Technique of Complete Heart Isolation with Continuous Cardiac Perfusion During Cardiopulmonary Bypass: New Opportunities for Gene Therapy.

Cardiopulmonary bypass (CPB) featuring complete heart isolation and continuous cardiac perfusion is a very promising approach for solving the problem of efficient gene delivery. In the technique presented here, separate pumps are used for the systemic and cardiac circuits. This system permits continuous isolated arrested heart perfusion through optimizing a number of delivery parameters including temperature, flow rate, driving pressure, ionic composition, and exposure time to the cardiac vessels. During complete cardiac isolation, the blood vector concentration trended from 11.51 ± 1.73 log genome copies (GCs)/cm3 to 9.84 ± 1.65 log GC/cm3 (p > .05). Despite restructuring a very high concentration to the heart, GCs were detectable in the systemic circuit. These values over time were near negligible by comparison but detectable 1.66 ± .26 during 20 minutes of recirculation and did not change (p > .05). After the completion of the recirculation interval and subsequent washing procedure, the initial systemic blood vector GC concentration slightly increased to 2.08 ± .38 log GCs/cm3 (p > .05). During the recirculation period, we supported flow via the cardiac circuit around 300 mL/min. In this technique of heart isolation with continuous cardiac perfusion, >99% of the vector remains in coronary circulation during recirculation period. The animal's non recirculation blood, or that in the system, was routinely tested during and after recirculation to contain much less than 1% of the original dose obtained via logging concentration of therapeutic over time. All of the sheep in this group recovered from anesthesia and received critical postoperative care, including all organ function, in the first 24-36 hours. Twenty-one sheep (84%) survived to euthanasia at 12 weeks. Average CPB time was 107 ± 19.0 minutes and cross-clamp time was 49 ± 7.9 minutes. This technology readily provides multiple pass recirculation of genes through the heart with minimal side effects of collateral expression of other organs.

Cardiac surgical delivery of the sarcoplasmic reticulum calcium ATPase rescues myocytes in ischemic heart failure.

BACKGROUND: The sarcoplasmic reticulum calcium ATPase (SERCA2a) is an important molecular regulator of contractile dysfunction in heart failure. Gene transfer of SERCA2a mediated by molecular cardiac surgery with recirculating delivery (MCARD) is a novel and clinically translatable strategy. METHODS: Ischemic heart failure was induced by ligation of OM1 and OM2 in 14 sheep. Seven sheep underwent MCARD-mediated AAV1-SERCA2a delivery 4 weeks after myocardial infarction, and seven sheep served as untreated controls. Magnetic resonance imaging-based mechanoenergetic studies were performed at baseline, 3 weeks, and 12 weeks after infarction. Myocyte apoptosis was quantified by Tdt-mediated nick-end labeling assay. Myocyte cross-sectional area and caspase-8 and caspase-9 activity was measured with imaging software, specific fluorogenic peptides, and immunohistochemistry. RESULTS: MCARD-mediated AAV1-SERCA2a gene delivery resulted in robust cardiac-specific SERCA2a expression and stable improvements in global and regional contractility. There were significantly higher stroke volume index, left ventricular fractional thickening, and ejection fraction at 12 weeks in the MCARD group than in the control group (30 ± 3 vs 21 ± 2 mL/m(2); 12% ± 5% vs 3% ± 3%; and 43 ± 4 vs 32 ± 4, respectively, all p < 0.05). Apoptotic myocytes were observed more frequently in the control group than in the MCARD-SERCA2a group (0.57.2 ± 0.16 AU vs 0.32.4 ± 0.08 AU, p < 0.05). MCARD-SERCA2a also resulted in decreased caspase-8 and caspase-9 expression and decreased myocyte area in the border zone of transgenic sheep compared with control sheep (14.6% ± 1.2% vs 2.9% ± 0.7%; 18.2% ± 1.9% vs 8.6% ± 1.4%; and 102.1 ± 3.8 µm(2) vs 88.1 ± 3.6 µm(2), all p < 0.05). CONCLUSIONS: MCARD-mediated SERCA2a delivery results in robust cardiac specific gene expression, improved contractility, and a decrease in both myocyte apoptosis and myocyte hypertrophy.

A pharmacokinetic analysis of molecular cardiac surgery with recirculation mediated delivery of ßARKct gene therapy: developing a quantitative definition of the therapeutic window.

BACKGROUND: Two major problems for translating gene therapy for heart failure therapy are: safe and efficient delivery and the inability to establish a relationship between vector exposure and in vivo effects. We present a pharmacokinetics (PK) analysis of molecular cardiac surgery with recirculating delivery (MCARD) of scAAV6-ßARKct. MCARD's stable cardiac specific delivery profile was exploited to determine vector exposure, half-life, and systemic clearance. METHODS AND RESULTS: Five naive sheep underwent MCARD with 10(14) genome copies of scAAV6-ßARKct. Blood samples were collected over the recirculation interval time of 20 minutes and evaluated with quantitative polymerase chain reaction (qPCR). C(t) curves were generated and expressed on a log scale. The exposure, half-life, and clearance curves were generated for analysis. qPCR and Western blots were used to determine biodistribution. Finally, all in vivo transduction data was plotted against MCARD's PK to determine if a relationship existed. Vector concentrations at each time point were (cardiac and systemic, respectively): 5 minutes: 9.16 ± 0.15 and 3.21 ± 0.38; 10 minutes: 8.81 ± 0.19 and 3.62 ± 0.37; 15 minutes: 8.75 ± 0.12 and 3.69 ± 0.31; and 20 minutes: 8.66 ± 0.22 and 3.95 ± 0.26; P < .00001. The half life of the vector was 2.66 ± 0.24 minutes. PK model data revealed that only 0.61 ± 0.43% of the original dose remained in the blood after delivery, and complete clearance from the system was achieved at 1 week. A PK transfer function revealed a positive correlation between exposure and in vivo transduction. Robust ßARKct expression was found in all cardiac regions with none in the liver. CONCLUSION: MCARD may offer a viable method to establish a relationship between vector exposure and in vivo transduction. Using this methodology, it may be possible to address a critical need for establishing an effective therapeutic window.

Reoperative cardiac surgery in Jehovah's witness patients with patent internal thoracic artery grafts: how far can we push the envelope?

Reoperative cardiac surgery in Jehovah's Witness (JW) patients with patent internal mammary arteries is a formidable surgical challenge. We have successfully performed 2 such cases using creative approaches. The first patient, a morbidly obese woman, presented with an acute coronary syndrome 4 years after off-pump coronary artery bypass grafting (CABG) with a hemoglobin of 10 gm/dL. She was stabilized with stenting of the culprit vessel; erythropoietin therapy was performed to increase her hemoglobin, and surgery was performed electively. The internal thoracic artery (ITA) was dissected and clamped, and intermittent cardioplegia was used for myocardial protection. The second patient needed aortic valve replacement 3 years after a previous CABG using an ITA. Limited dissection was used at redo operation without exposing the ITA. Aortic valve replacement was performed under cold fibrillatory arrest with an open ITA. Successful reoperative cardiac surgery in JW patients requires preoperative preparation using a multidisciplinary team approach and flexible operative planning.

Minimally invasive cardiac surgery using a flexible aortic clamp.

Minimally invasive surgery for mitral valve disease has been performed using a variety of technologies, some of which are complex, have a steep learning curve, and are expensive. We have adopted a simple cost-effective approach over the last 7 years to perform a variety of minimally invasive procedures with excellent outcomes. There have been no strokes, no mortalities, and no episodes of limb ischemia in our series. No patient has required reoperation.

Resection of a large atrial hemangioma using a bloodless surgical technique: a case report.

We present a biatrial hemangioma in a Jehovah's Witness patient. Hemangioma is extremely rare, accounting for 1% to 2% of benign cardiac tumors. Complete resection of a large hemangioma is mandatory due to its potentially life-threatening risk. In Jehovah's Witness patients, it is necessary to employ bloodless surgery protocols to maximize the patient's outcome. Our patient had undergone 6 weeks of monitoring and erythropoietin therapy prior to surgery, raising her hemoglobin level from 11.6 g/dL to 16.8 g/dL. Intraoperative bloodless surgical protocols as well as a continuous blood circuit were utilized. The patient's hemoglobin level on postoperative day one was 14.5 g/dL; one year postsurgery, the patient was symptom free.

Efficient myocyte gene delivery with complete cardiac surgical isolation in situ.

BACKGROUND: Previously, we used cardiopulmonary bypass with incomplete cardiac isolation and antegrade administration of vector for global cardiac gene delivery. Here we present a translatable cardiac surgical procedure that allows for complete surgical isolation of the heart in situ with retrograde (through the coronary venous circulation) administration of both vector and endothelial permeabilizing agents to increase myocyte transduction efficiency. METHODS: In 6 adult dogs the heart was completely isolated with tourniquets placed around both vena cavae and cannulas and all pulmonary veins. On cardiopulmonary bypass, the aorta and pulmonary artery were crossclamped, and the heart was isolated. Crystalloid cardioplegia at 4 degrees C containing 10(13) particles of adenovirus encoding LacZ and 15 microg of vascular endothelial growth factor was infused retrograde into the coronary sinus and recirculated for a total of 30 minutes. The dogs were then weaned from cardiopulmonary bypass and allowed to recover. With a catheter, 3 control dogs underwent retrograde infusion of the same cocktail without cardiac isolation or cardiopulmonary bypass. RESULTS: Beta-galactosidase activities in the cardiopulmonary bypass group were several orders of magnitude higher in both the right and left ventricles when compared with those in the control group (P < .05). X-gal staining from the cardiopulmonary bypass group showed unequivocal evidence of myocyte gene expression globally in a significant proportion of cardiac myocytes. No myocyte gene expression was observed in the control group. CONCLUSION: A novel cardiac surgical technique has been developed. This approach with cardiac isolation and retrograde delivery of vector through the coronary sinus results in efficient myocyte transduction in an adult large animal in vivo.

Global cardiac-specific transgene expression using cardiopulmonary bypass with cardiac isolation.

BACKGROUND: The available techniques for intravascular gene delivery to the heart are inefficient and not organ-specific. Yet, effective treatment of heart failure will likely require transgene expression by the majority of cardiac myocytes. To address this problem, we developed a novel cannulation technique that achieves efficient isolation of the heart in situ using separate cardiopulmonary bypass (CPB) circuits for the heart and body in dogs. METHODS: The arterial inflow and venous effluent from the two circuits were physically isolated. The efficiency of separation was 98% to 99% in three preliminary experiments using Evans Blue dye-labeled albumin. In 6 dogs, the cardiac circuit was perfused with oxygenated crystalloid cardioplegia at 37 degrees C containing approximately 4 x 10(11) particles of an adenovirus encoding LacZ (AdCMVLacZ) with a perfusion pressure of 170 to 200 mm Hg for 15 minutes allowing virus to recirculate through the heart approximately 15 times. Cross-clamp time was 26 +/- 2 minutes and CPB time was 90 +/- 3 minutes. RESULTS: Five animals survived and were euthanized at 7 days. Beta-galactosidase activities measured using a chemiluminescent assay were three orders of magnitude higher in all areas of the heart than in the liver. Histological analyses revealed heterogeneous X-Gal staining of myocytes in all areas of the myocardium. CONCLUSIONS: Despite using a constitutive promoter, this technique yields relatively cardiac-specific transgene expression and is potentially translatable to clinical applications. Future studies will allow for further optimization of the conditions necessary for vector-mediated gene delivery to the heart.