Wild-type and mutant HCN channels in a tandem biological-electronic cardiac pacemaker.

BACKGROUND: Biological pacemakers (BPM) implanted in canine left bundle branch function competitively with electronic pacemakers (EPM). We hypothesized that BPM engineered with the use of mE324A mutant murine HCN2 (mHCN2) genes would improve function over mHCN2 and that BPM/EPM tandems confer advantage over either approach alone. METHODS AND RESULTS: In cultured neonatal rat myocytes, activation midpoint was -46.9 mV in mE324A versus -66.1 mV in mHCN2 (P < 0.05). mE324A manifested a positive shift of voltage dependence of gating kinetics of activation and deactivation compared with mHCN2 (P < 0.05) in myocytes as well as Xenopus oocytes. In intact dogs in complete atrioventricular block, saline (control), mHCN2, or mE324A virus was injected into left bundle branch, and EPM were implanted (VVI 45 bpm). Twenty-four-hour ECGs were monitored for 14 days. With EPM discontinued, there was no difference in duration of overdrive suppression among groups. However, basal heart rates in controls were less than those in mHCN2, which did not differ from those in E324A (45 versus 57 versus 53 bpm; P < 0.05). When spontaneous rate fell below 45 bpm, EPM intervened at that rate, triggering 83% of beats in control, contrasting (P < 0.05) with 26% (mHCN2) and 36% (mE324A). On day 14, epinephrine (1 microg/kg per minute IV) induced a 50% heart rate increase in all mE324A, one third of mHCN2, and one fifth of control (P < 0.05 mE324A versus control or mHCN2). CONCLUSIONS: mE324A induces faster, more positive pacemaker current activation than mHCN2 and stable, catecholamine-sensitive rhythms in situ that compete with EPM comparably but more catecholamine responsively than mHCN2. BPM/EPM tandems function reliably, reduce the number of EPM beats, and confer sympathetic responsiveness to the tandem.

A common ankyrin-G-based mechanism retains KCNQ and NaV channels at electrically active domains of the axon.

KCNQ (KV7) potassium channels underlie subthreshold M-currents that stabilize the neuronal resting potential and prevent repetitive firing of action potentials. Here, antibodies against four different KCNQ2 and KCNQ3 polypeptide epitopes show these subunits concentrated at the axonal initial segment (AIS) and node of Ranvier. AIS concentration of KCNQ2 and KCNQ3, like that of voltage-gated sodium (NaV) channels, is abolished in ankyrin-G knock-out mice. A short motif, common to KCNQ2 and KCNQ3, mediates both in vivo ankyrin-G interaction and retention of the subunits at the AIS. This KCNQ2/KCNQ3 motif is nearly identical to the sequence on NaV alpha subunits that serves these functions. All identified NaV and KCNQ genes of worms, insects, and molluscs lack the ankyrin-G binding motif. In contrast, vertebrate orthologs of NaV alpha subunits, KCNQ2, and KCNQ3 (including from bony fish, birds, and mammals) all possess the motif. Thus, concerted ankyrin-G interaction with KCNQ and NaV channels appears to have arisen through convergent molecular evolution, after the division between invertebrate and vertebrate lineages, but before the appearance of the last common jawed vertebrate ancestor. This includes the historical period when myelin also evolved.

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers.

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Tyrosine kinase inhibition differentially regulates heterologously expressed HCN channels.

The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that are permeable to Na(+) and K(+). There are four members of this channel family, three of which, HCN1, HCN2, and HCN4, are expressed in the heart. Current evidence suggests that the HCN ion channel subunit family is the molecular correlate of the alpha subunit of the cardiac pacemaker current i(f). Our previous work has shown that HCN4 is the dominant isoform expressed in the rabbit sinoatrial (SA) node and that changes in tyrosine phosphorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node i(f) conductance with no change in voltage dependence. In the present study we investigate the actions of genistein, a tyrosine kinase inhibitor, on heterologously expressed HCN currents in Xenopus oocytes. Genistein had no effect on HCN1-induced currents, but reduced whole-cell currents induced by HCN2 or HCN4 and slowed activation kinetics at voltages near the midpoint of activation. In the case of HCN2 there was also a negative shift in the voltage dependence of activation that accompanies the current reduction. We have shown previously that HCN2 is the dominant isoform expressed in rat ventricular myocytes. The above results predict that genistein should reduce i(f) in the rat ventricle and cause a negative shift of voltage dependence and kinetics of activation. We tested this hypothesis by studying the effects of genistein on isolated rat ventricular myocytes. Genistein significantly reduced i(f) current density (pA/pF) (control: 12.2+/-1.8; genistein: 3.5+/-0.5; washout: 7.7+/-0.8; n=10), and caused a negative shift of the midpoint of activation by 14 mV (-133+/-1 mV for genistein and -119+/-1 mV for washout, n=7) with no change in slope factor. Our results thus suggest that i(f) in the heart and i(f)-like currents in other tissues can be regulated differentially by tyrosine phosphorylation based on isoform expression patterns.