Frank P. Ittleman

Protection of human left ventricular myocardium from cutting injury with 2,3-butanedione monoxime.

To prevent dissection injury when cutting strip preparations from human left ventricular papillary muscle tissue, dissections were carried out with 2,3-butanedione monoxime (30 mM) added to Krebs-Ringer solution and followed by washout with normal solution. Eleven muscle strip preparations were dissected from left ventricular papillary muscle tissue of five patients undergoing mitral valve replacement surgery. The average muscle strip length was 6.8 ± 1.4 mm, and cross-section area was 0.49 ± 0.16 mm2. Peak twitch tension was 2.02 ± 1.33 g/mm2 and ranged from 0.67 to 5.5 g/mm2 at an extracellular calcium concentration of 2.5 mM (21° C, 0.16 Hz). In one muscle strip, which was stored in Krebs-Ringer plus 2,3-butanedione monoxime solution for 20 hours, peak twitch tension in normal Krebs-Ringer solution was 1.85 g/mm2. When temperature was increased from 21° C, there was a continuous increase in peak twitch tension (by 38%) up to about 28° C; then peak twitch tension decreased so that at 37° C (n =3) average peak twitch tension was lower than at 21° C by 47%. The force-frequency relation exhibited a broad force plateau between 40 and 120 beats/min at 37° C. The plateau was markedly narrowed at 30° C and 24° C. Thermopile heat measurements revealed appropriate waveform characteristics in high-resolution single-beat heat records indicating minimal surface cell damage. Thus, cardioplegia with 2,3-butanedione monoxime protects human left ventricular myocardium from dissection injury facilitating dissection and preservation of strip preparations with extraordinarily low cross-sectional areas and high peak twitch tensions. These preparations are suitable for myothermal and mechanical measurements.

Energetics of isometric force development in control and volume-overload human myocardium. Comparison with animal species.

Alteration in crossbridge behavior and myocardial performance have been associated with myosin isoenzyme composition in animal models of myocardial hypertrophy or atrophy. In the hypertrophied human heart, myocardial performance is altered without significant changes in myosin isoenzymes. To better understand this discrepancy, isometric heat and force measurements were carried out in 1) control and volume-overload human myocardium, 2) control, pressure-overload, and hyperthyroid rabbit myocardium, and 3) control and hypothyroid rat myocardium. In control human myocardium, peak isometric twitch tension was 44.0 +/- 11.7 mN/mm2, and maximum rate of tension rise was 69.2 +/- 21.0 mN/sec.mm2. In volume-overload human myocardium, peak twitch tension and maximum rate of tension rise were reduced by 55% (p less than 0.05) and 65% (p less than 0.05), respectively. The average force-time integral of the individual crossbridge cycle, calculated by myothermal techniques, was increased by 85% (p less than 0.005) in volume-overload human myocardium. In control and hormonally altered myocardium, both across and within species (control human, control rat, control rabbit, hypothyroid rat, and hyperthyroid rabbit), there was a close relation between the crossbridge force-time integral and the percentage of V3-type myosin isoenzyme in the myocardium. However, hemodynamically altered (volume-overload human and pressure-overload rabbit) myocardium did not follow this relation. Across and within species, there were significant correlations between maximum rate of tension rise and average tension-dependent heat rate (r = 0.97, p less than 0.001) and between maximum rate of tension fall and average tension-independent heat rate (r = 0.82; p less than 0.025). Furthermore, there were close inverse relations between these heat rates and the crossbridge force-time integral. In addition, there was an inverse relation between tension-independent heat and the crossbridge force-time integral. Across and within species total myocardial energy turnover was significantly correlated with the crossbridge force-time integral (relative total heat, r = -0.84, p less than 0.02; relative total-activity related heat, r = -0.88, p less than 0.01). The present findings indicate that 1) factors separate from myosin isoenzymes account for the altered crossbridge cycle in volume-overload human and pressure-overload rabbit myocardium, 2) changes in excitation-contraction coupling processes accompany changes in the crossbridge cycle within and across species, and 3) the force-time integral of the crossbridge cycle is a major determinant of total myocardial energy turnover.

Thin Filament-Based Modulation of Contractile Performance in Human Heart Failure

Background—The contribution of the sarcomere’s thin filament to the contractile dysfunction of human cardiomyopathy is not well understood. Methods and Results—We have developed techniques to isolate and functionally characterize intact (native) thin filaments obtained from failing and nonfailing human ventricular tissue. By use of in vitro motility and force assays, native thin filaments from failing ventricular tissue exhibited a 19% increase in maximal velocity but a 27% decrease in maximal contractile force compared with nonfailing myocardium. Native thin filaments isolated from human myocardium after left ventricular assist device support demonstrated a 37% increase in contractile force. Dephosphorylation of failing native thin filaments resulted in a near-normalization of thin-filament function, implying a phosphorylation-mediated mechanism. Tissue expression of the protein kinase C isoforms &agr;, &bgr;1, and &bgr;2 was increased in failing human myocardium and reduced after left ventricular assist device support. Conclusions—These novel findings demonstrate that (1) the thin filament is a key modulator of contractile performance in the failing human heart, (2) thin-filament function is restored to near normal levels after LVAD support, and (3) the alteration of thin-filament function in failing human myocardium is mediated through phosphorylation, most likely through activation of protein kinase C.

Valves of the deep venous system: an overlooked risk factor

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.

Myosin from failing and non-failing human ventricles exhibit similar contractile properties.

In non-failing human myocardium, V1 myosin comprises a small amount (<10%) of the total myosin content, whereas end-stage failing hearts contain nearly 100% V3 myosin. It has been suggested that this shift in V1 myosin isoform content may contribute to the contractile deficit in human myocardial failure. To test this hypothesis, myosin was isolated from human failing and non-failing ventricles, and non-failing atria. Performance was assessed in in vitro motility and isometric force assays. Consistent with prior reports, a small amount of V1 myosin was present in both non-failing (6.2 +/- 1.0%) and failing (3.5 +/- 1.4%) ventricular tissues. No difference in isometric force or unloaded shortening velocity was observed for failing and non-failing ventricular myosin irrespective of myosin isoform content. Atrial tissue expressing predominantly V1 myosin (66.7 +/- 4.1%) generated half the force but greater velocity compared with ventricular tissue, expressing predominantly V3 myosin. In additional experiments, rabbit cardiac myosin was used in a calcium regulated assay system to determine if V1 and V3 isoforms differentially affect thin filament activation. Half-maximal calcium activation was similar for the two cardiac isoforms. A 1:9 mixture of V1/V3 myosin, simulating isoform composition in non-failing human myocardium, was indistinguishable from 100% V3 myosin (simulating the failing state) with regard to velocity of shortening and average force. These data suggest that the myosin isoform shift reported in human myocardial failure does not significantly contribute to the contractile deficit of this disease.

Atrial septal defect with right to left shunt despite normal pulmonary artery pressure.

A 74 year old woman had right to left shunting through an atrial septal defect despite normal right heart pressures. Acute volume expansion temporarily reduced the shunt. Contrast echocardiography and angiography demonstrated that this shunting occurred almost exclusively from the inferior vena cava. At surgery a redundant flap of septum secundum was found that was adjacent to the inferior vena cava orifice, intercepting its blood return like a spinnaker and shunting it into the left atrium.

Late thoracic outlet syndrome secondary to pseudarthrosis of the clavicle

Successful resolution of a late thoracic outlet syndrome secondary to pseudarthrosis of the clavicle was accomplished by surgical resection of the pseudoarthrosis and mid 3 cm of the clavicle, following conservative therapy which did not alleviate the symptoms. The patient was a 28-year-old woman. Arteriography demonstrated the lesion. A shoulder dislocation preceded the thoracic outlet syndrome; a congenital pseudarthrosis may have been present.

The detection of isolated tumor cells in bone marrow comparing bright-field immunocytochemistry and multicolor immunofluorescence.

BackgroundThe detection of isolated tumor cells in bone marrow by immunocytochemistry (ICC) has been reported to predict progression of early-stage breast cancer. The most common staining procedure uses bright-field ICC with cytokeratin (CK) antibodies to label isolated tumor cells. However, this method can result in false-positive staining events. We used multicolor immunofluorescence (IF) to develop a more specific assay for detecting isolated tumor cells in marrow samples from breast cancer patients.MethodsWe compared ICC and IF side by side for detection of cancer cells and false-positive staining events on bone marrow aspirates from breast cancer patients, bone marrow from healthy donors, and healthy donor blood spiked with cancer cells. The primary target for isolated tumor cell detection was CK for both methods. IF used an additional set of antibodies to label hematopoietic cells (HCs).ResultsThe detection rate of CK+ events in breast cancer patient bone marrow aspirates was 18 (58%) of 31 for ICC and 21 (68%) of 31 for IF. However, with IF, 17 of 21 CK+ cases were stained with HC markers and thus were identified as false-positive events. A surprisingly high CK+ event rate was observed in healthy donor blood and marrow. In all healthy donor samples, CK+ events were readily identified as HCs by IF. Detection sensitivity of spiked cancer cells in donor blood was similar for both methods.ConclusionsThere is a high frequency of CK+ events in blood and marrow, and it is important to note that this is observed both in patients with and those without cancer. IF with multiple HC markers allows straightforward discrimination between CK+ cells of hematopoietic and nonhematopoietic origin.

Traumatic Rupture of the Right Hemidiaphragm: Case Report of an Unusual Means of Diagnosis

It has been reported that 10 to 20% of all traumatic diaphragmatic ruptures are missed on initial evaluation, only to be discovered at a later date (1). In the chronic state an abnormal chest X-ray, symptomatic visceral incarceration, or barium contrast studies often lead to the correct diagnosis. This is a case of chronic diaphragmatic injury secondary to trauma being discovered when a diagnostic thoracentesis yielded viscous bile. With the needle left in situ a dye study was performed which demonstrated not only a normal cholangiogram, but also a chronic diaphragmatic rupture with partial herniation of the liver. We feel that this case represents a very unusual presentation of an uncommon injury.

The effects of aging on the intimal region of the human saphenous vein: insights from multimodal microscopy and quantitative image analysis

We hypothesized that structural remodeling associated with advancing age occurs in human saphenous veins. To address this hypothesis, we have identified structural remodeling in human saphenous veins by applying histochemistry, fluorescence staining and quantitative image analysis to specifically assess intimal area, intimal cellularity and intimal collagen content and organization. Saphenous veins were collected from patients undergoing coronary artery bypass graft surgery. Area measurements and cellularity were quantified using the image analysis software Stereo Investigator, employing planimetry and counting frames, respectively. Collagen content and organization were quantified in MetaMorph image analysis software based on measurements of color (hue, saturation, and intensity) from polarized light images. Intimal area and cellularity showed no statistically significant increases with age; in contrast, total collagen content showed a significant decrease with advancing age. Furthermore, collagen fiber types also demonstrated a statistically significant alteration with age; increases in age resulted in decreases in larger collagen fibers. No significant changes in small collagen fibers were identified. These results raise the possibility that age-associated structural alterations in total collagen content, specifically collagen fiber size, could be a factor in the etiology of age-associated venous diseases.