Eugenio Cingolani

Intracoronary cardiosphere-derived cells after myocardial infarction: evidence of therapeutic regeneration in the final 1-year results of the CADUCEUS trial (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction).

OBJECTIVES This study sought to report full 1-year results, detailed magnetic resonance imaging analysis, and determinants of efficacy in the prospective, randomized, controlled CADUCEUS (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction) trial. BACKGROUND Cardiosphere-derived cells (CDCs) exerted regenerative effects at 6 months in the CADUCEUS trial. Complete results at the final 1-year endpoint are unknown. METHODS Autologous CDCs (12.5 to 25 × 10(6)) grown from endomyocardial biopsy specimens were infused via the intracoronary route in 17 patients with left ventricular dysfunction 1.5 to 3 months after myocardial infarction (MI) (plus 1 infused off-protocol 14 months post-MI). Eight patients were followed as routine-care control patients. RESULTS In 13.4 months of follow-up, safety endpoints were equivalent between groups. At 1 year, magnetic resonance imaging revealed that CDC-treated patients had smaller scar size compared with control patients. Scar mass decreased and viable mass increased in CDC-treated patients but not in control patients. The single patient infused 14 months post-MI responded similarly. CDC therapy led to improved regional function of infarcted segments compared with control patients. Scar shrinkage correlated with an increase in viability and with improvement in regional function. Scar reduction correlated with baseline scar size but not with a history of temporally remote MI or time from MI to infusion. The changes in left ventricular ejection fraction in CDC-treated subjects were consistent with the natural relationship between scar size and ejection fraction post-MI. CONCLUSIONS Intracoronary administration of autologous CDCs did not raise significant safety concerns. Preliminary indications of bioactivity include decreased scar size, increased viable myocardium, and improved regional function of infarcted myocardium at 1 year post-treatment. These results, which are consistent with therapeutic regeneration, merit further investigation in future trials. (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction [CADUCEUS]; NCT00893360).

Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block

TBX18 gene therapy reprogrammed cardiomyocytes to become pacemaker cells in a pig model of heart block. Reprogrammed Heart Cells Set the Pace Pacemakers have revolutionized the care of patients with slow or abnormal heart rhythms. But these devices can fail by becoming infected or nonfunctional. For these patients, Hu and colleagues created biological pacemakers to serve as a “bridge to device,” providing temporary, hardware-free rhythmic support. Gene transfer of the human embryonic transcription factor T-box 18 (TBX18) to ventricular cardiomyocytes converted, or “reprogrammed,” these cells into pacemaker cells—cells that fire electrical impulses and therefore determine an individual’s heart rate. The authors previously demonstrated TBX18 gene transfer in mice but, in this study, showed that it is feasible in a large-animal model by restoring normal heart rate in pigs with complete heart block. Because the pig’s heartbeat and size are similar to humans, this study represents an important step in translation of this genetic reprogramming approach to the clinic. Somatic reprogramming by reexpression of the embryonic transcription factor T-box 18 (TBX18) converts cardiomyocytes into pacemaker cells. We hypothesized that this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications, and therefore tested whether adenoviral TBX18 gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. Biological pacemaker activity, originating from the intramyocardial injection site, was evident in TBX18-transduced animals starting at day 2 and persisted for the duration of the study (14 days) with minimal backup electronic pacemaker use. Relative to controls transduced with a reporter gene, TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity. Induced sinoatrial node cells could be identified by their distinctive morphology at the site of injection in TBX18-transduced animals, but not in controls. No local or systemic safety concerns arose. Thus, minimally invasive TBX18 gene transfer creates physiologically relevant pacemaker activity in complete heart block, providing evidence for therapeutic somatic reprogramming in a clinically relevant disease model.

Creation of a Genetic Calcium Channel Blocker by Targeted Gem Gene Transfer in the Heart

Calcium channel blockers are among the most commonly used therapeutic drugs. Nevertheless, the utility of calcium channel blockers for heart disease is limited because of the potent vasodilatory effect that causes hypotension, and other side effects attributable to blockade of noncardiac channels. Therefore, focal calcium channel blockade by gene transfer is highly desirable. With a view to creating a focally applicable genetic calcium channel blocker, we overexpressed the ras-related small G-protein Gem in the heart by somatic gene transfer. Adenovirus-mediated delivery of Gem markedly decreased L-type calcium current density in ventricular myocytes, resulting in the abbreviation of action potential duration. Furthermore, transduction of Gem resulted in a significant shortening of the electrocardiographic QTc interval and reduction of left ventricular systolic function. Focal delivery of Gem to the atrioventricular (AV) node significantly slowed AV nodal conduction (prolongation of PR and AH intervals), which was effective in the reduction of heart rate during atrial fibrillation. Thus, these results indicate that gene transfer of Gem functions as a genetic calcium channel blocker, the local application of which can effectively modulate cardiac electrical and contractile function.

Gene Therapy to Inhibit the Calcium Channel β Subunit Physiological Consequences and Pathophysiological Effects in Models of Cardiac Hypertrophy

Calcium cycling figures prominently in excitation-contraction coupling and in various signaling cascades involved in the development of left ventricular hypertrophy. We hypothesized that genetic suppression of the L-type calcium channel accessory &bgr;-subunit would modulate calcium current and suppress cardiac hypertrophy. A short hairpin RNA template sequence capable of mediating the knockdown of the L-type calcium channel accessory &bgr;-subunit gene was incorporated into a lentiviral vector (PPT.CG.H1.&bgr;2). Transduction of ventricular myocytes in vivo with the active short hairpin RNA partially inhibited the L-type calcium current. In neonatal rat cardiomyocytes, L-type calcium channel accessory &bgr;-subunit gene knockdown reduced calcium transient amplitude. Similarly, [3H]leucine incorporation was attenuated in PPT.CG.H1.&bgr;2-transduced neonatal rat cardiomyocytes compared with nonsilencing controls in a phenylephrine-induced hypertrophy model. In vivo gene transfer attenuated the hypertrophic response in an aortic-banded rat model of left ventricular hypertrophy, with reduced left ventricular wall thickness and heart weight/body weight ratios in PPT.CG.H1.&bgr;2-injected rats at four weeks post transduction. Fractional shortening was preserved in rats treated with PPT.CG.H1.&bgr;2. These findings indicate that knockdown of L-type calcium channel accessory &bgr;-subunit is capable of attenuating the hypertrophic response both in vitro and in vivo without compromising systolic performance. Suppression of the calcium channel &bgr; subunit may represent a novel and useful therapeutic strategy for left ventricular hypertrophy.

Non-cell-autonomous effects of vector-expressed regulatory RNAs in mammalian heart cells

In mammalian cells, small regulatory RNA molecules are able to modulate gene expression in a cell-autonomous manner. In contrast, this mechanism of gene regulation can occur systemically in plants and nematodes. The existence of similar cell-to-cell transmission in mammalian cells has been explored, but generalizibilty and mechanistic insights have remained elusive. Here, we show that small regulatory RNA molecules are capable of a non-cell-autonomous effect between primary cardiac myocytes through a gap-junction-dependent mechanism. Co-culture experiments showed that both Dicer-processed small-interfering RNAs (siRNAs) and Drosha-processed microRNAs (miRNAs) were capable of target gene knockdown and physiological effects in a non-cell-autonomous manner. Target gene siRNA molecules were detected in recipient cells, indicating transfer of the primary effector molecule. All of these effects were abrogated by dominant-negative molecular suppression of gap junction function. Our results show that both siRNAs and miRNAs are capable of a non-cell-autonomous effect between mammalian cells through gap junctions. The recognition of this biological process raises the novel therapeutic prospect of a bystander effect after gene transfer to tissues bearing gap junctions and for cell engineering with a view to creating regulatory RNA donor cells that exert their influence throughout a syncytium.

Gene Transfer of Connexin43 Mutants Attenuates Coupling in Cardiomyocytes. Novel Basis for Modulation of Cardiac Conduction by Gene Therapy

Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector–mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.

Intracoronary Cardiosphere-Derived Cells After Myocardial Infarction: Evidence of Therapeutic Regeneration in the Final 1-Year Results of the CADUCEUS Trial

OBJECTIVES This study sought to report full 1-year results, detailed magnetic resonance imaging analysis, and determinants of efficacy in the prospective, randomized, controlled CADUCEUS (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction) trial. BACKGROUND Cardiosphere-derived cells (CDCs) exerted regenerative effects at 6 months in the CADUCEUS trial. Complete results at the final 1-year endpoint are unknown. METHODS Autologous CDCs (12.5 to 25 × 10(6)) grown from endomyocardial biopsy specimens were infused via the intracoronary route in 17 patients with left ventricular dysfunction 1.5 to 3 months after myocardial infarction (MI) (plus 1 infused off-protocol 14 months post-MI). Eight patients were followed as routine-care control patients. RESULTS In 13.4 months of follow-up, safety endpoints were equivalent between groups. At 1 year, magnetic resonance imaging revealed that CDC-treated patients had smaller scar size compared with control patients. Scar mass decreased and viable mass increased in CDC-treated patients but not in control patients. The single patient infused 14 months post-MI responded similarly. CDC therapy led to improved regional function of infarcted segments compared with control patients. Scar shrinkage correlated with an increase in viability and with improvement in regional function. Scar reduction correlated with baseline scar size but not with a history of temporally remote MI or time from MI to infusion. The changes in left ventricular ejection fraction in CDC-treated subjects were consistent with the natural relationship between scar size and ejection fraction post-MI. CONCLUSIONS Intracoronary administration of autologous CDCs did not raise significant safety concerns. Preliminary indications of bioactivity include decreased scar size, increased viable myocardium, and improved regional function of infarcted myocardium at 1 year post-treatment. These results, which are consistent with therapeutic regeneration, merit further investigation in future trials. (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction [CADUCEUS]; NCT00893360).

Biological pacemaker created by percutaneous gene delivery via venous catheters in a porcine model of complete heart block

BACKGROUND Pacemaker-dependent patients with device infection require temporary pacing while the infection is treated. External transthoracic pacing is painful and variably effective, while temporary pacing leads are susceptible to superinfection. OBJECTIVE To create a biological pacemaker delivered via venous catheters in a porcine model of complete heart block, providing a temporary alternative/adjunct to external pacing devices without additional indwelling hardware. METHODS Complete atrioventricular (AV) nodal block was induced in pigs by radiofrequency ablation after the implantation of a single-chamber electronic pacemaker to maintain a ventricular backup rate of 50 beats/min. An adenoviral vector cocktail (K(AAA) + H2), expressing dominant-negative inward rectifier potassium channel (Kir2.1AAA) and hyperpolarization-activated cation channel (HCN2) genes, was injected into the AV junctional region via a NOGA Myostar catheter advanced through the femoral vein. RESULTS Animals injected with K(AAA) + H2 maintained a physiologically relevant ventricular rate of 93.5 ± 7 beats/min (n = 4) compared with control animals (average rate, 59.4 ± 4 beats/min; n = 6 at day 7 postinjection; P <.05). Backup electronic pacemaker utilization decreased by almost 4-fold in the K(AAA) + H2 group compared with the control (P <.05), an effect maintained for the entire 14-day window. In contrast to the efficacy of gene delivery into the AV junctional region, open-chest, direct injection of K(AAA) + H2 (or its individual vectors) into the ventricular myocardium failed to elicit significant pacemaker activity. CONCLUSIONS The right-sided delivery of K(AAA) + H2 to the AV junctional region provided physiologically relevant biological pacing over a 14-day period. Our approach may provide temporary, bridge-to-device pacing for the effective clearance of infection prior to the reimplantation of a definitive electronic pacemaker.

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening.

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ∼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.

Prevention of esophageal thermal injury during radiofrequency ablation for atrial fibrillation

Pulmonary vein isolation using radiofrequency ablation is an effective therapy in patients with atrial fibrillation. However, the esophagus descends in close proximity to the posterior wall of the left atrium and renders this structure susceptible to thermal injury. Esophageal ulceration has been hypothesized to be a precursor to left atrial–esophageal fistula, a procedural complication associated with poor prognosis. In this review, we have analyzed and summarized the published data regarding esophageal thermal injury during catheter ablation for atrial fibrillation and strategies to minimize risk of this complication. While esophageal temperature monitoring can be useful, multiple factors such as patient characteristics and specific strategies for radiofrequency energy delivery also merit consideration.