Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction.

Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.

Down regulation of immuno-detectable cardiac connexin-43 in BALB/c mice following acute fasting.

Acute starvation effects for connexin-43 protein expression, in the heart, had not been previously explored. Hence we examined acute fasting on the myocardial immuno-histochemical expression of connexin-43 in 3 groups of 8-week old female BALB/c mice. Groups consisted of control mice (n=5), fasting for 24 h (N=5) and 48 h (N=3). Under light microscopy all control fed cases revealed the presence of some immuno-detectable staining for connexin-43 that is either present or weakly observed in some or all of the regions of interest, that include the cross-sectional left ventricular sub-endocardium, mid-myocardium and papillary muscle. Whereas mice that underwent 24 or 48 h of acute starvation, connexin-43 expression was either difficult to detect visually (N=3) or was completely absent (N=5) at 40x magnification using a light microscope. In starved mice with no membrane staining for connexin-43 we observed an increase in the intracellular accumulation of cytoplasmic connexin-43 expression.

Usefulness of cardiac calcification on two-dimensional echocardiography for distinguishing ischaemic from nonischaemic dilated cardiomyopathy: a preliminary report.

BACKGROUND: Aortic valve calcification (AVC) and/or mitral annulus calcification (MAC) is considered to be a marker of atherosclerosis and has been demonstrated to predict cardiovascular morbidity and mortality. AIM: We hypothesized that the presence of cardiac calcification by echocardiography can be used in the differential diagnosis between ischaemic (DCMI+) and nonischaemic dilated cardiomyopathy (DCMI-). METHODS: We evaluated 62 patients with DCM (38 males, mean age 66 +/- 10 years, LVEF < 40%), without any prior history of myocardial infarction or coronary intervention, who were undergoing coronary angiography for aetiological diagnosis. DCMI+ was considered present when a > or = 70% stenosis of at least one coronary artery was found. AVC, MAC, aortic wall and papillary muscle calcifications were semiquantitatively assessed by two-dimensional echocardiographic examination with a calcium score ranging from 0 (no calcifications) to 8 (calcium in all four sites). RESULTS: DCMI+ was found in 20 out of 62 patients. As expected, there were no differences in LVEF and LV end-diastolic diameters between DCMI+ and DCMI--patients (29 +/- 8% versus 31 +/- 10% and 66 +/- 6 versus 68 +/- 8 mm, respectively; not significant). Regional wall motion abnormalities and conventional risk factors for atherosclerosis, such as hypertension and hypercholesterolaemia, were significantly more frequent in the DCMI+ compared to the DCMI- group. On the other hand, the calcium echo score was 4.6 +/- 2 (range 1.7-7.3) in DCMI+ patients and 0.8 +/- 0.95 (range 0-4) in DCMI--patients (P < 0.05). A calcium score > or = 3 was observed in 18 out of 20 (90%) DCMI+ patients and only in three of 42 (8%) DCMI--patients. CONCLUSIONS: The assessment of cardiac calcification by two-dimensional echocardiography could represent a simple, noninvasive and inexpensive approach to assess the aetiology (ischaemic versus nonischaemic) of dilated cardiomyopathy.

Dynamics of crossbridge-mediated activation in the heart.

Both intracellular calcium and strongly bound crossbridges contribute to thin filament activation in the heart, but the magnitude and the duration of the effects due to crossbridges are not well characterized. In this study, crossbridge attachment was altered in tetanized ferret papillary muscles and changes in the rate constant for the recovery of force (k (TR)) and unloaded shortening velocity (V (U)) were measured to track thin filament activation. k (TR) decreased as the time the muscles spent at low levels of crossbridge attachment (shortening deactivation) increased (0.02 s=17.9+/-2.3 s(-1), 0.32 s=3.3+/-0.4 s(-1); half-time=0.052 s; P<0.05). Furthermore, the deactivation was reversible and k (TR) recovered when muscles were allowed to regenerate force isometrically during the same tetanus. V (U) also decreased when the preceding load was lower (isometric load, V (U)=1.93+/-0.26 muscle lengths/s (ML/s); zero load, V (U)=0.93+/-0.14 ML/s, P<0.05) and as the length of time the muscle spent unloaded increased (>60% decline after 0.3 s). In addition, V (U) recovered when the muscle was allowed to regenerate force isometrically. These results indicate that crossbridge attachment increases thin filament activation as reflected in measurements of V (U) and k (TR). This 'extra' activation by crossbridges appears to be a dynamic process that decays during unloaded shortening and redevelops during isometric contraction.

Not just a plasma membrane protein: in cardiac muscle cells alpha-II spectrin also shows a close association with myofibrils.

Spectrin and its associated proteins are essential for the integrity of muscle cells and there is increasing evidence for their involvement in signalling pathways as well as having a structural function in mediating stress. Spectrin is a multigene family and it is essential to determine which isoforms are present and their location in the cell. In heart muscle, we have found that one spectrin isoform, alphaII-spectrin, is strongly represented and, using immunofluorescence, we show that it lies within the contractile fibres near the Z-disc as well as on the cardiomyocyte plasma membrane. Electron microscopy of immunogold-labelled cryosections reveals statistically significant clustering of gold particles near the Z-disc, within and close to the edge of myofibrils. betaII-spectrin and ankyrin-R and G are both known to occupy this region. We suggest that alphaIIbetaII spectrin tetramers with ankyrin organise and/or stabilise cardiac muscle cell membrane components relative to the contractile apparatus.

In situ measurements of crossbridge dynamics and lattice spacing in rat hearts by x-ray diffraction: sensitivity to regional ischemia.

BACKGROUND: Synchrotron radiation has been used to analyze crossbridge dynamics in isolated papillary muscle and excised perfused hearts with the use of x-ray diffraction techniques. We showed that these techniques can detect regional changes in rat left ventricle contractility and myosin lattice spacing in in situ ejecting hearts in real time. Furthermore, we examined the sensitivity of these indexes to regional ischemia. METHODS AND RESULTS: The left ventricular free wall of spontaneously beating rat hearts (heart rate, 290 to 404 bpm) was directly exposed to brief high-flux, low-emittance x-ray beams provided at SPring-8. Myosin mass transfer to actin filaments was determined as the decrease in reflection intensity ratio (intensity of 1,0 plane over the 1,1 plane) between end-diastole and end-systole. The distance between 1,0 reflections was converted to a lattice spacing between myosin filaments. We found that mass transfer (mean, 1.71+/-0.09 SEM, n=13 hearts) preceded significant increases in lattice spacing (2 to 5 nm) during systole in nonischemic pericardium. Left coronary occlusion eliminated increases in lattice spacing and severely reduced mass transfer (P<0.01) in the ischemic region. CONCLUSIONS: Our results suggest that x-ray diffraction techniques permit real-time in situ analysis of regional crossbridge dynamics at molecular and fiber levels that might also facilitate investigations of ventricular output regulation by the Frank-Starling mechanism.

Shortening of cardiac action potentials in endotoxic shock in guinea pigs is caused by an increase in nitric oxide activity and activation of the adenosine triphosphate-sensitive potassium channel.

OBJECTIVE: To investigate the roles of nitric oxide and adenosine triphosphate (ATP)-sensitive potassium channels (KATP) in the shortening of cardiac action potential in endotoxic shock. DESIGN: Prospective animal study with concurrent controls. SETTING: University animal research laboratory. SUBJECTS: Adult Hartley guinea pigs, weighing 300-400 g. INTERVENTIONS: Guinea pigs were anesthetized and mechanically ventilated for 6 hrs. Lipopolysaccharide (LPS) or saline (sham group) were given intravenously. Drug effects were examined at the end of 6 hrs. MEASUREMENTS AND MAIN RESULTS: Plasma nitrate concentration was measured hourly, while guanosine 3',5'-cyclic monophosphate (cGMP) content and action potential duration at 90% of repolarization (APD90) of papillary muscle were examined every 2 hrs in the 6-hr endotoxemia in both the sham and the LPS-treated groups. The basal levels of these three variables showed no difference in the two groups. In the sham group, these variables did not change significantly (n = 14 for plasma nitrate determination; n = 5 for cGMP content measurement; n = 5-14 for APD90 measurement; all p > .05). But in the LPS-treated group, both plasma nitrate concentration and cGMP content of papillary muscle showed time-dependent increases and they were significantly higher than those in the sham group (at the 6th hr, plasma nitrate: 42.6 +/- 7.7 vs. 21.8 +/- 3.1 micromol/L, both n = 14, p < .01; cGMP: 1.52 +/- 0.15 vs. 0.73 +/- 0.08 pmol/mg protein, both n = 5, p < .01). In contrast, APD90 revealed a time-dependent decrease compared with that in the sham group (at the 6th hr, 137.1 +/- 52 vs. 188.2 +/- 4.8 msecs, both n = 14, p < .001). In the following 60-min in vitro recording of action potentials after the end of 6-hr endotoxemia, the shortened APD90 in the LPS-treated group did not recover and remained shorter compared with that in the sham group, in which the APD90 showed no significant changes (at the 60th min, 165.1 +/- 5.7 vs. 200.2 +/- 3.8 msecs, each n = 14, p < .01). However, in the presence of glibenclamide, a specific KATP blocker (100 micromol/L; n = 10), the APD90 could be reversed almost completely to the same value as that in the sham group (n = 14) (196.6 +/- 3.5 vs. 200.2 +/- 3.8 msecs; p > .05), despite glibenclamide having no effect on the APD90 in the sham group. In the LPS-treated group, NG-nitro-L-arginine methyl ester (1 mmol/L; n = 4), methylene blue (10 micromol/L; n = 5), and aminoguanidine (100 micromol/L; n = 4) significantly prolonged the shortened APD90 (192.5 +/- 3.1, 195.0 +/- 3.3, and 176.5 +/- 3.3 msecs, respectively; p < .01, p < .01, and p < .05, respectively, compared with that without these agents, 165.1 +/- 5.7 msecs, n = 14). These agents had negligible effects on the APD90 in the sham group (all p > .05). Furthermore, 8-bromoguanosine-3',5'-cyclic monophosphate (500 micromol/L; n = 5) decreased APD in intact papillary muscle (mean reduction of APD90, 13.5 +/- 3.5%, n = 5; p < .05), an effect abolished by pretreatment with glibenclamide (100 micromol/L; n = 5) that did not have an effect by itself. CONCLUSIONS: In this experimental model, we provide reasonably convincing evidence to suggest that in endotoxic shock, an increase in nitric oxide activity may activate KATP, which plays a major role in the shortening of APD, presumably through a cGMP-dependent pathway.

Cardiac excitation-contraction coupling in the portal hypertensive rat.

Basal contractility and responses to beta-adrenoceptor activation are compromised in hearts from rats with chronic portal vein stenosis. Here we report the effect of partial ligation of the portal vein on myocardial G protein expression, beta-adrenoceptor-G protein coupling, and excitation-contraction coupling (ECC). Contractility (dT/dt) was reduced 30-50% in right and left ventricles, but the rate of relaxation (-dT/dt) was unaffected. Isoproterenol-induced positive inotropism was diminished, but there was no difference in ED(50). The concentration-dependent increase in -dT/dt was unaffected. G(s)alpha and G(i)alpha expression, cholera toxin- and pertussis toxin-induced ADP-ribosylation, and formation of the agonist-receptor-G(s) complex were unaffected by portal vein stenosis. Of the components of ECC examined, the caffeine-sensitive sarcoplasmic reticulum Ca(2+) pool was reduced 35%, although the Ca(2+) uptake and release processes were unchanged; the apparent density of L-type Ca(2+) channels decreased 60% with no change in affinity; the dihydropyridine Ca(2+) channel agonist BAY K 8644 produced relative changes in dT/dt that were similar in both groups, suggesting normal function in the remaining Ca(2+) channels; and Na(+)/Ca(2+) exchange was reduced 50% in the portal vein stenosis group. These data suggest that the effect of portal vein stenosis on the myocardium is the result of alterations to ECC.

An improved method for exchanging troponin subunits in detergent skinned rat cardiac fiber bundles.

We describe a method for the removal of endogenous troponin (Tn) complex from bundles of detergent-treated cardiac fibers. After 70 min treatment with cTnT-cTnI most of the endogenous Tn complex was removed from fiber bundles. Complete reconstitution of the Tn complex was achieved by reconstituting with cardiac troponin C (cTnC) in fully relaxing conditions. Ca(2+)-dependent maximum force of the fibers treated with cTnT-cTnI or cTnT-cTnI(33-211), which was used to aid in the visualization of the troponin exchange, decreased to 85-90% of the force developed by fibers before the treatment. SDS-PAGE analysis of the cTnT-cTnI(33-211) and the cTnT(77-289)-cTnI(33-211) treated fiber bundles demonstrated that 70-80% of the endogenous Tn subunits were removed. After reconstitution with cTnC, approximately 80-85% of the Ca(2+)-regulated force was restored in cTnT-cTnI/cTnI(33-211) treated fibers. Our results demonstrate that by minimizing the prolonged exposure of skinned cardiac fiber bundles to rigor conditions, successful exchange of all three subunits of the Tn complex can be accomplished with minimal loss of function.

Cell death in acromegalic cardiomyopathy.

BACKGROUND: Prolonged untreated acromegaly leads to a nonspecific myopathy characterized by ventricular dysfunction and failure. However, the mechanisms responsible for the alterations of cardiac pump function remain to be defined. Because cell death is implicated in most cardiac disease processes, the possibility has been raised that myocyte apoptosis may occur in the acromegalic heart, contributing to the deterioration of ventricular hemodynamics. METHODS AND RESULTS: Ten acromegalic patients with diastolic dysfunction and 4 also with systolic dysfunction were subjected to electrocardiography, Holter monitoring, 2-dimensional echocardiography, cardiac catheterization, and biventricular and coronary angiography before surgical removal of a growth hormone-secreting pituitary adenoma. Endomyocardial biopsies were obtained and analyzed quantitatively in terms of tissue scarring and myocyte and nonmyocyte apoptosis. Myocardial samples from papillary muscles of patients who underwent valve replacement for mitral stenosis were used for comparison. The presence of apoptosis in myocytes and interstitial cells was determined by confocal microscopy with the use of 2 histochemical methods, consisting of terminal deoxynucleotidyl transferase (TdT) assay and Taq probe in situ ligation. Acromegaly was characterized by a 495-fold and 305-fold increase in apoptosis of myocytes and nonmyocytes, respectively. The magnitude of myocyte apoptosis correlated with the extent of impairment in ejection fraction and the duration of the disease. A similar correlation was found with the magnitude of collagen accumulation, indicative of previous myocyte necrosis. Myocyte death was independent from the hormonal levels of growth hormone and insulin-like growth factor-1. Apoptosis of interstitial cells did not correlate with ejection fraction. CONCLUSIONS: Myocyte cell death, apoptotic and necrotic in nature, may be critical for the development of ventricular dysfunction and its progression to cardiac failure with acromegaly.

Peroxynitrite irreversibly decreases diastolic and systolic function in cardiac muscle.

Much of the damaging action of nitric oxide in heart may be due to its diffusion-limited reaction with superoxide to form peroxynitrite. Direct infusion of peroxynitrite into isolated perfused hearts fails to model the effects of in situ formation because the bulk of peroxynitrite decomposes before reaching the myocytes. To examine the direct effects of peroxynitrite on the contractile apparatus of the heart, we exposed intact and skinned rat papillary muscles to a steady state concentration of 4-microM peroxynitrite for 5 min, followed by a 30-min recovery period to monitor irreversible effects. In intact muscles developed force fell immediately to 26% of initial force, recovering to 43% by 30 min. Resting tension increased by 600% immediately, and was still elevated 500% by 30 min. Nitrotyrosine immunochemistry showed that peroxynitrite can induce tyrosine nitration at low concentrations and is capable of penetrating 200-380 microm into the papillary muscle after a 5-min infusion. Decomposed peroxynitrite had no effect on either intact or skinned muscle developed force or resting tension. Our results show that peroxynitrite directly damages both developed force and resting tension of isolated heart muscle, which can be extrapolated to systolic and diastolic injury in intact hearts.

Spatial relationship of the C-terminal domains of dystrophin and beta-dystroglycan in cardiac muscle support a direct molecular interaction at the plasma membrane interface.

Dystrophin and beta-dystroglycan are components of a complex of at least nine proteins (the dystrophin-glycoprotein complex) that physically link the membrane cytoskeleton in skeletal and cardiac muscle, through the plasma membrane, to the extracellular matrix. Mutations in the dystrophin gene, which result in an absence or a quantitative or qualitative alteration of dystrophin, cause a subset of familial dilated cardiomyopathies as well as Duchenne and Becker muscular dystrophy. Biochemical studies on isolated skeletal muscle molecules indicate that dystrophin is bound to the glycoprotein complex via beta-dystroglycan, with the C-terminus of beta-dystroglycan binding to the cysteine-rich domain and first half of the C-terminal domain of dystrophin. Ultrastructural labeling has demonstrated a close spatial relationship between dystrophin and beta-dystroglycan in intact skeletal muscle, but no previous ultrastructural labeling studies have examined the dystrophin/beta-dystroglycan interaction in cardiac muscle. In the present study, we have applied complementary immunoconfocal microscopy and double immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution visualization of labeled membrane components in thin section and in platinum-carbon replicas, to investigate the spatial relationship between dystrophin and beta-dystroglycan in rat cardiac muscle. When immunogold probes of two different sizes for the two proteins were used, "doublets" representing side-by-side antibody labeling were demonstrated in en face views at the level of the plasma membrane. The results support the conclusions that dystrophin and beta-dystroglycan directly interact at the cytoplasmic face of the rat cardiac muscle plasma membrane.

Contractile properties of thin (actin) filament-reconstituted muscle fibers.

Selective removal and reconstitution of the components of muscle fibers (fibrils) is a useful means of examining the molecular mechanism underlying the formation of the contractile apparatus. In addition, this approach is powerful for examining the structure-function relationship of a specific component of the contractile system. In previous studies, we have achieved the partial structural and functional reconstitution of thin filaments in the skeletal contractile apparatus and full reconstitution in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with plasma gelsolin, an actin filament-severing protein. Under these conditions, no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted by polymerization on the short actin fragments remaining at the Z line, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached as high as 30% of the original level in skeletal muscle, while it reached 140% in cardiac muscle. The augmentation of tension in cardiac muscle is mainly attributable to the elongation of reconstituted filaments, longer than the average length of thin filaments in an intact muscle. These results indicate that a muscle contractile apparatus with a high order structure and function can be constructed by the self-assembly of constituent proteins. Recently, we applied this reconstitution system to the study of the mechanism of spontaneous oscillatory contraction (SPOC) in thin (actin) filament-reconstituted cardiac muscle fibers. As a result, we found that SPOC occurs even in regulatory protein-free actin filament-reconstituted fibers (Fujita & Ishiwata, manuscript submitted), although the SPOC conditions were slightly different from the standard SPOC conditions. This result strongly suggests that spontaneous oscillation is intrinsic to actomyosin motors. We here summarize the contractile properties of the reconstitution system.

Cyclic AMP-receptor responses to hypergravity.

BACKGROUND: Altered gravity (G) encountered during spaceflight causes physiologic changes in humans and in experimental animals. In addition to weightlessness (0G) in space, sharply increased G forces are exerted on the spacecraft during the lift-off and reentry phases. Previous studies showed major changes in cAMP-associated activity of rat heart muscle after spaceflight, indicating that (hormone) signaling pathways may have been affected. HYPOTHESIS: The present study was designed to test the hypothesis that cAMP-related cellular responses of exocrine glands after simulated hypergravity (centrifugation at 1.7G) differ from the effects of 0G. METHODS: A portion of the parotid and lachrymal gland tissue was fixed for morphologic and immunocytochemical study, and another was used for biochemical determinations. A short-term tissue culture was established from each gland to determine the effects of stimulation by norepinephrine. Heart muscle (ventricle) was also studied. Soluble and particulate fraction extracts of tissue homogenates were prepared, photoaffinity labeled with the [32P]8-N3-analog of cAMP, proteins separated by electrophoresis and the cAMP-reactive proteins (cARP) identified by autoradiography. RESULTS: Differences were seen in protein banding patterns of the gland extracts and in altered cARP distribution in the 1.7G samples of heart ventricle and exocrine gland tissues, when compared with 1G controls. In the heart, cARP increased in the soluble fraction, while the particulate fraction extract showed no change. In acinar cells of the parotid, labeled cARP had accumulated, but decreased after stimulation to the level of the 1G controls. Immunogold labeling showed an increased content of amylase in the secretory granules of the 1.7G animals, while morphologic observation revealed few changes in the structure of parotid acinar cells. The response in the lachrymal gland was translocation of an isoform of cARP from the particulate to the cytoplasmic compartment. CONCLUSIONS: Changes distinct from those due to 0G, but specific for hyper-G were found in cARP activity, protein synthesis, as well as in an apparent inhibition of regulated secretion.

Progression of left ventricular hypertrophy does not change the sarcoplasmic reticulum calcium store in the spontaneously hypertensive rat heart.

The spontaneously hypertensive rat (SHR) is characterized by elevated blood pressure and the development of left ventricular hypertrophy. During compensatory hypertrophy in the SHR, (26 weeks) when baseline contractile function is normal or increased, the inotropic response to beta-adrenergic stimulation is impaired. We recently showed by electron probe microanalysis (EPMA) that the amount of Ca2+ stored in the sarcoplasmic reticulum (SR) following sympathetic stimulation is not decreased in the 26-week-old SHR heart. However, with disease progression, cardiac function declines further in the SHR and the response to beta-adrenergic stimulation is more impaired. To determine whether a decreased availability of SR Ca2+ is responsible for the severely depressed inotropic response in the older SHR, we used EPMA to measure directly the amount of Ca2+ stored in the SR following activation of the beta-adrenergic pathway in papillary muscles from 76-week-old SHR and Wistar-Kyoto (WKY) controls. In order to determine if there are other alterations in ion homeostasis, we also compared elemental content of A-band and mitochondria. Papillary muscles from 76-week-old SHR and WKY were stimulated by 10 microM isoproterenol and then rapidly frozen during relaxation. The elemental content of the junctional SR. A-band and mitochondria was measured by EPMA. We observed no significant difference in SR Ca2+ content between SHR and WKY. There was also no strain-dependent difference in mitochondrial or A-band Ca2+. Overall, these results indicate that the impaired response to beta-adrenergic stimulation in the SHR at 76 weeks is not due to altered availability of SR Ca2+.

Cardiac sarcoplasmic reticular function in rats with chronic heart failure following myocardial infarction.

Sarcoplasmic reticular function of rats with chronic heart failure (CHF) following coronary artery ligation was examined. The coronary artery ligation produced 43% infarction of the left ventricle and increased left ventricular end-diastolic pressure 8 weeks after the operation, suggesting the development of CHF by this period. The developed force transients of the skinned fiber of coronary artery-ligated rats were decreased when the skinned fiber was preloaded for 0.25-0.5 min with 10(-5)M Ca2+ (53-70%) and when preloaded with 10(-6)M Ca2+ and then exposed to 0.1-1 mM caffeine (39-87%). The results suggest that the rate of Ca2+ uptake by the sarcoplasmic reticulum (SR) and its ability to release Ca2+ were reduced in the failing heart. [3H]Ryanodine binding activities in homogenates and SR-enriched fractions were significantly reduced in the coronary artery-ligated group (32% and 21%, respectively). The results suggest that the amount of Ca2+ released from SR decreased due to decreased Ca2+ uptake rate of SR and down-regulation of the SR Ca(2+)-release channel, which contributes to cardiac dysfunction in failing hearts following acute myocardial infarction.

[A comparative analysis of the intracellular potassium content for the cardiomyocyte in papillary muscle tissue and in a primary heart culture]. / Sravnitel'nyi analiz vnutrikletochnogo soderzhaniia kaliia dlia kardiomiotsita v tkani papilliarnoi myshtsy i v pervichnoi kul'ture serdtsa.

This work was aimed to examine potassium contents in heart myocytes. The intracellular concentration of this element was measured with electron probe microanalysis (EPMA). The cardiomyocyte was studied in samples of the papillary muscle tissue and in heart primary culture. The low temperature fixation was applied to the sample preparation. As a result, cells of the primary heart culture were shown to be separated into three kinds of myocytes, defined by their different shape, size and cellular potassium content. The rod cardial cell, to be used for electrophysiological assays, has potassium and sodium concentrations close to those of the papillary muscle. The difference in potassium concentrations is to be explained presumably by the injury of cell membrane during the primary culture preparation. Healing over the membrane is likely to parallel with the membrane damage resulting from a continuous destruction of the cellular assembly in primary culture.

Dose-dependent alteration of rat cardiac sodium current by isoproterenol: results from direct measurements on multicellular preparations.

Conflicting results have been reported in literature about the influence of beta-adrenergic stimulation on the fast cardiac sodium current (INa+). To elucidate these mechanisms in multicellular preparations we used the loose-patch-clamp technique to evaluate the effect of the beta-adrenergic agonist isoproterenol 1-1000 nmol/l. Isoproterenol enhanced INa+ at all membrane potentials by elevation of the maximal available INa+ . Only at the high concentration of 1 micromol/l was INa+ slightly depressed after depolarizing conditioning clamps. The most marked increase of the maximal available INa+ was 30+/-9% after application of 100 nmol/l isoproterenol. To learn about the mechanisms in view of sodium channel modulation we combined isoproterenol with the sodium channel blocker lidocaine (47 micromol/l). Under these circumstances the effects of both drugs were completely independent. This investigation shows clearly that low concentrations of isoproterenol increase INa+ in multicellular preparations by a gating-independent mechanism.

Decreased inotropic response to beta-adrenergic stimulation and normal sarcoplasmic reticulum calcium stores in the spontaneously hypertensive rat heart.

Cardiac hypertrophy in the spontaneously hypertensive rat has been shown to be accompanied by a diminished inotropic response to beta-adrenergic stimulation. This diminished response has been attributed to abnormalities in various components of the beta-adrenergic signaling system. There is also evidence that regulation of intracellular Ca2+ cycling may be altered in the hypertrophied heart of the spontaneously hypertensive rat. We proposed that the diminished response to beta-adrenergic stimulation may reflect abnormalities in Ca2+ cycling, specifically alterations in the ability of the sarcoplasmic reticulum to effectively release and resequester Ca2+. We have used the unique combination of functional measurements on isolated, isometrically contracting papillary muscles from hearts of 26-week-old spontaneously hypertensive rats and their Wistar-Kyoto controls, together with electron probe microanalysis measurements of sarcoplasmic reticulum Ca2+ content in the same muscles after rapid freezing, to determine the availability of Ca2+ for activation of contraction, following beta-adrenergic stimulation. We observed a significant decrease in the inotropic response to beta-adrenergic stimulation in papillary muscles from the spontaneously hypertensive rats, as compared with Wistar-Kyoto controls, however in these same muscles, frozen during relaxation, there was no evidence of an accompanying decrease in the size of the sarcoplasmic reticulum Ca2+ store. In an additional group of muscles which were frozen during contraction, the amount of Ca2+ remaining in the sarcoplasmic reticulum after stimulated release was also not different in the two strains. These results indicate that the decreased inotropic response to beta-adrenergic stimulation in hypertrophied hearts of the spontaneously hypertensive rat is unlikely to be due to decreased availability of Ca2+ for activation of contraction. Additionally, to determine whether there is intracellular Ca2+ overload in the cardiac muscle cells of hearts of spontaneously hypertensive rats, we measured the amount of Ca2+ in mitochondrial and at the level of the myofilaments by electron probe microanalysis. These results indicate that intracellular Ca2+ overload does not accompany cardiac hypertrophy in the spontaneously hypertensive rat. This study therefore shows no correlation between altered intracellular Ca2+ cycling and the decreased inotropic response to isoproterenol in the spontaneously hypertensive rat at 26 weeks of age.

Troponin C-mediated calcium sensitization by levosimendan accelerates the proportional development of isometric tension.

The effects of various calcium sensitizers on myosin-actin crossbridge kinetics were evaluated in intact, paced guinea-pig papillary muscle by analysing the velocity of the development of isometric tension (dT/dt) in detail. The effect on association (the whole sequence of events from troponin onward) and dissociation rates of crossbridges was estimated from the rising phase and from the early decay phase of the normalized dT/dt curve. Levosimendan, a calcium sensitizer acting through troponin C, accelerated the proportional association rate and decelerated the dissociation rate of crossbridges. The effect of levosimendan on crossbridge kinetics occurred before the peak twitch tension was achieved. Thus, the compound did not change the actual relaxation phase of twitch tension. Since the effect on the association was more pronounced than on the dissociation of crossbridges, levosimendan shifted the entire twitch tension curve to the left. Based on the dissociation rate analysis levosimendan seems to act preferentially as a calcium sensitizer at low concentrations. At high concentrations the phosphodiesterase III (PDE III) inhibitory properties of levosimendan modulated its effect on the early relaxation processes. In contrast, PDE III inhibition is probably the primary mechanism of action for MCI-154. Pimobendan, and EMD 53998 at low concentrations, whereas their direct effects on crossbridge kinetics contributed to the positive inotropic action at high concentrations. The calcium sensitizing mechanisms of these compounds seemed to be based almost exclusively on the decelerating effect on dissociation of crossbridges.