Initial attempts at biological pacing focused on sympathomimetic modulation of existing pacemaker current. Edelberg et al. [20] reasoned that since β-adrenergic catecholamines can increase sinus rate, overexpressing β-adrenergic receptors should enhance heart rate. Hence, they incorporated the gene encoding the β₂-adrenergic receptor into a plasmid and injected this into porcine atrium in situ. Although the transfection was transient, they demonstrated both a faster atrial rate and an enhanced atrial rate response to catecholamine infusion in the injected animals. Concerns about this approach were: first, therapy would rely on the ability of the existing sinoatrial node (or other endogenous pacemaker) to respond to catecholamines. If the node were in any way damaged or diseased the response might be unpredictable. Second, catecholamines can be arrhythmogenic. However, as detailed below, a recent variant on this approach involves manipulating adenylyl cyclase to influence cAMP levels.

Miake et al. provided the first proof of concept experiments that alteration of ion channel function could create a biological pacemaker [21]. They reasoned that if repolarizing current were reduced, this would permit inward currents to depolarize the membrane during electrical diastole, resulting in a functioning biological pacemaker. To this end, they replaced three amino acid residues in the pore of the Kir2.1 gene that regulates the ion current, I_K1 and created a dominant negative construct. This was presumed to form multimeric, nonfunctional channels with endogenously expressed wild-type Kir2.1 and/or Kir2.2. The construct and green fluorescent protein (GFP) were packaged in a replication-deficient adenoviral vector which was injected into the left ventricular cavity of guinea pigs. After 3–4 days I_K1 was about 80 % reduced in ­isolated ventricular myocytes, and ventricular ectopic rhythms were seen on ECG. Recently this approach has been combined with HCN over-expression [22]. The major concern about reducing outward repolarizing currents was that it might excessively prolong repolarization, as was borne out in later experiments [23].

Proof of concept that increasing I_f in myocytes would lead to increases in pacemaker rate was provided by our group [24–26]. We elected to overexpress the HCN channel as the biological pacemaker because it not only carries the primary pacemaker current but because I_f activates on hyperpolarization and turns off at depolarized potentials. Therefore it has no effect to prolong action potential duration. Further, it directly binds cAMP [9], providing inherent autonomic responsiveness. After showing in rat ventricular myocytes in culture that I_f could be overexpressed by administering the HCN2 gene using a plasmid as the vector [24], we asked whether the channel expressed was autonomically regulated. Here, we infected ventricular myocytes with an adenoviral construct incorporating HCN2 and found cAMP regulation of the current was preserved, similar to that for wild-type I_f [25]. We also found that a β subunit, MiRP1, administered via a plasmid, increased pacemaker current density and kinetics of exogenously expressed HCN2 in xenopus oocytes [26] and in myocytes [27]. The final in vitro experiment involved HCN2 overexpression in neonatal myocytes in culture using an adenoviral vector. Spontaneous rate increased, suggesting a similar approach might result in biological pacemaking in situ [24, 25]. These results are demonstrated in Fig. 24.2.

Our initial attempt at proof of concept in vivo involved injecting HCN2 into left atrial myocardium [28]. We used an adenoviral vector incorporating HCN2, with a second adenovirus expressing green fluorescent protein (to facilitate visualization of transfected cells). In terminal experiments we used vagal stimulation to induce sinoatrial slowing and/or AV nodal block, and studied the subsequent escape rhythms. The approach was successful and we pace-mapped the ectopic rhythms to the site of injection of the construct. We then isolated and studied cells from that region, which showed I_f at least 100-fold greater than native current [28].

Although this approach successfully demonstrated functional escape rhythms, we believed it important to test the concept in the ventricle. Hence, under fluoroscopic control, we injected the same construct into the left bundle branch system of intact dogs [29]. We again used vagal stimulation to slow sinus rate or induce atrioventricular block and found that HCN2-injected dogs developed escape rhythms whose rates were about 25 % faster than the idioventricular rates of control or sham injected (GFP without HCN2) animals (Fig. 24.3a, b) [29]. We then isolated left bundle branch fibers from the injection site and studied them with microelectrode techniques. They showed a significantly faster automatic rate than those generated at the same sites by tissues from control or sham-injected animals [29]. Current recordings from cells disaggregated from the same sites showed approximately 100-fold overexpression of I_f in HCN2 injected animals (Fig. 24.3c). Finally, immunohistochemistry demonstrated increased fluorescence with an anti-HCN2 antibody.

In a subsequent study [30] we demonstrated that an HCN2 biological pacemaker implanted in the left bundle branch could function smoothly in tandem with an electronic pacemaker set at VVI 45 bpm. The biological pacemaker significantly reduced the incidence of electronic beats (Fig. 24.3d) while the electronic pacemaker’s memory function provided a record of pacemaker functionality. Finally, a recent study confirms that an HCN2 based biological pacemaker responds to natural physiological stimuli, in this case using arousal to induce an increase in rate [31].

Other experiments focused on fine-tuning pacemaker properties using mutant or chimeric constructs. For example, we have used the E324A mutant channel, incorporating a single point mutation at position 324 of the parent mHCN2 and found that this elicits a sequence of complex changes: first, there is less channel and current expression with the mutant than the parent HCN2; however there is a positive shift of both activation and cyclic AMP responsiveness: the result is expressed as a baseline rate not very different from that with HCN2 alone, but there is a greater rate response to catecholamines [30]. We also employed a chimeric HCN1/HCN2 channel which combined the transmembrane region of HCN1 with the cytoplasmic N- and C-termini of HCN2. The rationale was that the HCN1 component would provide faster gating kinetics while the HCN2 C-terminus, with that cAMP binding domain, would provide a robust cAMP response. This prediction proved correct, but the resultant biological pacemaker generated excessive rates, resulting in periods of ventricular tachycardia [32]. The HCN basis for the tachycardia was confirmed by the efficacy of ivabradine to terminate it.

Another group has employed an HCN1 mutation that deletes a portion of the S3–S4 linker, shifting activation positively [33]. The resultant pacemaker, implanted in atrial tissue, exhibited a good range of rates in a porcine model of SAN dysfunction and a demonstrable catecholamine response. This occurred despite the poor inherent catecholamine response of the HCN1 channel subunit, suggesting contributions from endogenous elements.

While interventions such as these provide means to optimize pacemaker constructs, no single gene construct based on HCN has yet achieved optimal results in terms of the basal or autonomically stimulated rate attained. This suggests that a combination gene approach may be necessary, and several initial attempts along that line have been pursued.

In considering combination gene approaches, Nature has given us at least two clues. The first derives from the observation that the sinus node expresses Ca²⁺-activated adenylyl cyclase (AC) isoforms [15, 16], which provides a possible intrinsic feedback mechanism between cytosolic Ca²⁺ changes and activation of transmembrane currents, in particular HCN based currents. We have pursued this by over-expressing AC1 (an isoform activated by Ca²⁺ in the submicromolar range [34]) in the canine left bundle branch in combination with HCN2. AC1+HCN2 expression resulted in a significantly greater escape rate and greater maximal rate following adrenergic agonists than HCN2 alone [35]. While we have not yet compared this to a pacemaker based on a different AC isoform, another group has studied the effect of AC6 over-expression (without HCN co-expression) [36]. This isoform, which is inhibited by Ca²⁺ in the micromolar range, was able to generate a biological pacemaker, but only in the setting of catecholamine stimulation.

A second idea for combination gene therapy derives from the observation that in animals with intrinsic high heart rate a Na channel, and in particular an isoform that is not inactivated at the typical membrane potential of the sinus node, contributes to diastolic depolarization [19, 37]. We explored this concept by overexpressing the Nav1.4 Na channel isoform along with HCN2 [38]. At low membrane potentials at which more than 50 % of cardiac Na channels are inactivated, cells expressing Nav1.4 generate action potentials having a high maximum rate of action potential upstroke [39, 40]. We administered combined SkM1/HCN2 to the left bundle branch of dogs in chronic heart block. Impulse initiation was improved over HCN2 or SkM1 alone, yielding basal rates of 70–100 bpm and maximal rates of 150–170 bpm [38]. While the mechanism underlying this combination approach requires further exploration, the result was a biological pacemaker operating in the normal physiological range and possessing a robust response to adrenergic stimuli.

Research on embryonic stem cells has been proceeding for over 25 years, having been initiated by biologists interested in maturation during early stages of development [41, 42]. Research on human embryonic stem cells is far more recent. The major contribution to date in human embryonic stem cell research on biological pacemaking has been provided by the Gepstein group [43–45]. They have used embryonic stem cells to create a cardiogenic line that has been implanted into the hearts of immunosuppressed pigs in complete heart block. Their implantation has resulted in stable idioventricular rhythms that have persisted for months, and whose initiating current appears consistent with I_f.

Yet, questions remain regarding the long-term consistency of function as well as whether the current providing pacemaker function is I_f or a family of currents. A potential limitation of this approach is that the cells must be driven down a cardiac lineage that results in spontaneously active cardiac-like cells (as opposed to, e.g., ventricular like cells), and in fact the resulting ­cardiac-like population is quite heterogeneous [46]. Even with a pure population of pacemaking cells, the magnitude and kinetics of the diastolic current generated will be determined by the endogenous channels expressed in the cell. In contrast, expression of exogenous genes makes it possible to consider channel mutagenesis and/or combination therapy to optimize pacemaker function, as described above. This is one of the considerations that led us to explore the use of engineered adult mesenchymal stem cells, as discussed in the next section.

In addition, a major concern with embryonic stem cells is the need for immunosuppression: most physicians and patients faced with a decision to pace biologically and immunosuppress, versus to pace electronically and not immunosuppress would elect the latter approach. An alternative approach that does not require immunosuppression (but does not address the issue of optimization of pacemaker function) is creation of autologous biological pacemakers using induced pluripotent stem cells (IPSCs) [47, 48]. Retroviral administration of transcription factors to adult cells reprograms them, resulting in pluripotent IPSCs that can then be driven down a cardiac lineage in a manner analogous to ESCs. As with ESCs, concerns remain regarding possible tumorgenicity and control of the terminally differentiated state. Some of these concerns for IPSCs may be alleviated by more recent approaches, including ones that result in the direct reprogramming of fibroblasts to automatic, differentiated cardiomyocytes without involving a pluripotent intermediate or the use of viruses [48, 49]. However, concerns remain about abnormal phenotype in these cells, as reflected in changes at the chromosomal and subchromosomal levels [50, 51].