 41% ± 5% smaller in nicotine-exposed cardiomyocytes vs sham in the presence of isoproterenol.
      Figure thumbnail gr6
      Figure 6Fraction of channels contributing to INaL expressed as percent of maximum peak current (INap) shown in and . Statistical significance: *P <.05, **P <.01 (vs sham Ctrl). Analysis of variance from 4–6 animals (n = 12). Ctrl = control; Iso = isoproterenol.
      We previously showed that changes in the amplitude of peak INa and INaL are not proportional because peak INa is largely dominated by current from NaV1.5 channels whereas nNaVs contribution accounts for 50% of the late current.
      • Biet M.
      • Barajas-Martinez H.
      • Ton A.T.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      About half of the late sodium current in cardiac myocytes from dog ventricle is due to non-cardiac-type Na(+) channels.
      The lower expression of NaV1.1 in nicotine-exposed myocytes we previously reported
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      should reduce their contribution to INaL and increase the proportionality between INaL and peak INa. Figure 6 confirms this is the case. INaL, when expressed as a fraction of peak INa, is larger in nicotine-exposed cardiomyocytes vs sham. In sham myocytes, isoproterenol increased this fraction. However, in nicotine-exposed myocytes the fraction remained the same and even showed a trend toward reduction. These results suggest that a uniform population of sodium channels, likely NaV1.5, contributed to INaL after exposure to nicotine.

      Discussion

      Our model shows that sudden death occurred frequently in nicotine-exposed kittens during the first 5 days after birth, an observation consistent with SIDS (Figure 1).
      • Haglund B.
      • Cnattingius S.
      Cigarette smoking as a risk factor for sudden infant death syndrome: a population-based study.
      We showed that prenatal exposure to nicotine inhibits INa adrenergic response, induces a long-lasting increase of peak INa, and hyperpolarizes its activation, thus reducing the voltage gap between the resting membrane potential and the action potential threshold.
      In cardiomyocytes from sham animals, isoproterenol shifted the activation of INa (and peak current) closer to the resting membrane potential, thus enabling the heart to adapt to cardiac demand. In nicotine-exposed rabbits, the activation voltage of INa was the same as in cells from sham animals exposed to isoproterenol, but in contrast the β-agonist had no effect. These results suggest that atrial cells from newborn kittens exposed to nicotine are hyperexcitable and do not respond to adrenergic stimulus. This increased excitability will potentiate the probability for the SAN to excite the surrounding atrium and reduce the impulse to the atrioventricular node through specialized conduction pathways. This may reduce the ability of the heart to accelerate at the onset of apnea, a finding consistent with a loss of cardiorespiratory reflex in SIDS. However, how the influence of nicotine on potassium currents active in the voltage range close to the resting membrane potential will mitigate the effects on INa on atrial excitability remains to be determined.
      The contribution of INaL in maintaining the plateau of the cardiac action potential
      • Dumaine R.
      • Antzelevitch C.
      Molecular mechanisms underlying the long QT syndrome.
      • Dumaine R.
      • Wang Q.
      • Keating M.T.
      • et al.
      Multiple mechanisms of Na+ channel-linked long-QT syndrome.
      and its link to SIDS are well established.
      • Schwartz P.J.
      • Priori S.G.
      • Dumaine R.
      • et al.
      A molecular link between the sudden infant death syndrome and the long-QT syndrome.
      Our results showing that nicotine increases INaL (Figure 5C vs Figure 5B) suggest that in utero exposure to nicotine will prolong the atrial action potential in the absence of ion channel mutations. Moreover, because isoproterenol had no effect on INaL in nicotine-exposed myocytes, this will hamper the possibility of the heart modulating APD during sleep. Although we focused on region atrial cells around the SAN, considering that exposure to nicotine quadrupled the amplitude of INaL, we speculate that a similar augmentation occurs in ventricle. In humans, the QT interval gradually prolongs and peaks at 6 to 11 weeks postnatal
      • Yoshinaga M.
      • Kato Y.
      • Nomura Y.
      • et al.
      The QT intervals in infancy and time for infantile ECG screening for long QT syndrome.
      before shortening to a childhood-level duration, in part due to a lower density or deficiency in several K+ currents in neonates.
      • Huynh T.V.
      • Chen F.
      • Wetzel G.T.
      • Friedman W.F.
      • Klitzner T.S.
      Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult rabbit ventricular myocytes.
      This maturation period overlaps with the most vulnerable period for SIDS (2 months). Therefore, the low or absent K+ currents would potentiate prolongation of the QT interval brought by INaL due to a reduced repolarization reserve and may promote arrhythmias during that period. This hypothesis is consistent with the QT prolongation often reported in SIDS infants. Moreover, the loss of response of INa to isoproterenol also supports, in part, the hypothesis that changes in the adrenergic response or catecholaminergic innervation of the heart may be involved in SIDS.
      • Schwartz P.J.
      Cardiac sympathetic innervation and the sudden infant death syndrome. A possible pathogenetic link.
      • Maron B.J.
      • Clark C.E.
      • Goldstein R.E.
      • Epstein S.E.
      Potential role of QT interval prolongation in sudden infant death syndrome.
      The size and limited number of SAN cells in newborn rabbit prevented us from acquiring pacemaker cells and reliable electrocardiograms. However, our results are consistent with INaL modulating the SAN rate in newborn but not in adult rabbit. In sham myocytes, isoproterenol tripled the amplitude of INaL in the range of resting membrane potentials of atrial cells. This is likely to accelerate the diastolic depolarization of pacemaker cells and increase sinus rhythm. In contrast, INaL was insensitive to isoproterenol in nicotine-exposed myocytes, and this will blunt the effect of the brain-mediated increase in sympathetic activity at the onset of sleep apnea. Despite the limitations of our atrial cell model, this mechanism nonetheless may explain why heart rhythm fails to accelerate at the onset of sleep apnea in SIDS. We believe that a similar mechanism may also contribute to conduction anomalies during SIDS.
      • Ottaviani G.
      • Matturri L.
      • Rossi L.
      • James T.N.
      Crib death: further support for the concept of fatal cardiac electrical instability as the final common pathway.
      Our results show that INaL expressed as a fraction of peak INa amplitude nearly doubled in sham cardiomyocytes exposed to isoproterenol (Figure 6). The ratio of INaL to peak INa changes due to the contribution of neuronal and cardiac Na+ channels
      • Biet M.
      • Barajas-Martinez H.
      • Ton A.T.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      About half of the late sodium current in cardiac myocytes from dog ventricle is due to non-cardiac-type Na(+) channels.
      because neuronal channels (especially NaV1.1 and NaV1.3) produce relatively larger late current than NaV1.5.
      • Smith R.D.
      • Goldin A.L.
      Functional analysis of the rat I sodium channel in xenopus oocytes.
      Our results thus suggest that isoproterenol is acting on a population of channels composed of neuronal and NaV1.5 isoforms in sham myocytes. In contrast, the fraction of late/peak current remained constant after exposure to isoproterenol in nicotine-exposed myocytes (Figure 5D), suggesting that isoproterenol targeted a uniform population of channels. This is consistent with the reduction in the expression NaV1.1 we reported,
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      thus leaving NaV1.5 as the major contributor to INaL in nicotine-exposed cardiomyocytes.
      The mechanism by which nicotine alters the response of INa to isoproterenol remains unknown. However, rats exposed to nicotine in utero show an initial reduction, followed by enhanced activity of cardiac β-adrenergic receptors
      • Navarro H.A.
      • Mills E.
      • Seidler F.J.
      • et al.
      Prenatal nicotine exposure impairs beta-adrenergic function: persistent chronotropic subsensitivity despite recovery from deficits in receptor binding.
      due to augmentation of adenyl cyclase activity
      • Chow F.A.
      • Seidler F.J.
      • McCook E.C.
      • Slotkin T.A.
      Adolescent nicotine exposure alters cardiac autonomic responsiveness: beta-adrenergic and m2-muscarinic receptors and their linkage to adenylyl cyclase.
      • Slotkin T.A.
      • Navarro H.A.
      • McCook E.C.
      • Seidler F.J.
      Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues.
      and potential desensitization of β-adrenergic receptors.
      • Larsen H.E.
      • Lefkimmiatis K.
      • Paterson D.J.
      Sympathetic neurons are a powerful driver of myocyte function in cardiovascular disease.
      These findings are consistent with changes in expression of neuronal and NaV1.5 channels
      • Ton A.T.
      • Biet M.
      • Delabre J.F.
      • Morin N.
      • Dumaine R.
      In-utero exposure to nicotine alters the development of the rabbit cardiac conduction system and provides a potential mechanism for sudden infant death syndrome.
      and may provide a basis to explain our results. Indeed, an increase in adenyl cyclase activity should augment production of cAMP and the activity of PKA. However, PKA has opposite effects on nNaV and NaV1.5. PKA decreases the activity of nNaVs
      • Li M.
      • West J.W.
      • Numann R.
      • Murphy B.J.
      • Scheuer T.
      • Catterall W.A.
      Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase.
      • Gershon E.
      • Weigl L.
      • Lotan I.
      • Schreibmayer W.
      • Dascal N.
      Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes.
      • Smith R.D.
      • Goldin A.L.
      Functional analysis of the rat I sodium channel in xenopus oocytes.
      but increases expression of NaV1.5.
      • Qu Y.H.
      • Rogers J.C.
      • Tanada T.N.
      • Scheuer T.
      • Catterall W.A.
      Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C.
      • Hallaq H.
      • Yang Z.
      • Viswanathan P.C.
      • et al.
      Quantitation of protein kinase A-mediated trafficking of cardiac sodium channels in living cells.
      Therefore, chronic adrenergic stimulation during in utero exposure to nicotine will favor expression of NaV1.5 and desensitization of β-adrenergic receptors. This may explain the larger INa, the lack of effect of isoproterenol, and the increased proportionality between INaL and peak INa.

      Study limitations

      The main limitation of our study is the lack of recordings of cardiac arrhythmias in newborn rabbits that we could compare to the ones observed in humans. This would further validate our model. Monitoring of ECGs in newborn rabbits using Holters proved to be very difficult as the mother chewed on the wiring or killed its kitten. However, mortality was clearly due to exposure to nicotine as there was no death in control neonates. Although our data showing a loss of cardiac response to adrenergic stimulus are consistent with a loss of the cardio-respiratory reflex, we cannot at this point prove with absolute certainty that death was due to failure of the neonate to awake from sleep apnea, as observed in human. More data are needed to link our findings to SIDS. Finally, we measured sodium currents in atrial cells close to the SAN. Although INa is relatively similar in various cardiac tissues, our data need to be validated in ventricular and SAN myocytes where a similar reduction in adrenergic response may promote conduction delays (ventricle) or bradycardia (pacemaker cells), which are arrhythmias also linked to SIDS.

      Conclusion

      Our results demonstrate that in utero exposure to nicotine interferes with cardiac excitability and may induce some of the arrhythmogenic changes linked to SIDS. This represents a new paradigm in our understanding of SIDS-related arrhythmias. Our data also raise awareness on the use of nicotine replacement therapies in pregnant women.

      Appendix. Supplementary data

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