Cytomorphometry of skeletal muscle: The influence of age and testosterone on the rat M. levator ani

Summary The levator ani muscle of the rat was examined by correlated light and electron microscopic morphometry. Corrections were made for shrinkage, compression, and differences in stretching. Age, castration, and subsequent testosterone treatment do not affect the fiber number, the filament lattice, and the size of the filaments and myonuclei. The fibers in intact growing males increase in width and length. The number of myonuclei rises, although relatively slower than the amount of contractile material.Castration, performed at six weeks, partially suppresses fiber growth. The increase of mean fiber width is more strongly inhibited than that of fiber length. Myonuclear multiplication is almost completely arrested in castrates, and the amount of contractile material per myonucleus is lower than in intact males of equal age.Testosterone, administered at about two months following orchidectomy, highly accelerates the transversal fiber growth, but fiber length is not significantly influenced. Between the fourth and seventh day of treatment a marked increase in myonuclear number occurs.Analysis of the frequency distribution of the individual fiber widths, which is logarithmic-normal in intact males, revealed that the hormonal influence on the net result of protein anabolism and catabolism markedly differs in the various fibers of a single muscle.With the technical assistance of Tineke J. Hoogenboezem.     

Cadmium-induced abnormality in strains of Euglena gracilis: morphological alteration and its prevention by zinc and cyanocobalamin

Euglena gracilis is susceptible to cadmium (Cd) at high concentrations. There are no comparative data on cytotoxicity or abnormality of CdCl₂ to E. gracilis Z and its achlorophyllous mutant SMZ. The present study examined the cytotoxicity of CdCl₂ under continual exposure at levels ranging from sub-ppm to ppm, and assessed the effects of zinc (Zn) or cyanocobalamin (VB₁₂) supplementation on the suppression of Cd-induced abnormal cell proliferation and hypertrophy. With Zn levels restricted to 1 ppm [as Zn⁺⁺], cell growth of both E. gracilis strains was reduced in proportion to Cd concentration. More abnormal cells (hypertrophied, V-shape and starfish-shape) were observed in both strains at sub-ppm levels of Cd. ZnSO₄ supplementation from 2 to 63 ppm significantly suppressed the incidence of Cd-induced abnormality. However, a significant increase in abnormal cells was observed following Zn supplementation at levels of 125 and 250 ppm, which produced remarkable differences in cell morphology. The incidence of abnormal cells varied with supplemented VB₁₂ levels ranging from 4 to 250 ppb in both E. gracilis strains.     

Zur Kenntnis der terminalen Strombahn im Myocard von Ratte und Katze

Zusammenfassung Das Verzweigungsmuster der terminalen Strombahn des Myocards wird an Zupfpräparaten von 67 Ratten- bzw. Katzenherzen untersucht. Die Gefäße wurden mit Farbsuspensionen gefüllt. Die Schwierigkeiten der vollständigen Darstellung des Kapillarbettes werden diskutiert. Durch aufeinanderfolgende Injektion mit mehreren Farben gelingt es, den arteriellen und venösen Kapillarschenkel unterschiedlich darzustellen. Zur genauen Analyse werden von einzelnen Muskelblättchen ausgedehnte Gefäßkarten hergestellt. Folgende Befunde wurden erhoben: Als feinste Zuleitungsgefäße münden die Präkapillaren in das Kapillarnetz. Ihre Wand enthält nur vereinzelte Muskelzellen. Die Präkapillaren verlaufen parallel oder senkrecht zu den Muskelfasern und teilen sich in gleichwertige Äste (dichotom) oder geben jeweils als Stammgefäß seitliche Äste ab. Die Arteriolen bzw. Präkapillaren anastomosieren nicht miteinander.Die gewöhnlich weiten Venolen bestehen im wesentlichen nur aus einem Endothelrohr. Sie verlaufen zunächst senkrecht zur Muskulatur und münden in abführende, meist längsgerichtete Venen ein. Anastomosen unter den Venolen sind selten.Zwischen Präkapillaren und Venolen sind Kapillarbahnen ausgespannt. Die Kapillaren verlaufen parallel zueinander im Abstand einer Muskelzellbreite und stehen über Queranastomosen miteinander in Verbindung.Die Kapillarwege von Präkapillaren zu Venolen variieren zwischen 100 m und 800 m. Messungen an zwei Herzen ergaben eine mittlere Strecke von 310 m (Ratte) bzw. 400 m (Katze).Die mittlere unverzweigte Kapillarstrecke beträgt im etwas kontrahierten Rattenherzen 65 m, im mäßig erschlafften Katzenherzen 110 m. Da sich höchstens 1/3 der Kapillaräste untereinander zu Maschen verbinden, entsteht ein sehr lockeres Netzwerk.Da benachbarte Kapillaren zu gleichen oder verschiedenen Präkapillaren und Venolen gehören können, sind mehrere Gefäanordnungen im Myocard unterscheidbar:Selten sind reine Gleichstromanordnungen (vgl. Krogh, 1918/19) oder Gegenstromanordnungen (vgl. Diemer, 1965) zu beobachten, bei denen die Kapillaren am gleichen Ort anfangen bzw. enden und benachbarte Kapillaren in gleicher oder entgegengesetzter Richtung durchströmt werden.Überwiegend finden sich asymmetrische Kapillaranordnungen (vgl. Grunewald und Lübbers, 1966), bei denen Kapillaranfänge bzw. -enden gegeneinander versetzt sind und benachbarte Kapillarabschnitte in gleicher oder entgegengesetzter Richtung durchströmt werden. Die morphologischen Befunde zeigen, daß physiologischen Untersuchungen ein gemischtes Versorgungsmodell für den Herzmuskel (Grunewald und Lübbers, 1966) zugrunde gelegt werden kann.

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The microcirculatory bed in the myocardium of the rat and the cat

Summary The branching modus of the microcirculatory bed is studied in isolated musclelayers from 67 hearts of rats or cats. The vessels are filled with different dye-suspensions. The methods to demonstrate the capillary bed by perfusion are discussed. It is shown that several dyes injected consecutively can mark the arterial and venous part of the capillary with different colours. For exact analysis maps are prepared of extensive parts of the microcirculatory bed.The precapillaries, the smallest vessels of the distributing system, are provided with only sporadic muscle cells. They run parallel to the muscle fibers or they cross them. They either divide into equal branches in a dichotomous manner or extend as trunk-vessels, from which the capillaries originate as branches. The arterioles and precapillaries don't anastomose with each other. The venous drainage begins with venoles which usually consist of only a wide endothelial tube. They cross the muscle fibers and open into veins which usually extend in the direction of the muscle layers. Anastomoses among the venoles are rare.Between precapillaries and venoles the capillary ways extend. The capillaries run parallel to each other in a distance of one muscle cell. They are connected by short transversal anastomoses. The length of the capillary way between precapillaries and venoles varies between 100 m and 800 m. According to measurements in two hearts there follows an average way of 310 m (rat) or 400 m (cat). The average length of the unbranched capillary segments was 65 m (rat) or 110 m (cat). As at most one third of the capillary branches are combined to mashes there is only a loose capillary-network.Adjacent capillaries may belong to the same or to different precapillaries and venoles, so that there can be differentiated between several different patterns of the microcirculatory bed:There are seldom found pure arrangements in such a way, that adjacent capillaries begin or end at the same place and that the blood flows in equal or contrary directions (cf. Krogh, 1918/19; Diemer, 1965).Mostly asymmetric arrangements are found with capillaries beginning or ending at staggered places and with equal or contrary flow directions in the capillary segments. The morphological results show that the base for physiological studies of the nutrition of the myocardium ought to be a mixed model of the capillary bed.

Die Arbeit entstand unter der Leitung von Herrn Prof. Dr. E. Lindner, Regensburg.     

HMG-CoA reductase inhibitor fluvastatin prevents angiotensin II-induced cardiac hypertrophy via Rho kinase and inhibition of cyclin D1

HMG-CoA reductase inhibitors, so called statins, decrease cardiac events. Previous studies have shown that HMG-CoA reductase inhibitors inhibit cardiomyocyte hypertrophy in vitro and in vivo by blocking Rho isoprenylation. We have shown that the G1 cell cycle regulatory proteins cyclin D1 and Cdk4 play important roles in cardiomyocyte hypertrophy. However, the relation between Rho and cyclin D1 in cardiomyocyte is unknown. To investigate whether HMG-CoA reductase inhibitors prevent cardiac hypertrophy through attenuation of Rho and cyclin D1, we studied the effect of fluvastatin on angiotensin II-induced cardiomyocyte hypertrophy in vitro and in vivo. Angiotensin II increased the cell surface area and [(3)H]leucine uptake of cultured neonatal rat cardiomyocytes and these changes were suppressed by fluvastatin treatment. Angiotensin II also induced activation of Rho kinase and increased cyclin D1, both of which were also significantly suppressed by fluvastatin. Specific Rho kinase inhibitor, Y-27632 inhibited angiotensin II-induced cardiomyocyte hypertrophy and increased cyclin D1. Overexpression of cyclin D1 by adenoviral gene transfer induced cardiomyocyte hypertrophy, as evidenced by increased cell size and increased protein synthesis; this hypertrophy was not diminished by concomitant treatment with fluvastatin. Infusion of angiotensin II to Wistar rats for 2 weeks induced hypertrophic changes in cardiomyocytes, and this hypertrophy was prevented by oral fluvastatin treatment. These results show that an HMG-CoA reductase inhibitor, fluvastatin, prevents angiotensin II-induced cardiomyocyte hypertrophy in part through inhibition of cyclin D1, which is linked to Rho kinase. This novel mechanism discovered for fluvastatin could be revealed how HMG-CoA reductase inhibitors are preventing cardiac hypertrophy.     

The significance of regulatory light chain phosphorylation in cardiac physiology

It has been over 35 years since the first identification of phosphorylation of myosin light chains in skeletal and cardiac muscle. Yet only in the past few years has the role of these phosphorylations in cardiac dynamics been more fully understood. Advances in this understanding have come about with further evidence on the control mechanisms regulating the level of phosphorylation by kinases and phosphatases. Moreover, studies clarifiying the role of light chain phosphorylation in short and long term control of cardiac contractility and as a factor in cardiac remodeling have improved our knowledge. Especially important in these advances has been the use of gain and loss of function approaches, which have not only testedthe role of kinases and phosphatases, but also the effects of loss of RLC phosphorylation sites. Major conclusions from these studies indicate that (i) two negatively-charged post-translational modifications occupy the ventricular RLC N-terminus, with mouse RLC being doubly phosphorylated (Ser 14/15), and human RLC being singly phosphorylated (Ser 15) and singly deamidated(Asn14/16 to Asp); (ii)a distinct cardiac myosin light kinase (cMLCK) and a unique myosin phosphatase targeting peptide (MYPT2) control phosphoryl group transfer;and (iii) ablation of RLC phosphorylationdecreases ventricular power, lengthens the duration of ventricular ejection, and may also modify other sarcomeric proteins (e.g., troponin I) as substrates for kinases and/or phosphatases. A long term effect of low levels of RLC phosphorylation in mouse models also involves remodeling of the heart with hypertrophy, depressed contractility, and sarcomeric disarray. Data demonstrating altered levels of RLC phosphorylation in comparisons of samples from normal and stressed human hearts indicate the significance of these findings in translational medicine.     

Compromised myocardial energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing SERCA2a

The sarcoplasmic reticulum calcium ATPase (SERCA) plays a central role in regulating intracellular Ca(2+) homeostasis and myocardial contractility. Several studies show that improving Ca(2+) handling in hypertrophied rodent hearts by increasing SERCA activity results in enhanced contractile function. This suggests that SERCA is a potential target for gene therapy in cardiac hypertrophy and failure. However, it raises the issue of increased energy cost resulting from a higher ATPase activity. In this study, we determined whether SERCA overexpression alters the energy cost of increasing myocardial contraction in mouse hearts with pressure-overload hypertrophy using (31)P NMR spectroscopy. We isolated and perfused mouse hearts from wild-type (WT) and transgenic (TG) mice overexpressing the cardiac isoform of SERCA (SERCA2a) 8 weeks after ascending aortic constriction (left ventricular hypertrophy (LVH)) or sham operation. We found that overexpressing SERCA2a enhances myocardial contraction and relaxation in normal mouse hearts during inotropic stimulation with isoproterenol. Energy consumption was proportionate to the increase in contractile function. Thus, increasing SERCA2a expression in the normal heart allows an enhanced inotropic response with no compromise in energy supply and demand. However, this advantage was not sustained in LVH hearts in which the energetic status was compromised. Although the overexpression of SERCA2a prevented the down-regulation of SERCA protein in LVH hearts, TG-LVH hearts showed no increase in inotropic response when compared with WT-LVH hearts. Our results suggest that energy supply may be a limiting factor for the benefit of SERCA overexpression in hypertrophied hearts. Thus, strategies combining energetic support with increasing SERCA activity may improve the therapeutic effectiveness for heart failure.     

Cells producing insulin-like androgenic gland hormone of the giant freshwater prawn, Macrobrachium rosenbergii, proliferate following bilateral eyestalk-ablation

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 μm in diameter), stain strongly with hematoxylin-eosin (H&;E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 μm in diameter), stain lightly with H&;E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction.     

On the local cardiac renin angiotensin system. Basic and clinical implications

In the present review we reevaluated the experimental and clinical evidence that there is a local renin angiotensin system in the heart as well as the presence of a functional intracrine component which is activated during pathological conditions like heart failure and hypertension. The implications of these findings for cardiology were discussed. The novel finding that cell swelling impairs cell coupling and impulse propagation through activation of ionic channels with consequent generation of cardiac arrhythmias and the evidence that AT1 receptors are mechanosensors able to alter the heart function independently of Ang II were discussed. Particular attention was given to the role of salt loading on the activation of a local cardiac renin angiotensin and its consequences.     

鳜早期发育阶段骨骼肌纤维的增生与肥大生长

骨骼肌是鱼体的主要组成部分,也是衡量鱼体生长发育的重要指标.为了解鳜(Siniperca chuatsi)早期发育阶段骨骼肌纤维的生长发育特征,通过制作孵化后1 ～41日龄个体骨骼肌背右侧第一肌节石蜡切片,利用图像分析软件统计该肌节中肌纤维的数目和面积,分析了鳜骨骼肌纤维的增生和肥大生长特征.结果表明,鳜早期发育阶段骨...     

Fetal cardiac troponin isoforms rescue the increased Ca2+ sensitivity produced by a novel double deletion in cardiac troponin T linked to restrictive cardiomyopathy: a clinical, genetic, and functional approach

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-ΔN100/ΔE101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-ΔN100/ΔE101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-ΔN100/ΔE101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.