Stem-like cells traffic from heart ex vivo, expand in vitro, and can be transplanted in vivo.

BACKGROUND: Cells with stem cell surface markers have been identified in heart tissue. Early indications suggest that these are cardiac progenitor cells that could contribute to cardiac repair/regeneration. Clinically relevant therapeutic strategies based on these cells will require improved methods for their isolation and characterization of determinants of their mobilization, proliferation and differentiation. METHODS: An ex vivo culture system was developed that promotes trafficking of progenitor-like cells from mouse ventricles to a culture surface. Cells that "trafficked" from cardiac tissue were phenotyped by flow cytometry and immunohistochemistry. RESULTS: Morphologically distinct cells spontaneously trafficked from mouse ventricular tissue, adhered in culture, and proliferated for up to 4 weeks in Dulbecco's minimal essential media supplemented with fetal calf serum. After 4 weeks in culture, cell number declined. Co-culture with unfractionated bone marrow restored the proliferation of these trafficked cells. A significant population of the trafficked cells expressed a phenotype consistent with that of a myogenic progenitor such as: c-kit+, Sca-1+, CD45-, CD34-, CD90.2-, MyoD1-, desmin-, muscle-specific actin-, and, infrequently, myogenin+. An expanded population of trafficked cells from ventricles of mice expressing green fluorescent protein (GFP+) and containing cardiac-derived progenitor cells were injected into the pericardial space of GFP- mice. GFP+ cells trafficked throughout the heart but retained a primitive undifferentiated morphology. However, when injected into the pericardial space of Apo-E-deficient mice with coronary vasculopathy, progenitor-like cells trafficked into myocardium, and GFP+ cells differentiated into vessel-lining endothelial cells and, rarely, smooth muscle and cardiomyocytes. CONCLUSIONS: Progenitor-like cells in the heart can be mobilized by tissue injury to spontaneously traffic from cardiac tissue and can expand in culture by co-culture with bone marrow. When re-infused by pericardiocentesis, these primitive cells traffic into heart, retain immature morphology, but are capable of undergoing injury-induced differentiation. The novel method described herein permits further characterization of cardiac-derived progenitor cells, which are a candidate for cardiac regeneration strategies.

Fate of infants with hypoplastic left heart syndrome listed for cardiac transplantation: a multicenter study.

BACKGROUND: Infants with hypoplastic left heart syndrome (HLHS) commonly undergo cardiac transplantation as primary management. METHODS: We examined outcomes of primary transplantation for unpalliated HLHS. We analyzed data from the 20 institutions of the Pediatric Heart Transplant Study Group, from January 1, 1993, through December 31, 1998, using actuarial and parametric survival analysis and competing outcomes analysis. RESULTS: During the 6 years studied, 1,234 patients were listed for cardiac transplantation; 262 patients (21.2%) had unpalliated HLHS. The number (and percentage) of patients with HLHS decreased from 58 (27% of patients listed) in 1993 to 30 (14%) in 1998. Overall, 25% of infants with HLHS died while waiting; primary cause of death was cardiac failure (50%). Of the remaining patients awaiting transplantation, 23 (9%) underwent Norwood/Fontan-type surgeries as interim palliation: 52% died. Ultimately, 175 patients underwent cardiac transplantation (67%); 50% received organs by 2 months after listing. Post-transplant actuarial survival was 72% at 5 years, with 76% of deaths (35/46) occurring within 3 months; early mortality was caused primarily by graft failure within the first 30 days after transplantation (in 54%). Among 1-month survivors, survival at 1 and at 5 years was 92% and 85%, respectively. Of the 262 patients listed with unpalliated HLHS, overall survival, taking into account mortality after listing and after transplantation, was 68% at 3 months and 54% at 5 years. CONCLUSIONS: Cardiac transplantation offers good intermediate survival for infants with unpalliated HLHS.

An improved echocardiographic rejection-surveillance strategy following pediatric heart transplantation.

BACKGROUND: The unique demands of cardiac transplantation in infancy have led to non-invasive rejection-surveillance strategies. ECHO-A is a multiparametric, two-dimensionally guided, M-mode analysis algorithm that assigns an empirically derived score for deviations of recipient parameters to age-adjusted, population-based normal values. A cumulative ECHO-A score > or =4 is highly predictive of endomyocardial biopsy Grade > or =3 and of cellular rejection. METHODS: This study determined whether modifying ECHO-A to score for deviations of recipient parameters from the recipient's baseline would improve the predictive power of ECHO-A. We reanalyzed 701 consecutive echocardiograms of 18 pediatric cardiac transplant recipients (median age at transplantation, 142 days) and based scoring on significant (Z score > or =1) deviation from the patients' baseline means (ECHO-B). RESULTS: Eight episodes of treated rejection occurred during the first year after transplantation (median, 1.4 years). Approximately 10% (72) of the analyses had ECHO-A scores > or =4 that were not associated with treated rejection and were considered false positives. We identified parameters that contributed to the false-positive evaluations and calculated patient-specific baseline mean +/- standard deviation. The ECHO-B, in comparison with ECHO-A, decreased the number of false positives from 72 to 10, increased specificity from 90% to 99%, and increased the positive predictive value about 4-fold (10% to 44%). With treated rejection episodes, ECHO-B increased ECHO-A scores in 7 of 8 recipients and increased the mean score from 6 to 8. CONCLUSIONS: analysis algorithm based on change from baseline improved the positive predictive power without reducing the negative predictive value of multiparametric quantitative analyses of echocardiograms following pediatric heart transplantation.

Sarcoplasmic reticulum calcium release is stimulated and inhibited by daunorubicin and daunorubicinol.

Cardiac effects of anthracyclines or their metabolites may include both the stimulation and inhibition of Ca(2+) release from sarcoplasmic reticulum. In this study, the ability of daunorubicin and its primary metabolite, daunorubicinol, to stimulate and inhibit Ca(2+) release from canine sarcoplasmic reticulum (SR) vesicles was investigated. It was observed that both daunorubicin and daunorubicinol were several fold more potent at inhibiting than they were at stimulating SR Ca(2+) release. Respective IC50 inhibition of daunorubicin and daunorubicinol for caffeine-induced calcium release was 1.2 and 0.6 microM, and for spontaneous Ca(2+) release was 3 and 1 microM. EC50's for daunorubicin- and daunorubicinol-induced calcium release were 30 and 15 microM, respectively. Inhibition of either spontaneous or caffeine-induced SR Ca(2+) release was inversely related to the amount of Ca(2+) loaded into the SR before exposure to daunorubicin or daunorubicinol. The free-radical scavenger dithiothreitol did not attenuate the ability of anthracyclines to inhibit SR Ca(2+) release. A nonquinone daunorubicin derivative, 5-iminodaunorubicin, was less potent than daunorubicin at inhibiting caffeine-induced Ca(2+) release. These data suggest anthracyclines and their metabolites may produce cardiotoxicity through free-radical independent, concentration-dependent effects on SR Ca(2+) release. These effects involve either inhibition or stimulation of SR Ca(2+) release and are partly dependent upon the presence of the quinone moiety.

Persistent effects of doxorubicin on cardiac gene expression.

During administration of the anthracycline antitumour agents, their cardiotoxicity can progress from cardiac dysfunction to heart failure. Cardiomyopathy can also develop years after receiving anthracyclines. To determine if persistent and/or progressive anthracycline effect(s) are referable to anthracycline effects on cardiac gene expression, steady-state mRNA levels were determined 4 days (n=8), 4 weeks (n=7) and 10 weeks (n=7) after doxorubicin (DOX; 2 mg/kg IV) in a well-characterized rabbit model. Levels of mRNA for alpha -actin, beta -myosin heavy chain and the calcium pump of the sarcoplasmic reticulum (SERCA2a) in the left ventricle (LV) were determined by Northern blot hybridization and expressed relative to an 18S constitutive marker. The mRNA levels for the high molecular weight subunit (cardiac isoform) of the ryanodine receptor (RyR2), sarcolemmal calcium channel (dihydropyridine receptor; DHPR), angiotensin-converting enzyme (ACE), angiotensin II receptor (ATR) and atrial naturetic peptide prohormone (ANP) were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis, and expressed relative to GAPDH, a constitutive marker. Histopathologic evidence for anthracycline-induced myocardial cell injury was absent (score <1) in all hearts examined except one (score=1.1; 4 weeks post-DOX), which was considered separately. Relative mRNA levels for beta -myosin heavy chain 4 days after DOX increased 1.9-fold compared to the vehicle-treated group, but by 4 weeks levels had returned to baseline. Relative mRNA levels for DHPR were increased 1.2-fold 4 days after DOX and were persistently increased 1.9- and 2.2-fold 4 and 10 weeks after DOX, respectively. The mRNA levels for ANP were first decreased (4.5-fold) 4 days after DOX. Four weeks after DOX, ANP message levels approached Control in seven out of eight rabbits. The one rabbit with early LV histopathology 4 weeks post-DOX had increased mRNA for DHPR (2.7-fold) and ANP (80-fold). Between 4 and 10 weeks after DOX, mRNA levels for ANP increased C 16-fold: evidence for late progression. In situ hybridization with specific riboprobes localized the persistent increase in DHPR and the progressive increase in ANP to myocytes. Thus, DOX alters steady-state mRNA levels in LV that are referable to both persistent and progressive anthracycline effects on myocellular gene expression.

Induction immunotherapy in pediatric heart transplant recipients: a multicenter study.

BACKGROUND: The efficacy and safety of induction immunotherapy with antithymocyte antibody preparations (IND) in pediatric heart transplantation is controversial. Experimentally, recipient age is an important determinant of immune responses. The effects on induction immunotherapy were determined by an analysis of outcomes of 465 pediatric (age <18 years) heart recipients that either did or did not receive IND in the first week post-transplant. METHODS: The outcomes of 2 groups who received either OKT3 (n = 101) or rabbit polyclonal antithymocyte serum (N/R-ATS, n = 105) were compared with 255 recipients who did not receive antithymocyte antibodies. The study population were all heart recipients enrolled in the Pediatric Heart Transplant Study Group (PHTS) between January 1993 and December 1995 and followed up to 36 months. RESULTS: Overall mortality and death due to rejection were lowest with N/R-ATS IND (8/105 and 1/105, respectively) compared with the no-induction group (58/255 and 8/ 255, respectively) or the OKT3 group (22/101 and 7/101, respectively) with significance of p = 0.001 and 0.06 respectively. Late mortality beyond 30 days after transplant was lowest with N/R-ATS IND compared with the OKT3 and no IND (p = 0.01). Induction did not affect cumulative infections, deaths due to infection, or the frequency of malignancies. Patients excluded from N/R-ATS induction had the highest mortality (18/ 43), suggesting that the protocol's exclusion criteria identified a high-risk group. To minimize potential effect(s) of exclusion bias, patients transplanted at centers participating in the N/R-ATS induction trial were reanalyzed with a post hoc intent-to-treat analysis assigning patients by center (IND or no IND) irrespective of actual treatment. With this analysis overall mortality was 18% for N/R-ATS centers, 21% for OKT3 centers, and 26% for centers not using IND (p = 0.3). The mortalities of recipients <6 months old at transplant were lowest at centers using N/R-ATS and OKT3 IND compared to centers not using IND (p = 0.04). Cumulative rejection (0.8 vs 1.2 rejection/pt/year, p = 0.01) and freedom from rejection death (99% vs. 93% at year 1, p = 0.02) of the N/R-ATS centers were lower compared to OKT3 centers but were not different from centers not using IND. CONCLUSION: Following orthotopic transplantation, induction immunotherapy can exert the enduring benefit of reducing late deaths, a possible surrogate for rejection severity, in recipients less than 6 months of age, while not being associated with higher rates of infectious or malignant complications. Since polyclonal anti-T cell antibody preparations appears superior to OKT3 induction in pediatric recipients, the efficacy of ATS induction should be further evaluated in a randomized prospective study in pediatric heart recipients.

Contractile failure in chronic doxorubicin-induced cardiomyopathy.

The mechanisms for the progression of the anthracycline-induced cardiomyopathy to contractile failure has not been defined. In vitro, doxorubicin (DOX) appears to modify calcium-mediated excitation-contraction coupling, which depresses cardiac contractility. This study characterizes the onset of contractile failure associated with the development of DOX-induced cardiomyopathy. Rabbits were treated with DOX (1 mg/kg i.v. twice weekly, 12-18 doses; DOX-treated group) and compared with a pair-fed Control group infuse with saline vehicle. The severity of the cardiomyopathy was determined by numerically-scored histopathology. Myocardial contractility was determined in thin fiber bundles from right ventricular (RV) papillary muscles and left atria that were removed and mounted on a force transducer in oxygenated Krebs-bicarbonate buffer (pH=7.4 at 30 degrees C) to record the amplitude (DT) and maximum rate (+dT/dt ) of isometric tension. Myofibrillar and calcium loading properties were determined by the calcium and caffeine-activated tension responses respectively in chemically-permeabilized fibers. With the onset of the cardiomyopathy (score <2) DT at low frequency (0.5 Hz) was depressed (0.61+/-0.01 mN/mg; n=14) compared to Control (0.93+/-0.09 mN/mg; n=15). Contractility at higher rates (1 Hz) was not different in this DOX-treated and Control groups. Maximum calcium and caffeine-activated force and the pCa to half-maximum force of permeabilized fibers were comparable in DOX-treated and Control groups. The loss of contractility of the DOX-treated group was related to reduction in sarcoplasmic reticulum calcium release channel density, as determined by Bmax for 3H-ryanodine binding in cardiac microsomal membrane fraction. Post-rest potentiation of contractility, as well as frequency-dependent (0.25-1.5 Hz) and post-extrasystolic potentiation of contractility were preserved in the DOX-treated group. In vitro, DOX depressed post-rest potentiation of contractility. Thus, the onset of contractile failure of the DOX-induced cardiomyopathy is characterized by effects consistent with disordered calcium-mediated excitation-contraction coupling and these effects are qualitatively different than in vitro effects of DOX.

Prospective evaluation of echocardiography for primary rejection surveillance after infant heart transplantation: comparison with endomyocardial biopsy.

Rejection surveillance is critical to successful heart transplantation, and infant recipients have necessitated the use of noninvasive measures. The survival, incidence of rejection, and accuracy of echocardiography in predicting treatable rejection, as detected on right ventricular endomyocardial biopsy, were prospectively studied in infants. The survival rate (82%) and rejection rate (1.4 rejection episodes per 100 patient days) of infants who underwent transplantation during the study were comparable to other studies that used heart biopsy only. Echocardiography was 98% accurate at predicting biopsy results. The sensitivity of echocardiography was 92%, and the specificity was 98%. We conclude that echocardiography can be used successfully for primary rejection surveillance in infants and that right ventricular biopsy is infrequently required.

Time-related increases in cardiac concentrations of doxorubicinol could interact with doxorubicin to depress myocardial contractile function.

1. The present study evaluated the time-dependency of acute anthracycline cardiotoxicity by varying the duration of exposure of rabbit isolated atria to doxorubicin and determining changes (1) in contraction and relaxation and (2) in atrial concentrations of doxorubicin and its C-13 hydroxy metabolite, doxorubicinol. 2. Following addition of doxorubicin (175 microM) to atria, contractility (dF/dt), muscle stiffness (resting force, RF) and relaxation (90% relaxation time, 90% RT) were monitored for a 3.5 h period. 3. Doxorubicin (175 microM) progressively diminished mechanical function (decreased dF/dt, increased RF and prolonged 90% RT) over 3 h. Doxorubicinol (1.8 microM), however, failed to produce time-related cardiac dysfunction; it depressed contractile function and increased muscle stiffness during the first 30 min without causing additional cardiac dysfunction during the remaining 3 h of observation. Doxorubicinol had no effect on 90% RT. 4. During treatment with doxorubicin, atria contained considerably more doxorubicin than doxorubicinol (ratio of doxorubicin to doxorubicinol ranged from 778 to 74 at 0.5 and 3 h, respectively). Elevations of doxorubicin and doxorubicinol in atria paralleled the degree of dysfunction of both contraction and relaxation; increases in muscle stiffness, however, were more closely associated with increases of doxorubicinol than doxorubicin. 5. To probe the relation between cardiac doxorubicinol and myocardial dysfunction further, without confounding effects of cardiac doxorubicin, concentration-response experiments with doxorubicinol (0.9-7.2 microM) were conducted. 6. Plots of doxorubicinol concentrations in atria vs contractility indicated that the cardiac concentration of doxorubicinol, at which contractility is reduced by 50%, is five fold lower in doxorubicin-treated than in doxorubicinol-treated preparations. Thus, doxorubicin and doxorubicinol appear to interact to depress contractile function.7. Cardiac concentrations of both doxorubicin and doxorubicinol, as observed in these studies, were found to stimulate markedly Ca2+ release from isolated SR vesicles, but 3 microM doxorubicinol promoted a 15 fold greater release rate than 3 microM doxorubicin.8. Our observations coupled with the previously reported finding that doxorubicinol inhibits Ca2+loading of SR, suggests that doxorubicinol accumulation in heart contributes to the time-dependent component of doxorubicin cardiotoxicity, through a mechanism that could involve perturbations of Ca2+ homeostasis.

Pattern of echocardiographic abnormalities with acute cardiac allograft rejection in adults: correlation with endomyocardial biopsy.

In patients who have undergone successful orthotopic heart transplantation, a noninvasive method for rejection surveillance would reduce the frequency of endomyocardial biopsy, guide the timing of biopsies, and allow for more frequent monitoring. This study identified the pattern of change in echocardiographically determined indexes of left ventricular mass, volume, and function that characterized biopsy-positive acute rejection in adult heart transplant patients receiving triple-drug immunosuppressive therapy and describes a simple computer-driven algorithm capable of identifying rejection with high sensitivity and specificity. Two-dimensional and M-mode echocardiography and Doppler color flow analyses were performed within 24 hours of endomyocardial biopsy. M-mode echocardiograms of the left ventricle were digitized and analyzed blinded to the biopsy interpretation, using a computer-assisted measurement format, for size, mass, and wall motion in systole and diastole. Twenty-nine studies were retrospectively analyzed to define the echocardiographic pattern characteristic for rejection. Left ventricular chamber size decreased, and indexes of diastolic function were significantly depressed in patients with biopsy evidence of moderate or severe rejection. However, no single parameter was sufficiently sensitive to detect all episodes of rejection partly because of differences between patients in the echocardiographic manifestations of acute rejection. To accommodate this patient variability, multiple echocardiographic parameters were clustered into a unique scoring algorithm (ECHO score). When applied prospectively to 49 studies, the likelihood that a patient would have an ECHO score not indicative of rejection but with moderate/severe rejection on biopsy was low (less than 3%) or a negative predictive value of 97.4%.(ABSTRACT TRUNCATED AT 250 WORDS)

Echocardiographic abnormalities with acute cardiac allograft rejection in children: correlation with endomyocardial biopsy.

In patients who have undergone successful orthotopic heart transplantation, echocardiography has the potential to be a noninvasive method for rejection surveillance that would reduce the frequency and guide the timing of endomyocardial biopsies, as well as allow for more frequent monitoring, with less cost and risk to the patient. To determine the applicability of echocardiography to detect moderate to severe rejection in children, 26 two-dimensional-guided M-mode echocardiograms with Doppler/color flow mapping were performed within 24 hours of endomyocardial biopsy. M-mode echocardiograms of the left ventricle were digitized and analyzed with a computer-assisted measurement format for left ventricular size, mass, and wall motion in systole and diastole. These echocardiographic parameters were clustered and analyzed by a unique echocardiographic scoring algorithm blinded to the biopsy interpretation. In eight cases in which findings of biopsies were consistent with moderate to severe rejection, left ventricular mass was increased and indexes of systolic and diastolic function were depressed compared with the remainder of the cases (n = 18), in which findings of biopsies included either no evidence or mild evidence of rejection. The echocardiographic score of the group with moderate to severe rejection was significantly greater than the score of the group that was normal or had mild rejection (5.4 +/- 0.7 vs 2.2 +/- 0.3; p < 0.001). With rejection prospectively defined as an echocardiographic score of greater than or equal to score 4, echocardiography achieved 88% sensitivity and 83% specificity in detecting moderate to severe rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Serial echocardiographic evaluation of cardiac graft rejection after infant heart transplantation.

The effects of cardiac graft rejection on infant myocardial function as assessed by echocardiography are largely unknown. To quantitate the myocardial response to rejection, serial echocardiographic studies were prospectively performed on 20 infants (less than 1 year of age at transplantation). Two-dimensional guided-M-mode tracings were digitized and quantified with a computer-assisted format. Rejection was diagnosed by clinical criteria, and 85% were graded as mild, that is without cardiac signs or symptoms. Echocardiographic analysis was blinded to rejection status, with studies available 4.2 +/- 2.9 days before rejection, on the day of rejection diagnosis, and 2.9 +/- 1.5 days after rejection treatment. Left ventricular mass increased acutely from 109% of predicted normal to 129% with rejection and decreased to 110% with therapy (p < 0.01). Left ventricular volume also tended to fall with rejection and increase with therapy. The left ventricular volume/mass ratio fell from 0.29 +/- 0.10 to 0.25 +/- 0.13 and increased to 0.37 +/- 0.15 (p < 0.05) with treatment. Systolic function was depressed by rejection as reflected in the posterior wall thickening fraction and velocity of wall thickening. Diastolic dysfunction was reflected in a decreased velocity of posterior wall thinning (-9.7 +/- 3.9 to -7.7 +/- 2.7 and recovery to -10.8 +/- 3.8 (1/second, p < 0.05) and depressed average velocity of cavity enlargement (41.2 +/- 9.6 to 36.4 +/- 8.9 and recovery to 40.7 +/- 8.6 mm/sec, p < 0.05). The utility of these echocardiographic measurements to predict rejection has not been prospectively compared with the endomyocardial biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramural left anterior descending coronary artery: significance of the depth of the muscular tunnel.

To establish whether an intramural left anterior descending coronary artery (LADA) is a simple anatomic or a singularly pathologic variant we studied 39 hearts, each with an intramural course of the LADA and no coronary artery disease, valvular derangement, cardiomyopathy, or congenital anomaly. Seventeen of the 39 hearts had no myocardial lesions, while 22 had gross and/or microscopic alterations in the myocardial territory supplied by the intramural LADA. The myocardial lesions consisted of one or more of the following: interstitial fibrosis, replacement fibrosis, contraction band necrosis, and/or increased vascular density in areas of focal fibrosis. The coronary anatomy of the 22 hearts with myocardial lesions (group 1) was compared with that of the 17 hearts without myocardial changes (group 2). Each of the group 1 hearts had an intramural LADA deeply placed within the ventricular wall and attenuation of potential collateral blood flow because of a co-existing intramural course of the posterior descending artery, other epicardial coronary arteries, and/or a diminutive right coronary artery. The myocardial changes in group 1 hearts and their absence in group 2 hearts suggest that the deep, intramural LADA of the group 1 hearts is abnormal rather than a simple anatomic variant of normal. Furthermore, the deep intramural LADA may be associated with sudden death since 13 of the 22 group 1 hearts were from sudden death victims. Six of these 13 persons died suddenly during vigorous exercise.

Doxorubicin cardiomyopathy is associated with a decrease in calcium release channel of the sarcoplasmic reticulum in a chronic rabbit model.

Doxorubicin is a highly effective cancer chemotherapeutic agent that produces a dose-dependent cardiomyopathy that limits its clinical usefulness. Clinical and animal studies of morphological changes during the early stages of doxorubicin-induced cardiomyopathy have suggested that the sarcoplasmic reticulum, the intracellular membrane system responsible for myoplasmic calcium regulation in adult mammalian heart, may be the early target of doxorubicin. To detect changes in the calcium pump protein or the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum during chronic doxorubicin treatment, rabbits were treated with intravenous doxorubicin (1 mg/kg) twice weekly for 12 to 18 doses. Pair-fed controls received intravenous normal saline. The severity of cardiomyopathy was scored by light and electron microscopy of left ventricular papillary muscles. Developed tension was measured in isolated atrial strips. In subcellular fractions from heart, [3H]ryanodine binding was decreased in doxorubicin-treated rabbits (0.33 +/- 0.03 pmol/mg) compared with control rabbits (0.66 +/- 0.02 pmol/mg; P < 0.0001). The magnitude of the decrease in [3H]ryanodine binding correlated with both the severity of the cardiomyopathy graded by pathology score (light and electron microscopy) and the decrease in developed tension in isolated atrial strips. Bmax for [3H]ryanodine binding and the amount of immunoreactive ryanodine receptor by Western blot analysis using sequence-specific antibody were both decreased, consistent with a decrease in the amount of calcium release channel of sarcoplasmic reticulum in doxorubicin-treated rabbits. In contrast, there was no decrease in the amount or the activity of the calcium pump protein of the sarcoplasmic reticulum in doxorubicin-treated rabbits. Doxorubicin treatment did not decrease [3H]ryanodine binding or the amount of immunoreactive calcium release channel of sarcoplasmic reticulum in skeletal muscle. Since the sarcoplasmic reticulum regulates muscle contraction by the cyclic uptake and release of a large internal calcium pool, altered function of the calcium release channel could lead to the abnormalities of contraction and relaxation observed in the doxorubicin cardiomyopathy.

Anthracycline-induced tension in permeabilized cardiac fibers: evidence for the activation of the calcium release channel of sarcoplasmic reticulum.

Anthracyclines, such as doxorubicin (DOX), are important cancer chemotherapeutic agents that are cardiotoxic. The mechanism for the cardiotoxicity is not well-defined. Recent studies have concluded that anthracyclines release calcium (Ca2+) from membrane fractions containing sarcoplasmic reticulum (SR). To determine whether anthracyclines release Ca2+ in situ from cardiac SR, the effects of DOX on Ca(2+)-activated contractions were analyzed in membrane-permeabilized and membrane-intact fibers from rabbit heart. DOX (10-120 microM) induced tension development in calcium-preloaded permeabilized fibers. DOX-induced tension required submicromolar Ca2+, and was blocked by ruthenium red (20 microM) and Triton X-100 treatment, characteristics shared by caffeine-induced tension referable to SR Ca(2+)-release. DOX (50 microM) did not alter the maximum Ca(2+)-activated tension or shift the Ca2+ concentration-tension relationship of permeabilized fibers, indicating no effect of DOX on the myofilaments. DOX (44-350 microM) depressed post-rest isometric contractility of membrane-intact fibers but did not inhibit steady-state contractility (at 1 Hz; 2.5 Mm Ca2+), similar to effects of caffeine and submicromolar ryanodine. The specific effects of DOX on post-rest contractility of membrane-intact fibers are consistent with DOX-induced Ca2+ release from the SR of membrane-permeabilized fibers. Thus, DOX alters SR Ca2+ release in situ which may contribute to the inotropic and lusitropic dysfunction observed with anthracyclines.

Daunorubicin-induced cardiac injury in the rabbit: a role for daunorubicinol?

This study evaluated potential contributions of daunorubicin and its principle metabolite, daunorubicinol, to the cardiotoxicity of daunorubicin therapy. Daunorubicin (15 mg/kg) or placebo (normal saline) was administered by iv bolus to New Zealand white rabbits and 3 to 4 days later, hearts were removed to measure contractility (dF/dt), concentrations of daunorubicin and daunorubicinol, and evidence of oxidative stress on glutathione and glutathione peroxidase. Contractile function of isolated atria and papillary muscles was depressed (p < 0.05). Daunorubicinol exceeded daunorubicin concentration in the heart (p < 0.005) with a ratio of metabolite to parent drug of 26 in atrial and 32 in ventricular tissue. There was a significant correlation between peak plasma (r = -0.63; p < 0.05) or cardiac concentration (r = -0.78; p < 0.02) of daunorubicinol, but not daunorubicin, and depression of dF/dt in papillary muscles. In separate in vitro studies, daunorubicinol at a concentration (5.5 micrograms/g tissue or 10 microM) approximating that observed ex vivo in heart inhibited Ca2+ uptake into cardiac sarcoplasmic reticulum vesicles by 39 +/- 3%, whereas 10 microM daunorubicin (14-fold higher than actual ex vivo cardiac concentrations) did not demonstrate any detectable inhibition. Daunorubicin treatment failed to significantly alter concentrations of GSH or GSSG or activities of glutathione peroxidase in the heart. Thus, cardiac dysfunction observed 3 to 4 days after a single dose of daunorubicin did not clearly relate to oxidative stress, but was associated with a cardiac concentration of daunorubicinol that appeared sufficiently high to impair Ca2+ metabolism.

Serum anti-tropo:anti-alpha-elastin antibody ratio assessing elastin turnover in scleroderma.

Serum antibodies to native (tropo) and denatured (alpha) elastins appear to correlate with the production and breakdown respectively of elastic tissue. Elastin may be degraded as a part of autoimmune diseases. This possibility was tested by measuring IgG antibodies to tropo- and alpha-elastins by ELISA in the sera of 111 patients with a variety of connective tissue diseases compared with 18 healthy individuals. Anti-alpha-elastin antibodies were significantly higher in sera from 18 scleroderma patients than from healthy controls (p less than 0.008). Conversely, anti-tropoelastin antibody levels for scleroderma patients (p less than 0.03) and for patients with a variety of other connective tissue diseases (p less than 0.02) were lower than in healthy controls. Low antibody levels to native elastin and high levels of antibodies to denatured elastin suggest a low synthesis: degradation ratio for elastin in scleroderma. Scleroderma may be a unique model for elastin turnover because of its heretofore unrecognized accelerated elastolysis.

Response of the hypertrophied left ventricle to global ischemia. Comparison of hyperkalemic cardioplegic solution with and without verapamil.

The hypertrophied left ventricle is at considerably greater risk for injury when subjected to global ischemia than is an otherwise normal heart. We evaluated the efficacy of verapamil, a calcium-channel blocking agent, as an adjunct to standard crystalloid cardioplegic solution in animals with left ventricular hypertrophy subjected to myocardial ischemia during cardiopulmonary bypass. Infracoronary aortic stenosis was produced in 15 mongrel puppies by plication of the noncoronary cusp of the aortic valve. Studies were conducted 3 to 4 months later. Left ventricular catheter-tip pressure transducers and major and minor axis ultrasonic dimension crystals were inserted, and the animals were then supported by cardiopulmonary bypass with 30 minutes of normothermic ischemia. Animals were randomized to receive either standard hyperkalemic crystalloid cardioplegic solution (n = 8) or the same solution with verapamil, 0.1 mg/kg (n = 7). After the 30 minutes of ischemia, the animals were supported on cardiopulmonary bypass for an additional 30 minutes and then separated from bypass. They were then studied for another 2 hours by measurement of myocardial adenosine triphosphate content, myocardial blood flow, systolic function with use of the end-systolic pressure/volume ratio, and compliance with use of the natural strain coefficient of the minor axis at 15 mm Hg end-diastolic pressure. There was a better recovery of systolic function in the animals treated with verapamil (89.2% versus 63.3%). The compliance as measured with use of the minor axis natural strain coefficient returned essentially to baseline in the group of animals treated with verapamil (0.236 +/- 0.038 before ischemia and 0.254 +/- 0.043 2 hours after ischemia), but it fell markedly in the control animals (0.219 +/- 0.027 before ischemia and 0.153 +/- 0.016 2 hours after ischemia). Myocardial adenosine triphosphate levels were not significantly different at any time during the study. Likewise, myocardial blood flow was not significantly different between groups. We conclude that the addition of verapamil to hyperkalemic cardioplegic solution improves recovery of both systolic and diastolic function after global ischemia in dogs with left ventricular hypertrophy resulting from aortic stenosis. The precise mechanism for this is unknown.

Dietary lipid modulation of connective tissue matrix in rat abdominal aorta.

Dietary lipid modulation of structural and passive mechanical properties of isolated rat abdominal aortic segments were assessed during the early developmental period. Rats were raised from conception to 90 days of age on semisynthetic diets containing various types and amounts of lipids. Aortic segments from three groups of rats fed high-fat diets (15%, wt/wt) consisting of olive oil, corn oil, or lard as the sole lipid sources were compared with those from rats fed a low-fat control diet containing corn oil (5%, wt/wt). Morphometric analysis of the tunica media demonstrated that rats raised on diets with a relatively low polyunsaturated fatty acid content (olive oil and lard) had greater numbers of elastic lamellae than rats raised on diets with opposite fatty acid indexes (high- and low-fat corn oil). Changes in elastin content of the tunica media, determined biochemically, paralleled those seen by morphometric analysis of the elastic lamellar number. Altered dietary fatty acid ratios were also associated with changes in smooth muscle cell number. In this regard, a decreased cellular density was observed in the olive oil and lard diets compared with the corn oil diet. The olive oil diet was unique amongst the dietary lipid regimens in raising, whereas the lard-containing diet lowered, indexes of aortic tissue elasticity. These results demonstrate an effect of chronic feeding of high dietary fat on the composition and biomechanical properties of the connective tissue matrix of abdominal aortic rings from young Sprague-Dawley rats.

Myxoid heart disease: an assessment of extravalvular cardiac pathology in severe mitral valve prolapse.

Because of the microscopic features of the affected leaflets in mitral valve prolapse (MVP), myxoid degeneration of the valve is a common pathologic designation applied to this condition. We undertook this study as a means of gaining an insight into the occurrence and prevalence of extravalvular cardiac alterations in hearts with severe MVP. Tissues of 24 hearts with severe myxomatous transformation of the mitral valve as the sole cardiac abnormality were examined. Eighteen of the 24 subjects with severe MVP died suddenly. Only two of these had pathologic evidence of severe mitral insufficiency. Twenty-four normal hearts served as controls. The two groups of hearts came from victims of homicide, suicide, accident, or natural death. Sections of the mitral valve, working myocardium, conduction system, and cardiac nerves and ganglia were studied by routine and special connective tissue and proteoglycan stains. Similar to the findings in severely affected mitral valves, prominent deposits of proteoglycans in neural and conduction tissue readily distinguished hearts with myxomatous valve changes from the control hearts. We conclude that the commonly recognized local derangement of valvular tissue in MVP is but one specific reflection of a more general myxomatous alteration in cardiac connective tissue.