Alan P. Kypson

Neovascularization after transmyocardial laser revascularization in a model of chronic ischemia

BACKGROUNDnThe mechanism of clinical improvement after transmyocardial laser revascularization (TMR) is unknown. One hypothesis holds that TMR causes increased myocardial perfusion through neovascularization. This study sought to determine whether angiogenesis occurs after TMR in a porcine model of chronic myocardial ischemia.nnnMETHODSnSix miniature pigs underwent subtotal left circumflex coronary artery occlusion to reduce resting blood flow to 10% of baseline. After 2 weeks in the low-flow state, dobutamine stress echocardiography and positron emission tomography were performed to document ischemic, viable myocardium. The animals then underwent TMR and were sacrificed 6 months later for histologic and immunohistochemical analysis.nnnRESULTSnHistologic analysis of the lased left circumflex region demonstrated many hypocellular areas filled with connective tissue representing remnant TMR channels. Histochemical staining demonstrated a highly disorganized pattern of neovascularization consistent with angiogenesis located predominantly at the periphery of the channels. Immunohistochemical analysis confirmed the presence of endothelial cells within neovessels. Vascular density analysis revealed a mean of 29.2+/-3.6 neovessels per high-power field in lased ischemic myocardium versus 4.0+/-0.3 (p<0.001) in nonlased ischemic myocardium.nnnCONCLUSIONSnThis study provides evidence that neovascularization is present long term in regions of ischemic, viable myocardium after TMR. Angiogenesis may represent the mechanism of clinical improvement after TMR.

Adenovirus-mediated gene transfer of the β2-adrenergic receptor to donor hearts enhances cardiac function

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of β2-adrenergic receptors (β2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human β2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1u2009×u20091010 p.f.u. of adenovirus encoding the β2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the β2-AR agonist, zinterol. Treatment with the β2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean β-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1u2009±u2009286 versus β2-AR, 6250.6*u2009±u2009432.5 mmHg/s; n = 10, *P < 0.02). β2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of β2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.

Molecular β-adrenergic signaling abnormalities in failing rabbit hearts after infarction

We studied alterations in the beta-adrenergic receptor (beta-AR) system of rabbit hearts during the development of heart failure (HF) after myocardial infarction (MI) to determine whether the molecular beta-AR abnormalities associated with human HF exist in this animal model. Rabbit HF was established 3 wk after left circumflex coronary artery (LCX) ligation by in vivo physiological measurements, and molecular beta-AR signaling was examined in tissue and cultured ventricular myocytes. We found that there was a significant global reduction in beta-AR density by approximately 50% in both ventricles of MI animals compared with sham-operated control animals and that functional beta-AR coupling was significantly reduced. Importantly, as found in human HF, myocardial protein levels and activity of the beta-AR kinase (beta-ARK1) and Galphai were found to be significantly elevated in MI rabbits, suggesting that these molecules are contributing to myocardial dysfunction. Thus the myocardial beta-AR system of this rabbit model of HF shares important biochemical characteristics with human HF and therefore is an ideal laboratory model to investigate novel therapeutic targets for the treatment of HF.We studied alterations in the β-adrenergic receptor (β-AR) system of rabbit hearts during the development of heart failure (HF) after myocardial infarction (MI) to determine whether the molecular β-AR abnormalities associated with human HF exist in this animal model. Rabbit HF was established 3 wk after left circumflex coronary artery (LCX) ligation by in vivo physiological measurements, and molecular β-AR signaling was examined in tissue and cultured ventricular myocytes. We found that there was a significant global reduction in β-AR density by ∼50% in both ventricles of MI animals compared with sham-operated control animals and that functional β-AR coupling was significantly reduced. Importantly, as found in human HF, myocardial protein levels and activity of the β-AR kinase (β-ARK1) and Gαi were found to be significantly elevated in MI rabbits, suggesting that these molecules are contributing to myocardial dysfunction. Thus the myocardial β-AR system of this rabbit model of HF shares important biochemical characteristics with human HF and therefore is an ideal laboratory model to investigate novel therapeutic targets for the treatment of HF.

Induction of angiogenesis after TMR: a comparison of holmium: YAG, CO2, and excimer lasers.

BACKGROUNDnTransmyocardial laser revascularization (TMR) is an emerging treatment for end-stage coronary artery disease. A variety of lasers are currently available to perform the procedure, although their relative efficacy is unknown. The purpose of this study was to compare changes in myocardial blood flow and function 6 months after TMR with holmium:yttrium-aluminum-garnet (holmium:YAG), carbon dioxide (CO2), and xenon chloride excimer lasers in a model of chronic ischemia.nnnMETHODSnMiniswine underwent subtotal (90%) left circumflex coronary stenosis. Baseline positron emission tomography and dobutamine stress echocardiography were performed to document hibernating myocardium in the left circumflex coronary artery distribution. Animals were then randomized to sham redo-thoracotomy (n = 5) or TMR using a holmium:YAG (n = 5), CO2 (n = 5) or excimer (n = 5) laser. Six months postoperatively, the positron emission tomography and dobutamine stress echocardiography studies were repeated and the animals sacrificed.nnnRESULTSnIn animals undergoing TMR with holmium: YAG and CO2 lasers, a significant improvement in myocardial blood flow to the lased left circumflex regions was seen. No significant change in myocardial blood flow was seen in sham- or excimer-lased animals. There was a significant improvement in regional stress function of the lased segments 6 months postoperatively in animals undergoing holmium:YAG and CO2 laser TMR that was consistent with a reduction in ischemia. There was no change in wall motion in sham- or excimer-lased animals. Significantly greater neovascularization was observed in the holmium:YAG and CO2 lased regions than with either the sham procedure or excimer TMR.nnnCONCLUSIONSnTransmyocardial laser revascularization with either holmium:YAG or CO2 laser improves myocardial blood flow and contractile reserve in lased regions 6 months postoperatively. These changes were not seen following excimer TMR or sham thoracotomy, suggesting that differences in laser energy or wavelength or both may be important in the induction of angiogenesis.

Ex Vivo Adenovirus-Mediated Gene Transfer To The Adult Rat Heart

OBJECTIVEnThe ability to transfer genes to adult myocardium may have therapeutic implications for cardiac transplantation. We investigated the feasibility of adenovirus-mediated transfer of marker genes LacZ and Luciferase, as well as the potentially therapeutic gene of the human beta2-adrenergic receptor in a rat heterotopic heart transplant model.nnnMETHODSnDonor hearts were flushed with 10(12) total viral particles of one of three transgenes. Hearts were harvested at various time points after transplantation. LacZ-treated hearts were assessed by histologic staining and Luciferase-treated hearts were assayed for specific luminescence activity. Hearts treated with beta2-adrenergic receptor underwent radioligand binding assays and immunohistochemistry with the use of an antibody specific for the human beta2-adrenergic receptor.nnnRESULTSnLacZ hearts revealed diffuse myocyte staining as opposed to none within controls at 5 days. Luciferase hearts demonstrated a mean activity of 970,000 +/- 220,000 arbitrary light units versus 500 +/- 200 for the controls (p = 0.001). Total beta2-adrenergic receptor densities (fmol/mg membrane protein) for hearts that received the beta2-adrenergic receptor transgene at 3, 5, 7, 10, and 14 days after infection were as follows: right ventricle, 488.5 +/- 126.8, 519.4 +/- 81.8,* 477.1 +/- 51.8,* 183.0 +/- 6.5,* and 82.7 +/- 19.1; left ventricle, 511.0 +/- 167.6, 1206.4 +/- 321.8,* 525.3 +/- 188.7, 183.5 +/- 18.6,* and 75.9 +/- 15.2 (*p < 0.05 vs control value of 75.6 +/- 6.4). Immunohistochemical analysis revealed diffuse staining of varying intensity within myocardial sarcolemmal membranes.nnnCONCLUSIONSnWe conclude that global overexpression of different transgenes is possible during cardiac transplantation and, ultimately, adenovirus-mediated gene transfer may provide a unique opportunity for genetic manipulation of the donor organ, potentially enhancing its function.

An experimental model of chronic myocardial hibernation

BACKGROUNDnHibernating myocardium describes persistently impaired ventricular function at rest caused by reduced coronary blood flow. However, a realistic animal model reproducing this chronic ischemic state does not exist. The purpose of this study was to explore whether chronic low-flow hibernation could be produced in swine.nnnMETHODSnMiniswine underwent 90% stenosis of the left circumflex coronary artery. Positron emission tomography and dobutamine stress echocardiography were performed 3 and 30 days (n = 6) or 14 days (n = 4) after occlusion to evaluate myocardial blood flow and viability. Triphenyl tetrazolium chloride assessed percent infarction. Electron microscopy was used to identify cellular changes characteristic of hibernating myocardium.nnnRESULTSnPositron emission tomography (13N-labeled-ammonia) 3 days after occlusion demonstrated a significant reduction in myocardial blood flow in the left circumflex distribution. This reduced flow was accompanied by increased glucose use (18F-fluorodeoxyglucose), which is consistent with hibernating myocardium. Thirty days after occlusion, positron emission tomography demonstrated persistent low flow with increased glucose use in the left circumflex distribution. Dobutamine stress echocardiography 3 days after occlusion demonstrated severe hypocontractility at rest in the left circumflex region. Regional wall motion improved with low-dose dobutamine followed by deterioration at higher doses (biphasic response), findings consistent with hibernating myocardium. The results of dobutamine stress echocardiography were unchanged 30 days after occlusion. Triphenyl tetrazolium chloride staining (n = 6) revealed a mean of 8% +/- 2% infarction of the area-at-risk localized to the endocardial surface. Electron microscopy (n = 4) 14 days after occlusion demonstrated loss of contractile elements and large areas of glycogen accumulation within viable cardiomyocytes, also characteristic of hibernating myocardium.nnnCONCLUSIONSnChronic low-flow myocardial hibernation can be reproduced in an animal model after partial coronary occlusion. This model may prove useful in the study of the mechanisms underlying hibernating myocardium and the use of therapies designed to improve blood flow to the heart.

Improved perfusion and contractile reserve after transmyocardial laser revascularization in a model of hibernating myocardium

BACKGROUNDnTransmyocardial laser revascularization (TMR) has been demonstrated effective for relieving angina, although prior studies have yielded inconsistent results regarding postoperative myocardial perfusion and function. This study evaluated long-term changes in myocardial perfusion and contractile reserve after TMR in a model of hibernating myocardium.nnnMETHODSnMiniswine had subtotal left circumflex coronary artery occlusion to reduce resting blood flow to 10% of baseline. After 2 weeks in the low-flow state, positron emission tomography and dobutamine stress echocardiography were performed to document ischemic, viable (hibernating) myocardium in the left circumflex distribution. Animals then had sham redo thoracotomy (n = 4) or TMR (n = 6). Six months later the positron emission tomography and dobutamine stress echocardiography studies were repeated.nnnRESULTSnMyocardial blood flow in the left circumflex distribution as measured by positron emission tomography was significantly reduced in all animals after 2 weeks in the low-flow state. In animals that had TMR, there was significant improvement in myocardial blood flow to the lased regions 6 months postoperatively. No significant change in myocardial blood flow was seen in sham animals at 6 months. Dobutamine stress echocardiography after 2 weeks of low-flow demonstrated severe hypocontractility at rest in the left circumflex region of all animals, with a biphasic response to dobutamine consistent with hibernating myocardium. In animals that had TMR, there was a trend toward improved resting function and significantly improved regional stress function in the lased segments 6 months postoperatively, consistent with a reduction in ischemia. Global left ventricular wall motion at peak stress improved significantly as well. There was no change in wall motion 6 months postoperatively in sham-operated animals.nnnCONCLUSIONSnThis study found improvements in myocardial perfusion and regional and global contractile reserve 6 months after TMR in a porcine model of hibernating myocardium. This improved perfusion and function likely accounts for the clinical benefits of the procedure.

Port-access approach for combined aortic and mitral valve surgery

A technique is described for combined aortic and mitral valve operation utilizing the minimally invasive Port-Access technique. Two patients are repaired with excellent chest wall healing and avoidance of sternotomy.

Minimally invasive tricuspid operation using port access

BACKGROUNDnPort-access techniques performed through a right mini-thoracotomy have been extensively described for both the mitral and aortic valves. However, reports of tricuspid valve operations using the port-access approach are rare. A technique for minimally invasive tricuspid valve operation using port access is described.nnnMETHODSnPort-access approach was applied to 33 consecutive patients undergoing tricuspid valve repair or replacement.nnnRESULTSnTwelve percent (4 of 33) underwent tricuspid replacement and 88% underwent repair (28 of 33). Perioperative mortality was 6% (2 of 33) and conversion to median sternotomy was 3% (1 of 33).nnnCONCLUSIONSnPort-access tricuspid operations are both feasible and safe with a low conversion rate to conventional median sternotomy.

Transmyocardial laser revascularization limits in vivo adenoviral-mediated gene transfer in porcine myocardium

OBJECTIVEnTransmyocardial laser revascularization (TMR) is emerging as a potential treatment option for patients with end-stage CAD, and adjuvant gene therapy may be helpful in further improving the results of the procedure. However, the effects of TMR on gene transfer are unknown.nnnMETHODSnSwine underwent left thoracotomy. TMR was performed to create five channels at 2-cm intervals in the anterolateral free wall of the left ventricle (LV) followed by injection of 1 x 10(9) plaque-forming units (pfu) of a replication-deficient adenovirus vector carrying the reporter gene beta-galactosidase (Ad.Pac beta-gal). An additional five direct injections of 1 x 10(9) pfu Ad.Pac beta-gal were made at 2-cm intervals in the posterolateral LV of each heart. Control animals underwent TMR alone/vehicle alone (n = 3) or empty virus alone/no treatment (n = 3) of the anterolateral/posterolateral LV, respectively.nnnRESULTSnELISA revealed significantly greater transgene expression in the direct Ad.Pac beta-gal injection versus TMR plus Ad.Pac beta-gal inject regions at both 3 (n = 6) (273.0 +/- 58.5 vs. 133.4 + 28.1 pg beta-gal/g protein, P = 0.02) and 7 days (n = 6) (180.0 + 59.9 vs. 56.7 + 18.1 pg beta-gal/g protein, P = 0.02) postoperatively. At 14 days postoperatively (n = 2), no transgene expression was detected in either region. No transgene expression was detected in any of the control regions at 3 days postoperatively. CD-18 staining revealed significantly greater inflammation in the TMR plus Ad.Pac beta-gal and TMR alone regions as compared to Ad.Pac beta-gal or vehicle (P < 0.001).nnnCONCLUSIONSnAdenoviral-mediated gene transfer in conjunction with TMR is possible, although TMR appears to limit the degree of transgene expression attained as compared to direct intramyocardial injection alone, likely due to the greater immune response observed with the former. These findings may have important implications for therapeutic strategies aimed at combining TMR with gene therapy for CAD.