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M F Wendt-Gallitelli

Publications and source records attributed to M F Wendt-Gallitelli.

18 recordsLinked to original sources
PubMed1992

Potentiation of contraction as related to changes in free and total intracellular calcium.

In voltage-clamped guinea-pig ventricular myocytes, we studied the potentiation of contraction in dependence on the concentration of intracellular calcium; ionized calcium [Ca2+]c was measured by Indo-1 microfluospectroscopy and total calcium (sigma Ca) by electronprobe microanalysis (EPMA). After a 15 min rest period, [Ca2+]c was approx. 90 nM and sigma Ca was below the detection limit (80 microM) in myoplasm (sigma Ca(myo)), junctional sarcoplasmic reticulum (sigma CaSR) and mitochondria (sigma Ca(Mito)). Post rest, repetitive clamp steps (1 Hz) potentiated extent and rate of shortening by 300%. In the literature, post-rest potentiation is attributed to the replenishment of SR with releasable calcium; by EPMA the postulated increase in sigma CaSR was measured directly. Post-rest, the peaks of systolic [Ca2+]c transients increased, however only by 40%. In addition, a moderate increase of end-diastolic [Ca2+]c was measured. In an other series of experiments, contraction was potentiated by 800% increase by means of paired voltage-clamp pulses (1 Hz, 36 degrees C, 2 mM [Ca2+]o). In the potentiated state, end-diastolic [Ca2+]c was 180 nM and sigma Ca(myo) was 0.65 mM. During systole, [Ca2+]c peaked within 20 ms to 950 nM. sigma Ca(myo) rose within 20 ms to 1.4 mM and fell within 40 ms to 1.1 and within 90 ms to 0.8 mM. In contrast, the time course of contraction was slow and peaked at a time (130 ms) when the [Ca2+]c and sigma Ca(myo) transients were finished. We suggest that Ca2+ bound to troponin C (TnC) controls only the onset but not the time course of myofilament interaction. From [Ca2+]c and sigma Ca(myo) we estimated a Ca2+ buffering capacitance of 1.5 mmol sigma Ca(myo) per pCa change, only a fraction of which can be attributed to Ca2+ binding sites on TnC. A model explaining the results requires the assumption of 0.6 mM additional slow, high affinity Ca2+ sites and 2 mM fast, low affinity Ca2+ sites. We discuss that end-diastolic Ca2+ binding to these sites contributes to the potentiation of contraction. Junctional SR. At the end of diastole sigma CaSR was 2.4 mM which is 4 times larger than sigma Ca(myo). This difference disappeared 20 ms after depolarization (sigma CaSR 1.1 mM), within another 20 ms it largely recovered (sigma CaSR 2.0 mM). These properties suggest that the junctional SR is a compartment suitable not only for Ca2+ release but also for rapid Ca2+ reuptake. Mitochondria. Paired-pulse potentiation increased end-diastolic sigma Ca(Mito) significantly (0.4 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Large and rapid changes of myofibrillar total calcium during the cardiac cycle. Electron probe microanalysis of voltage-clamped guinea-pig ventricular myocytes.

At 36 degrees C and 2 mM [Ca2+]o, single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). The same paired-pulse protocol was applied to another group of myocytes from which contraction was recorded and [Ca2+]i was estimated by microfluospectroscopy (50 microMNa-Indo-1). In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 /+- 220 nM (mean +/- SEM) and fell within 100 ms to about 450 nM. sigma Camyo, the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in 17 potentiated myocytes. During diastole, sigma Camyo was 2.6 +/- 0.4 mmol/kg dry weight (dw), which can be converted to 0.65 mM (mmoles per liter myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. A time-course for systolic sigma Camyo was determined by shock-freezing 13 cells at different times after start of depolarization to +5 mV. sigma Camyo was 5.5 +/- 0.3 mmol/kg dw (1.4 mM) after 15-25 ms, 4.6 +/- 0.5 mmol/kg dw (1.1 mM) after 30-45 ms, and 3.1 mmol/kg dw (0.8 mM) after 60-120 ms. The fast time-course of sigma Camyo suggests that calcium binds to and unbinds from troponin C at a fast rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Total and free myoplasmic calcium during a contraction cycle: x-ray microanalysis in guinea-pig ventricular myocytes.

1. At 36 degrees C and 2 mM [Ca2+]o single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. With a paired-pulse protocol applied at 1 Hz, a first pulse to +5 mV was followed by a second pulse to +50 mV. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron-probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). 2. The same paired-pulse protocol was applied to another group of myocytes from which contraction and [Ca2+]i was estimated by microfluospectroscopy (50 microM-Na5-Indo-1). Potentiation moderately reduced diastolic sarcomere length from 1.85 to 1.82 microns and increased diastolic [Ca2+]i from about 95 to 180 nM. In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 +/- 220 nM (mean +/- S.E.M.) and fell within 100 ms to about 450 nM. 3. Sigma Camyo, the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in seventeen potentiated myocytes. During diastole, sigma Camyo was 2.6 +/- 0.4 mmol (kg dry weight (DW]-1 which can be converted to 0.65 mM (mmoles per litre myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. 4. A time course for systolic sigma Camyo was determined by shock-freezing thirteen cells at different times after start of depolarization to +5 mV. Sigma Camyo was 5.5 +/- 0.3 mmol (kg DW)-1 (1.4 mM) after 15-25 ms, 4.6 +/- 0.5 mmol (kg DW)-1 (1.1 mM) after 30-45 ms, and 3.1 mmol (kg DW)-1 (0.8 mM) after 60-120 ms. The fast time course of sigma Camyo suggests that calcium binds to and unbinds from troponin C at a fast rate. Hence, it is the slow kinetics of the cross-bridges that determines the 130 ms time-to-peak shortening. 5. Mitochondria of potentiated cells contained during diastole a total calcium concentration, sigma Camito, of 1.3 +/- 0.2 mmol (kg DW)-1 (0.4 mM). During the initial 15-25 ms of systole, sigma Camito did not change, however, during 30-45 ms sigma Camito rose to 3.7 +/- 0.5 mmol (kg DW)-1 (1.2 mM). The data suggest that sigma Camito can follow sigma Camyo with some delay, thereby participating in both slow diastolic and fast systolic changes in total calcium (sigma Ca), at least under the given conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions.

By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).

Animals↗
PubMed1986

Ca-pools involved in the regulation of cardiac contraction under positive inotropy. X-ray microanalysis on rapidly-frozen ventricular muscles of guinea-pig.

Electron probe microanalysis of rapidly-frozen small ventricular trabeculae of guinea-pig demonstrates that the distribution of total intracellular calcium varies under positive inotropy depending on the type of inotropic intervention. The sarcoplasmic reticulum (SR) (or part of it) localized at the level of the z-lines reveals high calcium accumulation at the end of diastole whenever a stimulus is followed by a contraction with a short time to peak of force. After paired pulse stimulation, only this cell compartment accumulates calcium at the end of diastole. Since this cell compartment is "Ca-empty" in muscles frozen during contraction, SR is considered to be the source of activator Ca. In several cases of inotropy (after application of ARL, caffeine or after lowering the extracellular Na+ concentration), calcium is also detectable on the mitochondria, suggesting that these organelles participate in slow regulation of cytosolic calcium. In some cases, total calcium located on the sarcomeres is increased. The interpretation of this finding is intriguing and requires the assumption of supplementary cytosolic Ca-sinks as yet unknown.

Animals↗
PubMed1985

Extra- and intracellular lanthanum: modified calcium distribution, inward currents and contractility in guinea pig ventricular preparations.

In guinea pig ventricular strips and isolated cells, 0.1 mM LaCl3 blocks contractility and shortens the action potential (AP) in less than 2 min ("early La-effect"). After 30 min, it prolongs the APs which trigger slow contractions ("late La-effect"). These results confirm earlier reports. X-ray microprobe analysis shows that La initially displaces only a small fraction of that Ca which is superficially bound to the sarcolemma. But, since this Ca is completely removed by Ca-free solutions within 2 min, we suggest that La blocks contractility not by displacing superficial Ca but by blocking the Ca inward current iCa. Blocking of iCa is analyzed with voltage clamp experiments. It is not La-specific, and can also be observed with other calcium channel blockers as well. When iCa has been blocked, the membrane can still generate 100-200 ms long plateaus via the sodium inward current iNa. During the late La-effect, the cells internalize La. Intracellular La is detected by x-ray microprobe analysis in cryosections of frozen muscles and as La-precipitates in EM images from freeze substituted preparations. Simultaneously, the cytosol gains Na and Ca, but the plasmalemmal and sarcoplasmic reticulum (SR) membranes are no longer occupied by Ca but by La. The late La-effect on the prolongation of the AP is La-specific. In the absence of extracellular La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional inward current which is insensitive to Ca-removal, is inactivated by holding potentials of -40 mV, and is TTX-sensitive. We suggest that the current flows through a fraction of original Na-channels that is modified by i.c. La with respect to inactivation and selectivity. We attribute the late re-occurrence of contractility to activator Ca entering from the bath. Ca-entry might be mediated via enhanced Na/Ca-exchange whose rate is increased by the i.c. Na-load. In addition, Ca may enter through the La-modified Na-channels due to their impaired selectivity. Since i.c. La is known to interfere with the Ca-sequestration by the SR, it is expected to impair relaxation.

Action Potentials↗
PubMed1979

Electron probe X-ray microanalysis and cryoultramicrotomy of unstained myocardial sarcoplasmic reticulum, in situ and fragmented.

Myocardial sarcoplasmic reticulum of cats in situ and fragmented sarcoplasmic reticulum (FSR) were analysed using X-ray microanalysis, cryoultramicrotomy and scanning transmission electron microscopy. 2 types of FSR vesicles can be distinguished morphologically and by their different elemental composition especially by different Ca loading. The Ca content of the sarcoplasmic reticulum can also be detected in situ.

Animals↗
PubMed1976

[Maximum velocity of load-free shortening Vmax, myocardial capacity and "contractility indices" in the hypertrophic myocardium].

Based upon literature and our own experimentation on Goldblatt rats, the significance of a decrease of the maximal shortening velocity of the myocardium at zero load (Vmax) in the hypertrophied, chronically pressure-loaded heart is discussed. In the hypertrophied myocardium with varying concentrations of the contractile structures, the developed tension under isometric conditions (sigma) and the maximum rate of tension development (d sigma/dtmax) can indicate significant deviations from the values of controls of the same age, without, however, making it possible to draw from these changes absolute conclusions about the elementary contractile process. With the enhanced concentration of contractile proteins, the mentioned isometric values, as well as the maximum instantaneous power (cross-sectionally related) of the myocardium can be increased during a stage of hypertrophy in which Vmax is already reduced. The decrease of Vmax shows a rough correlation with the reduction of the specific ATPase activity of actomyosin and is already observed at moderate degrees of hypertrophy (30 to 50%). The time course of the change of both parameters in experimental hypertrophy suggests a causal relation between the changes of those two parameters and the failure of the myocardium in later stages of a chronic overload. Under the condition of reliable estimation, Vmax allows for, also with changed actomyosin concentration, an assessment of the elementary contractile process. On the other hand, Vmax does not present a sufficient measure for the cross-sectionally related power capacity of the hypertrophied myocardium. The possible dissociation between the unloaded shortening velocity and the cross-sectionally related power capacity could, in certain cases, explain an unsatisfactory correlation between Vmax and the clinical state of the heart. The so-called empirical indices of contractility, which are not always clearly related to basic physiological characteristics of the myocardium, should be interpreted with particular reserve in relation to the hypertrophied cardiac muscle.

Actomyosin↗
PubMedDate not supplied

Binding of calcium to myoplasmic buffers contributes to the frequency-dependent inotropy in heart ventricular cells.

In guinea-pig ventricular cells, the Ca2+ buffer capacity of the myoplasm was estimated from the ratio of ionized calcium (from Indo-1 fluorescence) through total calcium (ionized plus bound calcium, from x-ray microprobe analysis). During post-rest potentiation (1 Hz paired-pulses in voltage-clamp), where diastolic sarcomere length remained nearly constant, Ca2+ buffer capacity slowly fell from 5500:1 to 700:1 suggesting that slow Ca2+ binding sites became saturated. We discuss that frequency-inotropy depends not only on the replenishment of intracellular stores with Ca2+, but also on binding of Ca2+ to these slow sites; the slow Ca2+ sites could complete with the fast activator sites on troponin C for systolic Ca2+, or they could enhance the Ca2+ affinity of the fast Ca2+ sites on troponin C by cooperative interaction.

Action Potentials↗
PubMedDate not supplied

Myocardial function in different models of cardiac hypertrophy. An attempt at correlating mechanical, biochemical, and morphological parameters.

Based on mechanical, biochemical and electron microscopic studies performed in the same stage of experimental cardiac hypertrophy, an attempt is made to define the significance of individual factors responsible for the alterations in myocardial function. Using swimming rats, it is demonstrated that a load-induced increase in cardiac mass is not necessarily connected with an impairment of contractile capability on a cellular level. Yet, also, the reduction of specific ATPase activity and unloaded shortening velocity in pressure-induced hypertrophy (goldblatt rats; aortic stenosis) seems to be the expression of adaptation rather than of cellular damage, at least in the earlier stages. Although there are distinct indications of alterations in Ca-dependent activation and deactivation, in the Goldblatt model electromechanical coupling does not seem to be the main cause of altered contraction parameters. The correlation between specific ATPase activity of actomyosin and unloaded shortening velocity as well as the persistance of decrease in shortening velocity, also under optimal electromechanical coupling conditions, point to an inner relationship between the two values. A discrepancy between unloaded shortening velocity on the one hand and developed tension on the other is mainly due to an increased content of contractile structures. In later stages, an increased connective tissue content influences both isometric and isotonic parameters.

Actomyosin↗
PubMedDate not supplied

Dynamics of the hypertrophied left ventricle in the rat. Effects of physical training and chronic pressure load.

Left ventricular hypertrophy of about 40% was produced in rats by narrowing one renal artery (Goldblatt II) and of about 6% by swimming-training for 2 hours a day for 14 weeks. The dynamics of the hypertrophied ventricles were investigated by means of the isovolumic systolic and diastolic pressure-volume relations, the stress development during afterloaded and isovolumic contractions, and the force-velocity relation. The following results were obtained: The performance of the whole hypertrophied ventricle is increased. The developed stress and the maximum rate of stress development are enhanced, probably as a consequence of the increased density of the contractile proteins. The maximum shortening velocity can be reduced at the same time.

Adaptation, Physiological↗
PubMedDate not supplied

Time course of electron microscopic alterations in the hypertrophied myocardium of Goldblatt rats.

In Goldblatt rats, electron microscopic investigations of morphological changes in heart tissue during the compensatroy stage of pressure induced cardiac hypertrophy were performed. Myocardial cellular size enlarged considerably within the first 4 weeks after renal artery coarctation. An enlargement of the T-tubular system is particularly striking. The ratio of myofibril volume to total cell volume is significantly increased from the 4th-24th week and can at least partially explain a rise in developed tension which occurs in spite of a frequent disarrangement of myofibrils. In later stages severe changes in the vessel walls take place and there is an augmentation of connective tissue which impairs a correlation between myocardial cell alterations and mechanical parameters.

Animals↗
PubMedDate not supplied

Presystolic calcium-loading of the sarcoplasmic reticulum influences time to peak force of contraction. X-ray microanalysis on rapidly frozen guinea-pig ventricular muscle preparations.

Guinea-pig ventricular small papillary muscles and trabeculae were rapidly frozen presystolically after prolonged rest following positive inotropic interventions which strongly influenced peak of force and time to peak force. The possible sources of activator calcium for the different types of contraction were investigated. After rest in the presence of noradrenaline (10(-5)mol/l) the first post-rest contraction showed a retarded activation and a "late" peak of force. Muscle strips frozen after a rest period of 5 min in a bath solution containing noradrenaline were cryosectioned and analyzed with X-ray microanalysis for elemental distribution: although at this time an applied stimulus would induce a potentiated contraction, intracellular membrane systems such as sarcoplasmic reticulum and mitochondria failed to reveal any accumulation of calcium. After rest in a low sodium Tyrode the first post-rest contraction showed an "early" peak of force. Muscles frozen after rest in a low sodium solution revealed intracellular Ca accumulation on the sarcoplasmic reticulum, in the network at the level of the Z-lines. The results support the hypothesis that 1. the sarcoplasmic reticulum (SR) accumulates calcium presystolically when "early" contractions follow stimulation; 2. the network of sarcoplasmic reticulum at the level of the Z-lines is a crucial source of activator calcium; 3. the activator calcium for late contractions is probably of extracellular origin.

Animals↗