Peter Danilo

Human Mesenchymal Stem Cells as a Gene Delivery System to Create Cardiac Pacemakers

Abstract— 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 If-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.

Defective Cardiac Ryanodine Receptor Regulation During Atrial Fibrillation

Background—Ca2+ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca2+ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. Methods and Results—Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (n=6 for left atrial, n=4 for right atrial) and sham instrumented controls (n=6 for left atrial, n=4 for right atrial). Right atrial tissue was also collected from humans with AF (n=10) and sinus rhythm (n=10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca2+ leak. Conclusions—SR Ca2+ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of AF.

Expression and Function of a Biological Pacemaker in Canine Heart

Background—We hypothesized that localized overexpression of the hyperpolarization-activated, cyclic nucleotide-gated (HCN2) pacemaker current isoform in canine left atrium (LA) would constitute a novel biological pacemaker. Methods and Results—Adenoviral constructs of mouse HCN2 and green fluorescent protein (GFP) or GFP alone were injected into LA, terminal studies performed 3 to 4 days later, hearts removed, and myocytes examined for native and expressed pacemaker current (If). Spontaneous LA rhythms occurred after vagal stimulation-induced sinus arrest in 4 of 4 HCN2+GFP dogs and 0 of 3 GFP dogs (P <0.05). Native If in nonexpressed atrial myocytes was 7±4 pA at −130 mV (n=5), whereas HCN2+GFP LA had expressed pacemaker current (IHCN2) of 3823±713 pA at −125 mV (n=10) and 768±365 pA at −85 mV. Conclusions—HCN2 overexpression provides an If-based pacemaker sufficient to drive the heart when injected into a localized region of atrium, offering a promising gene therapy for pacemaker disease.

Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates.

Background—We hypothesized that administration of the HCN2 gene to the left bundle-branch (LBB) system of intact dogs would provide pacemaker function in the physiological range of heart rates. Methods and Results—An adenoviral construct incorporating HCN2 and green fluorescent protein (GFP) as a marker was injected via catheter under fluoroscopic control into the posterior division of the LBB. Controls were injected with an adenoviral construct of GFP alone or saline. Animals were monitored electrocardiographically for up to 7 days after surgery, at which time they were anesthetized and subjected to vagal stimulation to permit emergence of escape pacemakers. Hearts were then removed and injection sites visually identified and removed for microelectrode study of action potentials, patch clamp studies of pacemaker current, and/or immunohistochemical studies of HCN2. For 48 hours postoperatively, 7 of 7 animals subjected to 24-hour ECG monitoring showed multiple ventricular premature depolarizations and/or ventricular tachycardia attributable to injection-induced injury. Thereafter, sinus rhythm prevailed. During vagal stimulation, HCN2-injected dogs showed rhythms originating from the left ventricle, the rate of which was significantly more rapid than in the controls. Excised posterior divisions of the LBB from HCN2-injected animals manifested automatic rates significantly greater than the controls. Isolated tissues showed immunohistochemical and biophysical evidence of overexpressed HCN2. Conclusions—A gene-therapy approach for induction of biological pacemaker activity within the LBB system provides ventricular escape rhythms that have physiologically acceptable rates. Long-term stability and feasibility of the approach remain to be tested.

Xenografted Adult Human Mesenchymal Stem Cells Provide a Platform for Sustained Biological Pacemaker Function in Canine Heart

Background— Biological pacemaking has been performed with viral vectors, human embryonic stem cells, and adult human mesenchymal stem cells (hMSCs) as delivery systems. Only with human embryonic stem cells are data available regarding stability for >2 to 3 weeks, and here, immunosuppression has been used to facilitate survival of xenografts. The purpose of the present study was to determine whether hMSCs provide stable impulse initiation over 6 weeks without the use of immunosuppression, the “dose” of hMSCs that ensures function over this period, and the catecholamine responsiveness of hMSC-packaged pacemakers. Methods and Results— A full-length mHCN2 cDNA subcloned in a pIRES2-EGFP vector was electroporated into hMSCs. Transfection efficiency was estimated by GFP expression. IHCN2 was measured with patch clamp, and cells were administered into the left ventricular anterior wall of adult dogs in complete heart block and with backup electronic pacemakers. Studies encompassed 6 weeks. IHCN2 for all cells was 32.1±1.3 pA/pF (mean±SE) at −150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving ≥700 000 hMSCs (≈40% of which carried current). Rhythms were catecholamine responsive. Tissues from animals killed at 42 days manifested neither apoptosis nor humoral or cellular rejection. Conclusions— hMSCs provide a means for administering catecholamine-responsive biological pacemakers that function stably for 6 weeks and manifest no cellular or humoral rejection at that time. Cell doses >700 000 are sufficient for pacemaking when administered to left ventricular myocardium.

Impact of Sex and Gonadal Steroids on Prolongation of Ventricular Repolarization and Arrhythmias Induced by IK-Blocking Drugs

BackgroundMechanisms for longer rate-corrected QT intervals and higher incidences of drug-induced torsade de pointes in women than in men are incompletely defined, although gonadal steroids are assumed to be important determinants of these differences. Methods and ResultsWe used microelectrode techniques to study isolated rabbit right ventricular endocardium from control male and female and castrated male (ORCH) and female (OVX) rabbits. Action potential duration to 30% repolarization (APD30) was significantly shorter in male than female and in ORCH than OVX at a cycle length of 500 ms. The IKs blocker chromanol 293B had no effect on APD in males or females. The IKr blocker dofetilide prolonged APD in female and ORCH more than in male and OVX. At 10−6 mol/L dofetilide (cycle length=1 second), the incidence of early afterdepolarizations was: female, 67%; ORCH, 56%; male, 40%; and OVX, 28%. Serum 17&bgr;-estradiol levels were unrelated to the effects of dofetilide, but as testosterone levels increased, the dofetilide effect to increase APD diminished, as did early afterdepolarization incidence. ConclusionsSex-related differences in basal right ventricular endocardial AP configuration persist in castrated rabbits, suggesting that extragonadal factors contribute to the differences in ventricular repolarization. In this model, drugs that block IKr but not IKs prolong repolarization in a way that suggests that protection from excess prolongation in males is attributable to testosterone, whereas the risk of excess prolongation of repolarization in females is related to sex-determined factors in addition to estrogen.

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 &mgr;g/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.

Evolution and resolution of long-term cardiac memory.

BACKGROUND Cardiac memory (CM) refers to T-wave changes induced by ventricular pacing or arrhythmia that accumulate in magnitude and duration with repeated episodes of abnormal activation. We report herein the kinetics of long-term CM and its association with the ventricular action potential. METHODS AND RESULTS Dogs were paced from the ventricles at rates of 110 to 120 bpm for approximately 3 weeks. CM characterized by gradual sinus rhythm T vector rotation toward the paced QRS vector evolved in all dogs regardless of pacing site (left ventricular [LV] anterior apex or base, posterior LV, or right ventricular free wall). Cardiac hemodynamics and myocardial flow (microsphere studies) were unaltered by the pacing. Recovery time for the memory T wave to return to control increased with duration of the previous pacing. The protein synthesis inhibitor cycloheximide markedly (P<.05) and reproducibly attenuated evolution of CM. When pacing was performed from the atrium, CM did not occur. Standard microelectrode techniques were used to study action potential from the LV free wall of control and CM dogs. CM was associated with increased action potential duration in epicardial and endocardial but not midmyocardial cells, significantly altering the transmyocardial gradient for repolarization. CONCLUSIONS CM is a dynamic process for which the final T vector is predicted by the paced QRS vector and which is associated with significant changes in epicardial and endocardial but not midmyocardial cell action potential duration, such that the transmural gradient of repolarization is altered. It is unaccompanied by evidence of altered hemodynamics or flow, requires a change in pathway of activation, and appears to require new protein synthesis.

Effects of adrenergic amines on electrophysiological properties and automaticity of neonatal and adult canine Purkinje fibers: evidence for alpha- and beta-adrenergic actions.

We determined age-related differences in automaticity and responsiveness of cardiac Purkinje fibers from adult and neonatal dogs to graded concentrations of epinephrine, isoproterenol, and phenylephrine. Purkinje fibers were studied with standard microelectrode techniques during superfusion with Tyrodes solution at 37°C. Control spontaneous rates fur adults and neonates did not differ significantly. There was a biphasic response to all agonists such that rate decreased at low and increased at high concentrations. The decrease was greater with phenylephrine and epinephrine than with isoproterenol. The increase in rate was greater with isoproterenol and epinephrine than with phenylephrine. Propranolol shifted the dose-response curves downward and to the right for all agonists; phentolamine, shifted the curves upward and to the left. Epinephrine and isoproterenol dose-response curves for the neonates were upward and to the left of those for adults. Phenylephrine curves were identical for adults and neonates. Thus there are a- and β-adrenergic effects on Purkinje fiber automaticity; the former decrease and the latter increase rate. Furthermore, the effects on automaticity of /3-adrenergic amines are greater in the neonates than in the adult.

Repolarization Gradients in the Canine Left Ventricle Before and After Induction of Short-Term Cardiac Memory

Background—Questions remain about the contributions of transmural versus apicobasal repolarization gradients to the configuration of the T wave in control settings and after the induction of short-term cardiac memory. Methods and Results—Short-term cardiac memory is seen as T-wave changes induced by altered ventricular activation that persists after restoration of sinus rhythm. We studied cardiac memory in anesthetized, open-chest dogs paced from the ventricle for 2 hours. Unipolar electrograms were recorded from as many as 98 epicardial and 144 intramural sites, and activation times and activation-recovery intervals (ARIs) were measured. In separate experiments, epicardial monophasic action potentials were recorded. We found no appreciable left ventricular intramural gradients in repolarization times (activation time+ARI) in either control conditions or after the induction of memory. In controls, there was a left ventricular apicobasal gradient, with the shortest repolarization times in anterobasal regions and longest repolarization times posteroapically. After induction of memory, repolarization times shortened uniformly throughout the ventricular wall. Monophasic action potential duration at 90% repolarization decreased by ≈10 ms after induction of memory. Conclusions—In the intact canine left ventricle at physiological rates, there is no transmural gradient in repolarization. Apicobasal gradients in repolarization time, with shortest repolarization times in anterobasal areas and longest repolarization times in posteroapical regions, are important in the genesis of the T wave. Repolarization times and monophasic action potentials at the 90% repolarization level shorten after the induction of memory. The deeper T wave in the ECG after induction of memory may be explained by the more rapid phase 3 of the action potential.