Myopathic Syrian Hamsters Research Articles

Reduced Ca2+ transport across sarcolemma but enhanced spontaneous activity in cardiomyocytes isolated from left atrium-pulmonary veins tissue of myopathic hamster

BackgroundSeveral lines of evidence point to a particularly important role of the left atrium (LA) in initiating and maintaining atrial fibrillation (AF). This role may be related to the location of pulmonary veins (PVs) in the LA. The aim of the present study was to investigate the action potential (AP) and ionic currents in LA-PV cardiomyocytes isolated from Bio14.6 myopathic Syrian hamsters (36-57 week-old) versus age-matched F1B healthy control hamsters.Methods and ResultsWhole-cell patch-clamp techniques were used to record AP in current-clamp mode and ionic currents in voltage-clamp mode. The results obtained show that in both healthy and myopathic LA-PV tissue spontaneously discharging cardiomyocytes can be found, but they are more numerous in myopathic (9/29) than in healthy hamsters (4/42, p < 0.05 by χ2 analysis). Myopathic myocytes have shorter AP duration (APD) with smaller ICa,L and INCX than the healthy control. The currents ITO, IK, IK1 and ICa,T are not significantly different in myopathic versus healthy cells.ConclusionsOur results indicate that in myopathic Syrian hamsters LA-PV cardiomyocytes are more prone to automatic rhythms. Also, they show altered electrophysiologic properties, which may be due to abnormal Ca2+ channels and may account for contractile dysfunction.

The ionic characteristics of left atrium-pulmonary vein myocardium of healthy and myopathic Syrian hamsters

The ionic characteristics of left atrium-pulmonary vein myocardium of healthy and myopathic Syrian hamsters

Oscillatory transient inward currents in ventricular myocytes of healthy versus myopathic Syrian hamster

The present experiments were performed in order to study abnormal action potential configuration and ion channel activity in ventricular myocytes obtained from 23 male myopathic Syrian hamsters (Biobreeders strain 14.6, 32-52 weeks old) compared with 10 age-matched healthy control hamsters (Biobreeders F1B) by means of whole-cell patch-clamp techniques. The results show that the myopathic myocytes had a longer action potential duration, a reduced transient outward K(+) current on depolarization and a smaller transient inward current on repolarization after prolonged depolarizing pulses (> 500 msec). However, the L-type Ca(2+) current and the inwardly rectifing K(+) current were not significantly different from those of healthy myocytes. The oscillatory transient inward currents could be diminished by treatment with ryanodine (0.01-1 micromol/L), a sarcoplasmic reticulum (SR) Ca(2+) release channel blocker, or with Na(+)-free superfusate. We conclude that the hereditary myopathic hamsters are less likely to develop delayed after depolarization-related transient inward currents and triggered arrhythmias owing to a smaller SR Ca(2+) content.

Relaxation is impaired in the diaphragm muscle of the cardiomyopathic Syrian hamster.

Relaxation was examined in diaphragm muscle strips of cardiomyopathic Syrian hamsters and control hamsters. Isotonic lengthening velocity and isometric tension decay were analyzed over the load continuum in response to twitch. For each load level (P), we measured the maximum extent of shortening (deltaL), the peak lengthening velocity (VL), and the peak rate of tension decline (-dP/dtmax). The kinetics of sarcomere length (SL) were simultaneously measured by laser diffraction. In an attempt to account for the influence of shortening and/or load on relaxation, we calculated the slopes of the VL - deltaL and -dP/dtmax - P relationships. In both groups, there was a direct relationship between (1) VL and deltaL and (2) -dP/dtmax and P. In myopathic hamsters, we observed a decrease in the slope of the VL - deltaL relationship (p < 0.05), a decrease in VL at any common value of both muscle and sarcomere extent of shortening, and an increase in the duration of overall lengthening. Isometric tension decay was significantly prolonged in myopathic muscle strips, while the -dP/dtmax - P relationship was not significantly different than in controls. At low-to-medium loads, SLs at the onset of tension decline were greater in myopathy. These data indicate that relaxation of the diaphragm was both slowed and prolonged in myopathic Syrian hamsters.

Diminished beta-adrenergic receptor responsiveness and cardiac dilation in hearts of myopathic Syrian hamsters (BIO 53.58) are associated with a functional abnormality of the G stimulatory protein.

Previous studies have demonstrated a diminution in the bioactivity of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (Gs) in hearts of the hypertrophic BIO 14.6 Syrian hamster. In this study, we measured functional activity and immunodetectable levels of Gs in a mutant strain of hamsters (BIO 53.58) that develop a dilated cardiomyopathy. Pathological studies demonstrated that 100-day-old BIO 53.58 hamsters had substantial ventricular dilation when compared with age-matched F1B controls. Additionally, these 100-day-old hamsters demonstrated diminished contractile response to beta-adrenergic receptor stimulation. The pathological and hemodynamic changes were associated with defective coupling of Gs to adenylyl cyclase as adenylyl cyclase activation was distinctly decreased in the presence of isoproterenol, fluoride ion, guanine nucleotides, and forskolin. Additionally, the ability of the alpha-subunit of Gs to reconstitute isoproterenol-stimulated adenylyl cyclase activity in S49 cyc- membranes was reduced approximately 65%. By contrast, cyc- complementation assays did not reveal a difference between the functional activity of Gs in hearts from 30-day-old BIO 53.58 hamsters and F1B controls. Furthermore, beta-adrenergic receptor stimulation of adenylyl cyclase in the membranes of the young BIO 53.58 hamsters was not significantly different from controls. The substantial alterations in Gs bioactivity in hearts of the 100-day-old BIO 53.58 hamsters was not associated with alterations in the immunodetectable levels of either alpha Gs or alpha Gi on Western Blots. These results suggest that G protein changes are associated with ventricular dilation in BIO 53.58 hamsters and that G protein levels are not always reflective of G protein bioactivity.

Alterations in the creatine kinase system in the myocardium of cardiomyopathic hamsters

Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.

Control of norepinephrine turnover in brown adipose tissue of Syrian hamsters.

Sympathetic nervous system activity in brown adipose tissue (BAT) of normal and myopathic Syrian hamsters was assessed by measuring norepinephrine turnover rate (NETR) using [3H]norepinephrine. Acute exposure of normal hamsters to cold (4 degrees C) for 4 or 24 h increased norepinephrine secretion but not resynthesis. By 3 days and at 2 wk in the cold, NETR increased but returned to a normal level by 6 wk. Hamsters were initially hypothermic (to 3 days) then normothermic (2 and 6 wk). Adaptation of normal or myopathic hamsters to high-fat diet or short photoperiod (4 h light, 20 h dark) for up to 12 wk did not alter NETR in BAT. Serum triiodothyronine (T3) concentration increased rapidly in the cold to reach a maximum level by 24 h at which it remained for 6 wk and was not correlated with changes in NETR in BAT. The high-fat diet did not alter T3 level in normal hamsters; it increased the low T3 level in myopathic hamsters. Short photoperiod induced a transient increase in T3 level in normal hamsters but not in myopathic hamsters. We conclude that the hypertrophied state of BAT in Syrian hamsters adapted to cold, to high-fat diet, or to short photoperiod is not maintained by elevated norepinephrine secretion and that some other factor(s) must be involved. A selective increase in concentration of BAT mitochondrial uncoupling protein in hamsters may be dependent on raised T3 level in serum and/or production in BAT, since both occur in response to cold acclimation but not in response to high-fat diet or short photoperiod.

Construction of cosmid genomic libraries for the normal and myopathic Syrian hamsters.

Cosmid genomic libraries from both normal and myopathic Syrian hamsters have been constructed. MboI was used to generate 35- to 50-kilobase DNA fragments which were isolated from a 5-25% NaCl gradient. The 35- to 50-kilobase DNA fragments were ligated to the cosmid vector pCV108 and packaged into Escherichia coli DK1. Approximately 3 X 10(5) - 4 X 10(5) clones were obtained per microgram of ligated DNA. Thirteen clones have been isolated from 2 X 10(5) colonies using a cardiac myosin heavy chain clone as a probe. Restriction maps of two of these clones are presented here.

Microvascular reactivity of the myopathic Syrian hamster cremaster muscle

The myopathic Syrian hamster is a genetic model of congestive heart failure that exhibits focal myocytolytic necrosis in both heart and skeletal muscle. Previous investigations of microvascular morphology in heart and skeletal muscle have shown severe arteriolar constrictions without fixed anatomical vessel lesions. This study tested the hypothesis that these constrictions indicate a hyperreactivity of the myopathic microvasculature in vivo and that the reactivity corresponds to the developmental course of myocyte pathology. The microcirculation of the cremaster muscle was studied in eight myopathic and six control hamsters in the active stage of necrosis (39-81 days of age) and five myopathic and six control hamsters in the later stage of muscle healing (150-213 days of age). The internal diameter of second order arterioles was measured during topically applied noradrenaline. The myopathic arterioles of the younger group constricted at significantly lower concentrations of noradrenaline (p less than 0.01) and constricted to 35-50% of their resting internal diameter over a narrower range of noradrenaline (p less than 0.01). This indicated both a reduced threshold to noradrenaline and an enhanced response to the agonist. Active myocytolytic necrosis was found in the contralateral cremaster muscle of each myopathic hamster. The older myopathic and control hamsters showed no significant differences in arteriolar responsiveness to applied noradrenaline and no active necrosis. These results indicate a relation between a hyperreactive microvasculature and active necrosis and a normal reacting microvasculature and diminished necrosis in the two phases of the disease. Thus a general correspondence between vascular responsiveness and myocyte pathology exists in this model of heart failure and muscular dystrophy.

Thyroxine 5'-deiodinase in brown adipose tissue of myopathic hamsters.

Myopathic Syrian hamsters (BIO 14.6) have less brown adipose tissue (BAT) than normal. The trophic response of this tissue to cold is smaller than normal and trophic responses to diet and to photoperiod are absent. The objective was to find out whether activity of thyroxine 5'-deiodinase in their BAT was increased normally in response to cold and thus whether a defect in endogenous production of 3,5,3'-triiodothyronine might underlie the attenuated trophic response. The effect of feeding a high-fat diet on activity of 5'-deiodinase was also studied. Cold acclimation increased thyroxine 5'-deiodinase activity in BAT of the myopathic hamster, but the total remained smaller than normal because of the smaller size. The cold-induced increase in concentration of mitochondrial uncoupling protein was also smaller than normal. The level of serum 3,5,3'-triiodothyronine was low in myopathic hamsters and remained lower than normal when they were cold-exposed or cold acclimated. Feeding the high-fat diet to myopathic hamsters resulted in a greater than normal suppression of thyroxine 5'-deiodinase activity than in normal hamsters; the normal increases in protein content and in concentration of mitochondrial uncoupling protein were absent. We conclude that the defective trophic response of BAT of the myopathic hamster is not secondary to defective regulation of its thyroxine 5'-deiodinase activity because this activity does not appear to be obligatorily linked to hypertrophy of BAT. The low level of serum 3,5,3'-triiodothyronine in the myopathic hamster may be secondary to reduced capacity for peripheral thyroxine deiodination in its BAT.

Cardiomyopathic and healthy acidotic hamster hearts: mitochondrial activity may regulate cardiac performance

A 50% decrease in adenine nucleotides and a 60% decrease in adenosine triphosphate concentration was found in glucose perfused myopathic Syrian hamster heart (240 days old) whereas there was an 18% decrease and 40% decrease respectively in acidotic healthy Syrian hamster heart re-equilibrated with a physiological medium. Correspondingly, there was a 60% decrease in cardiac performance with both models. Developed pressure fell when the phosphorylation potential decreased to less than or equal to 2; however, the heart recovered if mitochondrial activity was activated. If a substrate such as pyruvate or ribose was used with either model cardiac performance returned to near normal, although adenine nucleotide and adenosine triphosphate concentrations were further depressed. With glucose as substrate cardiomyopathic hearts, healthy acidotic hearts, and healthy acidotic hearts re-equilibrated with glucose as substrate had low pyruvate concentrations; limited availability of pyruvate depressed mitochondrial activity. Like the myopathic hearts the re-equilibrated acidotic hearts had high myocardial pyruvate concentrations, above normal ratios of phosphocreatine to creatine, and near normal oxygen consumption, developed pressure, dP/dt, and cyclic adenosine monophosphate concentrations when re-equilibrated with a medium containing pyruvate or ribose as substrate, although adenosine triphosphate and adenine nucleotide concentrations were severely depressed. When adenosine triphosphate values fell from 24 to 2 mumol X g-1 dry weight in the pyruvate or ribose perfused and normal functioning heart the heart stopped beating with no progressive fall in performance before termination of the metabolic processes.

Multiple cardiac contractile protein abnormalities in myopathic syrian hamsters (BIO 53:58)

Hearts of genetically myopathic male hamsters (BIO 53 : 58) were studied at 1 month, 2 months, 3 months, 4 to 5 months and 7 months of age. The time course of alterations in the cardiac myofibrillar ATPase activity, the relationship of myofibrillar ATPase activity to free [Ca2+], myosin ATPase activity and the distribution of heavy chain myosin isoenzymes were evaluated. Mg2+-Ca2+ ATPase activity of cardiac myofibrils in myopathics was increased in 4 month and 7 month-old hamsters. Elevated Mg2+ ATPase activity was found as early as in 2-month-old hamster. However, there was no loss in the regulation of the myopathic myofibrillar assembly as measured by the PCa response (10(-7) M to 10(-4) M Ca2+). Scans of SDS electrophoresis slab gels of cardiac myofibrillar proteins from control (C) and myopathic animals (M) did not show any differences at any age group (1, 4 and 7 months). There was a significant decrease in myosin Ca2+ ATPase activity and actin activated Mg2+-ATPase activity at 4 to 5 months and 7 months of age in the myopathic hearts. At all ages in normal and myopathic animals cardiac myosin consisted of three isoenzymes, V1, V2 and V3. At all ages in controls and at 1 to 3 months in myopathics, V1 predominated and the isoenzyme distribution was V1 greater than V2 greater than V3. However, in myopathics at 4 to 5 months, the distribution was V1 = V3 greater than V2 and at 7 months was V3 greater than V2 greater than V1. Our experiments suggest alterations in different components of the contractile protein system that occur at different stages of myopathy.

Sarcolemmal alterations during the development of genetically determined cardiomyopathy.

The purpose of this study was to identify alterations in specific enzyme and Ca2+ binding activities in cardiac sarcolemmal fractions from UM-X7.1 myopathic Syrian hamsters during the development of cardiomyopathy. Experimental and healthy control animals were examined from 25 to 200 days of age. Sarcolemmal Na+, K+-ATPase activity was depressed in the myopathic hamsters throughout the time course of this study. Sarcolemmal ATP-independent Ca2+ binding was found to be depressed in experimental animals as early as 55 days of age. Ca2+ -stimulated, Mg2+ -dependent ATPase activity was depressed in the experimental animals by 90 days of age and this decrease in enzyme activity was accompanied by a decrease in ATP-dependent Ca2+ binding capacity of the sarcolemmal membranes. Mg2+ -ATPase and Ca2+ -ATPase activities were only affected in the latter stages of the disease (155 to 200 days old). NaF, epinephrine and Gpp(NH)p stimulation of the sarcolemmal adenylate cyclase activity was also observed to be attenuated during the latter stages of the disease. These defects in adenylate cyclase system of the sarcolemmal fraction appeared specific since basal adenylate cyclase activity was not altered at any age studied. The results demonstrate that the earliest lesions in sarcolemmal activity in myopathic hamster heart occur in Na+, K+-ATPase and ATP-independent Ca2+ binding capacity. These defects correspond temporally to the initial stages of cardiac necrotic development in this strain of myopathic hamster.

Numerical densities of intramembrane particles in the cardiac sarcolemma of normal and myopathic Syrian hamsters

The purpose of this study was to examine the density of intramembrane particles in the cardiac sarcolemma of normal and myopathic Syrian hamsters in a search for a morphological marker of a putative membrane defect. Hereditary cardiomyopathy in hamsters is manifested by progressive multifocal skeletal and cardiac muscle necrosis. Although pharmacological and biochemical data suggest a molecular defect in the cardiac sarcolemma, there has been no morphological observation that would substantiate this suggestion. We quantified numerical densities of intramembrane particles in freeze-fractured sarcolemmal membrane protoplasmic and extracellular faces from the left and right ventricular myocardium of female 25-day-old hamsters of the U.M.-X 7.1 cardiomyopathic line compared with sex- and age-matched randomly bred Syrian hamsters. Intramembrane particle numerical densities significantly increased above control values in cardiomyopathic hamsters for protoplasmic face (from 2188 ± 61 to 2454 ± 89 μm −2 in left ventricle ( P < 0.025) and from 2255 ± 83 to 2574 ± 56 μm −2 in right ventricle ( P < 0.001)). There was no significant difference between intramembrane particle densities of normal and cardiomyopathic hamsters for extracellular face (707 ± 45 and 687 ± 49 μm −2 in the left ventricle and 742 ± 41 and 746 ± 28 μm −2 in the right ventricle). These results implicate an increase of protoplasmic face-associated integral proteins in the sarcolemmal membrane of cardiomyopathic hamster. The significance of this finding is not known. It may represent an adaptive change related to a molecular defect in the sarcolemmal membrane of cardiomyopathic hamster.

Diminished effects of l-thyroxine on some functions and enzymes of the heart in myopathic hamsters

The effects of chronic administration of l-thyroxine on some functions of the heart in normal and myopathic Syrian hamsters (strain BIO 14.6) were investigated. In contrast to normal controls, in myopathic hamsters administration of low doses of l-thyroxine for 4 weeks failed to induce tachycardia, increase the total activity of NaK-ATPase (EC 3.6.1.3) within the cells or improve contractility of the heart muscle. In addition, l-thyroxine treatment decreased the activity of adenyl cyclase in the myopathic and that of soluble protein kinases in both normal and myopathic hearts. Some mechanism(s) that mediate the action of l-thyroxine in the myocardium therefore may be altered or deficient in this form of hereditary cardiomyopathy of the hamster.

Depressed myosin ATPase activity in hearts of myopathic hamsters: Dissociation from neutral protease activity

Myosin was isolated from the hearts of 210-day-old genetically myopathic Syrian hamsters (Bio 53:58) and its properties compared to the myosin from random bred controls of the same age. The Ca 2+ ATPase was depressed in the myosin from the hearts of myopathic animals. The K +-EDTA ATPase and the total-SH content did not differ significantly in the two groups. Myosin from myopathic animals showed a loss of LC 2. Analyses of myofibrils and actomyosin revealed that the LC 2 was present in these preparations from the myopathic animals and the loss of LC 2 was taking place during the purification of myosin. Despite the presence of LC 2 in the myofibrils and actomyosin, the corresponding ATPase activities were depressed in the myopathic hearts. The loss of LC 2 in myopathic cardiac myosin was found to be due to high levels of neutral protease activity co-purifying with myosin. The protease fraction from myopathic hearts degraded the cardiac LC 2 of control hamster and dog cardiac myosin, however, the Ca 2+ ATPase remained unchanged.

Post-natal development of the normal and cardiomyopathic Syrian hamster heart: A quantitative electron microscopic study

Quantitative ultrastructural analysis has been performed on 1-, 16- and 30-day-old normal (RB) and myopathic (14.6) Syrian hamster hearts. Normal developmental patterns for mitochondrial, myofibrillar, nuclear and sarcoplasmic reticular volume fractions have been determined and compared with structural development observed in the left ventricle of 14.6 hamsters. Myofibrillar development appears to be altered in the myopathic heart. Developmental changes in the sarcolemma and T-tubule network have been examined. The development of the calcium-handling membranes, the sarcoplasmic reticulum and mitochondria, has been studied in both strains and divergent patterns were noted. This study provides the first quantitative description of ultrastructural alterations in the early post-natal development of the cardiomyopathic Syrian hamster heart.

Myocardial contractility in relation to hypertrophy and failure in myopathic Syrian hamsters

Myocardial contractility of left ventricular papillary muscles from normal Syrian hamsters and those with hereditary cardiomyopathy has been studied. The animals were divided into five groups corresponding to different pathological stages and the mechanical parameters correlated with these stages. Developed force and dF dt corrected for cross-sectional area were depressed in all myopathic groups. However, developed force and dF dt , uncorrected for cross-sectional area, were restored to normal in the hypertrophied muscles in the myopathic animals at 220 and 320 days of age in the absence of congestive failure (i.e. without edema). In the early myolytic and healing stage and in the terminal edematous stages of the myopathy, force-velocity curves were depressed at all loads. However in hypertrophied and compensated hearts at 220 to 320 days, the force velocity curves were similar to controls when load was expressed as a fraction of P 0. Assuming developed force to reflect the number of contractile sites in parallel, it was concluded that the number of normally active contractile sites per unit area was reduced at all stages of the disease. In the hypertrophied, compensated phase, it was postulated that the qualitative nature of these sites remain normal despite their quantitative reduction.

Calcium binding by cardiac relaxing system isolated from myopathic syrian hamsters (strains 14.6, 82.62 and 40.54)

Cardiac relaxing system (CRS) was isolated from control (BIO-RB) and cardiomyopathic (BIO 14.6, BIO 82.62 and BIO 40.54) hamsters. Both dual-beam spectrophotometric and Millipore filter methods were employed to study calcium binding by CRS. The four groups (control and cardiomyopathic hamsters) were killed at three ages: Age I = 60 ± 9 days, Age II = 216 ± 7 days, and Age III = 262 ± 12 days. The latter two ages were in congestive heart failure. CRS isolated from Age I did not demonstrate diminished capacity or rate of calcium binding, whereas both Ages II and III revealed a decrease in rate and maximal capacity of calcium binding compared to controls. It is suggested that an aberration of intracellular calcium metabolism is characteristic of congestive heart failure associated with the cardiomyopathic process.