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We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell-cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell-cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of approximately 0.6 to 1.7 Hz (1.09 +/- 0.05 Hz). Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 microM 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1-5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (I(j)) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (V(j)) dependence. I(j) records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical V(j) dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical I(j) with pronounced V(j) gating when the Cx45 side was negative. Symmetrical I(j) suggests that the dominant functional channel is homotypic, while the asymmetrical I(j) suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (gamma(j)) of 30-80 pS. The macroscopic I(j) obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical V(j) dependence, while single channel events revealed gamma(j) of the size range 40-100 pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.

Pacemaker current (If) exists in both neonatal and adult ventricles, but activates at more negative voltages in the adult. This study uses whole-cell patch clamp to investigate the factors that may contribute to the maturational shift of If, comparing neonatal rat ventricular myocytes that were cultured for 4-6 days either alone, in co-culture with sympathetic nerves, or with neurotransmitters chronically present in culture. If recorded from nerve-muscle co-cultures had a significantly more negative and shallower activation-voltage relation than that from control muscle cultures, which was reflected in the midpoint potential (V50) and slope factor (K) of activation. This effect of innervation was prevented by the sustained presence in the culture of the alpha1-adrenergic antagonist prazosin (Pz) at 10(-7) M. In parallel experiments, myocytes treated with noradrenaline (NA) at 10(-7) M or neuropeptide Y (NPY) at 10(-7) M during culture had the same If activation as control cells, but cells treated with NA and NPY together had a significantly more negative and shallower activation curve. Maximum conductance and reversal potential were unchanged. The effect of chronic exposure to NA + NPY was prevented by the sustained presence of either Pz or the NPY Y2 selective antagonist T4-[NPY(33-36)]4 (3.5 x 10(-7) M) in the culture, indicating a requirement for both alpha1-adrenergic and NPY Y2 activation. Substituting NA with the alpha1A-adrenergic selective agonist A61603 (5(-10) x 10(-9) M), in the presence of NPY, did not alter If, suggesting the involvement of alpha1B- rather than alpha1A-adrenoceptors. Further, sequential exposure to NPY followed by NA was effective in reproducing the action of chronic simultaneous exposure to these agonists, but sequential exposure to NA followed by NPY was ineffective. The results are consistent with past studies indicating that NPY affects the functional expression of the alpha1B-adrenergic cascade and suggest that sympathetic innervation induces a negative shift of If in ventricle via a combined action at alpha1B-adrenergic and NPY Y2 receptors. This effect of innervation probably contributes to the developmental maturation of If activation.

1. Newborn rabbit sino-atrial node (SAN) myocytes were recently found to express a tetrodotoxin (TTX)-sensitive Na+ current. We now report that the dose-response relation indicates that this SAN Na+ channel has unusually high TTX sensitivity, with half-maximal inhibition (26 +/- 5 nM) which is more typical of neuronal than cardiac tissue. 2. Additional characterization used mu-conotoxin GIIIA and Cd2+ as relatively selective blockers of the skeletal and cardiac isoforms, respectively. mu-Conotoxin GIIIA had no effect on the current recorded from SAN myocytes, but the Cd2+ sensitivity was unexpectedly high for a neuronal isoform (half-maximal inhibition = 185 +/- 8 microM). 3. Analysis of the time constant of inactivation did not reveal evidence of multiple inactivation processes, with the data well fitted by a single, relatively rapid exponential (inactivation time constant = 0.58 +/- 0.03 ms at 0 mV). 4. In situ hybridization with anti-sense cDNA probes was used to test for expression of neuronal type I, II and III Na+ channel isoforms. Myocardial cells in newborn SAN tissue exhibited clear hybridization to the type I, but not the type II or III probes. No hybridization was observed in adult SAN tissue with any of the three probes. 5. It is concluded that the newborn SAN expresses a neuronal type I-like Na+ channel isoform, and that this probably accounts for the unusual characteristic of high sensitivity to both TTX and Cd2+.

1. Single cells were isolated from the sinus node region of rabbits (2 days old to adult) to study the age-dependent contribution of the sodium current (iNa) to pacemaker activity. 2. Experiments were conducted in 50 mM Na(+)-Ca(2+)-free solution. All newborn cells (2-19 days) exhibited a TTX-sensitive, Mn(2+)-insensitive fast inward Na+ current (peak current density 115.5 +/- 11.9 pA pF-1 at 0 mV). Fifty per cent of young cells (20-40 days) possessed the current, but only one in ten adult cells. Current density decreased with development independently of cell capacitance. 3. Newborn cells exhibited a noticeable window current. With development, the position of the activation curve was shifted in the positive direction, while the inactivation was unaltered, resulting in reduced overlap of the two curves and hence less window current. 4. In newborn cells, 3 microM TTX significantly reduced all measured parameters of spontaneous action potentials, slowing rate by 63%. In contrast, there was no significant effect of TTX on rate or most of the same parameters in adult cells. 5. These results indicate that cells of the sinus node region exhibit a substantial TTX-sensitive current at birth. With development, both the density and frequency of occurrence of this current within the sinus node decrease, as does its contribution to automaticity.

1. The dose dependence of the cholinergic agonist acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline (Iso) were determined for the hyperpolarization-activated current (If) and the L-type Ca2+ current (ICa,L) in single cells isolated from the rabbit sino-atrial (SA) node. 2. ACh inhibited If by a negative shift of its activation curve with a maximal effect of -9.9 mV; half-maximal effect was produced by 0.019 microM ACh. High ACh concentrations were required to inhibit ICa,L only partially (31% inhibition at 300 microM). 3. In contrast, If and ICa,L responded to Iso over a similar dose range, with concentrations for half-maximal enhancement of 0.0136 and 0.0070 microM, respectively. 4. The effects on spontaneous activity of ACh (range 0.001-0.03 microM) and Iso (range 0.001-1 microM) were investigated. ACh decreased the slope of diastolic depolarization at concentrations similar to those inhibiting If (> 50% at 0.03 microM). Iso enhanced diastolic depolarization at concentrations similar to those affecting both If and ICa,L (half-maximal effect at 0.027 microM). 5. In a ramp-clamp protocol simulating diastolic depolarization, the threshold for activation of inward nifedipine-sensitive current was -41.22 +/- 0.68 mV. Although enhancing ICa,L, Iso did not affect this threshold. 6. Half-maximal ACh concentrations for inhibition of automaticity and If are similar and are lower than the threshold concentrations for modulation of ICa,L; this argues against a role of ICa,L in direct muscarinic modulation of pacemaking. In contrast, modulation of If, ICa,L and automaticity occur at similar Iso concentrations. The difference between maximum diastolic potential (-61.95 +/- 0.93 mV) and the threshold for Iso-stimulated ICa,L (-39.54 +/- 1.03 mV) suggests that this current plays a role only at later stages of diastolic depolarization.

1. Single sodium channel currents were recorded from canine ventricular myocytes in cell-attached patches. The relative rates of single-channel activation vs. inactivation as well as the voltage dependence of the rate of open-channel inactivation were studied. 2. Ensemble-averaged sodium currents showed relatively normal activation and inactivation kinetics, although the mid-point of the steady-state inactivation (h infinity) curve was shifted by 20-30 mV in the hyperpolarizing direction. This shift was due to the bath solution, which contained isotonic KCl to depolarize the cell to 0 mV. 3. Steady-state activation showed less of a voltage shift. The threshold for eliciting channel opening was around -70 mV and the mid-point of activation occurred near -50 mV. 4. The decline of the ensemble-averaged sodium current during a maintained depolarization was fitted by a single exponential function characterizing the apparent time constant of inactivation (tau h). The apparent rate of inactivation was voltage dependent, with tau h decreasing e-fold for a 15.4 mV depolarization. 5. The relative contributions of the rates of single-channel activation and inactivation in determining the time course of current decay (tau h) were examined using the approach of Aldrich, Corey & Stevens (1983). Mean channel open time (tau o) showed significant voltage dependence, increasing from 0.5 ms at -70 mV to around 0.8 ms at -40 mV. At -70 mV tau h was much greater than tau o, while at -40 mV the two time constants were similar. 6. The degree to which the kinetics of single-channel activation contribute to tau h was studied using the first latency distribution. The first latency function was fitted by two exponentials. The slow component was voltage dependent, decreasing from 19 ms at -70 mV to 0.5 ms at -40 mV. The fast component (0.1-0.5 ms) was not well resolved. 7. Comparing the first latency distribution with the time course of the ensemble-averaged sodium current at -40 mV showed that activation is nearly complete by the time of peak inward sodium current. However, at -70 mV, activation overlaps significantly with the apparent time course of inactivation of the ensemble-averaged current. 8. Using the methods of Aldrich et al. (1983) we also measured the apparent rate of open-channel closing (a) and open-channel inactivation (b). Both rates were voltage dependent, with a showing an e-fold decrease for an 11 mV depolarization and b showing an e-fold increase for a 30 mV depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)