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(Circulation Research. 1997;80:688-698.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of Myocardial Crossbridge Cycling by Hypoxic Endothelial Cells

A Potential Mechanism for Matching Oxygen Supply and Demand?

Ajay M. Shah, Alexandre Mebazaa, Zhao-Kang Yang, Giovanni Cuda, Edward B. Lankford, Chris B. Pepper, Steven J. Sollott, James R. Sellers, James L. Robotham, , Edward G. Lakatta

From the Department of Cardiology (A.M.S., Z.-K.Y., C.B.P.), University of Wales College of Medicine, Cardiff, UK; Laboratory of Cardiovascular Science (A.M.S., A.M., S.J.S., E.G.L.), Gerontology Research Center/National Institute on Aging, National Institutes of Health, Baltimore, Md; Pulmonary Anesthesia Laboratory (A.M., J.L.R.), Johns Hopkins Medical Institutions, Baltimore, Md; Laboratory of Molecular Cardiology (G.C., J.R.S.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md; Cardiovascular Section (E.B.L.), University of Pennsylvania School of Medicine, Philadelphia; and Department of Anesthesiology (A.M.), Lariboisiere Hospital, Paris, France.

Correspondence to Ajay M. Shah, MD MRCP, Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK. E-mail shaham2{at}cf.ac.uk

	Abstract

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Abstract Previous studies have shown that cardiac endothelial cells release substances that influence myocardial contraction. Since PO₂ is an important stimulus that modulates endothelial function, we investigated the effects of acute moderate hypoxia and reoxygenation on the release of cardioactive factors by endothelial cells. Endothelial cells cultured from several vascular beds were superfused with normoxic (equilibrated with room air; PO₂, 160 mm Hg) or hypoxic (PO₂, 40 to 50 mm Hg) physiological buffer solution, and the superfusates were reequilibrated to a PO₂ of 160 mm Hg and then tested for their effects on various myocardial assays. Endothelial cell viability and buffer ionic composition were unaltered after the superfusion procedures. The superfusates of hypoxic endothelial cells induced rapid, potent, reversible inhibition of isolated cardiac myocyte contraction without reducing cytosolic Ca²⁺ transients. This activity was not lost after heating (95°C) and was present in low molecular weight (M_r, <500) superfusate fractions. Hypoxic endothelial superfusate reduced unloaded shortening velocity of human skinned soleus muscle fibers. It markedly depressed in vitro actin motility over cardiac myosin and reduced the rate of actin-activated cardiac myosin ATPase activity but had no effect on corresponding smooth muscle myosin assays. Reoxygenation of hypoxic endothelial cells resulted in loss of this inhibitory activity. These data indicate that cultured endothelial cells respond to acute moderate hypoxia by releasing an unidentified substance(s) that inhibits myocardial crossbridge cycling, independent of Ca²⁺ or other second messenger signaling pathways. Such a mechanism could have important implications for the regulation of oxygen supply-demand balance in the heart and be relevant to conditions such as myocardial hibernation.

Key Words: cardiac contraction • hypoxia • endothelial cell • hibernation • myofilament

	Introduction

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An accumulating body of data supports the existence of a paracrine pathway for the regulation of cardiac contractile function by endothelial cells, analogous to endothelial regulation of vascular tone and blood flow (reviewed in Reference 1¹ ). Both the coronary vascular and endocardial endothelium release several diffusible agents, eg, nitric oxide, endothelin, and prostaglandins, that modify cardiac myocyte function.^{2 3 4 5 6 7 8 9} Endothelial cells also possess enzymatic activities (in particular, angiotensin-converting enzyme/kininase activity) that can alter local levels of angiotensin II and bradykinin and thereby potentially influence myocardial function. Although most of the above data are derived from in vitro experimental approaches (eg, bioassay studies, isolated papillary muscle, and isolated heart experiments), recent cardiac catheterization laboratory–based studies support the presence of a similar paracrine pathway in human subjects in vivo.¹⁰ This endothelial pathway is likely to act in concert and to interact with other cardiac regulatory mechanisms, such as neurohumoral pathways, the Frank-Starling response, and the effects of heart rate, coronary flow, and loading.

In addition to agents such as nitric oxide and endothelin, endothelial cells appear to release other as-yet-unidentified cardioactive substances. In bioassay studies in which the coronary effluent of isolated perfused rat hearts was tested for its effects on isolated rat cardiac trabeculae, Winegrad and colleagues^{7 11} reported the presence of both positive and negative inotropic substances. The positive inotropic activity was attributed to endothelin,⁷ but the nature of the negative inotropic activity remains unknown. These authors further reported an association between coronary flow rate, ambient PO₂, and the relative levels of positive and negative inotropic substances present in coronary effluent.¹¹ In our own previous studies, a different experimental approach was used in which cultured endothelial cells were continuously superfused with physiological buffer solution and the superfusate was then tested for its effect on contraction of isolated cardiac myocytes. These studies demonstrated the tonic release by cultured vascular and endocardial endothelial cells of endothelin (which exerted positive inotropic effects)⁵ and an unidentified stable negative inotropic substance(s) that exerted its effects by reducing myofilament responsiveness to Ca²⁺.¹² The latter activity was not attributable to cardioactive endothelial agents, such as nitric oxide, adenosine, or prostanoids, nor did it involve second messenger signaling pathways, such as cGMP, cAMP, and protein kinase C. Although the chemical nature of this negative inotropic activity remains unknown, recent studies¹³ suggest that it is similar or identical to the cardiodepressant activity reported by Winegrad and colleagues in their experiments on the coronary effluent of isolated hearts.

The paracrine release of cardioactive substances by endothelial cells could potentially allow for sensitive modulation of regional myocardial contractile function, responsive to local signals. Physiological stimuli that induce both acute and chronic changes in endothelial cell function include flow-related shear stress and other mechanical forces, PO₂ and pH, and numerous receptor-mediated agonists. Of particular relevance to the present study is the influence of PO₂ on endothelial function. Moderate reductions in PO₂ (30 to 80 mm Hg in buffer-perfused systems) induce significant acute alterations in endothelial function.^{14 15 16 17 18 19} In many vascular beds, including the coronary vasculature, moderate hypoxia induces the release of vasodilator prostanoids^{16 17 19} and nitric oxide.^{15 16 17 18} Endothelium-mediated hypoxic coronary vasodilatation contributes to the cardiac hyperemic response.²⁰ These effects may be regarded as acute feedback responses that increase oxygen supply by increasing vascular flow.

In the present study, we investigated the effects of acute moderate hypoxia (PO₂, 40 to 50 mm Hg) on the release of cardioactive substances by endothelial cells. Pure cultures of normoxically or hypoxically superfused endothelial cells were used as the donor tissue, and various normoxic myocardial preparations were used as the assay tissue. We report that in response to moderate hypoxia, endothelial cells cultured from several different vascular beds release a highly potent and stable low molecular weight substance(s) that rapidly and reversibly inhibits actin-activated cardiac myosin ATPase activity, crossbridge cycling, and thus contraction. This inhibitory activity is specific for striated muscle myosin and is independent of sarcolemmal receptors, intracellular messengers, cytosolic Ca²⁺, or pH. These findings may have important implications for the role of endothelial cells in the regulation of myocardial oxygen supply-demand balance and be relevant to clinical conditions such as myocardial hibernation.

	Materials and Methods

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Endothelial Cell Culture
Endothelial cells were isolated from the following sources as described previously^{3 5 8 12} : sheep right ventricular endocardium, sheep conduit pulmonary artery, porcine aorta, and porcine right ventricular endocardium. Briefly, cells were isolated using 0.1% collagenase (type 1A, Sigma Chemical Co) and plated in medium 199 (Sigma) supplemented with 10% fetal bovine serum (Sigma), 10% newborn serum IV (Collaborative Research), L-glutamine (2 mmol/L), benzylpenicillin (50 U/mL), and streptomycin (50 µg/mL). Cells were transferred between passages 2 and 5 to siliconized stirrer vessels or roller bottles containing 3 to 4 mL of microcarrier beads and maintained on beads for 9 weeks before the experiments. Endothelial cell purity was confirmed before and after passage to beads by the presence of factor VIII–related antigen, uptake of rhodamine-labeled acetylated low-density lipoprotein in >95% of cells, and the total absence of cytokeratin.⁸

Endothelial Cell Superfusion
Paired identical aliquots of confluent cells on microcarrier beads (3 to 4 mL=10 to 14x10⁶ cells) were washed with HEPES-buffered physiological saline (mmol/L: NaCl 137, KCl 4.9, MgSO₄ 1.2, NaH₂PO₄ 1.2, glucose 15, HEPES 20, and CaCl₂ 1, pH 7.4). Beads were placed in cartridges with 0.8-µm filters and continuously superfused with this solution at 1 mL/min (37°C) for 7 hours. Both cartridges were initially superfused with normoxic physiological saline (equilibrated with room air; Po₂, 160 mm Hg). After 1 hour, the solution superfusing one cartridge was changed to hypoxic physiological saline (equilibrated with 95% N₂/5% O₂; PO₂, 40 to 50 mm Hg), whereas the other cartridge remained superfused with normoxic solution. In some experiments, after 3 hours of hypoxic superfusion, the solution was changed back to normoxic saline (ie, cells were reoxygenated). Matched single-pass superfusates of both cartridges were collected over consecutive 60-minute intervals. The ionic composition (Na⁺, K⁺, Mg²⁺, Cl^-, and Ca²⁺), osmolality, and pH of superfusates were not significantly altered during the periods of study. Endothelial viability was also unaltered during either normoxic or hypoxic superfusion, or after reoxygenation, as assessed by trypan blue exclusion and the absence of LDH in the superfusates. Precise control of temperature, pH, and PO₂ during endothelial perfusion was critical, with significant variability in results being noted in pilot studies if these parameters were not optimally maintained. Superfusates were studied either fresh or after storage at -70°C for 4 months; results were essentially similar for fresh or stored superfusates. Before any testing on assay tissues, all superfusates were carefully reequilibrated for temperature, PO₂ (160 mm Hg), and pH. Thus, regardless of the conditions at the time of superfusate collection, all myocardial assays were performed under identical conditions (in particular, PO₂ at 160 mm Hg).

Cardiac Myocyte Isolation and Assessment of Function
Ventricular myocytes were isolated from adult Wistar rats using previously described methods involving Ca²⁺-free collagenase digestion of isolated hearts.^{21 22} Two inverted fluorescence microscopes were used: one set up for dual excitation and the other for dual emission. Myocytes were thus loaded with one of the following fluorescent probes as described previously: indo 1-AM or fura 2-AM for assessment of intracellular Ca²⁺,^{13 22} indo 1 free acid for assessment of cytosolic Ca²⁺,²³ and SNARF 1-AM for assessment of cytosolic pH.²⁴ After the loading procedure, myocytes were kept at room temperature for 1 to 6 hours in HEPES-buffered saline until use.

Single adherent myocytes were studied in a chamber on the stage of the microscope according to previously established criteria (ie, they were rod-shaped and free of membrane blebs or granulation, with <1 spontaneous contractile wave per minute and a stable contraction pattern).²² The chamber was superfused with HEPES-buffered saline or test solutions (PO₂, 160 mm Hg) at 0.5 to 1 mL/min. Experiments were performed at room temperature (25°C) to minimize cell leakage of fluorescent probes. Myocytes were field-stimulated at 0.5 Hz unless stated otherwise. Cell length was monitored either by a custom-designed photodiode array system or by video edge detection (Crescent Electronics).^{13 22} The amplitude of unloaded twitch contraction is reported as percent reduction in diastolic (resting) cell length. Indo 1 fluorescence was excited at 350 nm, and the 410/490-nm fluorescence emission ratio was used as an index of intracellular Ca²⁺. Fura 2 fluorescence was excited at 340 and 380 nm, and emission was measured at 510 nm; the ratio of 510-nm fluorescence after excitation at these two wavelengths (340/380-nm ratio) provided an index of intracellular Ca²⁺. Calculations of cytosolic Ca²⁺ were only made in cells loaded with indo 1 free acid; calibration was precluded in indo 1-AM– or fura 2-AM–loaded cells because of the uncertain subcellular compartmentation of these probes.^{22 23} SNARF fluorescence was excited at 530 nm, and the 590/640-nm emission ratio was used to calculate cytosolic pH. Data files (fluorescence and cell length) of 6 to 10 consecutive steady state beats recorded at intervals were averaged for analyses.

For assessment of the steady state relationship between cell shortening and intracellular Ca²⁺ in intact single myocytes, cells were pretreated with the irreversible sarcoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (0.2 µmol/L for 15 minutes) and then repetitively stimulated at 10 Hz for 10 to 20 seconds.²⁵ This procedure results in reproducible tetani in which a stable elevation of cytosolic Ca²⁺ is accompanied by a stable shortening of the cell for the period of stimulation.^{13 25}

In Vitro Motility and ATPase Assays
The direct interaction between actin filaments and myosin molecules, independent of regulatory proteins and Ca²⁺, was studied by means of in vitro motility assays.^{26 27} Cardiac and smooth muscle myosin purified from rat heart and turkey gizzard, respectively, were either used immediately or stored at -30°C for 4 weeks in a 1:1 (vol/vol) mixture of glycerol and 500 mmol/L KCl, 10 mmol/L phosphate buffer (pH 7.0), 1 mmol/L MgCl₂, and 1 mmol/L DTT. Actin was purified from rabbit psoas muscle.²⁶ Motility assays were performed as follows: Myosin, dissolved in 0.5 mol/L NaCl, 10 mmol/L MOPS (pH 7.0), 0.1 mmol/L EGTA, and 1 mmol/L DTT, was introduced into a flow cell on a glass slide. After 60 seconds, the flow cell was washed with 2 to 3 vol of the same buffer containing 0.5 mg/mL bovine serum albumin and then with a lower ionic strength buffer to wash off unbound myosin. Two volumes of 20 nmol/L rhodamine phalloidin–labeled actin^{26 27} in 20 mmol/L KCl, 10 mmol/L MOPS (pH 7.2), 5 mmol/L MgCl₂, 0.1 mmol/L EGTA, and 10 mmol/L DTT was applied to the flow cell. After 30 to 60 seconds, the reaction was started by adding 2 vol of motility buffer (20 mmol/L KCl, 10 mmol/L MOPS [pH 7.2], 5 mmol/L MgCl₂, 1 mmol/L ATP, 0.1 mmol/L EGTA, 10 mmol/L DTT, 0.7% methylcellulose, 2.5 mg/mL glucose, 0.1 mg/mL glucose oxidase, and 0.02 mg/mL catalase). The glass slide was placed on a temperature-controlled stage (25°C) of a fluorescence microscope, and the sliding of actin filaments over myosin was recorded on video from several fields on each slide. A custom-designed automated motion analysis system was used to quantify the rate of translocation.²⁶ In this assay, actin filaments have a range of sliding velocities, which were plotted in histograms (eg, Fig 9). Each histogram represents pooled data from at least four assays and at least 50 filaments per test solution.

View larger version (67K): [in this window] [in a new window]   Figure 9. Effect of endothelial cell superfusates on actin-activated cardiac or smooth muscle myosin ATPase activity. HMM indicates heavy meromyosin; H-EC, hypoxic endothelial superfusate; and N-EC, normoxic endothelial superfusate. Dilutions of superfusates in assay buffer are as indicated. Control assays were performed using an identical dilution of HEPES buffer. Five determinations were performed for each sample using different batches of cardiac and smooth muscle myosin. *P<.05 vs control ATPase activity.

Cardiac myosin subfragment 1 or smooth muscle heavy meromyosin ATPase assays were performed by detection of radiolabeled P_i product.²⁸ Reaction buffer (5 mmol/L MgCl₂, 15 mmol/L Tris [pH 7.5], and 0.1 mmol/L EGTA) was added to equal amounts of myosin and actin to reach the final concentration of 20 to 100 µmol/L. While the solution was stirred at 25°C, the reaction was started by adding 10 mmol/L ATP (final concentration, 1 mmol/L). Aliquots (100 µL) were taken out at 0.5, 1, 1.5, 2, 4, and 8 minutes, and the reaction was stopped by adding 0.6 mL of a solution containing 5 mol/L H₂SO₄, 6% silicotungstic acid, and 1 mmol/L KP_i. After addition of 1 mL of 1:1 isobutanol/benzene and 200 µL of 5% ammonium molybdate, the solution was vortexed vigorously, and 0.5 mL of the top phase, containing the radiolabeled inorganic phosphate product, was counted in a scintillation counter.

Skinned-Fiber Studies
Skinned striated muscle fibers were studied as described previously.²⁹ Briefly, human soleus muscle bundles were placed overnight at 2°C in "skinning solution" of the following composition (mmol/L): K⁺ 140, EGTA 4, imidazole 30, ATP 5, Mg²⁺ 1, leupeptin 0.1, and 2-mercaptoethanol 1, pH 7.1. They were then stored in a solution that also contained 50% glycerol at -20°C. For experiments, single fibers were dissected and mounted in T clips photoetched from aluminum foil. They were suspended in a relaxing solution (pCa 8.0) between a force transducer (Akers AE801, SensoNor) and a servo motor system (model 300B, Cambridge Technologies, Inc). Fully activating solution (pCa 4.3) was of the following composition (mmol/L): EGTA 4, creatine phosphate 15, ATP 5, P_i 1, free Mg²⁺ 1, free K⁺ 100, and free Na⁺ 10, pH 7.1, with an ionic strength of 0.2 mol/L. Imidazole in concentrations >30 mmol/L was the pH buffer. Methyl sulfonate was the major anion and was in excess of 70 mmol/L.

Sarcomere uniformity was maintained by periodic rapid unloaded shortenings followed by rapid relengthening. Control of muscle length was performed using custom software.²⁹ Force and length data were sampled at 5 kHz and saved on computer disk for off-line analysis using custom software. Isometric force was measured before each shortening. Maximum velocity of shortening was determined by the Edman slack test,³⁰ using nine very rapid shortening steps at varying sizes between 4% and 12% of total fiber length. For stiffness measurements, sinusoidal length perturbations with an amplitude of <0.05% of the total fiber length were imposed on the muscle using driving frequencies between 0.02 Hz and 1 kHz. Data were sampled at up to 16 kHz. Analysis of stiffness involved taking the fast Fourier transform of the length and force signals and computing their magnitude ratio and phase difference at each frequency. The magnitude ratio of the force amplitude to the amplitude of the length change was normalized to fiber length and cross-sectional area. Determination of this dynamic stiffness at each frequency allowed the description of its impedance spectra over the entire frequency range. At high frequencies approaching 1 kHz, the length changes exceed the crossbridge cycling rate, and stiffness is determined by the number of crossbridges in a high force state. At frequencies below 0.1 Hz, crossbridges can cycle in response to small length changes, but the measured stiffness increases as the imposed length changes exceed their cycling rate.

At the end of experiments, ß-MHC composition of each fiber was determined by SDS-glycerol gel electrophoresis. Only fibers with 100% ß-MHC (ie, no fast MHC) were included for study.

Materials
Fluorescence probes were purchased from Molecular Probes. Thapsigargin, W7, KT5823, KT5926, and pertussis toxin were from Calbiochem, and Centricon MWCO 500 cutoff filters were from Amicon. All other chemicals and reagents were from Sigma.

Statistics
Data are expressed as mean±SE except where indicated otherwise. Comparisons were made by Student's paired t test using absolute values (even where data are expressed as percent changes) or by unpaired t test as appropriate.

	Results

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Superfusates of Hypoxic Endothelial Cells Inhibit Myocyte Contraction
Addition of the superfusate of hypoxic endothelial cells to isolated rat cardiac myocytes resulted in a rapid inhibition of contraction and a marked decrease in resting (diastolic) myocyte length. Contractile inhibition ranged from total abolition of twitch (Fig 1A) to more modest inhibition of 50% (Fig 1B). These effects were not accompanied by significant changes either in the cytosolic Ca²⁺ transient or in diastolic Ca²⁺ levels. Washout of the hypoxic endothelial cell superfusate resulted in rapid recovery of myocyte shortening, but there was often a transient further reduction in diastolic length, which tended to recover more slowly.

View larger version (12K): [in this window] [in a new window]   Figure 1. Representative examples of the range of effects of hypoxic endothelial cell superfusate (H-EC) on isolated rat cardiac myocytes loaded with indo 1 free acid. A, Effect of very potent superfusate, collected after 3-hour hypoxia. B, Effect of less potent superfusate, collected after 1-hour hypoxia, on another myocyte from same batch. Top panels show slow-speed chart recordings of myocyte contraction. Bottom panels show expanded-scale Ca2+ transients and contractions from the indicated time points.

Fig 2 presents pooled data indicating the effects of superfusate of hypoxic endothelial cells on myocyte contractile parameters and the response to washout in 25 myocyte assays. Depression of myocyte contraction was associated with significantly reduced velocities of myocyte shortening and relengthening (relaxation) and large increases in the time to peak twitch shortening and the time from peak shortening to 50% relaxation (relaxation half-time). However, there were no changes in diastolic or systolic fluorescence ratio (an index of intracellular Ca²⁺) or in the time to peak systolic fluorescence. All parameters except diastolic myocyte length recovered rapidly upon washout of the hypoxic superfusate. The diastolic length did recover to baseline values, followed more prolonged washout (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 2. Percent change (mean±SE) in myocyte contractile parameters upon exposure to hypoxic endothelial superfusate (H-EC) and after a 5-minute washout period (W) (n=25 myocyte assays). PS indicates peak twitch shortening; Vs, velocity of twitch shortening; Vr, velocity of twitch relengthening; tPS, time to peak twitch shortening; tR0.5, time from peak to 50% twitch relengthening; FRd, diastolic fluorescence ratio; FRs, peak systolic fluorescence ratio; and tFRs, time to peak systolic fluorescence ratio. *P<.05 versus basal conditions.

As described previously,¹² the identically collected superfusate of normoxic endothelial cells induced a small depression of myocyte contraction (Fig 3). To compare the normoxic and hypoxic endothelial cell effects, matched superfusates of normoxic and hypoxic endothelial cells were collected, identical in every respect (ie, endothelial cell source, passage number, duration of superfusion, day of collection, and conditions of storage) except for the difference in PO₂ during cell superfusion. Individual pairs of matched superfusates were tested in random order each on a single myocyte, with an intervening washout period sufficiently long for myocyte function to return to basal values. Fig 4 shows mean data from seven such paired myocyte assays. In contrast to normoxic superfusate, hypoxic endothelial superfusate induced much greater reduction in twitch amplitude (42% to 100%), reduced shortening velocity, markedly prolonged the time to peak shortening and relaxation half-time, and decreased myocyte diastolic length. After serial dilutions of potent hypoxic endothelial cell superfusate, a "hypoxic" effect (ie, depressed contraction with a reduction in myocyte diastolic length) was not converted to a "normoxic" effect (ie, depressed contraction with an increase in diastolic length), suggesting that the difference between these two effects was not simply one of concentration or dose (Table 1).

View larger version (14K): [in this window] [in a new window]   Figure 3. Representative example of the effect of normoxic endothelial cell superfusate (N-EC) on an isolated rat cardiac myocyte loaded with indo 1 free acid. Top, Slow-speed chart recording of myocyte contraction. Bottom, Expanded-scale Ca2+ transients and contractions from the indicated time points.

View larger version (29K): [in this window] [in a new window]   Figure 4. Comparison of effect of matched normoxic (N-EC) and hypoxic (H-EC) endothelial cell superfusates on paired myocyte assays (n=7). See text for experimental details. PS indicates peak twitch shortening; Vs, velocity of twitch shortening; tPS, time to peak twitch shortening; tR0.5, time from peak to 50% twitch relengthening; and FRA, fluorescence ratio amplitude. *P<.05 vs N-EC.

View this table: [in this window] [in a new window]   Table 1. Effects of Serial Dilution of Hypoxic Endothelial Cell Superfusate

Effect of Duration of Hypoxia and Reoxygenation
To assess the time course of appearance of characteristic hypoxic endothelial cell superfusate activity over and above normoxic endothelial superfusate, individual myocytes were first exposed to normoxic endothelial superfusate and then to matched hypoxic endothelial superfusate without an intervening washout. The relative change following exposure to the hypoxic cell superfusate was taken as the net "hypoxic effect." Fig 5A shows that a hypoxic effect was apparent within the first hour of hypoxic superfusion, and maximal inhibitory activity was generally observed by the second or third hour.

View larger version (11K): [in this window] [in a new window]   Figure 5. Effect of duration of endothelial cell hypoxia (A) and reoxygenation (B) on cardiac myocyte contraction. The percent change in myocyte peak shortening (PS) and in amplitude of the fluorescence transient (FR) represents the net effect of hypoxic or reoxygenated cell superfusate relative to matched normoxic cell superfusate. Numbers next to error bars indicate the number of myocyte assays. H-1 through H-5 indicates the duration of endothelial hypoxia; R-1 through R-3, the duration of reoxygenation in hours. *P<.05.

Fig 5B shows the effects of reoxygenation of endothelial cells after 3 hours of hypoxia, a time point when "hypoxic" inhibitory activity was maximal. These data are derived from a single endothelial cell superfusion study, to avoid potential complicating effects of variability between cell superfusions. Myocytes were exposed first to matched normoxic endothelial superfusate as described above, so that the results represent the net effect of hypoxia or reoxygenation. Endothelial cell reoxygenation resulted in a rapid loss of inhibitory activity within the first hour of reoxygenation. Thus, the production/release of myocyte inhibitory activity by endothelial cells was rapidly responsive both to hypoxia and reoxygenation.

Influence of Endothelial Cell Source
Similar effects on myocyte contraction were observed with the superfusates of cultured sheep right ventricular endocardial endothelial cells, sheep conduit pulmonary artery endothelial cells, porcine aortic endothelial cells, and porcine right ventricular endocardial endothelial cells (Table 2). The data for pig aortic and endocardial endothelial cells represent the net effect of hypoxic superfusate relative to normoxic superfusate (as described above). Since normoxic superfusate reduces twitch contraction by 20% on average (Fig 3), the net effect of hypoxic superfusate was similar for all four cell types. No differences in pattern of myocyte response were observed between experiments using endothelial cells cultured on beads for between 1 and 9 weeks (data not shown).

View this table: [in this window] [in a new window]   Table 2. Effect of Hypoxic Endothelial Cell Superfusates on Myocyte Contraction

Effect on Ca²⁺-Myofilament Interaction in Intact Myocytes
Inhibition or abolition of myocyte contraction by hypoxic endothelial cell superfusate in the absence of a parallel reduction in cytosolic Ca²⁺ transients suggests a subcellular mechanism involving alterations in myofilament properties. The relationship between cytosolic Ca²⁺ and the myofilaments in intact single cardiac myocytes was assessed during twitch contraction³¹ and steady state tetani,^{13 25} using myocyte shortening as an index of myofilament activation. Fig 6 shows typical phase-plane plots of instantaneous myocyte length versus cytosolic Ca²⁺ during twitch contraction before and during exposure to endothelial superfusates. In the presence of hypoxic endothelial superfusate, myocyte length at rest and during the rising phase of the Ca²⁺ transient was markedly shorter, but there was little further reduction in length during the shortening phase of the twitch. By contrast, in the presence of normoxic endothelial superfusate, myocyte length was longer at all points during the twitch.

View larger version (11K): [in this window] [in a new window]   Figure 6. Representative phase-plane plots of instantaneous myocyte length vs cytosolic Ca2+ during twitch contraction. A, Effect of hypoxic endothelial cell superfusate (H-EC) on Ca2+-myofilament interaction. B, Effect of normoxic endothelial cell superfusate (N-EC) on Ca2+-myofilament interaction. "Loops" proceed counterclockwise, with shortening denoted by the ascending limb and relaxation (relengthening) by the descending limb. C indicates baseline control in each case. See text for explanation.

Fig 7 shows the effects on tetanic contraction. In the presence of hypoxic endothelial superfusate, myocyte length was markedly reduced at rest, with little further shortening during the tetanus despite unaltered peak tetanic Ca²⁺. By contrast, in the presence of normoxic endothelial superfusate, myocyte length was greater at rest and during tetanic contraction. In five myocytes, hypoxic superfusate decreased the amplitude of tetanic shortening by 87.3±5.6% and reduced resting length by 4.2±0.9 µm (both P<.05) but did not alter the amplitude of the Ca²⁺ transient (-0.6±1.4%, P=NS).

View larger version (13K): [in this window] [in a new window]   Figure 7. Representative examples of effect of hypoxic (H-EC, panel A) and normoxic (N-EC, panel B) endothelial cell superfusates on steady state Ca2+-myofilament interaction in intact single myocytes. Top panels show tetanic elevation of Ca2+; the bottom panels, tetanic shortening. C indicates baseline control in each case.

Nature and Subcellular Mechanism of Hypoxic Superfusate Activity
The inhibitory activity of hypoxic endothelial cell superfusates was stable at -70°C for >3 months; over 50% of the experiments reported above were performed using stored superfusates. Activity was not destroyed by heating at 95°C for >30 minutes (Table 3). The activity was present exclusively in molecular weight fractions of <500 after ultracentrifugation through Centricon MWCO 500 cutoff filters (Table 3).

View this table: [in this window] [in a new window]   Table 3. Analysis of Signaling Pathway of Hypoxic Endothelial Cell Superfusate

Pending full chemical characterization of the substance(s) responsible for the effects of hypoxic endothelial superfusate, studies were undertaken to further explore its subcellular action. Myofilament properties are generally altered by extrinsic biological agents through receptor- or enzyme-mediated changes in activity/levels of intracellular messengers, eg, Ca²⁺, cAMP, cGMP, protein kinase C, myosin light chain kinase, and pH.³² In myocytes loaded with SNARF-1, no change in cytosolic pH was observed during superfusate-induced contractile inhibition (change, -0.01±0.00 pH units; n=6 myocytes; P=NS). Hypoxic endothelial superfusate effects were not abolished by inhibitors of cAMP-dependent protein kinase, cGMP-dependent protein kinase, myosin light chain kinase, protein kinase C, or pertussis toxin–sensitive G proteins, used at doses previously shown to be effective at inhibiting these pathways (Table 3).^{12 33 34 35}

Hypoxic Endothelial Superfusates Inhibit In Vitro Actin Motility and Actin-Activated Cardiac Myosin ATPase Activity
Since a primary involvement of common subcellular signaling pathways appeared unlikely, a direct action on myofilaments was investigated. We studied the effects of the active low molecular weight (<500) fraction of hypoxic endothelial superfusate. In Fig 8A and 8B, histograms are shown of in vitro actin sliding velocities over cardiac myosin in the presence either of control HEPES-buffered physiological saline (diluted 1/10 in motility buffer) or of hypoxic endothelial superfusate (diluted 1/25 to 1/30 in identical motility buffer). Any single coverslip could be used for only one assay, since washout of test solution usually resulted in some loss of filaments. Comparisons were therefore performed in parallel assays. In the presence of hypoxic superfusate (1/30 dilution or lower), the majority of actin filaments became virtually immobile (velocities 0.2 µm/s are indistinguishable from zero in this assay), indicating inhibition of crossbridge cycling. Washout of hypoxic superfusate from coverslips resulted in an immediate recovery of actin motility, but this effect was not quantified because we could not totally exclude the possibility that immobile filaments were preferentially washed off. Normoxic endothelial superfusates studied in a similar manner had no effect on actin sliding velocity over cardiac myosin (Fig 8C). In contrast to the cardiac myosin assays, identical hypoxic superfusate fractions had no measurable effect on actin translocation over smooth muscle myosin even at low dilution (1/10) (Fig 8D).

View larger version (61K): [in this window] [in a new window]   Figure 8. Effect of endothelial cell superfusates on in vitro actin sliding velocity over cardiac or smooth muscle myosin. H-EC indicates hypoxic endothelial superfusate; N-EC, normoxic endothelial superfusate. Superfusates were diluted in motility buffer as indicated. Control assays were performed using an identical dilution of HEPES buffer in motility buffer. Each histogram represents pooled data from at least four assays and at least 50 filaments per test solution.

Fig 9A shows the effects of endothelial superfusates or control HEPES-buffered physiological saline (each diluted 1/25) on the rate of actin-activated cardiac myosin subfragment 1 ATPase activity. Hypoxic superfusate caused potent inhibition of actin-activated cardiac myosin ATPase, whereas normoxic superfusate had no effect. In parallel assays performed with smooth muscle heavy meromyosin, no inhibition of actin-activated myosin ATPase was observed either with hypoxic or normoxic endothelial superfusate (Fig 9B).

Effect of Hypoxic Endothelial Cell Superfusate on Skinned Fibers
In order to characterize muscle mechanics in more detail, the effects of the active low molecular weight (<500) fraction of hypoxic endothelial superfusate (diluted 1/10 to 1/40 in activating or relaxing solution) were studied on skinned fibers. We studied human soleus muscle fibers, which contain ß-MHC as do cardiac fibers. In contrast to cardiac fibers, soleus fibers contain skeletal actin and different isoforms of C-protein and troponin subunits. However, since cardiac myosin seemed to be the essential component in the effects observed in the in vitro motility studies, we elected to use this source of human tissue since human cardiac fibers were not available to us.

In fully activated fibers, a 1/30 dilution of hypoxic endothelial superfusate significantly decreased maximal velocity of shortening to 51±9.5% of control values (n=7, P=.025) but caused no change in maximal isometric tension (123±34.0% of control values). The high-frequency (1-kHz) stiffness was also unaltered by hypoxic endothelial superfusate (87±13.7% of control values). This could be an indication that the number of attached crossbridges was unaltered from maximal Ca²⁺ activation, if individual crossbridge stiffness was unchanged. There was no evidence of activation of fibers when exposed to an identical dilution (1/30) of hypoxic endothelial superfusate in relaxing solution (data not shown).

The dynamics of crossbridge cycling were assessed by comparing the entire impedance spectrum (dynamic stiffness) for a fiber in relaxing solution and activating solution and with hypoxic endothelial superfusate diluted in activating solution (Fig 10). The impedance of the relaxed fiber is very low and subject to considerable noise because of the very low force variation. Fiber activation increases the impedance at high frequencies, whereas impedance remains low at frequencies where crossbridges cycle. Exposure to activating solution containing a 1/10 dilution of hypoxic endothelial superfusate markedly increased the impedance magnitude across the entire frequency spectrum. There was a nearly complete loss of the dip of impedance magnitude at 0.1 to 0.2 Hz, which is present in the normally activated fiber.

View larger version (14K): [in this window] [in a new window]   Figure 10. Representative effect of hypoxic endothelial cell superfusate (H-EC, 1/10 dilution in activating solution) on impedance across the frequency spectrum of a skinned soleus muscle fiber. Similar spectra were observed in three other fibers. The impedance magnitude is the ratio of the amplitude of sinusoidal force to the amplitude of sinusoidal length change, normalized to fiber length.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that upon exposure to acute moderate hypoxia, cultured endothelial cells release an unidentified substance(s) that potently but reversibly inhibits actomyosin crossbridge cycling and thus myocardial contraction via a direct action on the myofilaments. Although the chemical identity of this substance(s) has not been determined, the activity is found in low molecular weight fractions (M_r, <500) and is resistant to heating at 95°C, suggesting that it is not a polypeptide. To the best of our knowledge, such a subcellular action has not previously been reported for a paracrine agent released by living cells. The activity is not attributable to generally recognized cardioactive or vasoactive factors released by endothelial cells (eg, nitric oxide, adenosine, endothelin, and prostanoids) on the basis of molecular weight, heat stability, lack of requirement for subcellular signaling pathways (eg, Ca²⁺, pH, cGMP, cAMP, protein kinase C, and myosin light chain kinase), and the efficacy in skinned fiber and in vitro motility assays.

Effects on Myocardial Contraction and Relation to Underlying Mechanism
In isolated cardiac myocytes, hypoxic endothelial superfusate inhibits cell shortening and reduces diastolic length. In the in vitro motility assay, actin translocation over cardiac myosin is dramatically reduced. In skinned soleus fibers, maximum unloaded shortening velocity is depressed, but maximal isometric force is not significantly altered. A mechanism that would account for these observations would be one in which the hypoxic superfusate inhibits the transition of affected crossbridges out of high force states and depresses actomyosin ATPase activity.^{32 36 37} Inhibition of crossbridge detachment would inhibit cycling and reduce myocyte and skinned fiber unloaded shortening and in vitro actin filament motility. Impaired detachment of strongly attached crossbridges would also explain the reduction in diastolic myocyte length, since these crossbridges could act as a ratchet by preventing relengthening during relaxation (the forces to restore the length of an unloaded myocyte are low compared with forces generated by crossbridges during contraction). Consistent with such a mechanism, assessment in skinned fibers of impedance across the frequency spectrum (0.02 to 1000 Hz) reveals a dramatic increase in impedance magnitude across the entire frequency spectrum, although the 1-kHz stiffness is unchanged. The latter observation may indicate that the total number of crossbridges is not increased with the hypoxic superfusate. The pattern of effect of the hypoxic superfusate rather resembles the latch state of smooth muscle, in which force is maintained but the crossbridges turn over more slowly.³⁸ Intracellular ATP depletion, which leads to energy-depletion rigor,^{39 40 41} is implausible in the present studies, since myocardial oxygenation was normal and cytosolic Ca²⁺ transients were entirely unchanged in the isolated myocyte studies. Furthermore, a muscle in rigor would not be expected to shorten at any significant rate. The response to hypoxic endothelial superfusate also differs from the effects of P_i, which increases actin sliding velocity in vitro²⁶ and reduces myofilament responsiveness to Ca²⁺.⁴²

An intriguing finding is that the action of hypoxic endothelial superfusate appears to be specific for striated muscle myosin, since assays using smooth muscle myosin show no effect of the superfusate. Consistent with the hypothesis that this specificity is related to the myosin, the superfusate is effective in skinned soleus fibers, which contain ß-MHC (as do cardiac fibers) but which contain different isoforms of C-protein and troponin subunits. It remains possible, however, that the precise pattern of effect may vary subtly between skinned cardiac and slow skeletal fibers in view of the above differences in myofilament composition.

The marked reduction in resting myocyte length with hypoxic superfusate is analogous to diastolic contracture.^{39 41 43} Traditionally, diastolic contracture has been considered to reflect irreversible myocardial damage and has been used as a functional marker of such damage.⁴³ The present study suggests that there are circumstances in which "diastolic contracture" does not reflect myocardial damage and may even be a feature of a cardioprotective process (see below). Intriguingly, an association between diastolic contracture and cardioprotection has been reported in ischemic preconditioning.⁴⁴

Endothelial Cell Response to Hypoxia
Endothelial cells have a much lower energy demand than beating myocardium and a large capacity for glycolytic energy production.⁴⁵ Respiration of cultured endothelial cells is reported to be independent of an exterior PO₂ of >3 mm Hg in the presence of glucose, and both high-energy phosphate content and cell viability are maintained for several hours during severe hypoxia (PO₂, <10 mm Hg).⁴⁵ On the other hand, moderate hypoxia (PO₂, 30 to 80 mm Hg in buffer-perfused systems) induces both acute and chronic changes in endothelial function, independent of metabolic effects. For example, luminal hypoxia induces the acute (within minutes) release of vasodilator prostanoids and nitric oxide in perfused arteries^{14 15} as well as the intact coronary circulation.^{16 17 18} Chronic moderate hypoxia causes increased endothelial expression of proteins such as endothelin-1,⁴⁶ glyceraldehyde-3-phosphate dehydrogenase,⁴⁷ and vascular endothelial growth factor.⁴⁸ These responses have been postulated to represent an "oxygen-sensing" function of endothelial cells^{47 49} ; the underlying subcellular mechanism(s) remains yet to be fully defined. No assessment of endothelial energetic status was made in the present study, but cells remained viable throughout the study. Our findings are thus consistent with another oxygen-sensing function of endothelial cells, namely, the acute release of a potent cardioactive substance(s) rapidly responsive both to reduction in PO₂ and to reoxygenation. We observed no specificity for endothelial cell type (at least in culture), since similar effects were observed with cells cultured from both cardiac and noncardiac vascular beds from two different species. However, no comment can be made about the responses of different vascular beds in vivo to a similar hypoxic stimulus.

Relevance to Regulation of Myocardial Oxygen Supply-Demand Balance
The findings of the present study may have implications for the regulation of oxygen homeostasis in the heart. Myocardial oxygen supply is determined by coronary flow rate and the oxygen content of coronary blood, whereas the major determinant of oxygen consumption is contractile function. A close coupling normally exists between oxygen consumption and coronary flow rate.⁵⁰ When metabolic demand increases, eg, during exercise, oxygen supply-demand balance is maintained by increases in coronary flow, which involve at least in part the release of adenosine and endothelium-derived vasodilators.⁵⁰ During acute reduction in regional coronary flow (eg, with coronary artery disease), however, oxygen homeostasis may also be facilitated by a stable downregulation of oxygen demand as a result of a proportional decrease in regional contractile function, so-called "perfusion-contraction matching."^{51 52} The latter has been shown in several in vitro and in vivo experimental models to occur without a sustained decrease in high-energy phosphates or phosphorylation potential or evidence of significant lactate production, such that metabolic integrity is preserved at a lower level of myocardial work.^{52 53 54 55 56} Restoration of coronary flow leads to recovery of contractile function. This phenomenon has been termed "myocardial hibernation"⁵⁷ ; the underlying mechanisms remain elusive.

The present study raises the possibility that coronary endothelial cells, sited at the interface between vascular oxygen supply and the oxygen-using tissue, could be involved in an adaptive feed-forward downregulation of contractile function in response to reduction in coronary flow. Inhibition of cardiac actomyosin ATPase and crossbridge cycling by endothelial cells in response to reduced PO₂ would result in decreased energy turnover and oxygen consumption, thus contributing to the setting of a new level of oxygen supply-demand balance. The gain of such a system could be determined at several locations, eg, the number of endothelial cells stimulated, the extent and duration of hypoxia, and the response of myofilaments and myocytes to the inhibitory substance. The relevant level of PO₂ or oxygen supply at which such a mechanism might operate in vivo is difficult to assess from our studies. The oxygen content of crystalloid buffer at a PO₂ of 40 to 50 mm Hg (as in the present study) is significantly lower than that of arterial blood at an equivalent PO₂,⁵⁸ so that direct extrapolations cannot be made. Furthermore, PO₂ varies over a wide range across the vascular bed because of the presence of significant longitudinal oxygen gradients.⁵⁹ The site of release of the inhibitory factor would therefore be pertinent with respect to the PO₂ level at which such a system might operate and its potential physiological or pathophysiological role(s).

In 1976, Frezza and Bing⁶⁰ suggested that PO₂-modulated alterations in myocardial contractile performance could serve a protective function against ischemic injury. After nuclear magnetic resonance studies in hypoxic isolated rat hearts, Matthews et al⁶¹ postulated that inotropic state may be directly responsive to PO₂ through some undefined mechanism. A recent study of the effects of moderate systemic hypoxia (arterial PO₂, 50 mm Hg) in ventilated rats reported a significant reduction in high-energy phosphate turnover, which was postulated to represent a combination of impaired ATP synthesis and reduced ATP utilization.⁶² These data are consistent with our hypothesis. However, it should be noted that reduction in coronary flow not only decreases oxygen supply but may have other consequences (eg, accumulation of metabolites), which could contribute to the overall effect on myocardial contraction.

Conclusions
Increasing evidence supports the presence of a paracrine pathway for the regulation of myocardial contractile function by endothelial cells, often through alterations of myofilament properties rather than changes in cytosolic Ca²⁺.¹ The present study documents another endothelium-derived activity that influences cardiac myofilaments. The release of this substance(s) during moderate hypoxia raises the possibility that endothelial cells may play an important role in the regulation of myocardial oxygen supply-demand balance. The endothelial response to local hypoxia may involve both feedback coronary vasodilatation^{16 17 18} to increase oxygen supply and feedforward contractile depression to decrease local energy utilization. The contribution of such a mechanism to the pathophysiology of "ischemic" syndromes where reduced coronary flow is implicated (eg, myocardial hibernation, ischemic preconditioning, and myocardial stunning) merits further investigation.

	Selected Abbreviations and Acronyms

 

DTT = 1,4-dithiothreitol LDH = lactate dehydrogenase MHC = myosin heavy chain SNARF = carboxy-seminaphthorhodafluor

	Acknowledgments

 
Dr Shah is the recipient of a UK Medical Research Council Clinical Senior Fellowship. This study was also supported by grants from the British Heart Foundation (Dr Shah), the Welsh Scheme for Development of Health & Social Sciences Research (Dr Shah), and National Heart, Lung, and Blood Institute grant RO-1 HL-39138-03 (Dr Robotham). We thank M. Abraham and C. Rix for skillful assistance with cell cultures.

Received October 1, 1996; accepted February 10, 1997.

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