Chung-Wah Siu

Functional Sarcoplasmic Reticulum for Calcium Handling of Human Embryonic Stem Cell‐Derived Cardiomyocytes: Insights for Driven Maturation

Cardiomyocytes (CMs) are nonregenerative. Self‐renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell‐based therapies. In adult CMs, Ca2+‐induced Ca2+ release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR) is key in excitation‐contraction coupling. Therefore, proper Ca2+ handling properties of hESC‐derived CMs are required for their successful functional integration with the recipient heart. Here, we performed a comprehensive analysis of CMs differentiated from the H1 (H1‐CMs) and HES2 (HES2‐CMs) hESC lines and human fetal (F) and adult (A) left ventricular (LV) CMs. Upon electrical stimulation, all of H1‐, HES2‐, and FLV‐CMs generated similar Ca2+ transients. Caffeine induced Ca2+ release in 65% of FLV‐CMs and ∼38% of H1‐ and HES2‐CMs. Ryanodine significantly reduced the electrically evoked Ca2+ transient amplitudes of caffeine‐responsive but not ‐insensitive HES2‐ and H1‐CMs and slowed their upstroke; thapsigargin, which inhibits the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump, reduced the amplitude of only caffeine‐responsive HES2‐ and H1‐CMs and slowed the decay. SERCA2a expression was highest in ALV‐CMs but comparable among H1‐, HES2‐, and FLV‐CMs. The Na+‐Ca2+ exchanger was substantially expressed in both HES2‐ and H1‐CMs relative to FLV‐ and ALV‐CMs. RyR was expressed in HES2‐, H1‐, and FLV‐CMs, but the organized pattern for ALV‐CMs was not observed. The regulatory proteins junctin, triadin, and calsequestrin were expressed in ALV‐CMs but not HES2‐ and H1‐CMs. We conclude that functional SRs are indeed expressed in hESC‐CMs, albeit immaturely. Our results may lead to driven maturation of Ca2+ handling properties of hESC‐CMs for enhanced contractile functions.

Bioartificial Sinus Node Constructed via In Vivo Gene Transfer of an Engineered Pacemaker HCN Channel Reduces the Dependence on Electronic Pacemaker in a Sick-Sinus Syndrome Model

Background— The normal cardiac rhythm originates in the sinoatrial (SA) node that anatomically resides in the right atrium. Malfunction of the SA node leads to various forms of arrhythmias that necessitate the implantation of electronic pacemakers. We hypothesized that overexpression of an engineered HCN construct via somatic gene transfer offers a flexible approach for fine-tuning cardiac pacing in vivo. Methods and Results— Using various electrophysiological and mapping techniques, we examined the effects of in situ focal expression of HCN1-&Dgr;&Dgr;&Dgr;, the S3-S4 linker of which has been shortened to favor channel opening, on impulse generation and conduction. Single left ventricular cardiomyocytes isolated from guinea pig hearts preinjected with the recombinant adenovirus Ad-CMV-GFP-IRES-HCN1-&Dgr;&Dgr;&Dgr; in vivo uniquely exhibited automaticity with a normal firing rate (237±12 bpm). High-resolution ex vivo optical mapping of Ad-CGI-HCN1-&Dgr;&Dgr;&Dgr;–injected Langendorff-perfused hearts revealed the generation of spontaneous action potentials from the transduced region in the left ventricle. To evaluate the efficacy of our approach for reliable atrial pacing, we generated a porcine model of sick-sinus syndrome by guided radiofrequency ablation of the native SA node, followed by implantation of a dual-chamber electronic pacemaker to prevent bradycardia-induced hemodynamic collapse. Interestingly, focal transduction of Ad-CGI-HCN1-&Dgr;&Dgr;&Dgr; in the left atrium of animals with sick-sinus syndrome reproducibly induced a stable, catecholamine-responsive in vivo “bioartificial node” that exhibited a physiological heart rate and was capable of reliably pacing the myocardium, substantially reducing electronic pacing. Conclusions— The results of the present study provide important functional and mechanistic insights into cardiac automaticity and have further refined an HCN gene–based therapy for correcting defects in cardiac impulse generation.

Mechanistic Role of If Revealed by Induction of Ventricular Automaticity by Somatic Gene Transfer of Gating-Engineered Pacemaker (HCN) Channels

Background— Although If, encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, is known to be functionally important in pacing, its mechanistic action is largely inferential and indeed somewhat controversial. To dissect in detail the role of If, we investigated the functional consequences of overexpressing in adult guinea pig left ventricular cardiomyocytes (LVCMs) various HCN1 constructs that have been engineered to exhibit different gating properties. Methods and Results— We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-&Dgr;&Dgr;&Dgr;, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7&Dgr;&Dgr;&Dgr;, and Ins HCN1 channels, respectively; EVY235-7&Dgr;&Dgr;&Dgr; and Ins encode channels in which the S3–S4 linkers have been shortened and lengthened to favor and inhibit opening, respectively. Ad-CGI-HCN1, Ad-CGI-HCN1-&Dgr;&Dgr;&Dgr;, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of If with comparable densities when fully open (≈−22 pA/pF at −140 mV; P>0.05) but distinctive activation profiles (V1/2=−70.8±0.6, −60.4±0.7, and −87.7±0.7 mV; P<0.01, respectively). Whereas control (nontransduced or Ad-CGI–transduced) LVCMs were electrically quiescent, automaticity (206±16 bpm) was observed exclusively in 61% of Ad-HCN1-&Dgr;&Dgr;&Dgr;–transduced cells that displayed depolarized maximum diastolic potential (−60.6±0.5 versus −70.6±0.6 mV of resting membrane potential of control cells; P<0.01) and gradual phase 4 depolarization (306±32 mV/s) that were typical of genuine nodal cells. Furthermore, spontaneously firing Ad-HCN1-&Dgr;&Dgr;&Dgr;–transduced LVCMs responded positively to adrenergic stimulation (P<0.05) but exhibited neither overdrive excitation nor suppression. In contrast, the remaining 39% of Ad-HCN1-&Dgr;&Dgr;&Dgr;–transduced cells exhibited no spontaneous action potentials; however, a single ventricular action potential associated with a depolarized resting membrane potential and a unique, incomplete “phase 4–like” depolarization that did not lead to subsequent firing could be elicited on simulation. Such an intermediate phenotype, similarly observed in 100% of Ad-CGI-HCN– and Ad-CGI-HCN1-Ins–transduced LVCMs, could be readily reversed by ZD7288, hinting at a direct role of If. Correlation analysis revealed the specific biophysical parameters required for If to function as an active membrane potential oscillator. Conclusions— Our results not only contribute to a better understanding of cardiac pacing but also may advance current efforts that focus primarily on automaticity induction to the next level by enabling bioengineering of central and peripheral cells that make up the native sinoatrial node.

Overexpression of HCN-encoded pacemaker current silences bioartificial pacemakers.

BACKGROUND Current strategies of engineering bioartificial pacemakers from otherwise silent yet excitable adult atrial and ventricular cardiomyocytes primarily rely on either maximizing the hyperpolarization-activated I(f) or on minimizing its presumptive opponent, the inwardly rectifying potassium current I(K1). OBJECTIVE The purpose of this study was to determine quantitatively the relative current densities of I(f) and I(K1) necessary to induce automaticity in adult atrial cardiomyocytes. METHODS Automaticity of adult guinea pig atrial cardiomyocytes was induced by adenovirus (Ad)-mediated overexpression of the gating-engineered HCN1 construct HCN1-DeltaDeltaDelta with the S3-S4 linker residues EVY235-7 deleted to favor channel opening. RESULTS Whereas control atrial cardiomyocytes remained electrically quiescent and had no I(f), 18% of Ad-CMV-GFP-IRES-HCN1-DeltaDeltaDelta (Ad-CGI-HCN1-DeltaDeltaDelta)-transduced cells demonstrated automaticity (240 +/- 14 bpm) with gradual phase 4 depolarization (143 +/- 28 mV/s), a depolarized maximal diastolic potential (-45.3 +/- 2.2 mV), and substantial I(f) at -140 mV (I(f,-140 mV) = -9.32 +/- 1.84 pA/pF). In the remaining quiescent Ad-CGI-HCN1-DeltaDeltaDelta-transduced atrial cardiomyocytes, two distinct immediate phenotypes were observed: (1) 13% had a hyperpolarized resting membrane potential (-56.7 +/- 1.3 mV) with I(f,-140 mV) of -4.85 +/- 0.97 pA/pF; and (2) the remaining 69% displayed a depolarized resting membrane potential (-27.6 +/- 1.3 mV) with I(f,-140 mV) of -23.0 +/- 3.71 pA/pF. Upon electrical stimulation, both quiescent groups elicited a single action potential with incomplete phase 4 depolarization that was never seen in controls. Further electrophysiologic analysis indicates that an intricate balance of I(K1) and I(f) is necessary for induction of atrial automaticity. CONCLUSION Optimized pacing induction and modulation can be better achieved by engineering the I(f)/I(K1) ratio rather than the individual currents.