Loss Of Myofibrils Research Articles

Protective Efficacy of Nardostachys jatamansi (Rhizomes) on Mitochondrial Respiration and Lysosomal Hydrolases during Doxorubicin Induced Myocardial Injury in Rats

Doxorubicin is highly effective in treatment for several forms of cancer. However, doxorubicin induces a cumulative and dose dependent cardiomyopathy that has been ascribed to redox-cycling of the molecule on the mitochondrial complex 1 generating in the process of increased oxidative stress. In the search of new potential cardioprotective agents, the present study is directed to explore the effect of ethanolic extract of Nardostachys jatamansi on the mitochondrial and lysosomal damage induced by doxorubicin in rats. Heart mitochondria were isolated from rats treated with doxorubicin (15 mg/kg, ip) a single dose, exhibited depressed rates of state 3 respiration, low respiratory control ratio (RCR), decreased Oxidative Phosphorylation ratio, Adenosine Triphosphate content and cytochromes (c, c1 ,b , aa 3). In addition the doxorubicin given rats showed significant changes in the lysosomal enzymes (Cathepsin-D, Acid phosphatase, β-D-glucoronidase, β-D-galactosidase and β-N-acetyl glucosaminidase) and membrane bound phosphatases. Also myocardial damage, as assessed by ultrastructural changes showed loss of myofibrils, mitochondrial swelling, and cytoplasmic vacuolization. Pretreatment with Nardostachys jatamansi (500 mg/kg body weight orally) for seven days ameliorated the observed abnormalities and significantly prevented th em itochondrial respiration, lysosomal integrity, membrane bound phosphatases and ultrastructural studies in doxorubicin induced rats. These findings suggest that the cardioprotective efficacy of Nardostachys jatamansi could be mediated possibly through its antioxidant effect as well as by the attenuation of the oxidative stress.

Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

Upregulated cardioprotective mechanisms protect diet restricted rats against doxorubicin‐induced cardiotoxicity

The clinical use of doxorubicin (Adriamycin®) is limited by its oxyradical-mediated cardio- and nephrotoxicity. Despite many beneficial health effects of moderate diet restriction (DR), the benefits of DR against drug-induced cardiotoxicity have been sparsely investigated. The objective was to test the hypothesis that moderate DR protects against doxorubicin-induced cardio- and nephrotoxicity and to investigate the cardioprotective mechanisms. Male Sprague-Dawley rats (250–275 g), maintained on DR [35% less than ad libitum (AL) food consumption, 42 days], were challenged with a normally LD100 dose of doxorubicin (DOX, 12 mg/kg, ip, 0.9% NaCl) on day 43. While all AL fed rats died between days 7 to 13, all the DR rats survived. Estimation of serum cardiac troponin-I and creatine kinase-MB, and cardiac tissue malondialdehyde revealed high cardiotoxicity during the 12 day time course in AL fed rats. Histopathology revealed focal rarefaction, loss of cardiac myofibrils, and cytoplasmic vacuolization early, becoming more diffuse as severity increased with time. In contrast, the DR rats showed minimal myocardial degeneration by day 8 and were completely devoid of any cardiotoxicity by serum chemistry by day 12. Cytochrome P450 reductase activity was unchanged by DR, suggesting no decrease in DOX redox cycling. Cardiac tissue catalase activity, ATP, the cardioprotective STAT3, and pSTAT3 were higher in the DR rats. Nephrotoxicity was elevated ~4-fold in the AL rats from day 4 to 12 compared to the DR rats. Thus indicating that, moderate DR renders high resiliency to the cardio- and nephrotoxic outcomes of DOX.

Functional Consequences of Hypertrophic and Dilated Cardiomyopathy-causing Mutations in α-Tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.

Protective effect of CardiPro against doxorubicin-induced cardiotoxicity in mice

The effect of CardiPro, a polyherbal formulation, with an antioxidant property, has been studied on doxorubicin (DXR)-induced cardiotoxicity in mice. CardiPro (150 mg/kg b.w., twice daily was administered orally for 7 weeks along with four equal injections (each containing 4.0 mg/kg b.w., DXR) intraperitoneally, once weekly (cumulative dose 16 mg/kg). After a 3-week post DXR treatment period, cardiotoxicity was assessed by noting mortality, volume of ascites, liver congestion, changes in heart weight, myocardial lipid peroxidation, antioxidant enzymes and histology of heart. DXR-treated animals showed higher mortality (50%) and more ascites. Myocardial SOD and glutathione peroxidase activity were decreased and lipid peroxidation was increased. Histology of heart of DXR-treated animals showed loss of myofibrils and focal cytoplasmic vacuolization. CardiPro significantly protected the mice from DXR-induced cardiotoxic effects as evidenced by lower mortality (25%), less ascites, myocardial lipid peroxidation, normalization of antioxidant enzymes and minimal damage to the heart histologically. Our data confirm the earlier reports that DXR cardiotoxicity is associated with the free radical-induced tissue damage. Administration of CardiPro, with an antioxidant property, protected the DXR-induced cardiotoxicity in mice.

A systematic study of a myocardial lesion: Colliquative myocytolysis

Background The term “myocytolysis” was first used to define the repair process of contraction band necrosis associated with an acute myocardial infarction. On the other hand, in the latter condition a “myofibrillolysis,” presenting edematous myocardial cells not involved by infarct necrosis, and without evidence of repair process was reported. The objective of this study is to establish the frequency, extent and meaning of this myocardial lesion. Materials and methods In 12 groups of patients for a total of 432 cases with and without coronary heart disease, “colliquative myocytolysis” – i.e., progressive vacuolization by loss of myofibrils until their total or subtotal disappearance associated with intramyocellular edema in absence of any cellular reaction – was graded in 16 histological slides of the different cardiac regions in each pathological case. Results Colliquative myocytolysis (CM) was present in more than 90% with a maximal extent in cases of irreversible congestive heart failure followed by transplanted heart cases (67%) with a survival greater than 1 week. In all other groups, the lesion was absent or minimal. Conclusions No correlation was found between CM and contraction band necrosis, gender, age, heart weight, myocardial fibrosis, coronary artery stenosis, clinical data. Colliquative myocytolysis is a specific histological marker of congestive heart failure, without relation to coronary blood flow, heart weight and myocardial fibrosis. Vacuolization of myocardial cells may be due to other causes (e.g., storage disease, etc.) or may be an artifact. There is no support for the belief that coronary ischemia or myocardial hypoxia is its causes.

Macroscopic–microscopic characterization of the passive mechanical properties in rat soleus muscle

The purpose of the study was to investigate changes in passive mechanical properties of the soleus muscle of the rat during the first year of life. These mechanical changes were quantified at a macroscopic (whole muscle) and a microscopic level (fiber) and were correlated with biochemical and morphological properties. Three passive mechanical tests (a relaxation test, a ramp stretch test and a stretch release cycle test) with different amplitudes and velocities were performed on isolated soleus muscles and fibers in rats at ages 1 (R1), 4 (R4) and 12 (R12) months. Mechanical parameters (dynamic and static forces, stresses and normalized stiffness) were recorded and measured. The morphological properties (size of fibers and muscles) for the three groups of rats were assessed by light microscopy which allowed us to observe the evolution of the fiber type (I, IIc and IIa) in the belly region and along the longitudinal axis of the muscle. In addition, biochemical analyses were performed at the level of the whole muscle in order to determine the collagen content. The results of the passive mechanical properties between the macroscopic (muscle) and microscopic (fiber) levels showed a similar evolution. Thus, an increase of the dynamic and static forces appeared between 1 and 4 months while a decrease of the passive tension occurred between 4 and 12 months. These mechanical changes were correlated to the morphological properties. In addition, the size of the three fibers type which grew with age could explain the increase of forces between 1 and 4 months. Furthermore, the biochemical analysis showed an increase of the collagen content during the same period which could also be associated with the increase of the passive forces. After 4 months, the passive tension decreased while the size of the fiber continued to increase. The biochemical analysis showed a decrease of the collagen content after 4 months, which could explain the loss of passive tension in the whole muscle. Concerning the similar loss at the fiber level, other assumptions are required such as a myofibril loss process and an increase of intermyofibrillar spaces. The originality of this present study was to compare the passive mechanical properties between two different levels of anatomical organization within the soleus muscle of the rat and to explain these mechanical changes in terms of biochemical and morphological properties.

Protective effect ofSpirulina against doxorubicin-induced cardiotoxicity

The generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin (DOX)-induced cardiotoxicity. The aim of the present study was to determine whether Spirulina, a blue-green algae, could serve as a cardioprotective agent during DOX treatment in a mouse model. Mice were treated with DOX (4 mg/kg bw, intraperitoneally), weekly, for 4 weeks. Spirulina was administered orally for 3 days twice daily, then for 7 weeks along with the four equal injections of DOX. Cardiotoxicity was assessed, at 3 weeks after the end of the DOX-treatment period, by mortality, volume of ascites, liver congestion, oxidative stress and ultrastructural changes of heart tissue. The DOX-treated animals showed higher mortality (53%) and more ascites. Myocardial damage, as assessed by ultrastructural changes, showed loss of myofibrils, cytoplasmic vacuolization and mitochondrial swelling. Myocardial superoxide dismutase and glutathione peroxidase activities were decreased and lipid peroxidation was increased. Pretreatment with Spirulina significantly protected the mice from DOX-induced cardiotoxic effects as evidenced from lower mortality (26%), less ascites, lower levels of lipid peroxidation, normalization of antioxidant enzymes and ultrastructural studies showing minimal damage to the heart. In vitro cytotoxic studies using ovarian cancer cells demonstrated that Spirulina did not compromise the anti-tumor activity of doxorubicin. These results suggest that Spirulina has a protective effect against cardiotoxicity induced by DOX and it may, therefore, improve the therapeutic index of DOX.

Arrhythmogenic right ventricular cardiomyopathy: Clinicopathologic correlation based on a revised definition of pathologic patterns

Different morphologic features of arrhythmogenic right ventricular cardiomyopathy (ARVC) have been described. However, it is still unclear whether they correspond to distinct forms of the same disease. A pathologic study was performed on a series of ARVC (15 from heart transplant and 12 from autopsy) from 2 Italian referral university hospitals. Based on both myocellular features and the nature of myocardial replacement, hearts were divided into 2 groups: infiltrative, with a lacelike pattern of transmural fatty infiltration and strands of normal residual cardiomyocytes (n = 11); and cardiomyopathic, with massive myocardial replacement by fibro fatty tissue and cardiomyopathic changes (such as hypertrophy and myofibril loss) of residual cardiomyocytes (n = 16). Hearts from the infiltrative group were mostly obtained at autopsy of patients who died suddenly. Fatty substitution was limited almost exclusively to the right ventricle. Mitral valve dysplasia (prolapse or cleft) was frequently present. Hearts from the cardiomyopathic group came mainly from heart transplants for congestive heart failure. Fibro fatty replacement was more extensive, usually biventricular. Active myocarditis and features suggestive of myocardial transdifferentiation were also observed. Despite these differences in clinical outcome and morphologic features, patients from the 2 groups showed similar mean age, sex distribution, occurrence of threatening ventricular arrhythmias, and prevalence of family history of sudden death, arrhythmias, or cardiomyopathy. Infiltrative and cardiomyopathic patterns represent different clinical and pathologic subsets of ARVC. Myocellular features are an important clue in the distinction between the two entities. The differentiation between the 2 patterns is feasible on endomyocardial biopsy and could give important prognostic information. HUM PATHOL 32:1078-1086. Copyright © 2001 by W.B. Saunders Company

Accidental monensin toxicosis in horses in Mozambique.

Horses on several farms in Mozambique were inadvertently fed with a concentrate containing 69 ppm monensin. The horses developed acute signs of toxicity and several died. The animals were depressed, anorectic and paretic before death. Epistaxis was observed in 1 case. Petechial haemorrhages were present in the muscles, heart, lungs, gastrointestinal tract and spleen in 3 horses necropsied. No significant histopathological cardiac and skeletal muscle lesions were seen, except in 1 case, in which there was focal loss of myofibrils.

Skeletal muscle abnormalities associated with occupational exposure to mercury vapours.

There is scarce information on the possible effects of chronic exposure to mercury on skeletal muscle. Dental personnel are frequently exposed to inhalation of metallic mercury vapours. The skeletal muscle of five technicians and one dentist (females, age 36-55) was studied. All of them presented symptoms of chronic mercury poisoning. Needle biopsy was taken from the quadriceps femoris muscle and samples were prepared for light microscope histochemistry and for transmission electron microscopy. Selective atrophy of type IIB muscle fibres was found in patients, and in one of them there was fibre grouping. Most of the muscles showed increased fibre area per capillary. Atrophy was confirmed by the ultrastructural study, demonstrating increase of intermyofibrillar spaces, loss of myofibrils or complete disappearance in some fibres, and sarcolemmal foldings. Splitting of the fibres was also found. Some capillaries were altered, showing endothelial infoldings into the lumen, thickened basement membrane and partial or total occlusion. The alterations found in muscle may be secondary to nerve damage, to ischemia caused by capillary lesion and/or to a direct effect of mercury on muscle fibre proteins.

Protective effects of Gingko biloba extract EGb 761 on myocardium of experimentally diabetic rats: I: Ultrastructural and biochemical investigation on cardiomyocytes

Chronic diabetes in man and animal models develops cardiomyopathic alterations which cannot be absolutely avoided by insuline therapy. Since diabetic damage is partly attributed to oxidative stress antioxidative treatment could be able to reduce the alterations. Aim of this study was to investigate the cardioprotective effects of EGb 761, known as a radical scavenger, against diabetic alterations in rats. The diabetes was induced by i.p. injection of 60 mg/kg body weight streptozotocin. Duration of diabetes was 4 months, the protected group received 100 mg/kg body weight EGb 761 with the drinking water over 3 months. Electron and light microscopic morphometry of left-ventricular samples revealed typical diabetic alterations consisting in decrease of volume fraction of myofibrils, SR and t-tubules and diminishing of cardiomyocyte diameter, increase of interstitial volume, mitochondrial size and volume fraction, and of vacuoles and of lipid drops. EGb treatment could gradually prevent the loss of myofibrils and reduction of myocyte diameter but has only little influence on interstitial and mitochondria volume. The diabetic-induced increase of lipid and vacuoles and the decrease of SR and t-tubules were not influenced. Biochemical parameters of oxidative stress: malondialdehyde (MDA) was only insignificantly altered by diabetes and EGb. The superoxide dismutase (SOD) activity was increased by diabetes and more increased by EGb treatment. Creatine kinase (CK) activity was diminished by diabetes but slightly increased by EGb. The polymerase chain reaction (PCR) of i-NOS was not different between the diabetic and protected diabetic groups.

Sarcomeric myosin heavy chain is degraded by the proteasome.

Cardiac myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The balance between these opposing metabolic processes ultimately determines the number of functional contractile units within each cardiac muscle cell. Although alterations in myofibrillar protein degradation have been shown to contribute to cardiac growth and remodeling, the intracellular proteolytic systems responsible for degrading myofibrillar proteins to their constitutive amino acids are currently unknown. Lactacystin, a recently developed, highly specific proteasome inhibitor, was used in this study to examine the role of the proteasome in myosin heavy chain (MHC) degradation in cultured neonatal rat ventricular myocytes. Cells were treated with growth medium alone or with lactacystin (1-50 microM) for up to 48 h. Lactacystin significantly increased the total protein/DNA ratio and markedly prolonged MHC half-life. Other proteasome inhibitors, namely carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (10 microM) and N-acetyl-L-leucyl-L-leucyl-norleucinal (100 microM), were also effective in suppressing MHC degradation. Lactacystin and other proteasome inhibitors also suppressed the markedly accelerated MHC degradation associated with Ca2+ channel blockade but did not prevent the disassembly and loss of myofibrils that accompanied contractile arrest. Thus, sarcomere disassembly precedes the degradation of MHC, which is at least in part mediated by the proteasome.

Carnitine Promotes Heat Shock Protein Synthesis in Adriamycin-induced Cardiomyopathy in a Neonatal Rat Experimental Model

In order to evaluate carnitine protective strategy and its relationship with heat shock protein induction, female Sprague-Dawley neonatal rats, body weight 40 g, were randomized into four groups: control, adriamycin, carnitine and carnitine–adriamycin. Adriamycin was injected i.v. at a dose of 27 mg/kg (0.1 ml). Carnitine was administered i.v. (20 mg/0.1 ml) before each subdose of adriamycin and thenper os(180 mg/kg) daily for 12 weeks. Body weight was recorded weekly. Ventricular wall thickness and cellular damage percentage were morphometrically and ultrastructurally determined, respectively. The determinations were realized monthly until the third month after treatment. The heat shock protein 25 content in the supernatant of the homogenized heart tissue was determined by Western blot analysis. Eight and 12 weeks after treatment, body weight and ventricular wall thickness decreased much more in adriamycin groups than in control and carnitine ones. At the same time, electron microscopic analysis of adriamycin left ventricular wall samples showed loss of myofibrils, swollen mitochondria and vacuoles. Carnitine–adriamycin treated rats resemble control groups more than adriamycin treated samples. Moreover,de-novosynthesis of heat shock protein was three times more induced in carnitine–adriamycin rats than in adriamycin ones. Carnitine may enhance the cell-protecting mechanism based on an induction of shock protein, and this first cellular response could reduce the severity of late adriamycin-cardiomiopathy.

Structural and functional changes in skeletal muscle in anorexia nervosa.

Protein-energy malnutrition in anorexia nervosa is an under-recognised cause of muscle dysfunction. To characterise the skeletal myopathy that occurs in patients with severe anorexia nervosa, muscle function and structure were comprehensively examined in eight young adult female patients with severe (40%) self-induced weight loss. All of the patients showed impaired muscle function on strength and exercise measurement. The maximum voluntary contraction force for the patient group was significantly less than predicted values. Electromyography revealed myopathy in five of the patients, four of whom also had electro-physiological evidence of neuropathy. However, muscle biopsy specimens consistently showed myopathic changes with severe type 2 fibre atrophy but with no evidence of neuropathic changes. Ultrastructurally, there was separation and segmental loss of myofibrils and most biopsy samples contained abundant glycogen granules; we have previously reported that one of the most consistent biochemical abnormalities in these patients is impaired ischaemic lactate responses to forearm exercise. The result of severe protein-energy malnutrition on the musculo-skeletal system is a metabolic myopathy. Although the patients admitted to a variety of abnormal dieting behaviours, such as over-exercising and self-induced vomiting, no association was found between any of these and quantitative histological changes in the muscle biopsy samples. It is recommended that myopathy in anorexia nervosa be treated by instituting an appropriate refeeding programme.

Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice.

Loss of myofibril organization is a common feature of chronic dilated and progressive cardiomyopathy. To study how the heart compensates for myofibril degeneration, transgenic mice were created that undergo progressive loss of myofibrils after birth. Myofibril degeneration was induced by overexpression of tropomodulin, a component of the thin filament complex which determines and maintains sarcomeric actin filament length. The tropomodulin cDNA was placed under control of the alpha-myosin heavy chain gene promoter to overexpress tropomodulin specifically in the myocardium. Offspring with the most severe phenotype showed cardiomyopathic changes between 2 and 4 wk after birth. Hearts from these mice present characteristics consistent with dilated cardiomyopathy and a failed hypertrophic response. Histological analysis showed widespread loss of myofibril organization. Confocal microscopy of isolated cardiomyocytes revealed intense tropomodulin immunoreactivity in transgenic mice together with abnormal coincidence of tropomodulin and alpha-actinin reactivity at Z discs. Contractile function was compromised severely as determined by echocardiographic analyses and isolated Langendorff heart preparations. This novel experimentally induced cardiomyopathy will be useful for understanding dilated cardiomyopathy and the effect of thin filament-based myofibril degeneration upon cardiac structure and function.

Characterisation of a cellular model of cardiomyopathy, in the rabbit, produced by chronic administration of the anthracycline, epirubicin.

The objective of this study was to assess the structural, mechanical and electrophysiological changes associated with chronic administration of epirubicin (Pharmorubicin), which is a cardiotoxic anthracycline antibiotic used in conventional cancer chemotherapy. New Zealand white rabbits (8 weeks) were treated with twice-weekly infusions of epirubicin (2 mg/kg) or saline for a period of 6 weeks, followed by a wash-out period of 2 weeks. Myocardial damage consistent with cardiomyopathy was observed in the epirubicin-treated animals; electron micrographs indicated myofibril loss together with separation of the intercalated disc and dilation of the sarcotubular system. Contractile function, as measured by mechanical shortening, action potentials and L-type Ca2+ currents were examined in ventricular cardiomyocytes, which were isolated by means of enzymatic dissociation using collagenase. There was an attenuation in the contractile response to isoprenaline in cardiomyocytes isolated from the hearts of epirubicin-treated rabbits compared to control rabbits. Cardiomyocytes isolated from epirubicin-treated rabbits had greater basal contractile amplitude (11.0+/-0.3 %dL, n=8) than control myocytes (8.2+/-0.3 %dL, n=9), but had similar maximum responses of 19.1+/-0.6 %dL, and 17.3+/-0.5 %dL, respectively, when stimulated with 1 microM isoprenaline. No differences were noted in the peak L-type Ca2+ current of myocytes isolated from the hearts of control and epirubicin-treated rabbits; however, in the latter, there was a prolongation of the action potential duration (396+/-25 ms) compared to that in controls (321+/-26 ms). These results demonstrate structural and mechanical alterations in ventricular cardiomyocytes which are compatable with a mild cardiomyopathy following chronic treatment with the anthracycline, epirubicin. The increase in basal contraction is likely to be due to more efficient coupling of electrical stimulation, and the depressed inotropic responsiveness following stimulation with isoprenaline indicates that there may be changes in cell membrane properties. Compared to control cardiomyocytes, cells isolated from the hearts of epirubicin-treated rabbits were more heterogeneous, with respect to cell dimensions, and had significantly different electromechanical properties.

Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat.

After cardioversion of sustained atrial fibrillation (AF), the electrical and contractile functions of the atria are impaired, and recurrences of AF frequently occur. Whether remodeling of the structure of atrial myocardium is the basis for this problem is not known. Sustained AF was induced by electrical pacing in 13 goats instrumented long-term. The goats were killed after 9 to 23 weeks, and the atrial myocardium was examined by light and electron microscopy. The changes were quantified in left and right atrial free walls, appendages, trabeculae, the interatrial septum, and the bundle of Bachmann. A substantial proportion of the atrial myocytes (up to 92%) revealed marked changes in their cellular substructures, such as loss of myofibrils, accumulation of glycogen, changes in mitochondrial shape and size, fragmentation of sarcoplasmic reticulum, and dispersion of nuclear chromatin. These changes were accompanied by an increase in size of the myocytes (up to 195%). There were virtually no signs of cellular degeneration, and the interstitial space remained unaltered. The duration of sustained AF did not significantly affect the degree of myolytic cell changes. Sustained AF in goats leads to predominantly structural changes in the atrial myocytes similar to those seen in ventricular myocytes from chronic hibernating myocardium. These structural changes may explain the depressed contractile function of atrial myocardium after cardioversion. This goat model of AF offers a new approach to study the cascade of events leading to sustained AF and its maintenance.

Analysis of Myofibrillar Organization and Degeneration by Fluorescence Confocal Microscopy

Myofibrillar degeneration is an important pathological process in progressive cardiomyopathy leading to heart failure. Loss of myofibrils in vivo has been observed in both adaptive cardiac responses (i.e. hypertrophy) as well as in chemotherapeutic use of antitumor drugs with cardiotoxic side effects (i.e. doxorubicin). The molecular mechanism(s) of myofibrillar degeneration are poorly understood in comparison with the sequence of events involved in myofibrillar assembly and organization. Maintenance of myofibril integrity is dependent upon a variety of factors, including contractile protein stoichiometry and protein kinase activity.The repeating sarcomeric architecture of myofibrils is well suited to structural analysis, since disruption of normal organization is easily visualized by fluorescence microscopy. Many antibodies are available for use in observation of myofibril structure (see Table at right). Confocal microscopy provides advantages in studies of myofilament organization by allowing for direct and accurate measurement of distances to within 0.2 βm and the ability to perform vertical sectioning through individual cells. This vertical (Z-axis) sectioning can be used to select the focal plane within the cell for observation, resulting in higher resolution images by reducing fluorescent signals from above and below the plane. Image analysis software enables the user to create projections of optically sectioned cardiomyocytes which can be rotated to reveal interior structural relationships previously unobserved with single sections or conventional epifluorescence microscopy. Examples of image analysis will highlight useful features such as distance measurement, periodicity, pixel intensity, colocalization of dual labels, and three dimensional reconstruction.

Functional and structural alterations with 24-hour myocardial hibernation and recovery after reperfusion. A pig model of myocardial hibernation.

Short-term myocardial hibernation of 3 hours resulting from a moderate resting coronary flow reduction has been reproduced in pigs. This study was designed to determine whether any structural changes accompany short-term hibernation caused by a moderate flow reduction maintained for 24 hours and whether any such structural alterations are reversible after reperfusion. A severe left anterior descending coronary artery (LAD) stenosis was created with a reduction of resting flow to approximately 60% of baseline and maintained for 24 hours. Regional coronary flow was measured by a flowmeter; wall thickening was determined by echocardiography, and local metabolic changes were measured. Of 17 pigs, 11 completed the study protocol of 24 hours. The LAD flow was reduced from 0.91 +/- 0.11 to 0.52 +/- 0.13 mL.min-1.g-1, a 43% mean decrease, at 15 minutes after the LAD stenosis and was maintained at 0.56 +/- 0.11 mL.min-1.g-1 at 24 hours. The reduction of regional coronary flow initially produced acute myocardial ischemia, as evidenced by reduced regional wall thickening (from 37.2 +/- 6.9% at baseline to 11.5 +/- 6.8%), regional lactate production (-0.34 +/- 0.28 mumol.g-1.min-1), and a decrease in regional coronary venous pH (from 7.41 +/- 0.035 at baseline to 7.30 +/- 0.030). At 24 hours, the reductions in coronary flow and wall thickening were maintained relatively constant and the rate-pressure product was relatively unchanged, but lactate production ceased and regional H+ concentration normalized, with a tendency toward a further reduction in regional oxygen consumption, from 3.10 +/- 0.90 mL.min-1.100 g-1 at 15 minutes after stenosis to 2.52 +/- 0.95 mL.min-1.100 g-1 at 24 hours (P = .06), indicating metabolic adaptation of the hypoperfused regions. Of 11 pigs, 6 were free of myocardial infarction; 3 had patchy necrosis involving 4%, 5%, and 6% of the area at risk; and 2 other pigs had a few scattered myocytes with necrosis, detected only by light and electron microscopy. Ultrastructural changes consisted of a partial loss of myofibrils and an increase in mitochondria and glycogen deposition. Regional wall thickening recovered 1 week after reperfusion in most pigs, and the ultrastructural changes reverted to normal. In this pig model, moderately ischemic myocardium undergoes metabolic and structural adaptations but preserves the capacity to recover both functionally and ultrastructurally after reperfusion.