Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes

Figures

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  Figure 1. Intracellular recordings of spontaneous pacemaker activity from an adult mouse SAN myocyte under control conditions and in 10 nM Isoproterenol (ISO) at 35 °C. (A) Recording of action potentials from primary pacemaker cells under control or baseline conditions. (B) Recording of action potentials from the same myocyte as in (A) 4 to 5 min after 10 nM ISO was added to the superfusate. (C) Numerical values for key parameters (overshoot (i), dV/dt (ii), interbeat interval (iii)) of action potentials provide an indication of the experimental variability in these amphotericin-mediated patch electrode recordings in this study. The measured mean value of the interbeat interval is about 359.3 ms (167 beats/min) with SD = 27.3 ms and SE = 4.9 ms (n = 33).

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  Figure 2. Families of whole cell or macroscopic transmembrane currents in an SAN primary pacemaker myocyte at 35 °C. (A) Transmembrane current recording in control conditions. (B) Transmembrane current recording in 10 nM ISO. The transmembrane current records in both (A,B) were obtained in normal Tyrodes in response to 1 s rectangular voltage clamp steps, that displaced the membrane potential from −100 to +40 mV from a holding potential (HP) of −60 mV. These were applied every 8 s in 10 mV increments. A 50 ms pre-pulse to −40 mV was included in all cases to inactivate the transient inward Na+ and activate Ca2+ currents. This protocol resulted in most of the time- and voltage-dependent outward currents being due to a delayed rectifier K+ conductance, IKr; and the slow development of inward currents being generated mainly by If. The superimposed family of currents in (B) provide a qualitative indication of the changes in these families of currents recorded 5 min after 10 nM ISO was added to the superfusate. All recordings were made in the amphotericin-mediated whole cell patch clamp configuration (see Methods).

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  Figure 3. Isochronal current-voltage (I–V) relations corresponding to the families of currents shown in Figure 2. (A) The two current-voltage (I–V) relationships were obtained by measuring the maximum outward or inward currents at the end of each 200 ms rectangular voltage clamp step. (B) The two superimposed I–V curves are analogous to those on the left except that each data point was obtained following a 1 s rectangular voltage step. Each data set is expressed in terms of current density based on the mean capacitance of each pacemaker myocyte in this series measuring 30.4 pF +/− 2.4, (n = 6). Note that in response to the 200 ms clamp steps ISO at 10 nM (red data points) may produce a small increase in inward current in the membrane potential range between approximately −20 to −50 mV; and that this ISO-induced inward current is more clearly observed at strongly hyperpolarized membrane potentials between −80 and −100 mV. The two superimposed I–V curves in the right panel also provide an indication that relatively large and long (1 s) depolarizations or hyperpolarizations can increase transmembrane current in the presence of 10 nM ISO.

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  Figure 4. General properties and reversal potential measurements for the hyperpolarization-activated current, If, in adult mouse SAN pacemaker myocytes. (A,B) Transmembrane current records and I–V in control Tyrodes solution. (C,D) Transmembrane current records and I-V in Ba, Ni solution. In these experiments, a paired-pulse clamp protocol (P1–P2) was used. In P1, each myocyte was held at −45 mV and progressively larger 2 s hyperpolarizing voltage clamp waveforms were applied in 10 mV increments in the voltage range −55 mV to −135 mV. As shown in (A,B), most current waveforms consisted of an instantaneous inward current followed, by time- and voltage-dependent development of inward current that increased in size and activated faster with progressive hyperpolarizations. To obtain conditions under which the current change was dominated by If, these measurements were repeated in the presence of BaCl2 (0.1 mM) (C,D) to block the inwardly rectifying background K+ current, IK1; and NiCl2 (0.5 mM) to markedly reduce transmembrane currents generated by either L-type Ca2+ channels or the Na+/Ca2+ exchange mechanism. Comparison of the families of currents in the left versus the right columns…

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  Figure 5. Analysis of the kinetics of activation and deactivation of If in an isolated adult mouse primary pacemaker myocyte. The kinetics of activation and deactivation of If as a function of membrane potential were determined using a family of 20 s rectangular voltage clamp steps. To study activation, hyperpolarizing steps from −70 to −120 mV from a holding potential of −35 mV were applied. For deactivation, a 10 s step to −120 mV was utilized to fully activate If; and then its deactivation kinetics were studied by applying 10 s steps to membrane potentials in the range −50 to −110 mV. As shown, (red traces superimposed on black original current change records) when individual traces in these families of If currents were fitted using a function consisting of either one (A), or the sum of two (B), exponential relationships; the double exponential function provided a better fit (particularly to the onset kinetics). Similarly, the time courses of the deactivating records for If were better described by double exponential than by single exponential functions (data not shown). Additional details are provided in the Results and in the Supplemental data section.

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  Figure 6. Plots of the voltage dependence for both the slow and the fast kinetic processes that underlie the activation and deactivation of If. As indicated in the inset, data depicting the fast time constant for If kinetics is shown in red while data based on the determination of the slow time constant at each membrane potential is shown in black. Activation and deactivation time constants obtained at the same membrane potential have been pooled to form each of these plots. Details about the fit functions for the fast and slow time constants are given in Equations (S5) and (S6) in the Supplementary Materials.

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  Figure 7. Determination of steady-state voltage dependence of activation of the transmembrane current, If. Steady-state I–V relations for If were recorded, and the voltage dependence of activation for If was derived from primary data as described in Results. (A) Representative raw data of I–V relationship displayed as a signal averaged (n = 4). (B) Conductance values converted from If currents using the equation given in Results (assuming that the instantaneous I–V relationship for If is linear). The resulting conductance-voltage relation was fitted to a Boltzmann function (defined as: G = Gmax/(1 + exp[(Vm − Vh)/s])) and plotted as shown in Panel B (red).

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  Figure 8. Current changes due to If during the pacemaker depolarization obtained using amphotericin-mediated patch electrode recordings at 35 °C in an adult mouse SAN pacemaker myocyte. (A) Steady-state activation relationship for If plotted as a function of membrane potential. (B) Illustration of the If current activated by the same voltage ramp protocol described in the Legend of Figure 7. (C) Plot of the same data as that in (B), after an approximately 30-fold increase in gain. This provides an indication of the maximal If current change in If that can be expected to occur within the range of membrane potentials (−40 mV to −70 mV). This range corresponds to the membrane potentials during the spontaneous pacemaker depolarization (see Figure 1A).

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  Figure 9. Ramp voltage clamp procedure to reveal the ISO-induced current changes during the action potential and pacemaker depolarization in adult mouse SAN. (A) Superimposed current records, each obtained after averaging 6 current traces generated by repetitive application of the action potential-like voltage ramp waveforms shown in Panel Aii. The baseline or control record is in black and the analogous averaged record obtained after 10 nM ISO application is in red. (B) Mean I-V relation for the ISO-induced current obtained as an averaged record from six different SAN myocyte recordings. Note: (i) the large increase in net inward current in the voltage range approximately +20 to −20 mV likely due to ISO-induced augmentation of ICa-L;) and (ii) that ISO at 10 nM also resulted in a consistent but very small increase in net outward current at membrane potentials between −20 and approximately −65 mV (see Section 4).

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  Figure 10. Mathematical simulations of SAN pacemaker activity which compare output from our original model (HZ-1) with that from the updated model (HZ-2) used in this study. In this figure, data from the original model [8,38] is shown in black (HZ-1) and output from the updated model (HZ-2) is shown in red. (A) Two superimposed traces of pacemaker depolarization and action potential cycles computed from HZ-1 and HZ-2 models. (B–E) Corresponding data for the Na+ current, INa; the L-type Ca2+ current, ICaL; the rapid delayed rectifier K+, IKr current, and the hyperpolarization-activated cation current, If, respectively. (F) Changes in intracellular Ca2+ measured as the difference between diastolic and systolic levels.

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  Figure 11. Illustration of the effects of ISO on SAN pacemaker activity and underlying transmembrane ionic currents. Output records generated using the baseline mathematical model is shown in black and data obtained after addition of 10 nM ISO is shown in red. (A) Superimposed action potentials and pacemaker depolarizations confirm that ISO application results in an approximately 50% increase in pacemaker firing rate, and also reveal a small hyperpolarization of the maximum diastolic potential. (B–E) Corresponding data for the Na+ current, INa; the L-type Ca2+ current, ICaL; the rapid delayed rectifier K+, IKr current, and the hyperpolarization-activated cation current, If, respectively. Note that application of ISO approximately doubles the size of INa and ICaL and that IKr also increases significantly. The data in Panel C is the focus of this study. Two prominent effects of ISO on If are shown: (i) there is a significant shift in the inward direction of the steady or background current level, and (ii) the changes in If during each action potential are increased substantially (4- to 5-fold). (F) Increase (approximately 30%) in intracellular Ca2+ by ISO.