Search

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

While the basic pathobiology of myocardial ischemic injury and reperfusion has been determined over the last 50 years, there are important, unresolved, or at least not completely elucidated, issues in the field. These include the relative contributions of different modes of cell injury and death to evolving myocardial infarcts; interactions of phenomena produced by reperfusion, including stunning and preconditioning; and potential new approaches for successfully combining adjuvant therapy with coronary artery opening. A model of myocardial ischemic and reperfusion injury is proposed involving the potential expression of apoptotic and oncotic pathways in the same perturbed cardiomyocytes. Promising new cardioprotective strategies for reducing lethal reperfusion injury are discussed, including ischemic postconditioning, activators of the reperfusion injury salvage kinase (RISK) pathway, inhibitors of protein kinase c-delta, and inhibitors of the mitochondrial membrane permeability transition pore (PTP). Major outstanding clinical challenges are also discussed, including the development of clinical care systems that can routinely deliver very timely coronary opening and reperfusion, perhaps combined with adjuvant therapy, and the development of strategies to retard adverse remodeling and congestive heart failure in patients with significant myocardial infarction and scarring, perhaps by refinements in stem cell therapy.

The authors report the case of a patient with isolated protein C deficiency detected later in life, presenting with a mobile aortic thrombus and splenic infarction. Only one such case has been previously described. This case emphasizes the importance of including the aorta in the search for a cause of systemic embolization and highlights the diagnostic options and management dilemmas. Although anticoagulation with subsequent reassessment of thrombus size can be considered for layered thrombi, mobile thrombi warrant early surgical intervention to minimize the risk for systemic embolization. This patient was treated surgically with encouraging results.

The vascular smooth muscle cell (SMC)-specific isoform of alpha-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.

In this histological study, we assessed the role of mesenchymal stem cells (MSCs) in the healing process that takes place during the subacute phase of myocardial infarction in dogs. Seven days after occlusion of the left anterior descending coronary artery, adult mongrel dogs received 100 x 10(6) 4'-6-diamidino-2-phenylindole (DAPI)-labeled allogenic bone marrow-derived MSCs by the transendocardial (TE, n=6) and intracoronary (IC, n=4) routes; control dogs (n=6) received no infusion. The dogs were euthanized at 21 days after occlusion. Hearts were excised and sliced from apex to base into four transverse sections, which were divided into nine segments. Paraffin sections from each segment were stained with hematoxylin and eosin, trichrome, picrosirius red, and antibodies against several extracellular matrix components. Frozen sections were immunostained for host cardiac phenotypical markers and analyzed by epifluorescence and deconvolution fluorescence microscopy (DFM). We found less unresolved necrotic myocardium and more extracellular matrix deposition in MSC-treated dogs than in controls 2 weeks after cell delivery. By DFM, no DAPI+ MSC nuclei were observed within native cardiac cells. MSCs delivered during the subacute phase of acute myocardial infarction positively affect healing, apparently by mechanisms other than differentiation into mature native cardiac cells.

In Western countries, autopsy rates for patients deceased in hospitals have dropped to record lows, while the average frequency of major errors in clinical diagnoses has more than doubled during the same time period. Meanwhile, the Institute of Medicine and the U.S. Department of Health and Human Services have called attention to the high frequency of errors affecting patient safety, bringing the issue of public safety to the forefront of public health concerns. Although autopsies represent a vital tool for the acquisition of new medical knowledge and for medical quality assurance, health care professionals, insurers, and politicians apparently have not chosen the right approach to solve the problem of declining autopsy rates. The present article reviews the current status of clinical autopsies and addresses causes and consequences of their neglect and appeal the urgent need to revise the policy for clinical autopsy.

During post-natal maturation of the mammalian heart, proliferation of cardiomyocytes essentially ceases as cardiomyocytes withdraw from the cell cycle and develop blocks at the G0/G1 and G2/M transition phases of the cell cycle. As a result, the response of the myocardium to acute stress is limited to various forms of cardiomyocyte injury, which can be modified by preconditioning and reperfusion, whereas the response to chronic stress is dominated by cardiomyocyte hypertrophy and myocardial remodeling. Acute myocardial ischemia leads to injury and death of cardiomyocytes and nonmyocytic stromal cells by oncosis and apoptosis, and possibly by a hybrid form of cell death involving both pathways in the same ischemic cardiomyocytes. There is increasing evidence for a slow, ongoing turnover of cardiomyocytes in the normal heart involving death of cardiomyocytes and generation of new cardiomyocytes. This process appears to be accelerated and quantitatively increased as part of myocardial remodeling. Cardiomyocyte loss involves apoptosis, autophagy, and oncosis, which can occur simultaneously and involve different individual cardiomyocytes in the same heart undergoing remodeling. Mitotic figures in myocytic cells probably represent maturing progeny of stem cells in most cases. Mitosis of mature cardiomyocytes that have reentered the cell cycle appears to be a rare event. Thus, cardiomyocyte renewal likely is mediated primarily by endogenous cardiac stem cells and possibly by blood-born stem cells, but this biological phenomenon is limited in capacity. As a consequence, persistent stress leads to ongoing remodeling in which cardiomyocyte death exceeds cardiomyocyte renewal, resulting in progressive heart failure. Intense investigation currently is focused on cell-based therapies aimed at retarding cardiomyocyte death and promoting myocardial repair and possibly regeneration. Alteration of pathological remodeling holds promise for prevention and treatment of heart failure, which is currently a major cause of morbidity and mortality and a major public health problem. However, a deeper understanding of the fundamental biological processes is needed in order to make lasting advances in clinical therapeutics in the field.

Objective: This study sought to characterize the inflammatory infiltrate in ascending thoracic aortic aneurysm in patients with Marfan syndrome, familial thoracic aortic aneurysm, or nonfamilial thoracic aortic aneurysm., Background: Thoracic aortic aneurysms are associated with a pathologic lesion termed "medial degeneration," which is described as a noninflammatory lesion. Thoracic aortic aneurysms are a complication of Marfan syndrome and can be inherited in an autosomal dominant manner of familial thoracic aortic aneurysm., Methods: Full aortic segments were collected from patients undergoing elective repair with Marfan syndrome (n = 5), familial thoracic aortic aneurysm (n = 6), and thoracic aortic aneurysms (n = 9), along with control aortas (n = 5). Immunohistochemistry staining was performed using antibodies directed against markers of lymphocytes and macrophages. Real-time polymerase chain reaction analysis was performed to quantify the expression level of the T-cell receptor beta-chain variable region gene., Results: Immunohistochemistry of thoracic aortic aneurysm aortas demonstrated that the media and adventitia from Marfan syndrome, familial thoracic aortic aneurysm, and sporadic cases had increased numbers of T lymphocytes and macrophages when compared with control aortas. The number of T cells and macrophages in the aortic media of the aneurysm correlated inversely with the patient's age at the time of prophylactic surgical repair of the aorta. T-cell receptor profiling indicated a similar clonal nature of the T cells in the aortic wall in a majority of aneurysms, whether the patient had Marfan syndrome, familial thoracic aortic aneurysm, or sporadic disease., Conclusion: These results indicate that the infiltration of inflammatory cells contributes to the pathogenesis of thoracic aortic aneurysms. Superantigen-driven stimulation of T lymphocytes in the aortic tissues of patients with thoracic aortic aneurysms may contribute to the initial immune response.

Background: In surgical series, a majority of benign cardiac tumors are myxomas. Of these, only about 2.5% are biatrial. Only 10 cases have been reported in the last 10 years. We present here a successful case in a 51-year-old man. A brief review of the literature is presented to place this case in context., Methods: The tumor was removed surgically via a midline sternotomy using cardiopulmonary bypass. Both left and right atrial extensions of the tumor mass were removed. The resection involved the entire septum, with a bovine patch used to reconstruct the atrial septum., Results: Patient recovered uneventfully. We advised follow-up evaluation using transthoracic echocardiography annually., Conclusions: Biatrial myxoma is a very rare condition, with diagnostic challenges, but is amenable to modern surgical approaches.

Background: We hypothesized that not all subtypes of alpha- and beta-adrenoreceptors undergo similar upregulation and redistribution in human myocardium after mechanical unloading with an assist device., Methods: We obtained core biopsy samples of the left ventricle in 19 patients before and after removal of a Jarvik or Thoratec left ventricular assist device (LVAD) to study the effect of mechanical unloading on the distribution of alpha- and beta-adrenoreceptors. Fresh, embedded tissue sections were incubated with receptor blockers and antibodies before the fluorescent labeling of receptors. Images were obtained by fluorescence deconvolution microscopy, and composite tissue renditions were made from the stacked images. Multiple adrenoreceptor subtypes were studied., Results: We saw a reversal of myocyte hypertrophy in all patients, but the upregulation of receptors was not seen in all post-LVAD tissue samples. Furthermore, we noted receptor relocalization from an initial punctate/clumped pattern to a normal homogeneous distribution in many patients. Significant differences were seen in the distribution of beta(2)- and alpha(1)-receptors and in alpha(1A) subtypes., Conclusions: In this study we show not only the expected reversal of myocyte hypertrophy and the increase in adrenoreceptors after ventricular unloading, but also the relocalization of specific receptor subtypes.

Due to up-regulation of the parathyroid gland calcium-sensing receptor (CaR), burned children have hypocalcemic hypoparathyroidism, and decreased myocardial contractility. Our aim was to localize the CaR in the heart and measure receptor density changes due to burns. Heart and aorta samples from sheep subjected to 40% burn or sham conditions were probed for CaR via fluorescence microscopy. CaR was localized to endocardial endothelium, myocardial microvasculature, and fibroblasts and vessels of the aortic adventitia. CaR was not found in cardiomyocytes or smooth muscle cells. No differences in density of CaR or beta-adrenergic receptors were noted. No differences in CaR distribution were seen in the myocardium or aorta, in contrast to the parathyroid where burn injury up-regulates CaR. We suggest that CaR has a local, tissue-specific role, and functions in vascular calcium sensing for intravascular calcium deposition or regulation of other calcium channels after trauma or burn.

This study assessed safety of transendocardial (TE) electromechanical-guided delivery of bone marrow mesenchymal stem cells (MSCs) after acute myocardial infarction (AMI) and compared intracoronary (IC) delivery with TE delivery. In a canine acute myocardial ischemia model, 100 x 10(6) MSCs were delivered 7 days after AMI via IC and TE routes. Functional assessment was performed by 2D echocardiogram, and detailed histopathologic analyses were performed to assess the impact of cell therapy in vascular density and fibrosis. Patterns of cell distribution in both delivery methods were also compared. There was a statistically significant reduction in the amount of myocardial ischemia in the TE group (P=0.007). Left ventricular ejection fraction (LVEF) increased 13% (mean) in the TE group (21-day follow-up) and was significantly better than that of the controls (P=0.01), but did not improve in the IC-delivery group. Dissimilar patterns of cell distribution were noted between the IC and TE groups. This study suggests that MSC treatment is probably safe and effective after AMI. In the comparison of TE and IC delivery, the TE group showed higher cell retention (clusters even in the injury center of the infarct) with an increased vascularity and greater functional improvement than did the IC group (no clusters; cells at the border of the infarct). The higher local cell density in the TE group may be important for therapeutic effectiveness.

Adult rat cardiomyocytes in culture respond to sub-lethal doses of lipopolysaccharides (LPS) by activation of pathways including the production of TNF-alpha and increased apoptosis. We and others have demonstrated a protective phenotype for neonatal rat cardiomyocytes to LPS. Concentrations of LPS far exceeding those necessary to induce TNF-alpha release do not induce apoptosis in the neonatal cells, although these cells are fully capable or inducing apoptosis in response to multiple other stimuli. In neonatal cells, we demonstrate that LPS treatment leads to a loss of mitochondrial membrane potential (Deltapsi) which is temporally associated with an increase in the level of uncoupling protein 3 (UCP3). Cells remain viable with no measurable increase in apoptotic or necrotic cell death. Many markers of mitochondrial biogenesis are also activated. LPS treatment stimulates an increase in the (i) transcription of mitochondrial transcription factor A (Tfam), (ii) nuclear accumulation of redox-sensitive nuclear respiratory factor 1 (NRF-1), and (iii) expression of peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1). We also observed that LPS increased intracellular autophagy. Autophagy was assessed by monitoring the levels of a mammalian protein specifically associated with autophagosomes, microtubule-associated light chain 3 (LC3). Furthermore, inhibition of autophagy in the presence of LPS stimulates markers of apoptosis. Our data suggest that the protective response of neonatal cells to LPS is multi-faceted at the level of the mitochondrion. Viable cells replace dysfunctional mitochondria by mitochondrial biogenesis and the extent of the damage limited by the rapid removal of damaged organelles by the stimulation of autophagy.

The major function of vascular smooth muscle cells (SMCs) is contraction to regulate blood pressure and flow. SMC contractile force requires cyclic interactions between SMC alpha-actin (encoded by ACTA2) and the beta-myosin heavy chain (encoded by MYH11). Here we show that missense mutations in ACTA2 are responsible for 14% of inherited ascending thoracic aortic aneurysms and dissections (TAAD). Structural analyses and immunofluorescence of actin filaments in SMCs derived from individuals heterozygous for ACTA2 mutations illustrate that these mutations interfere with actin filament assembly and are predicted to decrease SMC contraction. Aortic tissues from affected individuals showed aortic medial degeneration, focal areas of medial SMC hyperplasia and disarray, and stenotic arteries in the vasa vasorum due to medial SMC proliferation. These data, along with the previously reported MYH11 mutations causing familial TAAD, indicate the importance of SMC contraction in maintaining the structural integrity of the ascending aorta.

Non-syndromic thoracic aortic aneurysms and dissections (TAADs) are inherited in an autosomal dominant manner in approximately 20% of cases. Familial TAAD is genetically heterogeneous and four loci have been mapped for this disease to date, including a locus at 16p for TAAD associated with patent ductus arteriosus (PDA). The defective gene at the 16p locus has recently been identified as the smooth muscle cell (SMC)-specific myosin heavy chain gene (MYH11). On sequencing MYH11 in 93 families with TAAD alone and three families with TAAD/PDA, we identified novel mutations in two families with TAAD/PDA, but none in families with TAAD alone. Histopathological analysis of aortic sections from two individuals with MYH11 mutations revealed SMC disarray and focal hyperplasia of SMCs in the aortic media. SMC hyperplasia leading to significant lumen narrowing in some of the vessels of the adventitia was also observed. Insulin-like growth factor-1 (IGF-1) was upregulated in mutant aortas as well as explanted SMCs, but no increase in transforming growth factor-beta expression or downstream targets was observed. Enhanced expression of angiotensin-converting enzyme and markers of Angiotensin II (Ang II) vascular inflammation (macrophage inflammatory protein-1alpha and beta) were also found. These data suggest that MYH11 mutations are likely to be specific to the phenotype of TAAD/PDA and result in a distinct aortic and occlusive vascular pathology potentially driven by IGF-1 and Ang II.

Objective: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling., Results: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences., Conclusion: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Context: It has become increasingly evident that adipose tissue is a multifunctional organ that produces and secretes multiple paracrine and endocrine factors. Research into obesity, insulin resistance, and diabetes has identified a proinflammatory state associated with obesity. Substantial differences between subcutaneous and omental fat have been noted, including the fact that omental fat produces relatively more inflammatory cytokines. Periadventitial fat, as a specific adipose tissue subset, has been overlooked in the field of atherosclerosis despite its potential diagnostic and therapeutic implications., Objective: To review (1) evidence for the role of adventitial and periadventitial fat in vessel remodeling after injury, (2) the relationship between adventitial inflammation and atherosclerosis, (3) the association between periadventitial fat and plaque inflammation, and (4) the diagnostic and therapeutic implications of these roles and relationships for the progression of atherosclerosis., Data Sources: We present new data showing greater uptake of iron, administered in the form of superparamagnetic iron oxide, in the periadventitial fat of atherosclerotic mice than in control mice. In addition, macrophage density in the periadventitial fat of lipid-rich plaques is increased compared with fibrocalcific plaques., Conclusions: There is a striking paucity of data on the relationship between the periadventitial fat of coronary arteries and atherosclerosis. Greater insight into this relationship might be instrumental in making strides into the pathophysiology, diagnosis, and treatment of coronary artery disease.

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low concentration (1.5 microg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 microg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 microg/ml) induced diffuse swelling or oncosis-diffuse cell swelling (DCS). To assess the underlying mechanism of sanguinarine-induced apoptosis and oncosis-BCD in K562 cells, we studied their response to pre-treatment with two chemical compounds: aurintricarboxylic acid (ATA) and cycloheximide (CHX). The pretreatment effects of both chemical compounds on apoptosis and oncosis-BCD were evaluated by measuring multiple parameters using quantitative morphology, electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling and annexin-V-binding. ATA, a DNA endonuclease inhibitor, efficiently prevented DNA nicking and inhibited apoptosis almost completely and oncosis-BCD by about 40%, while CHX, a protein synthesis inhibitor, failed to inhibit both apoptosis and oncosis-BCD. These results demonstrate, first, the importance of endonuclease in sanguinarine-induced apoptosis and to some extent in oncosis-BCD and, second, that this inhibition does not require de novo protein synthesis.

Severe burn causes immunosuppression, and the eschar remains a perfect culture medium for microbial growth. The resulting sepsis is a common complication of burns with a high mortality. The skin produces a number of molecules including antimicrobial peptides (AMPs) that act in the first line of host defense. Previous studies from our laboratory suggested decreased expression of human beta-defensin-2 (HBD-2) in burned wounds. Here, we have expanded our work by identifying HBD-1, HBD-2, HBD-3 and human neutrophil peptide (HNP) in normal and burn skin samples using fluorescence deconvolution microscopy. In normal skin, HBD-1 was localized to the perinuclear region of keratinocytes, while HBD-2 was seen primarily in the stratum basale of the epidermis. HBD-3 was found in dendritic cells of the stratum spinosum. HNP was distributed in a somewhat random pattern in the papillary dermis. In burned skin, in which the epidermis had been destroyed or disrupted, the presence of HBD-1 was localized to dermal glandular structures and hair shafts. HBD-2 was found in both the upper portions of the remaining keratin layers, and localized to lower, f-actin containing, acini-like structures, a pattern also evident with HBD-3. We conclude that although the upper layers of skin are destroyed and disrupted by burn, cells in the lower portions of the skin demonstrate an ability to synthesize most of the AMPs, thereby maintaining some barrier against infection. The results of these studies further contribute to an understanding of the role of AMPs in the pathophysiology of cutaneous burn and the possibility of using these sites for upregulation of AMP synthesis in the prevention of burn sepsis.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Sepsis induced by exposure to lipopolysaccharide (LPS) can be life-threatening and lead to multiple-organ dysfunction. Sepsis-associated cardiac dysfunction is a primary cause of mortality. The response of isolated cardiac myocytes to LPS exposure is poorly understood. Cultured neonatal rat ventricular cardiomyocytes were used to evaluate the response to LPS exposure. Other authors have reported that LPS exposure at doses sufficient to induce tumor necrosis factor alpha (TNF-alpha) production and apoptosis in adult cardiomyocytes do not induce apoptosis in neonatal cardiomyocytes. We therefore hypothesized that neonatal cardiomyocytes have innate protective mechanisms that protect from septic damage. Cultured neonatal rat ventricular cardiomyocytes were stimulated by exposure to LPS for varying lengths of time. NFkappaB signaling pathways, TNF-alpha production, and Akt activation were monitored. We also assessed the induction of apoptosis in these cells by monitoring caspase-3 activity. LPS rapidly stimulates nuclear translocation of NFkappaB and Akt activation. TNF-alpha production is also stimulated. However, high doses of LPS are unable to induce apoptosis in these cells, and protection is not a function of Akt activation. LPS treatment also stimulated the levels of cyclooxygenase-2 and the production of downstream metabolites, specifically PGE2 and 15deoxyDelta12-14PGJ2 (15dPGJ2). Specific inhibition of cyclooxygenase-2 activity induced apoptosis in the presence of LPS, whereas direct exposure to 15dPGJ2 at pharmacological levels induced apoptosis. Neonatal rat ventricular cardiomyocytes have innate protective mechanisms that prevent apoptotic cell death after LPS exposure. Metabolic products of arachidonic acid metabolized by the cyclooxygenase pathway can be potentially apoptotic or antiapoptotic. The balance of these products within these cells may define the cellular response to LPS exposure.

We report a case of ascending and transverse aortic arch aneurysm repair with aortic valve replacement in a patient with MAGIC syndrome (mouth and genital ulcers with inflamed cartilage), the unusual setting of both Behçet's disease and relapsing polychondritis.

Objective: The objective of this study was to observe the effects of cell culture on cellular polarity in cardiomyocytes as influenced by cytoskeletal proteins., Methods: Cardiomyocytes from adult and neonatal rats were isolated and grown on 2 different extracellular matrices--laminin and a complex, fibroblast-derived extracellular matrix, cardiogel. The location of a number of proteins was visualized by means of fluorescence deconvolution microscopy, using specific fluorescent probes for alpha-adrenergic receptors, beta-adrenergic receptors, the sarcolemmal L-type calcium channel, and the sodium + potassium adenosine triphosphatase pump protein. Intracellular migration of these proteins during the first 4 days of culture was followed and microscopic stacked images were reconstructed. A fluorescein isothiocyanate-labeled probe for actin was used to ensure that cardiomyocytes were being examined, based on protein patterns., Results: We examined 2 types of myocyte: freshly isolated neonates and cultured adult cardiomyocytes that undergo dedifferentiation. Initial, perinuclear clumping (endoplasmic reticulum/Golgi-associated) of the probes with an ensuing spread to the cytoplasm and periphery, accompanied by a better organization and more rapid response to biochemical stimuli, was seen on the complex matrix., Conclusions: A complex matrix overcomes cell polarity at a faster rate than myocytes cultured on a simple matrix, although both culture matrices were able to support cell growth and differentiation, and single-layer cultures are a good method by which structural and biochemical data can be obtained. The use of a native, complex matrix is preferable to employing a simple, single protein, although temporal aspects of cell growth must be considered regarding the particular aspect of the cell structure development/biochemical pathways that the researcher intends focusing on.

Objective: The histopathologic abnormality underlying ascending aortic aneurysm and dissection is medial degeneration, a lesion that is described as the noninflammatory loss of smooth muscle cells and elastic fibers. This study sought to determine whether inflammatory cells are present in medial degeneration and assess any possible contribution of these cells to apoptosis of smooth muscle cells., Methods: Aortic specimens were obtained from patients undergoing prophylactic surgical repair of an ascending aortic aneurysm (n = 9) and type A dissection (n = 7), along with control patients dying of causes unrelated to aortic disease (n = 5). Immunohistochemical staining was performed to evaluate the presence of lymphocytes and macrophages, and markers of apoptosis were assessed in the aortas of patients with ascending aortic aneurysm and dissection., Results: Immunohistochemical study indicated significantly more CD3+ cells in the aortas of patients with aneurysms or dissections than in control aortas (P = .020 and P = .0022, respectively). In addition, aortas of patients with aneurysms or dissections had more CD68+ cells (P = .01 and P = .005, respectively). CD3+ cells were localized in the media and surrounding the vasa vasorum in the adventitia. Cells yielding a positive result on in situ terminal transferase-mediated deoxyuridine triphosphate nick end-labeling were found in increased numbers in the aortas of patients with aneurysms or dissections relative to control aortas (P = .005 and P = .002, respectively). Furthermore, Fas and FasL were increased in the aortic samples from patients with aneurysms and dissections relative to control aortas., Conclusion: The coexistence of inflammatory cells with markers of apoptotic vascular cell death in the media of ascending aortas with aneurysms and type A dissections raises the possibility that activated T cells and macrophages may contribute to the elimination of smooth muscle cells and degradation of the matrix associated with thoracic aortic aneurysms and dissections.

We describe a case of primary lymphoma of the thoracic aorta that presented as an aneurysm of the descending thoracic aorta. The aortic tumor was successfully resected with 2-year disease-free survival.

Myocardial ischemic injury results from severe impairment of coronary blood supply and produces a spectrum of clinical syndromes. As a result of intensive investigation over decades, a detailed understanding is now available of the complexity of the response of the myocardium to an ischemic insult. Myocardial ischemia results in a characteristic pattern of metabolic and ultrastructural changes that lead to irreversible injury. Recent studies have explored the relationship of myocardial ischemic injury to the major modes of cell death, namely, oncosis and apoptosis. The evidence indicates that apoptotic and oncotic mechanisms can proceed together in ischemic myocytes with oncotic mechanisms and morphology dominating the end stage of irreversible injury. Myocardial infarcts evolve as a wavefront of necrosis, extending from subendocardium to subepicardium over a 3- to 4-hour period. A number of processes can profoundly influence the evolution of myocardial ischemic injury. Timely reperfusion produces major effects on ischemic myocardium, including a component of reperfusion injury and a greater amount of salvage of myocardium. Preconditioning by several short bouts of coronary occlusion and reperfusion can temporarily salvage significant amounts of myocardium and extend the window of myocardial viability. Ongoing research into the mechanisms involved in reperfusion and preconditioning is yielding new insights into basic myocardial pathobiology.

Background: The use of left ventricular (LV) assist devices (LVADs) can improve performance and recovery of failing human hearts., Aim: Following our alpha-adrenergic receptor work, we hypothesized that mechanical unloading in patients with low output syndrome and LV failure would yield similar results with beta-adrenergic receptors ((beta)AR), that being increased numbers and intra-myocytic relocalization., Methods: (beta)AR density and localization were investigated by fluorescence deconvolution microscopy and compared at LVAD insertion and removal in 13 heart failure patients, the patients therefore acting as their own control. (beta)AR densities and distribution were determined in snap frozen sections of human core biopsy left ventricular apical tissue. Samples were probed with tagged CGP 12177 for visualization of (beta)AR and challenged with cold agonists and antagonists. (beta)AR density was measured by two independent methods. Localization of receptors was examined in reconstructed, deconvoluted, stacked section images., Results: There was an increase in (beta)AR density following ventricular unloading in most of the patients, and also significant normalization in the location of the receptors in the myocardium comparing pre- and post-LVAD tissue., Conclusions: These findings suggest that supporting an ailing heart via unloading initiates mechanisms and pathways responsible for myocardial recovery and repair. With appropriate pharmacological support, patients with LVAD might recover to the point where they no longer depend on eventual organ transplantation, and (beta)AR number, type, and distribution in pre-LVAD myocardial tissue, could predict outcome with regard to recovery, repair, and improvement in cardiac function.

Cardiac alpha one adrenoreceptors (alpha(1)AR) are known to mediate positive inotropism in human ventricular myocardium. In the early stages of heart failure, ventricular contractility is maintained by adrenergic stimulation, rennin-angiotensin activation, and other neurohormonal and cytokine system responses. As the disease progresses, however, these compensatory mechanisms cease to provide benefit; ventricular dilation and fibrosis occur and cardiac function deteriorates. When ventricular contractility becomes severely depressed and is no longer responsive to inotropic support, insertion of a left ventricular assist device (LVAD) may allow the left ventricle to rest, remodel, and recover some contractile function. Our previous work has demonstrated that the myocardium has the capacity to repair itself during a period of unloading, after which some patients are able to resume a normal lifestyle and no longer need a cardiac transplant.

Background: The heart undergoes repair and initiates protective mechanisms via ventricular unloading. We examined the presence of 2 markers in pre-unloaded and post-unloaded human cardiac tissue that are important indicators of cardiac failure, tumor necrosis factor-alpha and inducible nitric oxide synthase. We also measured 2 nuclear transcription factors, NFkappaB50 and NFkappaB65, comparing quantities and localizations to determine if mechanical unloading reduced their presence, as these markers are also thought to be indicators of impending heart failure. Amounts and localizations in patients that had been diagnosed with either ischemic or non-ischemic cardiomyopathy were compared after mechanical unloading with a left ventricular assist device. To establish that unloading had been achieved, levels of atrial natriuretic protein were determined., Methods: Core biopsies were harvested at assist device implantation and removal. Fluorescence deconvolution microscopy image reconstructions of fluorescence probes were correlated with data obtained by western Blot and electrobility shift assays., Results: Statistically significant differences in localization and amounts of tumor necrosis factor and nitric oxide synthase were seen between pre- and post-assist device samples. Amounts of tumor necrosis factor and nitric oxide synthase in ischemic tissue were increased at the time of assist device removal, but decreased in dilated or idiomyopathic samples. Ventricular unloading resulted in reduced levels of natriuretic protein, with the greatest reduction being seen in ischemic tissue. Both NFkappaB50 and NFkappaB65 increased in ischemic tissue, but only NFkappaB50 in non-ischemic samples., Conclusions: Changes in localization of the factors and altered levels of cytokine and nitric oxide synthase indicate that the heart switches to a "protective and repair" mode, and mechanical unloading allows this transition to occur. Observed changes were dependent on the etiology of the disease.

We describe a computational model of human T cell regulatory dynamics. We used this model to simulate changes in T cell pool numbers and for studying feedback and feed-forward responses in and among these pools. The pools identified were the bone marrow stem cell compartment, early and late thymocyte compartments and the peripheral compartment of mature T lymphocytes. Simulated data showed variable intercompartmental strengths indicative of a range of sensitivities to feedback regulation and respective variable feed-forward responses. The results compare well to known clinical and experimental data, rendering the computational model a good basis for further research in T cell development and regulation.

A new mathematical model is presented to simulate various changes of cell pools in the T cell immune system with validation procedures imitating viral infections. The present paper focuses on changes during the course of an acute progressive HIV-1 infection. Parameters are optimized by a direct search method and the stability of the model is studied. Mathematical modeling supports the hypothesis that the differentiation blockade is one major reason for the depletion of CD4 cells and the proliferation of CD38 cells in HIV-1 infection. The model appears to be a useful basis for further simulating disturbances of the T cell immune system in other viral infections as well as to elucidate the pathogenesis of various immunological diseases including the development of malignant lymphomas.

The development of T-lymphocytes (T cells) constitute one of the basic and most vital processes in immunology. Conventional mathematical models, being based on the systems theory, fail to sufficiently distinguish the constituents of thymocytes and are thus of limited significance. On the basis of some well thought-out definitions and concepts, a continuous model was designed to describe T cell maturation in the thymus. A partial differential equation was first derived through the analysis of an infinitesimal element of the flow of thymocytes. A computation scheme was designed to determine the growth field in the thymus based upon available experimental data. The corresponding algorithm proved quite simple. A numerical example is given that focuses on the DN stage of the T cell development. The model opens a window for investigating the thymic microenvironment. The potential of the model in studying lymphomagenesis is discussed.

A computational model was recently designed to simulate cellular changes in the T cell immune system. The model was validated by simulating cell changes in viral infections which target the same CD4+ T cell, yet cause either hyperplastic, aplastic or neoplastic responses. Respective case material for comparison was available from human infections with human herpesvirus-6 (HHV-6), human immunodeficiency virus (HIV-1) or human T cell leukemia virus (HTLV-1). Starting with cell values for a healthy human individual, factorial changes that influence the individual course of the various infections were determined by an algorithm search procedure. Such factorial differences determining a clinical course with aplasia, hyperplasia or neoplasia are outlined and further discussed in this paper.

Acute infection with human herpesvirus-6 induces physiological cell proliferation in persons without major immune deficiency. It thus can serve as a parameter to validate a mathematical model designed to simulate cell proliferation under physiological and pathological conditions. Such a mathematical model is presented to simulate various cell changes of the T-cell immune system during the course of HHV-6 infection. Model development follows several steps, beginning with a basic model containing physiological T-cell pools to the introduction of infectious stimuli in the final model. A search algorithm designed to optimize the system parameters, as well as initial variables of the model, is presented. The results of simulation runs for acute HHV-6 infection of the final computational model correspond well to the data, as documented in human patients; they suggest that the computational model presented for the simulation of T-cell levels in a given viral infection may well serve as a tool for similar studies of other viral infections, including those that lead to cellular aplasia or neoplasia., (Copyright 2003 Wiley-Liss, Inc.)

Based on a previously developed theory of dysregulative lymphoma pathogenesis, an advanced mathematical model was developed for simulation of thymic T cell populations and their differentiation stages. The model subsequently was tested in comparison to thymic changes in mice with Moloneyvirus infection and leukemia development. Numerical examples are given, which suggest that the model is useful to study T cell proliferation, differentiation, and may probably also be useful to simulate T cell changes in various lymphoproliferative diseases.

Members of the T lymphocyte lineage belong to a highly reactive cell system engaged in the control of internal homoeostasis and bodily intactness. It fulfills its commitments in close communication with the cellular and non-cellular microenvironment and with neuroendocrine regulatory mechanisms. The tools of such communication are various substances and compounds identified as cytokines, chemokines, hormones, growth factors, neuropeptides and components of the extracellular matrix. In a previous publication (22) we described the major players in the network regulation of T cell development and function as a basis for a computational modeling study. The present paper summarizes normal reference values for serum and/or plasma concentrations of these factors in man.

Based upon a previously developed theory of dysregulative lymphoma pathogenesis, a computer model is designed in order to simulate cell changes occurring in disturbances of the T cell immune system and in lymphoproliferative diseases. The model is based upon the concept that factors identified as proliferation factors, differentiation factors and inhibition factors exert a network regulation upon development and function of the T cell system, and that selective disturbances of these factors may lead to hyperplastic, aplastic or neoplastic diseases. The resulting computer model (TCM-1) was validated by comparing it with data from human diseases such as acute HHV-6 (viral) infection, chronic persistent HHV-6 infection, progressive HIV1 infection and HTLV-1 infection, and comparing the simulation results with the actual cell data in the human patients. All these infections target the same T cell population (i.e. CD4 + T helper cells), yet cause different prototypical reactions (hyperplastic, aplastic, neoplastic). The described computer model, which was successfully used to simulate changes in the benign lymphoproliferative disease, Canale-Smith syndrome, will serve as the basis model for further supplementation to accommodate identified factorial influences such as by cytokines, chemokines and others.

The T lymphocyte lineage, from initial stem cells to peripheral mature T-cells, are members of a highly reactive cell system engaged in the control of internal homoeostasis and body intactness. It fulfills its commitments in close communication with the cellular and non-cellular microenvironment and with neuroendocrine regulatory mechanisms. Tools of such communication are various substances and compounds identified as cytokines, chemokines, hormones, growth factors and neuropeptides. Any pathogenetical model of functional aberration and disease development in the T-cell system must take into account the delicate network control exerted by the above mechanisms. In the following we describe the major players in the network regulation of T-cell development and function as a basis for a computational modeling study.

Syndecan-4 (syn-4), a transmembrane heparan sulfate-containing proteoglycan, is unique among the four members of the syndecan family in its specific cellular localization to complex cytoskeletal adhesion sites, i.e., focal adhesions. During early phenotypic redifferentiation of neonatal cardiomyocytes in culture, immunolocalization reveals syn-4 to be heavily concentrated in the perinuclear endoplasmic reticulum-Golgi region, with little found at the peripheral regions. Subsequently, syn-4 becomes localized to a cytoskeletal adhesion complex unique to striated muscle, the costamere. Soon after redifferentiation of myofibrils in cultured neonatal cardiomyocytes, syn-4 is present only in costameres, not in focal adhesions. In cultured adult cardiomyocytes, it is present in both costameres and focal adhesions-the latter in two distinct regions of the spread cardiomyocytes, reflecting localization with two types of actin-containing filaments. The fact that syn-4 is observed early in the costameric regions, as opposed to later in the focal adhesions, suggests that it may play an initial role in early adhesion/signal transduction mechanisms in close proximity to the contractile apparatus, as well as in transmission of contractile force to the collagenous extracellular matrix (ECM) which surrounds the cardiac myofibers in situ. With respect to possible regulatory mechanisms of syn-4, we localized syn-4 with both the epsilon isoform of protein kinase C and the tyrosine kinase pp60(csrc) in costameric regions. These findings suggest that syn-4 may not only play a role in cellular adhesion and contractile force transmission, it may also, through ser, thr, and tyr phosphorylation, be part of an interactive signal transduction mechanism in myocardial functioning via these adhesive cytoskeletal complexes., (Copyright 2002 Wiley-Liss, Inc.)

Objective: The objective of this study was to simulate changes in the human T cell system representing Canale-Smith syndrome using a dynamic computer model of T cell development and comparing with available human data., Study Design: Physiological stepwise maturation and function of T lymphocytes in the computer model is altered by introducing functional disturbances following lymphotropic virus infection. In the present model, acute and chronic persistent infection with the human herpesvirus-6 (HHV-6) was simulated, and ensuing changes in T cell populations were compared with those measured in human patients., Results: Using our computer model we previously found that simulated acute HHV-6 infection produced T cell computer data, which resembled an infectious mononucleosis-like disease in patients. Simulated chronic persistent infection, instead, resulted in variable cell changes comparing well to patients with chronic fatigue syndrome. In one setting, however, persistent immature lymphocytosis was observed similar to what initial has been described in this journal as Canale-Smith syndrome., Conclusion: Using a computer model developed by us we were able to produce simulations that resemble the immune system features of Canale-Smith syndrome. Further understanding of these simulation results may possibly guide future investigations into this disorder.

Objective: We sought to determine whether pretreatment with a sodium/hydrogen-exchange inhibitor (EMD 96 785) improves myocardial performance and reduces myocardial edema after cardioplegic arrest and cardiopulmonary bypass., Methods: Anesthetized dogs (n = 13) were instrumented with vascular catheters, myocardial ultrasonic crystals, and left ventricular micromanometers to measure preload recruitable stroke work, maximum rate of pressure rise (positive and negative), and left ventricular end-diastolic volume and pressure. Cardiac output was measured by means of thermodilution. Myocardial tissue water content was determined from sequential biopsy. After baseline measurements, hypothermic (28 degrees C) cardiopulmonary bypass was initiated. Cardioplegic arrest (4 degrees C Bretschneider crystalloid cardioplegic solution) was maintained for 2 hours, followed by reperfusion-rewarming and separation from cardiopulmonary bypass. Preload recruitable stroke work and myocardial tissue water content were measured at 30, 60, and 120 minutes after bypass. EMD 96 785 (3 mg/kg) was given 15 minutes before bypass, and 2 micromol was given in the cardioplegic solution. Control animals received the same volume of saline vehicle. Arterial-coronary sinus lactate difference was similar in both animals receiving EMD 96 785 and control animals, suggesting equivalent myocardial ischemia in each group., Results: Myocardial tissue water content increased from baseline in both animals receiving EMD 96 785 and control animals with cardiopulmonary bypass and cardioplegic arrest but was statistically lower in animals receiving EMD 96 785 compared with control animals (range, 1.0%-1.5% lower in animals receiving EMD 96 785). Preload recruitable stroke work decreased from baseline (97 +/- 2 mm Hg) at 30 (59 +/- 6 mm Hg) and 60 (72 +/- 9 mm Hg) minutes after cardiopulmonary bypass and cardioplegic arrest in control animals; preload recruitable stroke work did not decrease from baseline (98 +/- 2 mm Hg) in animals receiving EMD 96 785 and was statistically greater at 30 (88 +/- 5 mm Hg) and 60 (99 +/- 4 mm Hg) minutes after bypass and arrest compared with control animals., Conclusions: Sodium/hydrogen-exchanger inhibition decreases myocardial edema immediately after cardiopulmonary bypass and cardioplegic arrest and improves preload recruitable stroke work. Sodium/hydrogen-exchange inhibition during cardiac procedures with cardiopulmonary bypass and cardioplegic arrest may be a useful adjunct to improve myocardial performance in the immediate postbypass or arrest period.

Objective: chronic aortic dissection has long been considered a risk factor for neurologic deficit following thoracoabdominal aortic aneurysm (TAA) surgery. We reviewed our experience with regard to aneurysm extent and the use of adjunct, (distal aortic perfusion/cerebrospinal fluid drainage), and examined the impact of these factors on neurologic deficit among chronic dissection and non-dissection cases., Methods: between February 1991 and March 2001, we repaired 800 aneurysms of the descending thoracic and thoracoabdominal aorta. Seven hundred and twenty-nine cases were elective; 196 chronic dissection, 533 non-dissection. 182/729 (24.9%) were TAA extent II. Among these, 61/182 (33%) involved chronic dissection. Adjunct was used in 507/729 (69.6%). We conducted detailed multivariate analyses to isolate the impact of chronic aortic dissection on neurologic morbidity, with other important risk factors taken into account., Results: overall, 32/729 (4.4%) patients had neurologic deficit upon awakening; 7/196 (3.6%) in chronic dissections, and 25/533 (4.7%) in non-dissections. Adjunct had a major effect, reducing neurologic deficit in TAA extent II from 10/36 (27.8%) to 10/146 (6.9%) (p=0.001). However, in univariate and multivariate analysis, chronic dissection did not increase the risk of neurologic deficit, regardless of extent or mode of treatment., Conclusion: in contrast to previous reports, we determined that chronic aortic dissection is not a risk factor in TAA patients., (Copyright 2002 Harcourt Publishers Limited.)

A literature search for HTLV-1-induced adult T-cell leukemia (ATL) at the National Library of Medicine resulted in 1003 publications which were evaluated with regard to HTLV-1 virus load, apoptosis and peripheral blood leukocyte changes during the latent period and leukemia development following virus infection. The data are presented in a comparable way to previous publications of infections with HHV-6 and HIV (which target the same CD4+ cell for infection) to be used for computer validation studies. After initial infection, HTLV-1 remains clinically latent for many years at low provirus copy numbers in CD4 cells. Once immune surveillance deteriorates and viral replication progresses, provirus copy numbers increase rapidly. Unlike other virus infections, apoptotic death of virus-infected "atypical" lymphocytes decreases with increasing viral load, thus favoring continued proliferation of these cells and further virus replication at the same time. Changes in the peripheral blood are characterized by coincident rises in oligoclonal lymphocyte populations including HTLV-1-positive CD4+ T-lymphocytes and their precursors with a progressive shift to immature cells as disease progresses. The pathogenesis of HTLV-1-induced adult T-cell leukemia is an example of dysregulative leukemogenesis ideal for validation of respective computer simulation models.

The saturated fatty acid palmitate induces apoptosis in neonatal rat cardiomyocytes. This apoptosis is associated with early mitochondrial release of cytochrome c and a subsequent loss of mitochondrial membrane potential. Recent reports implicate a role for reactive oxygen species (ROS) in palmitate-induced apoptosis. We studied the role of ROS in palmitate-induced apoptosis in the neonatal rat cardiomyocyte and report no evidence of ROS involvement. ROS production, nitric oxide production, and nuclear factor-kappaB activation were not increased above those observed using the nonapoptotic fatty acid oleate. Indeed, the production of ROS was significantly higher in cells treated with oleate. Furthermore, the presence of antioxidants and ROS scavengers did not attenuate the induction of apoptosis by palmitate. Variations in the fatty acid-to-albumin ratio from 2:1 to 7:1 had no effect on the absence of ROS production or on the extent of apoptosis. No evidence was found for an increase in oxidative protein modification in palmitate-treated cells. Our results lead us to conclude that oxidative stress does not play a role in palmitate-induced apoptosis.

Background: Extensive aortic aneurysms (ascending aorta, aortic arch, and descending or thoracoabdominal aorta) require innovative surgical techniques. Some surgeons advocate a single procedure with long periods of profound hypothermia, whereas others use a staged approach. We adopted a two-staged procedure (elephant trunk technique) in 1991 for elective repair of extensive aortic aneurysms., Methods and Results: Between February 1991 and May 2000, we performed a total of 1146 aortic aneurysm operations. Of these, 182 (15.9%) operations were first- or second-stage elephant trunk procedures, performed in a total of 117 patients. Stage 1 was completed in all 117 patients. Stage 2 was completed in 65 (55.6%) of 117 patients. Thirty-day mortality rate for the first stage was 5.1% (6 of 117). Mortality rate during the interval between operations was 3.6% (4 of 111), of which 75% (3 of 4) were the result of aneurysm rupture. Thirty-day mortality rate for the second stage was 6.2% (4 of 65). A total of 43 patients did not return for second-stage repair. Among these patients, within an average period of 3.4 years (range, 1.5 months to 4.9 years), 13 of 43 (30.2%) died, 4 of 13 (30.8%) as the result of rupture. Two of 117 (1.7%) first-stage patients had postoperative stroke. No spinal cord dysfunction occurred in second-stage patients., Conclusions: Extensive aortic aneurysms can be repaired with acceptable morbidity and mortality rates through the use of the elephant trunk technique. Death was most commonly the result of rupture, both in interval patients awaiting scheduled second-stage repair and in patients who did not return. After the first stage, prompt treatment of the remaining segment is crucial to the success of staged repair.

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function., (Copyright 2001 Harcourt Publishers Ltd.)