Diastolic Cell Length Research Articles

Frequency-dependent effects of omecamtiv mecarbil on cell shortening of isolated canine ventricular cardiomyocytes.

Omecamtiv mecarbil (OM) is a myosin activator agent developed for the treatment of heart failure. OM was reported to increase left ventricular ejection fraction and systolic ejection time, but little is known about the effect of heart rate on the action of OM. The present study, therefore, was designed to investigate the effects of OM on unloaded cell shortening and intracellular Ca2+ ([Ca2+]i) transients as a function of the pacing frequency. Isolated cardiomyocytes were stimulated at various frequencies under steady-state conditions. Cell length was monitored by an optical edge detector and changes in [Ca2+]i were followed using the Ca2+-sensitive dye Fura-2. At the pacing frequency of 1Hz, OM (1-10μM) significantly decreased both diastolic and systolic cell length, however, fractional shortening was augmented only by 1μM OM. Time to peak tension and time of 90% relaxation were progressively increased by OM. At the frequency of 2Hz, diastolic cell length was reduced by 10μM OM to a larger extent than systolic cell length, resulting in a significantly decreased fractional shortening under these conditions. OM had no effect on the parameters of the [Ca2+]i transient at any pacing frequency. The results suggest that supratherapeutic concentrations of OM may decrease rather than increase the force of cardiac contraction especially in tachycardic patients.

Negative Inotropic Effect of Rapamycin on Isolated Human Cardiomyocytes

Rapamycin is an increasingly important immunosuppressive drug and reduces restenosis after coronary stenting, but its effects on cardiac contractility are largely unknown. We investigated the acute inotropic effects of rapamycin on isolated human cardiomyocytes. Cardiomyocytes were enzymatically isolated from right atrial appendages obtained during routine coronary artery bypass surgery. Cell morphology was examined by confocal microscopy. Cell contraction was recorded after electrical stimulation. Rapamycin elicited a concentration-dependent decrease in fractional cell shortening ranging from 14.3 +/- 2.6% at 10(-8) M rapamycin to 26.4 +/- 4.2% at 10(-5) M. Rapamycin also caused a concentration-dependent decrease in diastolic cell length. Contractile performance of isolated cardiomyocytes was well preserved, as evidenced by the profound positive inotropic effects of high extracellular calcium concentration and the beta-adrenoreceptor agonist isoproterenol. The acute negative inotropic effect of rapamycin on human cardiomyocytes might be due to altered calcium homeostasis through the binding of rapamycin to FKBP12.6 and its regulatory function on the ryanodine receptor, with increased calcium leakage from the sarcoplasmic reticulum.

Osmolality- and Na+-dependent effects of hyperosmotic NaCl solution on contractile activity and Ca2+ cycling in rat ventricular myocytes

Hypertonic NaCl solutions have been used for small-volume resuscitation from hypovolemic shock. We sought to identify osmolality- and Na(+)-dependent components of the effects of the hyperosmotic NaCl solution (85 mOsm/kg increment) on contraction and cytosolic Ca(2+) concentration ([Ca(2+)](i)) in isolated rat ventricular myocytes. The biphasic change in contraction and Ca(2+) transient amplitude (decrease followed by recovery) was accompanied by qualitatively similar changes in sarcoplasmic reticulum (SR) Ca(2+) content and fractional release and was mimicked by isosmotic, equimolar increase in extracellular [Na(+)] ([Na(+)](o)). Raising osmolality with sucrose, however, augmented systolic [Ca(2+)](i) monotonically without change in SR parameters and markedly decreased contraction amplitude and diastolic cell length. Functional SR inhibition with thapsigargin abolished hyperosmolality effects on [Ca(2+)](i). After 15-min perfusion, both hyperosmotic solutions slowed mechanical relaxation during twitches and [Ca(2+)](i) decline during caffeine-evoked transients, raised diastolic and systolic [Ca(2+)](i), and depressed systolic contractile activity. These effects were greater with sucrose solution, and were not observed after isosmotic [Na(+)](o) increase. We conclude that under the present experimental conditions, transmembrane Na(+) redistribution apparently plays an important role in determining changes in SR Ca(2+) mobilization, which markedly affect contractile response to hyperosmotic NaCl solutions and attenuate the osmotically induced depression of contractile activity.

Treprostinil potentiates the positive inotropic effect of catecholamines in adult rat ventricular cardiomyocytes

Prostanoids have been shown to improve exercise tolerance, hemodynamics and quality of life in patients with pulmonary arterial hypertension (PAH). We investigated whether treprostinil exerts direct contractile effects on cardiomyocytes that may explain partly the beneficial effects of these drugs. Ventricular cardiomyocytes from adult rats were paced at a constant frequency of 0.5 to 2.0 Hz and cell shortening was monitored via a cell edge detection system. Twitch amplitudes, expressed as percent cell shortening of the diastolic cell length, and maximal contraction velocity, relaxation velocity, time to peak of contraction and time to reach 50% of relaxation were analyzed. Treprostinil (0.15 - 15 ng ml(-1)) slightly increased contractile dynamics of cardiomyocytes at clinically relevant concentrations. However, the drug significantly improved cell shortening of cardiomyocytes in the presence of isoprenaline, a beta-adrenoceptor agonist. Treprostinil exerted this effect at all beating frequencies under investigation. Treprostinil mimicked this potentiating effect in a Langendorff preparation as well. The potentiating effect of treprostinil on isoprenaline-dependent cell shortening was no longer seen after phosphodiesterase inhibition. Long-term cultivation of cardiomyocytes with treprostinil did not modify load free cell shortening of these cells, but reduces the duration of contraction. We conclude that the clinically used prostanoid treprostinil potentiates the positive inotropic effects of catecholamines in adult ventricular cardiomyocytes. This newly described effect may contribute to the beneficial clinical effects of prostanoids in patients with PAH.

Nitric oxide's role in the heart: control of beating or breathing?

Beneficial actions of nitric oxide (NO) in failing myocardium have frequently been overshadowed by poorly documented negative inotropic effects mainly derived from in vitro cardiac preparations. NO's beneficial actions include control of myocardial energetics and improvement of left ventricular (LV) diastolic distensibility. In isolated cardiomyocytes, administration of NO increases their diastolic cell length consistent with a rightward shift of the passive length-tension relation. This shift is explained by cGMP-induced phosphorylation of troponin I, which prevents calcium-independent diastolic cross-bridge cycling and concomitant diastolic stiffening of the myocardium. Similar improvements in diastolic stiffness have been observed in isolated guinea pig hearts, in pacing-induced heart failure dogs, and in patients with dilated cardiomyopathy or aortic stenosis and have been shown to result in higher LV preload reserve and stroke work. NO also controls myocardial energetics through its effects on mitochondrial respiration, oxygen consumption, and substrate utilization. The effects of NO on diastolic LV performance appear to be synergistic with its effects on myocardial energetics through prevention of myocardial energy wastage induced by LV contraction against late-systolic reflected arterial pressure waves and through prevention of diastolic LV stiffening, which is essential for the maintenance of adequate subendocardial coronary perfusion. A drop in these concerted actions of NO on diastolic LV distensibility and on myocardial energetics could well be instrumental for the relentless deterioration of failing myocardium.

Hydrogen peroxide enhances iron-induced injury in isolated heart and ventricular cardiomyocyte in rats.

To explore the cardiac effects of iron with or without hydrogen peroxide, the isolated perfused rat heart and enzymatically isolated ventricular cardiomyocyte were used. It was shown that treatment with cell-permeable iron (Fe-HQ) for 10 min reduced the contractile amplitude and velocity and end diastolic cell length in the cardiomyocyte and increased the contents of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary effluent and malondialdehyde (MDA) in the myocardium. The left ventricular developed pressure (LVDP), +/-dP/dtmax, and heart rate and coronary flow are showed a biphasic phase, an increase at first followed by a decline. Treatment with hydrogen peroxide for 10 min following Fe-HQ augmented the effect of iron with an increase in coronary LDH and CK release and myocardial MDA content, and decrease in LVDP, +/-dP/dtmax and heart rate. Perfusion of reduced glutathione with hydrogen peroxide counteracted these effects of Fe-HQ and hydrogen peroxide while dimethyl sulfoxide had no effect on the injury induced by Fe-HQ and hydrogen peroxide in the isolated rat heart. This suggests that augmentation of myocardial injury as a result of an increase in intracellular iron by hydrogen peroxide might involve the dysfunction of sulfydryl group containing proteins but not the hydroxyl radicals.

Brief rapid pacing depresses contractile function via Ca(2+)/PKC-dependent signaling in cat ventricular myocytes.

The purpose of this study is to determine the effects of brief rapid pacing (RP; approximately 200-240 beats/min for approximately 5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (-44 +/- 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca(2+) current (I(Ca,L)). During RP, baseline and peak intracellular Ca(2+) concentration ([Ca(2+)](i)) increased markedly. After RP, Ca(2+) transients were similar to control. The effects of RP on cell shortening were not prevented by 1 microM calpain inhibitor I, 25 microM L-N(5)-(1-iminoethyl)-orthinthine, or 100 microM N(G)-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca(2+)] (0.5 mM) during RP or exposure to chelerythrine (2-4 microM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-beta. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (-46 +/- 7%). Western blots indicated that cat myocytes express PKC-alpha, -delta, and -epsilon as well as PKC-beta. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca(2+)/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.

A N-terminal PTHrP peptide fragment void of a PTH/PTHrP-receptor binding domain activates cardiac ET(A) receptors.

1. Adult ventricular cardiomyocytes show an unusual structure-function relationship for cyclic AMP-dependent effects of PTHrP. We investigated whether PTHrP(1 - 16), void of biological activity on classical PTHrP target cells, is able to mimic the positive contractile effect of PTHrP(1 - 34), a fully biological agonist on cardiomyocytes. 2. Adult ventricular cardiomyocytes were paced at a constant frequency of 0.5 Hz and cell contraction was monitored using a cell-edge-detection system. Twitch amplitudes, expressed as per cent cell shortening of the diastolic cell length, and rate constants for maximal contraction and relaxation velocity were analysed. 3. PTHrP(1 - 16) (1 micromol l(-1)) mimicked the contractile effects of PTHrP(1 - 34) (1 micromol l(-1)). It increased the twitch amplitude from 5.33+/-0.72 to 8.95+/-1.10 (% dl l(-1)) without changing the kinetic of contraction. 4. PTH(1 - 34) (10 micromol l(-1)) affected the positive contractile effect of PTHrP(1 - 34), but not that of PTHrP(1 - 16). 5. RpcAMPS (10 micromol l(-1)) inhibited the positive contractile effect of PTHrP(1 - 34), but not that of PTHrP(1 - 16). 6. The positive contractile effect of PTHrP(1 - 16) was antagonized by the ET(A) receptor antagonist BQ123. 7. Sarafotoxin 6b and PTHrP(1 - 16), but not PTHrP(1 - 34), replaced (3)H-BQ123 from cardiac binding sites. 8. We conclude that N-terminal PTHrP peptides void of a PTH/PTHrP-receptor binding domain are able to bind to, and activate cardiac ET(A) receptors.

Positive inotropic effects of calcium sensitizers on normal and failing cardiac myocytes.

Calcium sensitizers increase myocardial contractile function without affecting Ca2+ homeostasis, which might be beneficial in the treatment of patients with heart failure. However, it remains uncertain whether Ca sensitizers induce quantitatively similar inotropic responses in control and failing hearts. To compare their effects in normal versus failing hearts at the cellular level, shortening mechanics and intracellular calcium ([Ca2+]i) transient were simultaneously measured in the left ventricular myocytes isolated from normal dogs (n = 8) and dogs with rapid pacing-induced heart failure (n = 7). CGP 48506 and EMD 57033 exerted a positive inotropic effect in a dose (0.1-3 microM)-dependent manner in both normal and heart failure myocytes. The percent increase of cell shortening magnitude was comparable between the two groups. CGP 48506 and EMD 57033 did not affect the diastolic cell length and resting [Ca2+]i level. They did not affect the duration of [Ca2+]i transient dynamics. Thus Ca2+ sensitizers exerted comparable positive inotropic effects without affecting the rest cell length and rest [Ca2+]i in normal and heart failure myocytes.

Rate-dependent changes in cell shortening, intracellular Ca(2+) levels and membrane potential in single, isolated rainbow trout (Oncorhynchus mykiss) ventricular myocytes.

The effects of increasing stimulation frequency (from 0.2 to 1.4 Hz) on the contractility, intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane potential of single ventricular myocytes isolated from the heart of rainbow trout (Oncorhynchus mykiss) were measured. Cell shortening, expressed as a percentage of resting cell length, was our index of contractility. The fluorescent Ca(2+) indicator Fura-2 was used to monitor changes in [Ca(2+)](i). Action potentials and L-type Ca(2+) currents (I(Ca)) were recorded using the whole-cell patch-clamp technique. Experiments were performed at 15 degrees C. Increasing the stimulation frequency caused a significant increase in diastolic [Ca(2+)](i) and a significant decrease in diastolic cell length and membrane potential. During systole, there was a significant fall in the amplitude of the [Ca(2+)](i) transient, cell shortening and action potential with a decrease in the duration of the action potential at both 20 % and 90 % repolarisation. Caffeine was used to assess the Ca(2+) content of the sarcoplasmic reticulum. We observed that sarcoplasmic reticulum Ca(2+) load was greater at 1.0 Hz than at 0.6 Hz, despite a smaller electrically evoked [Ca(2+)](i) transient. The amplitude of I(Ca) was found to decrease with increased stimulation frequency. At 0.6 Hz, electrically evoked [Ca(2+)](i) transients in the presence of 10 mmol l(-)(1) caffeine or 10 micromol l(-)(1) ryanodine and 2 micromol l(-)(1) thapsigargin were reduced by approximately 15 %. We have described the changes in contractility, [Ca(2+)](i) and action potential configuration in a fish cardiac muscle system. Under the conditions tested (0.6 Hz, 15 degrees C), we conclude that the sarcoplasmic reticulum contributes at least 15 % of the Ca(2+) associated with the [Ca(2+)](i) transient. The rate-dependent decrease in contraction amplitude appears to be associated with the fall in the amplitude of the [Ca(2+)](i) transient. This, in turn, may be influenced by changes in the action potential configuration via mechanisms such as altered Ca(2+) efflux and Ca(2+) influx. In support of our conclusions, we present evidence that there is a rate-dependent decrease in Ca(2+) influx via I(Ca) but that the Ca(2+) load of the sarcoplasmic reticulum is not reduced at increased contraction frequencies.

(−)-Enantiomer EMD 57439 Antagonizes the Ca2+ Sensitizing Effect of (+)-Enantiomer EMD 57033 on Diastolic Function but Not on Systolic Function in Rabbit Ventricular Cardiomyocytes

EMD 53998 (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one), the racemic mixture of (+)-enantiomer EMD 57033 and (−)-enantiomer EMD 57439, is a prototype of Ca2+ sensitizers that act via a central and/or down-stream mechanism in cardiac E-C coupling. In rabbit ventricular cardiomyocytes loaded with indo-1/AM, EMD 53998 and EMD 57033 shifted the relationship between Ca2+ transients and cell shortening (systolic function) to the left to the same extent as compared with that of elevation of [Ca2+]o. EMD 57439 did not elicit a positive inotropic effect (PIE). The PIE of EMD 57033 was associated with a more pronounced decrease in the diastolic cell length than that of EMD 53998, whereas the systolic effects of these compounds were equivalent. These results indicate that weak phosphodiesterase (PDE) III inhibition may exert a differential action on diastolic and systolic function. Thus, EMD 57439 antagonizes the Ca2+-sensitizing effect of EMD 57033 on diastolic function with no effect on systolic function, which may lead to a decease in diastolic cell length of a lesser extent with the racemate EMD 53998 compared with (+)-enantiomer EMD 57033.

Propofol and ketamine only inhibit intracellular Ca2+ transients and contraction in rat ventricular myocytes at supraclinical concentrations.

The cellular mechanisms that mediate the cardiodepressant effects of intravenous anesthetic agents remain undefined. The objective of this study was to elucidate the direct effects of propofol and ketamine on cardiac excitation-contraction coupling by simultaneously measuring intracellular calcium concentration ([Ca2+]i) and shortening in individual, field-stimulated ventricular myocytes. Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening (video edge detection) were monitored simultaneously in individual cells that were field-stimulated at 0.3 Hz. Baseline [Ca2+]i (mean +/- SEM) was 80 +/- 12 nM, and resting cell length was 112 +/- 2 microm. Field stimulation increased [Ca2+]i to 350 +/- 23 nM, and the myocytes shortened by 10% of diastolic cell length. Both intravenous anesthetic agents caused dose-dependent decreases in peak [Ca2+]i and shortening. At 300 microM, propofol prolonged time to peak concentration and time to 50% recovery for [Ca2+]i and shortening. In contrast, changes in time to peak concentration and time to 50% recovery in response to ketamine were observed only at the highest concentrations. Neither agent altered the amount of Ca2+ released from intracellular stores in response to caffeine. Propofol but not ketamine, however, caused a leftward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. These results indicate that both intravenous anesthetic agents have a direct negative inotropic effect, which is mediated by a decrease in the availability of [Ca2+]i. Propofol but not ketamine may also alter sarcoplasmic reticulum Ca2+ handling and increase myofilament Ca2+ sensitivity. The effects of propofol and ketamine are primarily apparent at supraclinical concentrations, however.

Calcium homeostasis in cardiomyocytes isolated from heat-shocked rats.

The cellular mechanism of heat shock-mediated cardioprotection is still under debate. Because heat pretreatment negatively affects the normoxic left ventricular contractile performance in vitro when the extracellular Ca2+ concentration ([Ca2+]o) is relatively low (0.65-1.25 mM), the intracellular Ca2+ homeostasis was studied in more detail in cardiomyocytes isolated from adult rats 24 h after heat stress (42 degrees C for 15 min) or anesthesia (control). Sensitivity to Ca2+ overload was assessed by exposure to veratridine (quiescent cells) or to [Ca2+]o ranging from 0.125 to 20 mM in quiescent and paced cardiomyocytes. The fraction of irreversibly hypercontracted cells was not different between groups. The fura-2 fluorescence ratio (I340/I380), which was used as a measure for cytoplasmic Ca2+ concentration ([Ca2+]i) in quiescent cells after exposure to [Ca2+]o (0.5-10 mM), was also not different between groups. Myofilament Ca2+ sensitivity was assessed in paced (0.5 Hz) cells by simultaneous measurement of [Ca2+]i transients and cell shortening. At stepwise increases of [Ca2+]o from 1 to 10 mM, these parameters were comparable between groups. The diastolic cell length shortened progressively and equally in both groups after increasing [Ca2+]o. However, within 2 min of return from 10 to 1 mM [Ca2+]o, cells from heat-shocked rats retained the same length, whereas cells from control rats contracted further (P = 0.05). These data suggest that heat stress improves relaxation after challenge with high [Ca2+]o.

Effects of TNF-alpha on [Ca2+]i and contractility in isolated adult rabbit ventricular myocytes.

The mechanism of the acute negative inotropic effect of tumor necrosis factor-alpha (TNF-alpha) was studied in enzymatically isolated adult rabbit ventricular myocytes. In cells loaded with fura 2 acetoxymethyl ester (AM) and paced intermittently at 0.2 Hz, TNF-alpha at doses < or = 10,000 U/ml caused a significant reduction in active cell shortening at 20 min, without reducing the amplitude of the accompanying intracellular Ca2+ concentration ([Ca2+]i) transient. Similar results were obtained in cells loaded with indo 1-AM and paced continuously at 0.2 Hz during exposure to TNF-alpha (10,000 U/ml). The effect of TNF-alpha on cell shortening could be prevented by the nitric oxide (NO) synthase blocker NG-nitro-L-arginine methyl ester (L-NAME) but not its inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). The NO scavenger hemoglobin also attenuated the effects of TNF-alpha. TNF-alpha also caused a significant increase in diastolic cell length without any change in diastolic [Ca2+]i. The effect on cell length was prevented by L-NAME but not D-NAME. In cells loaded with the pH indicator seminaphthorhodafluor-AM, TNF-alpha did not alter pH sufficiently to account for the negative inotropic effect. These data suggest that high doses of TNF-alpha can acutely induce NO synthesis in isolated myocytes and reduce contractility by decreasing myofilament [Ca2+]i responsiveness. The mechanism of this altered myofilament [Ca2+]i response is unknown but does not appear to be pH mediated.

Modulation of sodium-hydrogen exchange activity in cardiac myocytes during acidosis and realkalinisation: effects on calcium, pHi, and cell shortening

The aim was to examine the effects of the Na+/H+ exchange inhibitor methylisobutylamiloride (MIA) as well as protein kinase C, a putative regulator of Na+/H+ exchange, on intracellular calcium, intracellular pH, and unloaded cell shortening in isolated guinea pig cardiac myocytes subjected to lactic acid induced acidosis followed by realkalinisation. Calcium transient amplitude and cell shortening were measured simultaneously in single isolated myocytes loaded with fura2-AM. Intracellular pH was measured in cells loaded with BCECF-AM. Exposure of cells to 5 min of lactate (20 mM) acidosis (pH 6.8) caused an increase in calcium transient amplitude and a decrease in cell shortening and intracellular pH. During realkalinisation (pH 7.3), the calcium transient gradually decreased while intracellular pH became more alkaline than pre-acidosis values. The cells underwent transient hypercontractility as evidenced by a marked increase in systolic cell shortening and a decrease in diastolic cell length. Inhibition of sodium/hydrogen exchange with MIA (1 microM) caused a significant attenuation of the increase in calcium transient amplitude during acidosis and further depressed cell shortening as well as intracellular pH. In addition, MIA significantly attenuated hypercontractility and abolished cell contracture upon realkalinisation. In contrast, phorbol 12-myristate 13-acetate (10(-12) M) exerted no effects on the response to acidosis; however, this treatment exacerbated cell hypercontractility and reduced functional recovery upon realkalinisation. Inhibition of Na+/H+ exchange activity during acidosis/realkalinisation enhances recovery of cell function.

8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes.

The role of cGMP in myocardial contraction is not established. Recent reports suggest that nitric oxide, released by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. We studied the effects of 8-bromo-cGMP (8bcGMP, 50 mumol/L) on contraction (cell shortening) and simultaneous intracellular Ca2+ transients (indo 1 fluorescence ratio) in intact adult rat ventricular myocytes (0.5 Hz and 25 degrees C) 8bcGMP reduced myocyte twitch amplitude and time to peak shortening (-19.6 +/- 4.2% and -17.6 +/- 1.3%, respectively) and increased steady-state diastolic cell length (+0.6 +/- 0.1 microns, mean +/- SEM, n = 8; all P < .05) but had no effect on shortening velocity, systolic or diastolic fluorescence ratio, or time to peak fluorescence ratio (all P = NS). In 7 of 13 myocytes, this negative inotropic effect was preceded by a transient positive inotropic effect, with small increases in twitch amplitude, shortening velocity, and cytosolic Ca2+ transient. Analysis of 8bcGMP effects on both the dynamic and steady-state relation between cell shortening and intracellular Ca2+ (during twitch contraction and tetanic contraction, respectively) indicated reduction in the myofilament response to Ca2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 mumol/L), an inhibitor of cGMP-dependent protein kinase, or by the presence of isoproterenol (3 nmol/L). 8bcGMP had no effect on cytosolic pH in cells (n = 4) loaded with the fluorescent probe carboxyseminaphthorhodafluor-1. These data indicate that cGMP may modulate myocardial relaxation and diastolic tone by reducing the relative myofilament response to Ca2+, probably via cGMP-dependent protein kinase.

Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in humans. Assessment by bicoronary sodium nitroprusside infusion.

In isolated mammalian cardiomyocytes, papillary muscle preparations, and ejecting hearts, nitric oxide (NO) or other cyclic GMP-elevating interventions increase diastolic cell length and reduce peak contractile performance by hastening onset of myocardial relaxation. In the present study, the effect of NO on left ventricular (LV) relaxation and diastolic distensibility was investigated in humans. The NO donor substance sodium nitroprusside was infused during cardiac catheterization in the global coronary bed of the LV of patients (n = 13) investigated for chest pain who were without evidence of obstructive coronary artery or other cardiac disease. Sodium nitroprusside was infused intracoronarily at a dosage (< or = 4 micrograms/min) that was previously shown to be devoid of systemic effects when infused into the brachial artery to investigate the reactivity of the forearm vascular bed. The effect of this global intracoronary infusion of the NO donor sodium nitroprusside was assessed by sequential LV angiograms and tip-micromanometer pressure recordings. During global intracoronary nitroprusside infusion, there was a decrease in heart rate from 78 +/- 11 to 76 +/- 12 beats per minute (P < .05), in LV peak systolic pressure from 161 +/- 18 to 146 +/- 18 mm Hg (P < .001), and in time to onset of LV relaxation (interval from Q wave on the ECG to LV dP/dtmin) from 432 +/- 36 to 419 +/- 36 milliseconds (P < .01). In 7 patients in whom adequate sequential LV angiograms could be obtained, LV end-diastolic volume increased from 158 +/- 34 to 165 +/- 40 mL (P < .05), whereas LV end-diastolic pressure fell from 18 +/- 5 to 12 +/- 3 mm Hg (P < .02), and in 5 of these 7 patients, a downward shift of the diastolic LV pressure-volume relation was observed. In 5 patients, a right atrial infusion of sodium nitroprusside was performed either before (n = 2) or after the global intracoronary infusion. The decrease in LV peak systolic pressure observed during right atrial infusion was significantly smaller (P < .01) than during global intracoronary infusion. The present study reveals reduced LV pressure development, an LV relaxation-hastening effect, and improved LV diastolic distensibility during global intracoronary infusion of the NO donor substance sodium nitroprusside. These effects appeared to be unrelated to systemic vasodilation or to pericardial constraint and could be explained by a direct myocardial effect of NO, probably through activation of guanylyl cyclase to increase cyclic GMP or through modification of other cellular proteins.

Mechanisms of excitation‐contraction coupling failure during metabolic inhibition in guinea‐pig ventricular myocytes.

1. The effects of complete metabolic inhibition on excitation-contraction coupling in heart were studied by exposing patch-clamped guinea-pig ventricular myocytes, loaded via the patch pipette with the Ca2+ indicator Fura-2 (0.1 mM), to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 microM) and 2-deoxyglucose (2-DG, 10 mM) while simultaneously recording membrane current, Fura-2 fluorescence, and cell motion. The patch pipette solution contained Cs+ and TEA (tetraethylammonium) to partially block K+ currents. 2. During voltage clamps from a holding potential of -40 mV to a test potential of 0 mV, complete metabolic inhibition decreased the Ca2+ current (ICa), activated the ATP-sensitive K+ current, modestly elevated diastolic [Ca2+]i and markedly reduced the [Ca2+]i transient without altering its voltage dependence. Active shortening was impaired and diastolic cell length decreased prior to large increases in diastolic [Ca2+]i, consistent with rigor induced by ATP depletion. Return of the [Ca2+]i transient to baseline and relaxation upon repolarization were also delayed. 3. Despite the depression of the peak [Ca2+]i transient induced by membrane depolarization during metabolic inhibition, the [Ca2+]i transient induced by a rapid exposure to 5 mM-caffeine was greater than control. The Na(+)-Ca2+ exchange current during the caffeine-induced [Ca2+]i transient was not affected by metabolic inhibition. 4. [Ca2+]i transients depressed by metabolic inhibition could be enhanced by augmenting ICa with elevated [Ca2+]o (10 mM) and Bay K 8644 (5 microM). 5. To study the relationship between the magnitude of ICa and the amplitude of the [Ca2+]i transient, ICa was modulated either by (a) voltage clamping the cell to different membrane potentials at constant [Ca2+]o or by (b) rapidly altering [Ca2+]o immediately prior to a voltage clamp to a fixed membrane potential. Under control conditions, the relationship between the size of ICa and the magnitude of the [Ca2+]i transient was the same whether ICa was modulated by altering membrane potential or [Ca2+]o, suggesting that membrane potential does not significantly modulate the Ca(2+)-induced Ca2+ release mechanism of cardiac excitation-contraction coupling. 6. After metabolic inhibition, however, the same ICa released less Ca2+ than under control conditions, consistent with some impairment of the Ca2+ release mechanism. 7. These results suggest that under conditions in which excitability is maintained by controlling membrane voltage and minimizing metabolically sensitive K+ currents, the decreased [Ca2+]i transient observed during metabolic inhibition severe enough to induce rigor is caused primarily by depression of ICa and not by depletion of intracellular Ca2+ stores. Additional factors also modestly hinder Ca2+ release from intracellular stores during metabolic inhibition.