Adam E. Saltman

The Use of Extracellular Matrix as an Inductive Scaffold for the Partial Replacement of Functional Myocardium

Regenerative medicine approaches for the treatment of damaged or missing myocardial tissue include cell-based therapies, scaffold-based therapies, and/or the use of specific growth factors and cytokines. The present study evaluated the ability of extracellular matrix (ECM) derived from porcine urinary bladder to serve as an inductive scaffold for myocardial repair. ECM scaffolds have been shown to support constructive remodeling of other tissue types including the lower urinary tract, the dermis, the esophagus, and dura mater by mechanisms that include the recruitment of bone marrow-derived progenitor cells, angiogenesis, and the generation of bioactive molecules that result from degradation of the ECM. ECM derived from the urinary bladder matrix, identified as UBM, was configured as a single layer sheet and used as a biologic scaffold for a surgically created 2 cm2 full-thickness defect in the right ventricular free wall. Sixteen dogs were divided into two equal groups of eight each. The defect in one group was repaired with a UBM scaffold and the defect in the second group was repaired with a Dacron patch. Each group was divided into two equal subgroups (n = 4), one of which was sacrificed 15 min after surgical repair and the other of which was sacrificed after 8 weeks. Global right ventricular contractility was similar in all four subgroups groups at the time of sacrifice. However, 8 weeks after implantation the UBM-treated defect area showed significantly greater (p < 0.05) regional systolic contraction compared to the myocardial defects repaired with by Dacron (3.3 ± 1.3% vs. −1.8 ± 1.1%; respectively). Unlike the Dacron-repaired region, the UBM-repaired region showed an increase in systolic contraction over the 8-week implantation period (–4.2 ± 1.7% at the time of implantation vs. 3.3 ± 1.3% at 8 weeks). Histological analysis showed the expected fibrotic reaction surrounding the embedded Dacron material with no evidence for myocardial regeneration. Histologic examination of the UBM scaffold site showed cardiomyocytes accounting for approximately 30% of the remodeled tissue. The cardiomyocytes were arranged in an apparently randomly dispersed pattern throughout the entire tissue specimen and stained positive for α-sarcomeric actinin and Connexin 43. The thickness of the UBM graft site increased greatly from the time of implantation to the 8-week sacrifice time point when it was approximately the thickness of the normal right ventricular wall. Histologic examination suggested complete degradation of the originally implanted ECM scaffold and replacement by host tissues. We conclude that UBM facilitates a constructive remodeling of myocardial tissue when used as replacement scaffold for excisional defects.

Myocardial recovery from ischemia is impaired in CD36-null mice and restored by myocyte CD36 expression or medium-chain fatty acids.

Long-chain fatty acid uptake, which provides a large part of myocardial energy, is impaired in human and murine hearts deficient in the membrane fatty acid translocase, FAT/CD36. We examined myocardial function in CD36-null mice using the working heart. Fatty acid oxidation and stores of glycogen, triglycerides, and ATP were reduced in CD36-deficient hearts and were restored to WT levels by rescue of myocyte CD36. Under normal perfusion conditions, CD36-null hearts had similar cardiac outputs and end-diastolic pressures as WT or transgenic hearts. After 6 min of ischemia, cardiac output decreased by 41% and end diastolic pressure tripled for CD36-null hearts, with no significant changes in WT or transgenic hearts. Null hearts also failed more frequently after ischemia as compared with WT or transgenics. To dissect out contribution of fatty acid uptake, a perfusate-lacking fatty acids was used. This decreased cardiac output after ischemia by 30% in WT hearts as compared with 50% for CD36-deficient hearts. End diastolic pressure, a negative index of myocardial performance, increased after ischemia in all heart types. Addition to the perfusate of a medium-chain fatty acid (caprylic acid) that does not require CD36 for uptake alleviated poor ischemic tolerance of CD36-null hearts. In summary, recovery from ischemia is compromised in CD36-deficient hearts and can be restored by CD36 rescue or by supplying medium-chain fatty acids. It would be important to determine whether the findings apply to the human situation where polymorphisms of the CD36 gene are relatively common.

The completely endoscopic treatment of atrial fibrillation: report on the first 14 patients with early results.

UNLABELLED We report the early results of a new completely endoscopic technique for the treatment of atrial fibrillation (AF). METHODS Fourteen patients underwent surgery solely for the treatment of AF. The thoracoscopic technique delivered microwave energy to the epicardial surface of the beating heart. Access was obtained through 3 right-sided and 3 left-sided thoracic ports. The AFx/Guidant Flex-10 catheter was employed to produce a box lesion around the pulmonary veins along with additional right- and left-sided lesions. The left atrial appendage was amputated. RESULTS Ten patients had paroxysmal fibrillation, 1 had persistent fibrillation, and 3 were in permanent AF. Mean age of the group was 60 years, and their mean duration of AF was 74 months. Half had undergone unsuccessful attempts at chemical and/or electrical cardioversion. There were no deaths. Two patients required conversion to open procedure to control bleeding from the left atrial appendage. Average procedure time was 221 minutes, with the last 2 procedures taking less than 2 hours. Median length of hospital stay was 6 days, with 7 patients staying less than 3 days. Seventy-one percent of patients were in sinus rhythm at discharge, 100% at 6 months follow-up, and 67% at 12 months. CONCLUSION Totally endoscopic microwave ablation of atrial fibrillation appears to be safe and truly minimally invasive. It is associated with a short length of stay, short procedure time, and acceptable rhythm results. This procedure has the potential to greatly expand the indications for surgery in patients suffering from AF and deserves longer-term investigation.

Preconditioning with PKC and the ATP-sensitive potassium channels: a codependent relationship

BACKGROUND Both potassium channel openers and protein kinase C have been shown to independently elicit the myoprotective preconditioning response. However, the in vivo dependency between the two is unknown. METHODS Thirty-seven sheep were divided into seven groups; animals received no pretreatment, pinacidil, pinacidil and potassium channel opener blocker glibenclamide, protein kinase C activator 4beta-phorbol-12,13-dibutyrate (PDBu), or PDBu and protein kinase C blocker chelerythrine. The last two groups underwent opposite blockade, chelerythrine + pinacidil, or glibenclamide + PDBu. All groups underwent 60 minutes of regional ischemia followed by 180 minutes of reperfusion. Regional function was assessed throughout the experiment, and at the conclusion of the study the infarct size (as a percentage of the area at risk) was determined. RESULTS Infarct size decreased in the groups receiving only pinacidil or PDBu (control: 54%+/-3%, pinacidil: 25% +/-2%, PDBu: 21%+/-3%; p<0.05 pinacidil or PDBu versus control). This preconditioning protection was lost when the direct blocker was given (58%+/-5%, glibenclamide + pinacidil; 70%+/-6%, chelerythrine + PDBu; p = not significant versus control). The preconditioning response was again attenuated when the opposite blockers were given (64%+/-5%, chelerythrine + pinacidil; 63%+/-1%, glibenclamide + PDBu; p = not significant versus control). There was no significant difference in regional function. CONCLUSIONS This study shows that both protein kinase C and potassium channels are necessary and codependent for preconditioning in the in vivo heart.

Nitric oxide–generating β-adrenergic blocker nipradilol preserves postischemic cardiac function

BACKGROUND Preconditioning protects the heart from ischemic injury, but some of its effects are reversed by beta-adrenergic blockade. We hypothesize that because nitric oxide is known to precondition the heart, the nitric oxide-generating beta-blocker nipradilol may simultaneously precondition and provide clinically relevant beta-blockade. METHODS Isolated, crystalloid-perfused rabbit hearts underwent 1 hour of left anterior descending coronary artery ischemia followed by 1 hour of reperfusion. Before ischemia, six hearts received nipradilol, six received the nitric oxide donor L-arginine, four hearts received the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester before L-arginine, nine underwent ischemic preconditioning, and six received beta-blockade by esmolol before ischemic preconditioning. Seven hearts received no pretreatment (control). Action potential duration and ventricular pressure were measured. Infarct size was determined at the end of reperfusion. RESULTS Both L-arginine and ischemic preconditioning prolonged action potential duration significantly at 60 minutes of reperfusion. Compared with control, infarct size was reduced by ischemic preconditioning (26%+/-4% versus 49%+/-3%, IPC versus control; p<0.01), L-arginine (24%+/-2%; p<0.01 versus control), and nipradilol (24%+/-2%; p<0.01 versus control). Only nipradilol preserved peak developed pressure during reperfusion. CONCLUSIONS Despite its properties as a beta-adrenergic blocking agent, nipradilol was able to precondition the heart, probably as a result of its ability to produce nitric oxide.

Both Metabolic Inhibition and Mitochondrial KATP Channel Opening Are Myoprotective and Initiate a Compensatory Sarcolemmal Outward Membrane Current

Background—Blockade of oxidative phosphorylation may activate ATP sensitive mitochondrial potassium (mitoKATP) channels. We examined whether both metabolic inhibition and mitoKATP channel openers protect both the whole organ and isolated cells from ischemia. Methods and Results—Using a Langendorff preparation, one group of isolated rabbit hearts were exposed to ischemic preconditioning (IPC) via 2 episodes of flow interruption. The second group of hearts was preconditioned with 2 episodes of either the metabolic inhibitor, sodium cyanide (NaCN), or the mitoKATP channel opener, diazoxide. The third group of hearts was exposed to the mitoKATP channel inhibitor, 5-hydroxydecanoic acid (5-HD) prior to preconditioning with NaCN, diazoxide or IPC. Controls had no drug infused. Then, ischemia was induced in all hearts by left anterior descending coronary artery occlusion and infarct size was determined. Compared with controls (40±3%), infarct size was significantly reduced in hearts preconditioned with NaCN, diazoxide or IPC (18±3%, 26±3%, 21±2%, respectively; P <0.05 versus control). These reductions were reversed by 5-HD (36±3%, 33±2%, 37±2%; NaCN, diazoxide, IPC, respectively). Secondly, whole cell patch clamped isolated guinea pig ventricular myocytes were preconditioned with 2 episodes of either NaCN or diazoxide followed by Tyrodes perfusion with membrane potential set to −70 mV. Control cells were exposed to Tyrodes solution. All cells were then clamped to −20 mV and exposed to NaCN, which caused induction of an outward potassium current. Compared with controls, the average time to induction of the outward current was significantly reduced in cells preconditioned with either brief application of NaCN (11.6±1.8 versus 5.1±1.0 minutes, control versus NaCN, P <0.05) or diazoxide (5.5±1.4 versus 2.0±0.8 minutes, control versus diazoxide, P <0.05). Conclusion—Preconditioning protects the heart through mitoKATP. This protection also alters a surface membrane current, which may be important in myocardial protection.

Thrombogenic Performance of a St. Jude Bileaflet Mechanical Heart Valve in a Sheep Model

The sheep model is preferred for chronic evaluation of prosthetic heart valves, surgical techniques, and endocardiographic studies. A bileaflet mechanical heart valve (MHV) was implanted into a sheep model to study its in vivo performance and to evaluate the thrombogenic potential of the valve. Transesophageal echocardiography and transcranial Doppler ultrasonography measurements were conducted before and after the valve implantation. Platelet activity state (PAS) assay measurements were also conducted before and after the implantation surgery. After sheep euthanasia, the MHV was explanted and scanning electron microscopy (SEM) was performed on the explanted valve to examine changes to the MHV surface. Tissue blocks were taken from the sheep brain, left ventricle, aorta, spleen, and lung lobes for histological examination. Our results indicated that after the MHV implantation, more embolic signals were detected in the sheep carotid artery, increasing monotonously as a function of implantation time. Echocardiographic parameters including blood aortic velocity, transvalvular pressure gradient, and velocity time integral increased. PAS increased significantly after valve implantation. SEM pictures demonstrated calcium and phosphate deposition on the valve surfaces. Histological examination demonstrated hemorrhage in the lung tissue, pulmonary thrombosis, and osteogenesis in heart tissue.

Amrinone preconditioning in the isolated perfused rabbit heart.

BACKGROUND Ischemic preconditioning (IPC) reduces infarct size in experimental preparations. IPC, however, is not without detrimental effects. We studied amrinone as a possible alternative to IPC. METHODS Isolated perfused rabbit hearts were given a 5-minute infusion of 10 micromol/L amrinone followed by a 5-minute washout (n = 6). The anterior descending artery was then occluded for 1 hour and reperfused for 1 hour. Six hearts underwent IPC, with two episodes of 5-minute global ischemia followed by 5-minute reperfusion before LAD occlusion; eight control hearts received no preconditioning. Left ventricular pressure and ischemic zone epicardial monophasic action potentials were continuously monitored. RESULTS IPC but not amrinone reduced peak pressure before anterior descending artery occlusion. Peak pressure fell significantly during ischemia and reperfusion in all hearts. End diastolic pressure rose significantly during reperfusion in control and IPC hearts but not in amrinone hearts. Action potentials shortened during ischemia in all hearts. They returned to preocclusion values in control hearts but lasted beyond preocclusion values in IPC and amrinone hearts. Both the incidences of ventricular fibrillation and infarct size were significantly reduced in amrinone hearts but not in IPC hearts. CONCLUSIONS Amrinone is not only a useful inotropic agent but is also a superior preconditioning agent when compared to IPC.

Pharmacological preconditioning with the adenosine triphosphate–sensitive potassium channel opener pinacidil

BACKGROUND Ischemic preconditioning (IPC) decreases infarct size after global or regional ischemia. Potassium channel openers also precondition but are subject to dose-limiting vasodilation. We compared the mechanical and electrophysiological effects of ischemic and pharmacological preconditioning in an isolated rabbit heart model. METHODS Rabbit hearts were preconditioned with either 10 micromol/L pinacidil alone (P-), 10 micromol/L pinacidil with 10 micromol/L phenylephrine (P+), or two cycles of global ischemia and reperfusion (IPC) before 1 hour of LAD occlusion. Left ventricular pressure, epicardial monophasic action potential duration (APD) and coronary flow were monitored throughout. Infarct size was determined at the end of reperfusion. RESULTS Regional ischemia uniformly decreased APD (p<0.05). During reperfusion, APDs were prolonged beyond preischemic values in all preconditioned groups (p<0.05). P- and P+ reduced the incidence of fibrillation. P- significantly increased coronary flow (+15%, p = 0.001), whereas IPC and P+ did not. However, IPC and P- significantly decreased systolic function (p<0.05) but P+ did not. In addition, IPC depressed diastolic function (p<0.05) but P- and P+ did not. Infarct size was reduced by all methods (p<0.05). CONCLUSIONS Pinacidil presents a safe and effective alternative to IPC for preserving the heart during regional ischemia. Its coronary vasodilatory effects are safely and effectively reversed by the addition of phenylephrine.

Effects of Ischemia on Epicardial Deformation in the Passive Rabbit Heart

BACKGROUND Surgically induced ischemia in the arrested heart can result in changes in the mechanical properties of the myocardium. Regions of ischemia may be characterized based on the amount of epicardial deformation for a given load. Computer aided speckle interferometry (CASI), which tracks the movement of clusters of particles, is developed as a technique for measuring epicardial deformation, thereby determining the perfusion status of the passive heart. MATERIALS AND METHODS Silicone carbide particles and retroreflective beads were dispersed randomly onto the epicardial surface of 11 isolated rabbit hearts to form speckle images. The hearts were arrested with hyperkalemic Krebs-Henseleit buffered solution. Each heart was then exposed to a series of intracavitary pressures, and at each pressure speckle images were acquired with a charge-coupled device (CCD) camera. Nine hearts were exposed to global ischemia, and two hearts were exposed to regional ischemia by occluding the second diagonal branch of the left anterior descending artery (LAD). The hearts were again loaded and the speckle images were acquired. CASI was used to determine the distribution of deformation field. RESULTS CASI was able to determine displacements with a spatial resolution of about 50 microns. Global ischemia resulted in a significant increase in the maximum principle strain and the first invariant of the 2-D strain tensor. In the regionally ischemic heart, a large difference in deformation between the ischemic and perfused regions was clearly observed. CONCLUSION Based on epicardial deformation, CASI is able to distinguish between perfused and ischemic myocardium, with a spatial resolution of 50 microns.