Donald G. Weilbaecher

Localization of a Gene Responsible for Arrhythmogenic Right Ventricular Dysplasia to Chromosome 3p23

BACKGROUND Arrhythmogenic right ventricular dysplasia (ARVD), a familial cardiomyopathy occurring with a prevalence of 1 in 5000, is characterized by replacement of myocytes with fatty and fibrous tissue. Clinical manifestations include structural and functional abnormalities of the right ventricle and arrhythmias, leading to a sudden death rate of 2.5% per year. Four loci have been mapped, but no gene has been identified as yet. METHODS AND RESULTS We identified a large family of >200 members with ARVD segregating as an autosomal dominant trait affecting 10 living individuals. The diagnosis of ARVD was based on international diagnostic criteria including history, physical examination, ECG, echocardiogram, right ventricular angiogram, endomyocardial biopsy, and 24-hour ambulatory ECG. Blood was collected for DNA from 149 family members. Analysis of 257 polymorphic microsatellite markers by genetic linkage excluded previously known loci for ARVD and identified a novel locus at 3p23. Analysis of an additional 20 markers further defined the region. A peak logarithm of the odds score of 6.91 was obtained with marker D3S3613 at theta=0% recombination. Haplotype analysis identified a shared region between markers D3S3610 and D3S3659 of 9. 3 cM. CONCLUSIONS A novel locus for ARVD has been mapped to 3p23 and the region narrowed to 9.3 cM. Identification of the gene will allow genetic screening and a specific diagnosis for a disease with protean nonspecific findings. It should also provide insight fundamental to understanding cardiac chamber-specific gene expression and/or the mechanism of myocyte apoptosis observed in this disease.

Assessment of Myocardial Viability With 99mTc Sestamibi in Patients Undergoing Cardiac Transplantation A Scintigraphic/Pathological Study

BACKGROUND 99mTc sestamibi and 201 Tl are tracers that allow equivalent detection of myocardial infarction. However, because sestamibi does not undergo as much time-dependent redistribution as does 201Tl, it has been considered suboptimal for the detection of myocardial viability. METHODS AND RESULTS Fifteen consecutive patients with ischemic cardiomyopathy who underwent orthotopic cardiac transplantation received an intravenous injection of 99mTc sestamibi at 1 to 6 hours before transplantation. Rotational tomography of the excised, intact, native hearts was performed to quantify the extent of myocardial hypoperfusion. The hearts were then sliced and reimaged on a gamma camera, followed by pathological quantification of the extent and severity of scarred and normal myocardium. Samples of normally and abnormally perfused myocardium underwent gamma well counting to determine tissue radioactivity and were examined under light microscopy for delineation of myocardial structure after trichrome staining. The mean extent of scintigraphic scar quantified through the use of rotational tomography was 45 +/- 14% of the left ventricle and correlated closely with pathological scar size (r = .89), despite a slight overestimation. Scintigraphic scar size determined with planar imaging of the individual myocardial slices also correlated closely with pathological scar size (r = .88). A good correlation existed between tissue 99mTc sestamibi activity determined through well counting and histological evidence of myocardial viability (r = .89). Most hypokinetic and 40% of akinetic/dyskinetic myocardial segments contained scintigraphically and histologically normal myocardium. CONCLUSIONS 99mTc sestamibi scintigraphy can be used to accurately quantify the extent of myocardial scarring. Furthermore, the relative sestamibi activity in perfusion defects, measured several hours after administration, is a good indicator of myocardial viability determined with microscopy.

The Locus of a Novel Gene Responsible for Arrhythmogenic Right-Ventricular Dysplasia Characterized by Early Onset and High Penetrance Maps to Chromosome 10p12-p14

Arrhythmogenic right-ventricular dysplasia (ARVD), a cardiomyopathy inherited as an autosomal-dominant disease, is characterized by fibro-fatty infiltration of the right-ventricular myocardium. Four loci for ARVD have been mapped in the Italian population, and recently the first locus was mapped in inhabitants of North America. None of the genes have been identified. We have now identified another North American family with early onset of ARVD and high penetrance. All of the children with the disease haplotype had pathological or clinical evidence of the disease at age <10 years. The family spans five generations, having 10 living and 2 dead affected individuals, with ARVD segregating as an autosomal-dominant disorder. Genetic linkage analysis excluded known loci, and a novel locus was identified on chromosome 10p12-p14. A peak two-point LOD score of 3.92 was obtained with marker D10S1664, at a recombination fraction of 0. Additional genotyping and haplotype analysis identified a shared region of 10.6 cM between marker D10S547 and D10S1653. Thus, a novel gene responsible for ARVD resides on the short arm of chromosome 10. This disease is intriguing, since it initiates exclusively in the right ventricle and exhibits pathological features of apoptosis. Chromosomal localization of the ARVD gene is the first step in identification of the genetic defect and the unraveling of the molecular basis responsible for the pathogenesis of the disease.

Assessment of Myocardial Viability With 99mTc-Sestamibi Tomography Before Coronary Bypass Graft Surgery Correlation With Histopathology and Postoperative Improvement in Cardiac Function

BACKGROUND Assessment of myocardial viability by 99mTc-sestamibi remains controversial. Accordingly, we investigated the use of sestamibi as a marker of myocardial viability, defined by histopathology, and for predicting improvement of myocardial function after coronary artery bypass graft surgery (CABG). METHODS AND RESULTS 99mTc-sestamibi perfusion tomography and radionuclide angiography were performed within 2 days before CABG in 21 patients with > or = 75% stenosis of the left anterior descending coronary artery and resting anterior wall dyssynergy. During CABG, transmural myocardial biopsies were obtained from the dyssynergic anterior wall and from normal myocardial segments to determine the extent of viable myocardium by histopathology. Improvement of regional left ventricular function was evaluated by radionuclide angiography at 6 to 8 weeks after CABG. There was a good correlation (r=.85, P<.001) between the quantified sestamibi activity and the extent of viable myocardium determined morphometrically. Among 21 biopsied dyssynergic myocardial segments, 11 improved their function after CABG and 10 failed to improve. Biopsied segments with improved postoperative function had significantly higher sestamibi activity (81+/-5% versus 49+/-16%, P<.0001) and significantly lower extent of interstitial fibrosis (7+/-4% versus 31+/-21%, P=.0002) than segments that failed to improve. A 55% threshold of 99mTc-sestamibi activity had positive and negative predictive values of 79% and 100%, respectively, for recovery of function after CABG in the biopsied segments. CONCLUSIONS Myocardial 99mTc-sestamibi activity correlates well with the extent of viable myocardium and predicts improvement in regional function after CABG. This lends support to the use of sestamibi as a myocardial viability agent.

Relation of the Contractile Reserve of Hibernating Myocardium to Myocardial Structure in Humans

BACKGROUND Although dobutamine echocardiography (DE) is widely used to assess myocardial viability in humans, little is known about the relation between contractile reserve and myocardial structure. METHODS AND RESULTS We evaluated 20 patients with coronary disease (64+/-13 years old, ejection fraction 28+/-7.5%) with DE (up to 40 micrograms . kg(-1). min(-1)), rest-redistribution (201)Tl single photon emission CT, and quantitative angiography before bypass surgery. During surgery, patients underwent transmural myocardial biopsies (n=37) guided by transesophageal echocardiography to determine the extent of interstitial fibrosis and intracellular and interstitial proteins by histopathology and immunohistochemistry. Among the 37 segments biopsied, 16 recovered function as assessed 2 to 3 months later. Segments with postoperative functional recovery had more wall thickening at low-dose DE (28% versus 3%, P<0.001), higher thallium uptake (69% versus 48%, P=0.03), and less interstitial fibrosis (2% versus 28%, P<0.001). Quantitative angiographic parameters did not predict recovery of function. Segments with DE viability (contractile reserve and/or ischemia) had less fibrosis (2.7% versus 28%, P<0.001), less vimentin and fibronectin (both P<0.01), more glycogen (P=0.016), and higher thallium uptake (64% versus 35.5%, P<0.05) than those without viability. Viable segments by both DE and thallium had less fibrosis (1%) than those viable by 1 of the 2 techniques (9%) or not viable by both (28%, P=0.005). Thickening at low-dose DE correlated well with the extent of interstitial fibrosis (r=-0.83, P<0.01). CONCLUSIONS Contractile reserve during DE correlates inversely with the extent of interstitial fibrosis and the amount of fibronectin and vimentin and directly with rest-redistribution thallium uptake.

Ultrastructure of human bronchiolo-alveolar cell carcinoma

Ultrastructural features were correlated with a series of special staining reactions in eight cases of bronchiolo‐alveolar carcinoma. Ultrastructurally, all tumors were similarly composed of large cells with abundant cytoplasm and small nuclei in close contact with each other. Straight membranes or complex interdigitations occurred within adjacent tumor cells, attached to each other by scattered desmosomes. Microvilli or cilia abutted from free surfaces of the cells, and were noted in different stages of evolution. Numerous organelles were seen in the cytoplasm, including prominent mitochondria and single or coalescent secretory vacuoles with granular matrix resembling mucin. Other cytosomes less commonly found were irregular, partially lamellated inclusions and dark, homogeneous structures without limiting membranes. The stroma of the tumors was rich in elastin and collagen. Both the number of secretory vacuoles in the cytoplasm of tumor cells and the amount of connective tissue fibrils in the stroma of the tumors correlated well with the findings in the series of special staining reactions. No definite ultrastructural feature was present to identify the tumors as originating from Type II alveolar epithelial cells, but the possibility exists that they arose in the bronchiole, from undifferentiated basal cells or mucinous cells per se. Our impression in these eight cases studied is consistent with the view that bronchiolo‐alveolar carcinomas are indistinguishable at the ultrastructural level from other bronchogenic adenocarcinomas.

Treatment of symptomatic peripheral atherosclerotic disease with a rotational atherectomy device

Narrowings 70 to 90% in diameter in 3 iliac, 4 superficial femoral and 2 popliteal arteries were crossed and atherectomized successfully in 6 patients using the Squibb Rotablator under angiographic guidance during surgical bypass procedures on these arteries. The Rotablator consists of a 1.25 to 4.5 mm diameter oblong burr with tiny diamond blades mounted on a flexible shaft, which tracks over a spring-tip guidewire and rotates at speeds greater than 120,000 rpm. All stenoses were reduced to less than or equal to 50% of the normal luminal diameter. No significant complications occurred. Of the 6 patients having the atherectomy procedure, 5 were reevaluated by duplex Doppler measurements 1.5 to 5.5 (mean 3.5) months after atherectomy and found to be patient with only mild residual flow disturbance. Repeat follow-up by angiography after a mean of 5.2 months, however, showed only 3 (37%) of the atherectomized segments in 3 patients to still be patent. All were symptomatically improved. Of the effluent particles analyzed, 90% were less than 8 microns in size, while only 5% reached 250 microns. With improvements in technique, the largest particles were 150 to 180 microns, constituting only 1.4% of effluent debris. Samples of the effluent from 2 patients were injected in vivo into the left coronary system of 2 pigs. There were no acute hemodynamic or electrocardiographic complications or pathologic evidence of muscle necrosis or vascular thrombosis 18 to 48 hours later. These preliminary results with respect to feasibility and safety of the Rotablator are promising.

Valvulitis involving a bioprosthetic valve in a patient with systemic lupus erythematosus

A 37-year-old man with systemic lupus erythematosus, who underwent an aortic valve replacement with a Carpentier-Edwards porcine valve for severe aortic insufficiency, was admitted to the hospital with pulmonary edema. Transesophageal echocardiography revealed severe aortic insufficiency arising from partial dehiscence of the valve sewing ring, as well as centrally from the valve cusp. In addition, marked thickening of the mitral valve was observed with severe eccentric regurgitation. At surgery, valvulitis of the native mitral and bioprosthetic aortic valves was demonstrated, with a perforation of the porcine valve cusp. After replacement of both valves, the patient had a stormy postoperative course with recurrent communications between the left ventricle and atrium requiring multiple surgeries and eventually died. This case illustrates the severity of valvulopathy and ensuing complications that can affect patients with systemic lupus erythematosus and demonstrates that the valvulopathy can affect bioprosthetic valves, a finding that has significant implications as to the type of valve replacement in these patients.

Platelet-mediated thrombosis in stenosed canine coronary arteries: Inhibition by nicergoline, a platelet-active alpha-adrenergic antagonist

The effects of nicergoline, a new agent that blocks alpha-adrenergic receptors and inhibits platelet phospholipase, were evaluated in a canine model of platelet-mediated coronary thrombosis. In 48 open chest dogs, the circumflex coronary artery was stenosed by plicating the artery wall with a suture. Thirty-four of the 48 dogs exhibited cyclic reductions in flow in the stenotic vessel, followed by a sudden return to control levels. The reductions in flow were unabated in all but two dogs after heparin administration (1,000 U/kg per h), unaffected by large doses of nitroglycerin and nifedipine and associated with platelet aggregates in the stenotic segment (demonstrated by histologic and electron microscopic examination). These observations support the conclusion that the flow reductions were caused by platelet aggregation rather than by fibrin deposition or vasospasm. Twenty dogs were monitored for 1 hour after heparin administration and then assigned to a control (n = 7) or nicergoline-treated (n = 13; 1 mg/kg intravenously) group. In control dogs, cyclic reductions in flow continued unchanged for another hour, whereas in the treated group they were markedly decreased in 1 dog and completely abolished in the other 12 dogs. Aspirin (30 mg/kg intravenously) suppressed flow reductions in all control dogs, confirming the primary role of platelet aggregation in the phenomenon. This study provides a modified model of platelet-mediated thrombosis in stenosed coronary arteries. Furthermore, the results indicate that nicergoline can effectively interfere with platelet function in vivo. The potent antithrombotic activity exhibited by nicergoline might enhance the therapeutic usefulness of this vasodilator.

Seeding of arteriovenous prostheses with homologous endothelium: A preliminary report

Endothelial cell seeding of expanded polytetrafluoroethylene (e-PTFE) arteriovenous prostheses was performed to compare seeding with homologous vs. autologous cells and to study the effect of homologous seeding with a larger vs. a smaller number of cells. Sixteen dogs were randomly assigned to four equal groups: I, control; II, light homologous seeded; III, heavy homologous seeded; and IV, autologous seeded. Bilateral femoral arteriovenous loop grafts were inserted in all. Efficiency of seeding was assessed by cell counts of instilled vs. retained cells. All grafts remained patent and were harvested approximately 8 weeks after implantation. Samples were evaluated grossly by scanning electron microscopy and light microscopy. There was a direct correlation between number of cells instilled and number retained for each graft; group III received and retained the largest number (p less than 0.001 and p less than 0.05, respectively). The amount of thrombus deposition on the lumen of all grafts was grossly the same. Endothelium was demonstrated in samples obtained from the midgraft of groups III and IV; in contrast no endothelium was seen in groups I and II. The percentage of endothelialized surface was not determined. No immunologic cellular reaction was detected in any of the samples. We conclude that in the animal laboratory it is possible to seed e-PTFE arteriovenous prostheses successfully with homologous cells and to improve the efficiency of seeding by implanting a larger number of cells obtained from an endothelial cell bank. The potential applications of this technique to the clinical field are discussed herein.