[DEVELOPMENT OF FUNCTIONAL AND MORPHOLOGICAL CHANGES OF A PANCREAS DEPENDING ON THE DURATION OF OBSTRUCTIVE CHOLESTASIS].

PURPOSE OF THE STUDY: To study the severity and timing of the development of functional (reversible) and morphological (irreversible) disturbances in the pancreas, depending on the duration of obstructive cholestasis. MATERIALS AND METHODS: Obstructive jaundice in the experiment 18 dogs modeled by applying the loop stranglehold on the common bile duct, followed by observation for 30 days. We measured total bilirubin and fractions aspartate aminotransferase activity (AST) and alanine aminotransferase (ALT), the contents alpha-amylase, pancreatic lipase, glucose, histological examination of the pancreas (magnification of 100 times and 400). RESULTS: On day 3 the common bile duct obstruction bilirubin increased from 7.1 to 286.8 µmol/l, ALT--from 0.17 to 4.18 µmol*h/l, alpha-amylase from 89 to 186 U/L and lipase--to 68 to 179 U/L. Then there was a slight decrease in the parameters studied with repeated their increase to 15 hours. Morphological changes in the first three days were characterized by reversible (swelling), impaired organ that 14-16 days passed in organic (irreversible) changes. CONCLUSION: Dynamics of fluctuations in the level of liver enzymes in the pancreas and obstructive cholestasis correlates with morphological abnormalities in the pancreas and fit into the concept of general biological organism's reaction to injury.

RNase1 prevents the damaging interplay between extracellular RNA and tumour necrosis factor-α in cardiac ischaemia/reperfusion injury.

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant, and the initial mechanistic trigger of myocardial "ischaemia/reperfusion (I/R) injury" remains greatly unexplained. Here we show that factors released from the damaged cardiac tissue itself, in particular extracellular RNA (eRNA) and tumour-necrosis-factor α (TNF-α), may dictate I/R injury. In an experimental in vivo mouse model of myocardial I/R as well as in the isolated I/R Langendorff-perfused rat heart, cardiomyocyte death was induced by eRNA and TNF-α. Moreover, TNF-α promoted further eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R with the massive production of oxygen radicals, mitochondrial obstruction, decrease in antioxidant enzymes and decline of cardiomyocyte functions. The administration of RNase1 significantly decreased myocardial infarction in both experimental models. This regimen allowed the reduction in cytokine release, normalisation of antioxidant enzymes as well as preservation of cardiac tissue. Thus, RNase1 administration provides a novel therapeutic regimen to interfere with the adverse eRNA-TNF-α interplay and significantly reduces or prevents the pathological outcome of ischaemic heart disease.

Telocytes in meninges and choroid plexus.

Telocytes (TCs) are a recently identified type of interstitial cells present in a wide variety of organs in humans and mammals (www.telocytes.com). They are characterized by a small cell body, but extremely long cell processes - telopodes (Tp), and a specific phenotype. TCs establish close contacts with blood capillaries, nerve fibers and stem cells. We report here identification of TCs by electron microscopy and immunofluorescence in rat meninges and choroid plexus/subventricular zone, in the vicinity of putative stem cells. The presence of TCs in brain areas involved in adult neurogenesis might indicate that they have a role in modulation of neural stem cell fate.

Stem cell-mediated natural tissue engineering.

Recently, we demonstrated that a fully differentiated tissue developed on a ventricular septal occluder that had been implanted due to infarct-related septum rupture. We suggested that this tissue originated from circulating stem cells. The aim of the present study was to evaluate this hypothesis and to investigate the physiological differentiation and transdifferentiation potential of circulating stem cells. We developed an animal model in which a freely floating membrane was inserted into each the left ventricle and the descending aorta. Membranes were removed after pre-specified intervals of 3 days, and 2, 6 and 12 weeks; the newly developed tissue was evaluated using quantitative RT-PCR, immunohistochemistry and in situ hybridization. The contribution of stem cells was directly evaluated in another group of animals that were by treated with granulocyte macrophage colony-stimulating factor (GM-CSF) early after implantation. We demonstrated the time-dependent generation of a fully differentiated tissue composed of fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells and new blood vessels. Cells differentiated into early cardiomyocytes on membranes implanted in the left ventricles but not on those implanted in the aortas. Stem cell mobilization with GM-CSF led to more rapid tissue growth and differentiation. The GM-CSF effect on cell proliferation outlasted the treat ment period by several weeks. Circulating stem cells contributed to the development of a fully differentiated tissue on membranes placed within the left ventricle or descending aorta under physiological conditions. Early cardiomyocyte generation was identified only on membranes positioned within the left ventricle.

Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling.

The human heart can be frequently affected by an organ-limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long-standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo-red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10-nm-thick filaments were strictly extra-cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.

Telocytes in human epicardium.

The existence of the epicardial telocytes was previously documented by immunohistochemistry (IHC) or immunofluorescence. We have also demonstrated recently that telocytes are present in mice epicardium, within the cardiac stem-cell niches, and, possibly, they are acting as nurse cells for the cardiomyocyte progenitors. The rationale of this study was to show that telocytes do exist in human (sub)epicardium, too. Human autopsy hearts from 10 adults and 15 foetuses were used for conventional IHC for c-kit/CD117, CD34, vimentin, S-100, τ, Neurokinin 1, as well as using laser confocal microscopy. Tissue samples obtained by surgical biopsies from 10 adults were studied by digital transmission electron microscopy (TEM). Double immunolabelling for c-kit/CD34 and, for c-kit/vimentin suggests that in human beings, epicardial telocytes share similar immunophenotype features with myocardial telocytes. The presence of the telocytes in human epicardium is shown by TEM. Epicardial telocytes, like any of the telocytes are defined by telopodes, their cell prolongations, which are very long (several tens of µm), very thin (0.1-0.2 µm, below the resolving power of light microscopy) and with moniliform configuration. The interconnected epicardial telocytes create a 3D cellular network, connected with the 3D network of myocardial telocytes. TEM documented that telocytes release shed microvesicles or exocytotic multivesicular bodies in the intercellular space. The human epicardial telocytes have similar phenotype (TEM and IHC) with telocytes located among human working cardiomyocyte. It remains to be established the role(s) of telocytes in cardiac renewing/repair/regeneration processes, and also the pathological aspects induced by their 'functional inhibition', or by their variation in number. We consider telocytes as a real candidate for future developments of autologous cell-based therapy in heart diseases.

Calcium-dependent signalling is essential during collateral growth in the pig hind limb-ischemia model.

We investigated the effect of pharmacological activation of the Ca(2+)-channel transient receptor potential cation channel, subfamily V, member 4 (TRPV4) on collateral growth in a pig hind limb-ischemia model thereby identifying subcellular mechanisms. Domestic pigs received femoral artery ligature and were randomly assigned to one of the following groups (each n=6): (1) 4alpha-phorbol 12,13-didecanoate (4alphaPDD) treatment; (2) treatment with an arterio-venous shunt (AV-shunt) distal to the occlusion; or (3) implantation of NaCl-filled minipump. Six sham-operated pigs acted as controls. Aortic and peripheral mean arterial pressure (MAP) measurements were performed to assess the collateral flow index (CFI). Tissue was isolated from M. quadriceps for immunohistochemistry and from isolated collateral arteries for quantitative real time PCR (qRT-PCR). Shortly after ligature the CFI dropped from 0.96+/-0.02 to 0.21+/-0.02 in all ligature-treated groups. In ligature-only-treated pigs CFI increased to 0.56+/-0.03 after 7days. Treatment with 4alphaPDD led to an enhancement of CFI compared with ligature alone (0.73+/-0.03). CD31-staining showed improved arteriolar density. Increased Ki67 staining in collaterals indicated proliferation. qRT-PCR and Western blot analysis showed upregulation or modulation of Ca(2+)-dependent transcription factors nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), Kv channel interacting protein 3, calsenilin (KCNIP3/CSEN/DREAM), and myocyte enhancer factor 2C (MEF2C) in 4alphaPDD- and AV-shunt-treated pigs compared with controls. Improved CFI after 4alphaPDD treatment identifies TRPV4 as an initial fluid shear-stress sensor and collateral remodelling and growth trigger. Subcellularly, modulation of Ca(2+)-dependent transcription factors indicates a pivotal role for Ca(2+)-signalling during arteriogenesis.

[Qualitative and quantitative changes of circulating in blood endotheliocyte precursor cells in patients with hypercholesterolemia].

Aim of this study was elucidation of diagnostic value of qualitative and quantitative parameters of circulating endotheliocyte precursor cells (EPC), in the state of apoptosis (apoptotic endotheliocytes A AE), as well as effect of main risk factors on the studied parameters in patients with coronary atherosclerosis and hypercholesterolemia. We examined 24 patients with ischemic heart disease (14 men, 10 women, mean age 58.4 +/- 2.3 years) with stenoses >75% in coronary arteries according to angiography data and hypercholesterolemia (main group). Control group comprised 15 healthy volunteers. Circulating EPC in peripheral blood were evaluated by cytofluorimetry. Functional parameters of isolated EPC were assessed after cultivation in human fibronectin. Adhesive properties were studied in cell culture by immunehistochemical method after 7 days of incubation. Proliferation capacity was assessed by number of EPC in 1 mm3 of medium at day 7 of incubation. Determination and counting of AE was carried out using cytofluorimetry with immunostaining of cells by anexin B and CDA31 after preliminary treatment of endotheliocyte culture with H2O2 and comparing the results with control culture of endotheliocytes. Number of circulating EPC in patients with IHD was 58.% less than in healthy persons. Adhesive capacity of EPC in patients with IHD was lowered by 43%. Number of EPC surviving in cell culture was 80.9% less than in control group. At the same time number of circulating AE in patients with IHD was 2.5 times greater. Reduction of number of EDC, adhesive capacity, and rise of number of AE correlated significantly with number of involved coronary arteries, presence of diabetes, and smoking status. Presence of arterial hypertension and blood cholesterol level did not correlate with quantitative and qualitative parameters of EPC. Thus qualitative and qualitative parameters of circulating EPC and apoptotic cells can be considered as markers of dysfunction of endothelium and predictors of atherosclerotic vascular lesions in patients with hypercholesterolemia.

Desynchronization: a novel model to induce heart failure.

BACKGROUND: Previous large animal heart failure models led to inhomogeneous results. Therefore, we developed a novel model combining rapid pacing with forced ventricular desynchronization. METHODS: Heart failure was induced in 20 pigs during a pacing period of 21 days. Group A (n = 10) received one right ventricular lead (220 bpm). In group B (n = 10), two leads were implanted in different right ventricular regions with beat-to-beat alternation of activation sites (each lead 110 bpm). Sham-operated pigs (n = 6) served as controls. Hemodynamics were invasively evaluated and tissue was analyzed by immunohistochemistry and zymography. RESULTS: Hemodynamics were significantly more impaired in group B with an increase of pulmonary capillary wedge and central venous pressure and a reduction of cardiac index (control 4.3 +/- 0.1 l/min/m (2); A 3.6 +/- 0.2; B 2.9 +/- 0.2, P < 0.05). Heart-to-body weight ratio was significantly higher in group B. Histological analyses showed a significant increase of cell diameters and interstitial fibrosis with significantly higher collagen contents in group B. CONCLUSION: The new model with a combination of rapid pacing and forced desynchronization of the ventricular contraction is superior to traditional heart failure models induced solely by rapid pacing.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

A distinct type of cell in myocardium: interstitial Cajal-like cells (ICLCs).

The existence of a novel type of interstitial cells in the heart, interstitial Cajal-like cells (ICLCs), had been described for the first time in 2005. Their identification was mainly based on ultrastructural criteria: very long (tens up to hundreds of micrometres) and moniliform prolongations, which are extremely thin (less than 0.2 microm), below the resolving power of light microscopy. Myocardial ICLCs were also identified by methylene-blue vital staining, silver impregnation, and immunoreactivity for CD 34, vimentin, CD117/c-kit, etc. Although a series of studies provided evidence for the existence of ICLCs in human atria and rat ventricles, further investigations in other laboratories, using additional techniques, are required to substantiate the consistency of these findings. Here we provide further evidence for the existence of ICLCs in human and mammalian hearts (by transmission and scanning electron microscopy, as well as confocal laser scanning microscopy). Noteworthy, we confirm that ICLCs communicate with neighbouring cells via shedding (micro)vesicles. Although these so-called ICLCs represent a distinct type of cells, different from classical interstitial cells of Cajal, or fibroblasts, their role(s) in myocardium remain(s) to be established. Several hypotheses are proposed: (i) adult stromal (mesenchymal) stem cells, which might participate in cardiac repair/remodelling; (ii) intercellular signalling (e.g. via shedding microvesicles); (iii) chemo-mechanical transducers and (iv) players in pacemaking and/or arrhytmogenesis, and so on.

Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarction.

An important goal in cardiology is to minimize myocardial necrosis and to support a discrete but resilient scar formation after myocardial infarction (MI). Macrophages are a type of cells that influence cardiac remodelling during MI. Therefore, the goal of the present study was to investigate their transcriptional profile and to identify the type of activation during scar tissue formation. Ligature of the left anterior descending coronary artery was performed in mice. Macrophages were isolated from infarcted tissue using magnetic cell sorting after 5 days. The total RNA of macrophages was subjected to microarray analysis and compared with RNA from MI and LV-control. mRNA abundance of relevant targets was validated by quantitative real-time PCR 2, 5 and 10 days after MI (qRT-PCR). Immunohistochemistry was performed to localize activation type-specific proteins. The genome scan revealed 68 targets predominantly expressed by macrophages after MI. Among these targets, an increased mRNA abundance of genes, involved in both the classically (tumour necrosis factor alpha, interleukin 6, interleukin 1beta) and the alternatively (arginase 1 and 2, mannose receptor C type 1, chitinase 3-like 3) activated phenotype of macrophages, was found 5 days after MI. This observation was confirmed by qRT-PCR. Using immunohistochemistry, we confirmed that tumour necrosis factor alpha, representing the classical activation, is strongly transcribed early after ligature (2 days). It was decreased after 5 and 10 days. Five days after MI, we found a fundamental change towards alternative activation of macrophages with up-regulation of arginase 1. Our results demonstrate that macrophages are differentially activated during different phases of scar tissue formation after MI. During the early inflammatory phase, macrophages are predominantly classically activated, whereas their phenotype changes during the important transition from inflammation to scar tissue formation into an alternatively activated type.

Mechanical load induced by glass microspheres releases angiogenic factors from neonatal rat ventricular myocytes cultures and causes arrhythmias.

In the present study, we tested the hypothesis that similar to other mechanical loads, notably cyclic stretch (simulating pre-load), glass microspheres simulating afterload will stimulate the secretion of angiogenic factors. Hence, we employed glass microspheres (average diameter 15.7 microm, average mass 5.2 ng) as a new method for imposing mechanical load on neonatal rat ventricular myocytes (NRVM) in culture. The collagen-coated microspheres were spread over the cultures at an estimated density of 3000 microspheres/mm2, they adhered strongly to the myocytes, and acted as small weights carried by the cells during their contraction. NRVM were exposed to either glass microspheres or to cyclic stretch, and several key angiogenic factors were measured by RT-PCR. The major findings were: (1) In contrast to other mechanical loads, such as cyclic stretch, microspheres (at 24 hrs) did not cause hypertrophy. (2) Further, in contrast to cyclic stretch, glass microspheres did not affect Cx43 expression, or the conduction velocity measured by means of the Micro-Electrode-Array system. (3) At 24 hrs, glass microspheres caused arrhythmias, probably resulting from early afterdepolarizations. (4) Glass microspheres caused the release of angiogenic factors as indicated by an increase in mRNA levels of vascular endothelial growth factor (80%), angiopoietin-2 (60%), transforming growth factor-beta (40%) and basic fibroblast growth factor (15%); these effects were comparable to those of cyclic stretch. (5) As compared with control cultures, conditioned media from cultures exposed to microspheres increased endothelial cell migration by 15% (P<0.05) and endothelial cell tube formation by 120% (P<0.05), both common assays for angiogenesis. In conclusion, based on these findings we propose that loading cardiomyocytes with glass microspheres may serve as a new in vitro model for investigating the role of mechanical forces in angiogenesis and arrhythmias.

Negative inotropic effect of rapamycin on isolated human cardiomyocytes.

Rapamycin is an increasingly important immunosuppressive drug and reduces restenosis after coronary stenting, but its effects on cardiac contractility are largely unknown. We investigated the acute inotropic effects of rapamycin on isolated human cardiomyocytes. Cardiomyocytes were enzymatically isolated from right atrial appendages obtained during routine coronary artery bypass surgery. Cell morphology was examined by confocal microscopy. Cell contraction was recorded after electrical stimulation. Rapamycin elicited a concentration-dependent decrease in fractional cell shortening ranging from 14.3 +/- 2.6% at 10(-8) M rapamycin to 26.4 +/- 4.2% at 10(-5) M. Rapamycin also caused a concentration-dependent decrease in diastolic cell length. Contractile performance of isolated cardiomyocytes was well preserved, as evidenced by the profound positive inotropic effects of high extracellular calcium concentration and the beta-adrenoreceptor agonist isoproterenol. The acute negative inotropic effect of rapamycin on human cardiomyocytes might be due to altered calcium homeostasis through the binding of rapamycin to FKBP12.6 and its regulatory function on the ryanodine receptor, with increased calcium leakage from the sarcoplasmic reticulum.

Atrial extracellular matrix remodelling in patients with atrial fibrillation.

UNLABELLED: Atrial fibrillation (AF) is the most frequent clinical arrhythmia. Atrial fibrosis is an important factor in initiating and maintaining AF. However, the collagen turnover and its regulation in AF has not been completely elucidated. We tested the hypothesis that the extracellular matrix changes are more severe in patients with permanent AF in comparison with those in patients in sinus rhythm (SR). Intraoperative biopsies from the right atrial appendages (RAA) and free walls (RFW) from 24 patients with AF undergoing a mini-Maze procedure and 24 patients in SR were investigated with qualitative and quantitative immunofluorescent and Western blot analyses. As compared with SR, all patients with AF exhibited dysregulations in collagen type I and type III synthesis/degradation. Tissue inhibitors of metalloproteinases (TIMP2) was significantly enhanced only in RAA-AF. As compared with SR, collagen VI, matrix metalloproteinases MMP2, MMP9 and TIMP1 were significantly increased while TIMP3 and TIMP4 remained unchanged in all AF groups. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a newly discovered MMPs inhibitor, was elevated in RFW as compared to RAA-AF (P<0.05) and RFW-SR (P<0.05). The level of transforming growth factor (TGF)-beta1 was higher in AF than SR. Smad2 and phosphorylated Smad2 showed an elevation in RFW-AF as compared to RFW-SR, RAA-AF, and RAA-SR groups (P<0.05). CONCLUSIONS: Atrial fibrosis in AF is characterized by severe alterations in collagen I and III synthesis/degradation associated with disturbed MMP/TIMP systems and increased levels of RECK. TGF-beta1 contributes to atrial fibrosis via TGF-beta1-Smad pathway by phosphorylating Smad2. These processes culminate in accumulations of fibrillar and non-fibrillar collagens leading to excessive atrial fibrosis and maintainance of AF.

Effects of local MCP-1 protein therapy on the development of the collateral circulation and atherosclerosis in Watanabe hyperlipidemic rabbits.

OBJECTIVE: The objective of our study was to quantify the arteriogenic potency of Monocyte Chemoattractant Protein-1 (MCP-1) under hyperlipidemic conditions. Additionally, we aimed to determine the effects of locally applied MCP-1 on systemic serum lipid levels as well as on atherosclerosis. METHODS: A total of sixty-four Watanabe rabbits was treated with either low dose MCP-1 (1 microg/kg/week), high dose MCP-1 (3.3 microg/kg/week) or PBS as a control substance. Substances were applied directly into the collateral circulation via an osmotic minipump with the catheter placed in the proximal stump of the ligated femoral artery. Either 1 week or 6 months after initiation of the treatment X-ray angiography was performed as well as measurements of collateral conductance using fluorescent microspheres. The extent of atherosclerosis was quantified in whole aortas using Sudan IV staining. RESULTS: One week after ligation of the femoral artery a significant increase in collateral conductance was observed in animals treated with high dose MCP-1 (control: 2.2+/-0.8 ml/min/100 mmHg vs. MCP-1 high dose: 8.9+/-2.0 ml/min/100 mmHg, P<0.05). Six months after femoral artery ligation no differences were found between the treated and the control group (PBS; 44.9+/-11.6 ml/min/100 mmHg, MCP-1; 47.8+/-11.5 ml/min/100 mmHg, P=NS). No influence was found on serum lipids or on the development of atherosclerosis in the present model. CONCLUSION: MCP-1 accelerates arteriogenesis upon femoral artery ligation under hyperlipidemic conditions. Six months after treatment these pro-arteriogenic effects of MCP-1 can no longer be observed. The present data do not show an effect of local MCP-1 treatment on serum lipids or on atherosclerosis. It should be noted however that a high standard deviation was observed for the data on atherosclerotic surface area, necessitating additional experiments in a different model of atherosclerosis.

Functional disturbances due to structural remodeling in the failing human heart.

In severely hypertrophied hearts structural remodeling occurs continuously and finally leads to heart failure. The remodeling process involves all structural components of the cardiomyocyte and all protein families and it consists of cellular enlargement accompanied by degeneration in addition to the occurrence of fibrosis. Nuclei are increased in size but the nuclear volume/cell volume ratio is reduced. Transcription and translation are downregulated for contractile and sarcomeric skeleton proteins but both are upregulated for cytoskeletal and membrane-associated proteins. The connexin43 content is significantly reduced. Chronic degeneration finally leads to cell death by ubiquitin-related autophagy, and acute ischemic cell death (oncosis) is also observed. Apoptosis seems to be of minor importance. The morphological alterations described here are the structural correlate of the typical clinical characteristics of heart failure in human patients: of reduced contractile function, of increased ventricular stiffness represented by an increased left end-diastolic pressure and of ventricular arrhythmia.

Tissue engineering of a differentiated cardiac muscle construct.

Cardiac tissue engineering is an emerging field. The suitability of engineered heart tissue (EHT) for both in vitro and in vivo applications will depend on the degree of syncytoid tissue formation and cardiac myocyte differentiation in vitro, contractile function, and electrophysiological properties. Here, we demonstrate that cardiac myocytes from neonatal rats, when mixed with collagen I and matrix factors, cast in circular molds, and subjected to phasic mechanical stretch, reconstitute ring-shaped EHTs that display important hallmarks of differentiated myocardium. Comparative histological analysis of EHTs with native heart tissue from newborn, 6-day-old, and adult rats revealed that cardiac cells in EHTs reconstitute intensively interconnected, longitudinally oriented, cardiac muscle bundles with morphological features resembling adult rather than immature native tissue. Confocal and electron microscopy demonstrated characteristic features of native differentiated myocardium; some of these features are absent in myocytes from newborn rats: (1) highly organized sarcomeres in registry; (2) adherens junctions, gap junctions, and desmosomes; (3) a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum; and (4) a basement membrane surrounding cardiac myocytes. Accordingly, EHTs displayed contractile characteristics of native myocardium with a high ratio of twitch (0.4 to 0.8 mN) to resting tension (0.1 to 0.3 mN) and a strong beta-adrenergic inotropic response. Action potential recordings demonstrated stable resting membrane potentials of -66 to -78 mV, fast upstroke kinetics, and a prominent plateau phase. The data indicate that EHTs represent highly differentiated cardiac tissue constructs, making EHTs a promising material for in vitro studies of cardiac function and tissue replacement therapy.