Hamster Myocytes Research Articles

Contractile dysfunction of cardiomyopathic hamster myocytes is pronounced under high load conditions

To understand the pathophysiology of hereditary cardiomyopathy, the contractile function of cardiomyopathic hamsters has been studied at the cellular level. However, most of the studies to date have described the cell shortening under the unloaded condition. Using a novel force–length measurement system for single cardiomyocytes, we studied the contractile function of cardiomyopathic hamster myocytes over a wide range of loading conditions. Cardiomyocytes were isolated from the ventricles of eight- to 10-week-old cardiomyopathic (CMP) hamsters (Bio TO-2 strain), as well as control (CTRL) Syrian hamsters. A pair of carbon fibers was attached to both ends of single cardiomyocytes and their contractile characteristics were recorded while changing the after-load by controlling the fiber motion. Under the unloaded condition, the shortening fraction (CMP 9.2 ± 0.5% vs. CTRL 10.7 ± 0.8%, P = 0.06) and maximum shortening velocity (CMP 98.2 ± 7.3 μm/s vs. CTRL 147.2 ± 6.5 μm/s, P < 0.05) were decreased in CMP hamster myocytes. The peak force under the isometric condition (CMP 35.8 ± 2.2 mN/mm 2 vs. CTRL 69.0 ± 8.4 mN/mm 2, P < 0.05) and external work (CMP 898 ± 130 J/m 3 vs. CTRL 3058 ± 576 J/m 3, P < 0.05) under physiologically loaded conditions were also decreased, but the differences were more pronounced under the loaded conditions. Calcium transients measured by Indo-1 revealed elevated diastolic level, decreased peak level, and slower diastolic decay in CMP myocytes thus being consistent with the observed contractile dysfunction. These results clearly indicate the importance of the loading conditions in evaluating the contractile function of CMP hamster myocytes, and may provide insights into the mechanism of contractile dysfunction in this disease.

Role of voltage-sensitive release mechanism in depression of cardiac contraction in myopathic hamsters.

We investigated excitation-contraction (EC) coupling in isolated ventricular myocytes from prehypertrophic cardiomyopathic (CM) hamster hearts. Conventional and voltage-clamp recordings were made with high-resistance microelectrodes, and cell shortening was measured with a video-edge detector at 37 degrees C. Contractions were depressed in myocytes from CM hearts, whether they were initiated by action potentials or voltage-clamp steps. As in guinea pig and rat, contraction in hamster myocytes could be triggered by a voltage-sensitive release mechanism (VSRM) or Ca(2+)-induced Ca(2+) release (CICR). Selective activation of these mechanisms demonstrated that the defect in EC coupling was primarily caused by a defect in the VSRM. However, activation and inactivation properties of the VSRM were not altered. When the VSRM was inhibited, the remaining contractions induced by CICR exhibited identical bell-shaped contraction voltage relations in normal and CM myocytes. Inward Ca(2+) current was unchanged. Thus a defect in the VSRM component of EC coupling precedes the development of hypertrophy and failure in CM hamster heart.

Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes.

-The influence of intracellular administration of angiotensin II (Ang II) on the inward calcium current (ICa) was investigated in single myocytes isolated from adult rat ventricle. Comparative studies were also made in ventricular cells of Golden hamsters. The ICa was measured in single cells using the whole-cell voltage clamp configuration. The results indicated that Ang II (10(-8) mmol/L) dialyzed into the rat myocytes reduced the peak ICa by 35+/-5.5% (n=20; P<0.05). Losartan (10(-7) mmol/L) added to the bath did not suppress the effects of Ang II, indicating that the peptide is acting intracellularly. Moreover, the intracellular dialysis of losartan (10(-6) mmol/L) or [Sar1Val5Ala8] Ang II (10(-6) mmol/L) did not change the effect of Ang II. Stimulation of ICa by exogenous cAMP or inhibition of protein kinase C did not alter the effect of Ang II on ICa. Zaprinast (100 micromol/L), an inhibitor of cGMP phosphodiesterase, when added to the bath solution increased appreciably the effect of Ang II on ICa (P<0.05). In ventricular myocytes of Golden hamsters, in which Ang II has a positive inotropic action, the intracellular administration of Ang II (10(-8) mmol/L) increased ICa by 36+/-2.4% (n=20; P>0.05). The effect of the peptide was not altered by the intracellular administration of losartan (10(-6) mmol/L), by [Sar1Val5Ala8] Ang II (10(-6) mmol/L), or by the inhibitor of protein kinase A. The inhibition of protein kinase C, however, prevented the effect of Ang II ICa in the hamster myocytes. The results particularly suggest that the activation of the cardiac renin-angiotensin system regulates ICa and myocardial contractility, an effect that varies with the species.

Opioid Peptide Gene Expression in the Primary Hereditary Cardiomyopathy of the Syrian Hamster: III. AUTOCRINE STIMULATION OF PRODYNORPHIN GENE EXPRESSION BY DYNORPHIN B

Prodynorphin mRNA and dynorphin B expression have been previously shown to be greatly increased in cardiac myocytes of BIO 14.6 cardiomyopathic hamsters. Here we report that exogenous dynorphin B induced a dose-dependent increase in prodynorphin mRNA levels and stimulated prodynorphin gene transcription in normal hamster myocytes. Similar responses were elicited by the synthetic selective kappa opioid receptor agonist U-50,488H. These effects were counteracted by the kappa opioid receptor antagonist Mr-1452 and were not observed in the presence of chelerythrine or calphostin C, two specific protein kinase C (PKC) inhibitors. Treatment of cardiomyopathic cells with Mr-1452 significantly decreased both prodynorphin mRNA levels and prodynorphin gene transcription. In control myocytes, dynorphin B induced the translocation of PKC-alpha to the nucleus and increased nuclear PKC activity without affecting the expression of PKC-delta, -epsilon, or -zeta. Acute release of either U-50,488H or dyn B over single normal or cardiomyopathic cells transiently increased the cytosolic Ca2+ concentration. A sustained treatment with each opioid agonist increased the cytosolic Ca2+ level for a more prolonged period in cardiomyopathic than in control myocytes and led to a depletion of Ca2+ from the sarcoplasmic reticulum in both groups of cells. The possibility that prodynorphin gene expression may affect the function of the cardiomyopathic cell through an autocrine mechanism is discussed.

Early fetal like slow Na+ current in heart cells of cardiomyopathic hamster.

Using the whole-cell voltage-clamp technique, early embryonic tetrodotoxin (TTX) and Mn(2+)-insensitive slow Na+ current was detected in 10-22 week old fetal human heart cells as well as in 1 day old and young cardiomyopathic hamster myocytes. This slow Na+ current in both heart cell preparations has the same kinetics and pharmacology. This type of slow Na+ current was absent in heart cells of newborn and young normal hamsters and became less present in myocytes of 19 and 22 week old human heart myocytes. Our results demonstrate that the slow Na+ channel does exist in early fetal human life and this type of channel continues to be functional after birth in myocytes of the hereditary cardiomyopathic hamster.

Rat uterine myometrium contains the gap junction protein connexin45, which has a differing temporal expression pattern from connexin43

OBJECTIVE: Our purpose was to determine whether myometrial cell lines and rat myometrial tissue contained additional gap junction proteins besides connexin43. STUDY DESIGNS: Syrian hamster myocytes (SHM-ER) and human SK-UT-1 myometrial cell lines were analyzed for intercellular coupling by microinjection of Lucifer yellow. These cell lines and myometrial tissue isolated from pregnant rats were analyzed for connexin expression by ribonucleic acid blotting and immunofluorescence. RESULTS: SHM-ER and SK-UT-1 cells showed functional gap junctional coupling by intercellular passage of microinjected dye. Both cell lines contained connexin43 and connexin45 messenger ribonucleic acids but did not contain any other detectable connexin messenger ribonucleic acids. Immunofluorescence confirmed the presence of connexin43 and connexin45 proteins in these cells. Connexin43 and connexin45 messenger ribonucleic acids and immunoreactive proteins were detected in pregnant rat myometrium. Connexin43 messenger ribonucleic acid levels increased dramatically at term. In contrast, connexin45 messenger ribonucleic acid was present in nonpregnant myometrium, remained relatively constant early in gestation, fell just before term, and more than doubled post partum. CONCLUSIONS: Rat uterine myometrium contains connexin45 and connexin43. Coexpression of connexin45 with connexin43 in uterine myometrium may regulate gap junctional coupling between these cells. The different temporal expression patterns suggest that connexin45 and connexin43 may have different roles or that the ratio of these connexins may be important in the increased cellular coupling coincident with the onset of labor. (Am J Obstet Gynecol 1996;175:853-8.)

Species differences in the activity of the Na(+)‐Ca2+ exchanger in mammalian cardiac myocytes.

1. Species differences in the activity of the exchanger were evaluated in isolated myocytes from rat, guinea-pig, hamster ventricles and human atria. Fluorescence measurements using fura-2 were carried out in conjunction with the whole-cell patch-clamp technique for simultaneous recording of membrane currents and intracellular Ca2+ concentration. 2. Ca2+ release from sarcoplasmic reticulum (SR) induced either by rapid application of caffeine or by Ca2+ current elicited inward Na(+)-Ca2+ exchange currents (INa-Ca). The magnitude of INa-Ca was largest in hamster, smallest in rat, with guinea-pig and human myocytes having intermediate values. The ratio of caffeine-induced exchanger current densities, normalized with respect to the peak Ca2+ release, was 4:2:1.5:1 for hamster > guinea-pig > or = human > or = rat myocytes. 3. The rates of Ca2+ removal in the presence of caffeine, which reflect primarily the Ca2+ extruding activity of the Na(+)-Ca2+ exchanger, followed the same order of hamster > guinea-pig > or = human > or = rat. 4. The kinetics of INa-Ca vs. Ca2+ transients were different among species. In rat myocytes, the kinetics of the INa-Ca and the Ca2+ transients were similar, with INa-Ca linearly proportional to intracellular Ca2+ concentration ([Ca2+]i). In hamster myocytes, the time course of INa-Ca tracked only the declining phase of the Ca2+ transient with INa-Ca having faster kinetics during the Ca2+ release. These findings suggest that the Ca2+ concentrations in the vicinity of the exchanger were significantly higher than those of the cytosol during Ca2+ release in hamster myocytes. 5. We concluded that there are significant species differences in the exchanger activity of cardiac myocytes, arising from differences in exchanger densities, their modulation and/or their spatial distribution with respect to the ryanodine receptors of cardiac myocytes.

T-type Ca2+ channels are abnormal in genetically determined cardiomyopathic hamster hearts.

Although there is substantial evidence of abnormal Ca2+ homeostasis in heart cells of the cardiomyopathic Syrian hamster (Bio 14.6 strain), the mechanism by which these myocytes become Ca(2+)-overloaded is not known. To elucidate the role of voltage-sensitive Ca2+ channels in the pathogenesis of myopathy, whole-cell Ca2+ currents were measured in myopathic and normal control cardiac myocytes. These studies demonstrate the presence of two voltage-sensitive Ca2+ channel types in ventricular myocytes isolated from 200- to 300-day-old cardiomyopathic and age-matched normal hamsters. The two Ca2+ channel types were identified by their unitary conductance properties and pharmacologic sensitivities. Both L-type and T-type Ca2+ channels were present in cardiomyopathic and normal cells. Current density through L-type Ca2+ channels was the same in cardiomyopathic and normal control myocytes. However, the mean current density of T-type Ca2+ channels in cardiomyopathic cells was significantly higher than in normal cells (myopathic, 12.3 +/- 1.8 pA/pF; normal, 5.8 +/- 1.1 pA/pF; n = 8; P < .01). The T-type Ca2+ current in cardiomyopathic myocytes was activated and inactivated at more negative potentials than in cells from normal hamster hearts. These findings demonstrate no abnormality of the dihydropyridine-sensitive voltage-dependent L-type Ca2+ channel. In contrast, the observed abnormalities in T-type Ca2+ channel function in cardiomyopathic hamster myocytes suggest that this alteration may be related to the pathogenesis of Ca2+ overload and the arrhythmias in this genetically determined form of cardiomyopathy.

Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line.

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

The effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in mammalian hearts.

The uptake of choline in mammalian hearts in the presence of amino acids was examined. Isolated hamster, guinea pig, rat and rabbit hearts were perfused with labeled choline in the presence and absence of amino acids. Neutral amino acids enhanced choline uptake in the hamster heart, but not in the guinea pig, rat and rabbit hearts. Phosphatidylcholine biosynthesis in these hearts was not affected by the presence of amino acids. Choline uptake in the hamster myocytes was also enhanced by neutral amino acid. The enhancement of choline uptake suggests a direct interaction between the amino acid and the transport of choline into the myocardiac cells. The different responses in choline uptake to neutral amino acids indicate that the regulation of choline uptake in the hearts may be different between mammalian species.

Effect of U-50,488H on the contractile response of cardiomyopathic hamster ventricular myocytes

We examined the effects of a selective κ opioid receptor agonist (U-50,488H) on the contractile properties of single ventricular myocytes from 127 day old control (F1B) and cardiomyopathic (BIO 14.6) hamsters. Myocytes in bicarbonate buffered solution with 1.5 mM [Ca 2+] were electrically stimulated with field electrodes in the bath. Length changes were monitored via myocyte edge tracking. Twitch amplitude and the velocity of cell shortening were less in the cardiomyopathic hamster myocytes than in age-matched hamsters (P≤0.05). There was a concentration-dependent effect of U-50,488H (0.1–20 μM) to decrease twitch amplitude and shortening velocity in both control and cardiomyopathic myocytes (P≤0.001). In cells loaded with the Ca 2+ indicator indo-1 the negative inotropic action of U-50,488H was associated with a decreased indo-1 fluorescence transient amplitude. There was no difference in the negative inotropic effect of U-50,488H on control and cardiomyopathic cells. Thus, the CM hamster does not demonstrate a different contractile response to U-50,488H.

Immunofluorescent studies for α‐actinin in cultured cardiomyopathic hamster heart cells

Primary cultures of cardiac myocytes from normal and genetically cardiomyopathic (CM) newborn hamsters (strain UM X7.1) were analyzed by indirect immunofluorescent microscopy after 3, 5, 7, and 9 days in culture. The cultures were fixed in cold acetone and immunostained by an indirect method using FITC-labelled anti-alpha-actinin to label the myofibrillar Z bands. Most normal and CM myocytes appeared round in shape after 3 days in culture. Normal cardiac myocytes began to exhibit cytoplasmic projections after 5 days in culture and their myofibrils usually showed parallel arrangements with respect to each other. The cardiac cells from CM hearts showed an obvious myofibril disarray. Moreover, projections formed later than normal. As the size of the cells increased, more and more projections formed in normal hamster myocytes during development. By contrast, most of the cardiomyopathic myocytes showed few projections even as late as 9 days in culture. Hence, the number of projections per cell was much less in cardiomyopathic myocytes than in normal, especially after 7 and 9 days in culture. These results suggest that cardiomyopathic cells have abnormal shapes in culture and, in particular, fail to form projections as in normal cells. Whether this unusual behavior is related to an abnormality of the membranes or cytoskeletal system in cardiomyopathic heart cells or to some other factor requires further study.

Intracellular elemental content of cardiac and skeletal muscle of normal and dystrophic hamsters.

To test the hypothesis that the genetic lesion causing muscular dystrophy might be reflected in an abnormal intracellular elemental content, the elemental content of individual cardiac and skeletal muscle fibers in 50-day-old male control and cardiomyopathic BIO 53.58 hamsters was determined. The technique of electron probe x-ray microanalysis of freeze-dried tissue was employed. No electrolyte content differences were found between control and diseased animals for nuclei, myofibrillar cytoplasm, or mitochondrially-enriched cytoplasm of cardiac myocytes. Sulfur was elevated in dystrophic cardiac myocytes and was the only element significantly different in heart tissue of control and cardiomyopathic animals. Sulfur was also elevated in dystrophic soleus muscle fibers. The pattern of electrolyte content of these cells reflected a mixture of normal cells and damaged cells with altered electrolyte content. In this hamster model, alteration of electrolyte content of myocytes appears to be a result of the disease process and not an inherent characteristic of muscular dystrophy. The elevated sulfur in dystrophic hamster myocytes reflects a biochemical lesion which deserves further study.