Effects of the Nitric Oxide Donor Sodium Nitroprusside on Intracellular pH and Contraction in Hypertrophied Myocytes
Background We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls.
Methods and Results pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37°C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH=7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10−6 mol/L SNP caused a decrease in the amplitude of cell contraction (72±7% of baseline, P<.05) that was associated with a decrease in pHi (−0.10±0.03 U, P<.05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (−0.01±0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na+-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells.
Conclusions SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na+-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.
Nitric oxide donors and stimulation of the release of NO from endothelial cells depress LV pressure development in association with an earlier onset of relaxation in human studies1 2 and isolated papillary muscle preparations.3 4 Both the exogenous NO donor nitroprusside5 and the induction of NOS in vitro in response to exposure to inflammatory cytokines depress the contractility of paced adult myocytes.6
The effects of NO on the myocardial contractile response are postulated to be predominantly mediated by the intracellular messenger cGMP via a depression of myofilament sensitivity to intracellular Ca2+.2 3 4 5 6 7 8 However, the underlying mechanism is unknown, and the contribution of alterations in cytosolic Ca2+ versus pHi is controversial.
The objective of this study was to test the hypothesis that the effects of the NO donor SNP on cell contraction are mediated by changes in pHi. Because intracellular signaling and recovery from intracellular acidosis may differ in adult normal and hypertrophied cells, we also wished to determine if the response to nitroprusside differed in adult hypertrophied versus normal myocytes. Therefore, we compared the effects of SNP as well as the cGMP analogue 8-bromo-cGMP on myocyte contraction, pHi, and intracellular Ca2+ in isolated normothermic adult ventricular myocytes from aortic-banded rats and normal age-matched controls. We also examined the effects of SNP on recovery from intracellular acidification, which depends on the stimulation of Na+-H+ exchange. Our findings indicate that SNP causes a negative inotropic effect in normal adult myocytes that is predominantly mediated by intracellular acidification, which decreases myofilament sensitivity to Ca2+; in contrast, this effect does not occur in adult hypertrophied cells.
Preparation of Aortic-Banded Rats
Weanling male Wistar rats (Charles River Breeding Laboratories) were banded at the age of 3 to 4 weeks (body weight, 75 to 100 g) by placing a 0.6-mm-ID stainless steel clip on the ascending aorta via a thoracic incision. Age-matched control rats underwent a sham operation. The rats were fed normal rat chow and water ad libitum and were used 12 weeks after banding. We have shown9 10 11 12 that at this stage after aortic banding, this model of pressure-overload hypertrophy is characterized by an increase in LV weight relative to sham-operated controls, concentric hypertrophy, and the absence of chamber dilatation.
Dissociation of LV Myocytes
LV myocyte isolation was performed13 by using a modification of the methods of Capogrossi et al14 and Haddad et al.15 Rats were anesthetized with an injection of pentobarbital sodium (65 mg/kg body wt IP), and the heart was rapidly excised and attached to an aortic cannula. Continuous retrograde coronary perfusion was initiated at a perfusion pressure of 70 cm H2O for control hearts and 100 cm H2O for hypertrophied hearts. The heart was first perfused with nominally Ca2+-free modified Krebs-Henseleit buffer of the following composition (in mmol/L): NaCl 123, KCl 5.4, MgSO4 1.2, NaH2PO4 1.2, NaHCO3 20, and glucose 11. This medium was not recirculated and was continuously gassed with 95% O2 and 5% CO2 (pH 7.4) at 37°C. After 3 minutes of the initial perfusion, the heart was perfused with recirculating Krebs-Henseleit buffer supplemented with 0.6 mg/mL collagenase (class II, Worthington Biochemical Corp), 0.04 mg/mL protease (type XIV, Sigma), and 1 mg/1 mL BSA for 20 to 40 minutes. The heart was then detached from the cannula, the LV was cut into small pieces, and dispersion of the myocytes was performed by gentle agitation of ventricular tissue through a serologic pipette in Krebs-Henseleit buffer containing 100 μmol/L CaCl2 and 1 mg/mL BSA. The resulting suspension was then gently forced through a 450-μm nylon screen filtration cloth into a 50-mL plastic tube and rinsed twice. After this the myocytes were resuspended in HEPES-buffered solution of the following composition (in mmol/L): NaCl 137, KCl 5.4, MgSO4 1.2, NaH2PO4 1.2, HEPES (free acid) 20, CaCl2 1.2, glucose 15, and 5% FBS. The myocytes were stored at 37°C for 1 hour.
Simultaneous Measurement of [Ca2+]i and Cell Contraction
[Ca2+]i was measured with the Ca2+-sensitive fluorescence indicator indo 1-AM16 (Molecular Probes, Inc) as described13 17 by using a modification of the method of duBell et al18 and Ikenouchi et al.19 First, 10 mL FBS was mixed with 234 μL of 25% pluronic F-127 (BASF Wyandotte Corp) dissolved in dimethyl sulfoxide. After this, 1 mL of 1 mmol/L indo 1-AM in dimethyl sulfoxide was added to 9 mL of an FBS–pluronic F-127 mixture, sonicated, and divided into 400-μL aliquots that were stored at 37°C. Myocytes were attached on coverslips with cell adhesive (Cell-Tak, Collaborative Research, Inc) and loaded with 5 μmol/L indo 1-AM in HEPES-buffered solution at room temperature for 30 minutes. The coverslip was rinsed with indo 1-AM–free buffer solution and placed in a flow-through heated (37°C) cell-superfusion chamber on the stage of an inverted microscope (Nikon). The instrumentation for fluorescence measurement has been described.16 17 19 20 The excitation source was a high-pressure mercury arc lamp that provided an intense emission peak at 360 nm. Further selection of this excitation was made with narrow-bandwidth interference filters. The excitation beam was chopped at 360 Hz to reduce bleaching, and the myocyte was illuminated via epifluorescent optics by using a Fluor ×40 objective lens (Nikon). The fluorescence light was collected by the objective lens and transmitted to a custom-modified spectrofluorometer (FM-1000, Rincon Scientific Instruments) for simultaneous measurement of both 400- and 500-nm wavelengths by using two separate photomultiplier tubes. The spectrofluorometer provided analog signals representing the fluorescence intensity at both wavelengths and the ratio of emitted fluorescence (400/500 nm). The subtraction of background autofluorescence was done by offsetting the photomultiplier tube outputs during the measurement of fluorescence from an unloaded myocyte at the beginning of each experiment. An adjustable iris was used to restrict the optical image to only one myocyte of interest in each experiment to minimize background fluorescence from other myocytes. The image of the beating myocyte was obtained by illumination via the 50-W standard microscope light source passed through a 645-nm band-pass filter. This wavelength was long enough not to interfere with the fluorescence detection at 400 and 500 nm. The motion of the myocyte was monitored by using a solid-state camera (GP-CD60, Panasonic) and a custom-modified video detector system20 (Crescent Electronics). The analog output of the cell-motion signal was monitored and recorded continuously with the analog signal of the [Ca2+]i-sensitive fluorescence ratio (F400/500 nm). Two platinum electrodes placed in the bathing fluid were connected to a stimulator (SD9G, Grass Instruments) and were used to stimulate the myocyte at 0.5 Hz with 3-ms pulses.
Myocytes from 21 hearts from sham-operated rats and 16 hearts from aortic-banded rats were studied. The yield of viable myocytes, defined as the percentage of rod-shaped myocytes with clear striations and exclusion of Trypan blue, was 60% to 70% in control myocytes and 50% to 60% in hypertrophied myocytes. To prevent bias when we selected a myocyte to be analyzed among myocytes in a microscopic field, we chose a rod-shaped myocyte with very clear striations, without any spontaneous cell-motion oscillations, and with visually moderate cell-motion amplitude of contraction (5.0 to 10.0 μm) at a pacing rate of 0.5 Hz. This selection procedure was used to counter the potential bias of selecting excessively vigorous rather than depressed myocytes. One to three experiments were performed in sequence from separate coverslips of myocytes isolated from one heart. We have recently documented the stability of both cell motion and [Ca2+]i for the 15-minute duration of the protocols used in the present study and have demonstrated minimal compartmentalization of indo 1 in intracellular organelles in chemically skinned myocytes.13 To determine the absolute values of peak-systolic and end-diastolic [Ca2+]i under baseline conditions in hypertrophied and control myocytes, we performed calibration studies by using a modification of the methods of Cheung et al21 and Borzak et al.22 These methods have been described in detail.13
Measurement of pHi
pHi was measured with the pH-sensitive indicator SNARF-1 AM (Molecular Probes, Inc).17 23 24 25 First, 50 μg SNARF-1 AM was added to 50 μL dimethyl sulfoxide that had been mixed with 450 μL FBS. Loading of SNARF-1 AM was done by exposing the myocytes on coverslips to a final concentration of 8 μmol/L SNARF-1 AM for 15 minutes at 37°C. The coverslip was rinsed with SNARF-1 AM–free solution and placed on a flow-through cell chamber as described above. Excitation was performed at 540 nm, and fluorescence emission was collected simultaneously at 580 and 640 nm by using the same optics system as described above except for the substitution of different dichroic mirror and interference filters. At the end of the experiment, the emission ratio from each cell was calibrated in situ by exposing cells to solutions of varying pH. Each solution contained (in mmol/L) K+ 140 (adjusted to keep [K+] constant), MgCl2 1.0, HEPES 4.0, EGTA 2.0, 2,3-butanedione monoxime 30, BAPTA-AM (Molecular Probes, Inc) 50 μmol/L, and nigericin (Sigma) 14 μmol/L and was titrated to varying pH values (6.7, 6.85, 7.0, 7.2, and 7.4) by using 1.0N KOH. Since changes in pH are not linear to the ratio of fluorescence emission,25 pHi was calculated by the equation pHi=(ax+c)/(1+bx), where x is the measured emission ratio, and a, b, and c are constant parameters.24 25 26
Effects of Nitroprusside on Cell Contraction and [Ca2+]i
Hypertrophied (n=16) and control (n=26) myocytes were superfused with oxygenated HEPES-buffered normal Tyrode’s solution of the following composition (in mmol/L): NaCl 137, KCl 3.7, MgCl2 0.5, HEPES (free acid) 4.0, CaCl2 1.2, glucose 11, and probenecid 0.5 (final pH, 7.40). Probenecid, a blocker of organic anion transport, was added because it inhibits the secretion of both indo 1 and fura 2 free acids from loaded cells.27 The myocytes were maintained at 36°C to 37°C and were paced at 0.5 Hz. After baseline data were recorded, the cells were superfused with an oxygenated HEPES-buffered solution containing SNP (Sigma) over a concentration range of 10−10, 10−8, and 10−6 mol/L. Each SNP concentration was studied in a separate group of myocytes (five to nine cells per group) in the presence of a 1.2-mmol/L [Ca2+] perfusate for 10 minutes. The analog cell-motion signals and the F400:500-nm analog signals were recorded simultaneously. The F400/500 values were converted to [Ca2+]i by using the procedure described in the previous section.
Effects of Nitroprusside on pHi
To determine the effects of 10−6 mol/L SNP on pHi, separate experiments were performed in hypertrophied (n=7) and control (n=7) myocytes loaded with SNARF-1. Myocytes were first superfused with the same HEPES-buffered solution that was used for baseline superfusion to eliminate the effects of Cl−/HCO3− exchange and Na+/HCO3− exchange on pHi. The superfusate was then switched to a solution with 10−6 mol/L SNP for 6 minutes. In additional experiments in hypertrophied (n=11) and control (n=14) myocytes, the effects of an NH4Cl pulse in the presence and absence of 10−6 mol/L SNP was examined by abrupt exposure and washout of HEPES-buffered solution containing 10.0 mmol/L NH4Cl that had been prepared by equimolar replacement of NaCl with NH4Cl. The abrupt exposure to NH4Cl initially increases pHi as basic NH3 rapidly enters the myocyte.24 25 pHi then falls as changed NH4+ enters the cell via K+ channels and dissociates. Upon washout of extracellular NH4Cl, intracellular acidosis is created as internal NH3 leaves the cell, causing the intracellular retention of H+. The initial recovery from this intracellular acidosis depends predominantly on forward Na+-H+ exchange.24 28 To determine the relationship between cell shortening and pHi in the absence of SNP, transient intracellular acidification was induced by a pulse and washout of NH4Cl in additional adult myocytes (n=26). The same protocol was performed sequentially in the myocytes to assess pHi and contraction since optical considerations do not permit simultaneous measurements of SNARF-1 fluorescence and detection of cell motion.
Contribution of Na+-H+ Exchange
To further assess the contribution of Na+-H+ exchange to SNP-induced acidification, additional SNARF-1–loaded control (n=5) and hypertrophied (n=5) myocytes were perfused with 10 μmol/L EIPA (Sigma), which inhibits Na+-H+ exchange. As described above, all experiments were performed using HEPES-buffered solutions with an extracellular pH of 7.40. The pHi was measured at baseline and after 6 minutes of exposure to 10 μmol/L EIPA, followed by 6 minutes of exposure to 10−6 mol/L SNP in the presence of EIPA. To directly examine the effects of inhibition of forward Na+-H+ exchange, control (n=7) and hypertrophied (n=6) myocytes were abruptly exposed to a solution containing 0 Na+–0 Ca2+ (no added Ca2+) in which N-methyl-d-glucamine (Sigma)24 was substituted for NaCl. Calcium was removed to prevent Ca2+ entry via Na+–0 Ca2+ exchange, which may modify pHi. After pHi showed stability (≈15 minutes), the cells were also perfused with 10−6 mol/L SNP for 6 minutes.
Effects of 8-Bromo-cGMP on pHi
To examine the potential role of cGMP in control and hypertrophied myocytes, the effects of the cGMP analogue 8-bromo-cGMP (10−5 mol/L; Sigma) on pHi were compared in control (n=7) and hypertrophied (n=5) myocytes loaded with SNARF-1. The same protocol was performed in an additional series of control (n=5) and hypertrophied (n=6) myocytes to measure cell contractions. The effect of 8-bromo-cGMP on recovery from the intracellular acidosis induced by an NH4Cl pulse was also examined in control (n=11) and hypertrophied (n=13) myocytes.
Two-way ANOVA with repeated measures was used to compare the values measured in response to exposure to SNP or 8-bromo-cGMP for control and hypertrophied myocytes. ANOVA for repeated measures was used to compare the responses of control and hypertrophied cells during recovery from the acidification induced by an NH4Cl pulse. An unpaired Student’s t test was used for comparisons of the values between the groups at baseline. A probability value of <.05 was considered significant. Results are expressed as mean±SEM.
Characteristics of Isolated Normal and Hypertrophied Myocytes
The extent of hypertrophy in the aortic-banded and sham-operated control rats is shown in Table 1⇓; Table 2⇓ shows the baseline function of LV control myocytes from sham-operated rats and hypertrophied myocytes from aortic-banded rats. Myocytes were superfused with oxygenated HEPES-buffered solution (pH 7.40) containing 1.2 mmol/L CaCl2 and were paced at 0.5 Hz at 37°C. Diastolic cell length was slightly increased in hypertrophied myocytes compared with controls. The amplitude of cell shortening and percent fractional cell shortening were not significantly different between the groups. The time to peak shortening and the time to 50% relengthening were slightly prolonged in the hypertrophied cells relative to the control cells. Under the experimental conditions of this study, systolic and diastolic [Ca2+]i values using the indicator indo 1 were similar between the two groups; these values are similar to those reported by us13 and others22 29 in adult rat myocytes. The values of pHi using the indicator SNARF-1 were similar in control (n=19) and hypertrophied (n=15) myocytes; these values are similar to measurements in isolated ventricular myocytes.19 25 26
Effects of SNP on Cell Shortening
The effects of SNP on fractional cell shortening were examined in the presence of 10−10, 10−8, and 10−6 mol/L SNP in separate groups of control and hypertrophied myocytes. SNP caused a maximal negative inotropic effect at a concentration of 10−6 mol/L in the control myocytes that was manifested as a reduction in the amplitude of contraction to 72±7% of baseline (P<.05). In contrast, 10−6 mol/L SNP did not depress fractional cell shortening in the hypertrophied myocytes (87±5% of baseline, NS). Fig 1⇓ illustrates that the depressant effect of SNP on cell shortening in the control myocytes was associated with an abbreviation of the duration of cell contraction and a slight increase in diastolic cell length, whereas these effects were not observed in the hypertrophied myocytes. SNP (10−6 mol/L) significantly decreased the time to 50% relengthening in the control but not the hypertrophied myocytes (Table 3⇓). The effects of 10−6 mol/L SNP on intracellular Ca2+ were examined by using the indicator indo 1 in the presence of 1.2 mmol/L Ca2+. There was no effect of 10−6 mol/L SNP on peak systolic [Ca2+]i in either control (99.4±1.0% of baseline, NS) or hypertrophied (100.2±0.1% of baseline, NS) myocytes, nor was there any effect on the time course of the [Ca2+]i transient (Fig 1⇓).
Effects of SNP on pHi
The effects of 10−6 mol/L SNP on pHi in control and hypertrophied myocytes are shown in Fig 2⇓. At baseline, pHi was similar in control and hypertrophied myocytes (7.10±0.01 versus 7.07±0.02 U, NS). SNP caused a significant fall in pHi (−0.10±0.03 U, P<.05) in the control myocytes. In contrast, the hypertrophied myocytes showed no change in pHi (−0.01±0.01 U, NS) in response to SNP.
Additional experiments were done to determine if a similar degree of intracellular acidification depresses contractility in adult myocytes when it is caused by an intervention other than SNP. Fig 3⇓ shows the relationship between pHi and fractional shortening expressed as a percent of baseline in normal and hypertrophied myocytes obtained during maximal acidification and after an initial 1 minute of recovery after an NH4Cl pulse in the absence of SNP. The mean values of pHi and fractional shortening are redisplayed for the normal myocytes described above, which were studied at baseline and in response to 10−6 mol/L SNP. These data show that there is a linear relationship between the decrease in pHi and the depression of contractility in both normal and hypertrophied myocytes. Thus, the absence of a depressant effect of SNP on contractility in the hypertrophied myocytes is not due to a difference in the relationship between intracellular acidification and contractility in normal and hypertrophied cells. Second, these data show that a similar magnitude of intracellular acidification caused by the intervention of an NH4Cl pulse compared with the intracellular acidification caused by SNP depresses contractility to a similar extent in normal myocytes.
To assess the potential contribution of Na+-H+ exchange on the effects of SNP, additional cells were exposed to 10 μmol/L EIPA for 6 minutes (to inhibit Na+-H+ exchange) and then exposed to 10−6 mol/L SNP for 6 minutes during continued perfusion with EIPA. EIPA alone did not modify baseline pHi in either the control (−0.01±0.01 U, NS) or hypertrophied (−0.01±0.02 U, NS) myocytes. In the presence of EIPA, 10−6 mol/L SNP failed to cause intracellular acidification in either the control or hypertrophied myocytes. To directly compare the contributions of forward Na+-H+ exchange on pHi in the control and hypertrophied myocytes, forward Na+-H+ exchange was abruptly inhibited by exposure to HEPES-buffered solution containing 0 Na+–0 Ca2+. Abrupt inhibition of forward Na+-H+ exchange caused a more rapid and larger intracellular acidification in control than hypertrophied myocytes (Fig 4⇓, left). Steady-state levels of pHi were reached after ≈15 minutes of inhibition of Na+-H+ exchange, after which exposure to 10−6 mol/L SNP caused no further acidification in control or hypertrophied myocytes (Fig 4⇓, right).
To further assess the potential contribution of an NO donor on recovery from intracellular acidosis, additional control and hypertrophied myocytes were exposed to an abrupt pulse and washout of NH4Cl in the presence and absence of 10−6 mol/L SNP. Abrupt exposure to NH4Cl initially increases pHi as basic NH3 rapidly enters the cell.25 26 Charged NH4+ then enters the cell via K+ channels and dissociates. Upon rapid washout of extracellular NH4Cl, intracellular acidosis is created as NH3 leaves the cell, causing the intracellular retention of H+. Recovery from this intracellular acidosis depends predominantly on forward Na+-H+ exchange.24 28 The rate of recovery from maximum intracellular acidification is similar in control and hypertrophied cells at baseline.26 In the presence of SNP, the rate of recovery from the intracellular acidification was depressed in the control compared with the hypertrophied myocytes (P<.004) (Fig 5⇓, left).
Effects of 6-Bromo-cGMP on Cell Shortening and pHi
The effects of NO on myocardial contractions are postulated to be partly mediated by the intracellular messenger cGMP, which activates protein kinase G and modulates cAMP levels via inhibition of cAMP phosphodiesterases. To examine this potential mechanism, additional control and hypertrophied myocytes were exposed to the cGMP analogue 8-bromo-cGMP (10−5 mol/L); steady-state changes in fractional cell shortening and [Ca2+]i were measured after 6 minutes of perfusion. Exposure to 8-bromo-cGMP caused a significant depression of fractional cell shortening in the control myocytes (74±9% of baseline, P<.05) in the absence of a change in peak systolic [Ca2+]i (101±1% of baseline, NS). In the control myocytes, 8-bromo-cGMP caused a significant fall in pHi (−0.04±0.01 U, P<.05), suggesting that its negative inotropic effects were related to intracellular acidification. In contrast, in the hypertrophied myocytes, 8-bromo-cGMP had no effect on fractional cell shortening (97±6% of baseline), peak systolic [Ca2+]i (104±1% of baseline), or pHi (−0.01±0.02 U).
We also studied the effects of 10−5 mol/L 8-bromo-cGMP on recovery from the intracellular acidification induced by an NH4Cl pulse. Like SNP, 8-bromo-cGMP depressed the rate of recovery from acidification in the control compared with the hypertrophied myocytes (Fig 5⇑, right). Thus, the effects of the NO donor SNP and 8-bromo-cGMP on cell shortening, pHi, and recovery from intracellular acidification are similar in normal adult myocytes, whereas these depressant effects on contractility and pHi are not observed in adult hypertrophied myocytes.
In the present study, the NO donor SNP depressed the amplitude of cell shortening and promoted an earlier onset of relaxation in isolated normal adult rat myocytes. The predominant mechanism of this depression of the contractile response was due to myofilament desensitization to calcium due to intracellular acidification rather than changes in intracellular calcium. Moreover, exposure to the cAMP analogue 8-bromo-cGMP caused similar changes in myocyte contractility and pHi in control myocytes. In contrast, the effects of SNP as well as 8-bromo-cGMP were strikingly different in adult hypertrophied myocytes from rats with chronic aortic banding. SNP and 8-bromo-cGMP had no significant effects on myocyte contractility or pHi in hypertrophied myocytes.
Effects of NO on Cardiac Contraction
In the human heart, acute exposure to the exogenous NO donor SNP via intracoronary infusion depresses LV developed pressure in association with an earlier onset of relaxation.1 2 Similar cardiodepressant effects have been observed in humans in response to the intracoronary infusion of substance P, which releases NO from the coronary endothelium.2 In clinical studies and studies using isolated hearts from experimental animals, interpretation of the direct effects of NO donors on contractile function is confounded by potential simultaneous changes in coronary blood flow, which modify coronary vascular turgor and load.
For this reason, several studies have examined the direct effects of NO donors on contractile function in isolated cardiac muscle preparations and cardiac myocytes. Smith et al3 have shown that SNP depresses isometric tension development and causes premature tension decline in isolated papillary muscles. Similar effects on the contractile function of isolated papillary muscles have been observed in response to the paracrine action of endogenous NO released from endocardial cells by stimulation with bradykinin and cytokines.3 4 Weyrich et al30 failed to observe an effect of either NO donors or NOS inhibitors on the contractile function of unstimulated isolated adult rat papillary muscles or myocytes. However, the majority of recent studies have confirmed that NO donors depress adult cardiomyocyte shortening and promote premature myocyte relengthening. Brady et al5 report that SNP depresses the amplitude of isolated myocyte shortening. The induction of NOS in vitro in response to exposure to inflammatory cytokines depresses the contractility of paced adult rat myocytes.6 The depressant effects of NO are even more pronounced in the presence of β-adrenergic stimulation. Both the exposure to NO donors and the induction of endogenous NOS activity by cytokines blunt the enhanced contractile response of isolated myocytes to β-adrenergic agonists,7 31 and similar inhibitory effects of NO on β-adrenergic stimulation have been observed in humans.32 The effects of NO on the myocardial contractile response are postulated to be predominantly mediated by the intracellular messenger cGMP, which activates protein kinase G and modulates cAMP levels via inhibition of cAMP phosphodiesterases7 8 ; however, non-cGMP effects, including the generation of reactive oxygen species, may also occur.33 34
Several downstream mechanisms could potentially contribute to the depressant effects of NO on myocardial contractile function. β-Adrenergic stimulation is well recognized as increasing the amplitude of myocyte contraction and the intracellular calcium transient via increases in the inward L-type calcium current, and NO depresses β-adrenergic–mediated increases in intracellular calcium.35 However, the mechanism of the depressant effect of NO donors on contractility in the absence of β-adrenergic stimulation is controversial. Recent studies in isolated myocytes show that cGMP, acting through protein kinase G or the inhibition of cAMP phosphodiesterase, may decrease the inward L-type calcium current,36 37 38 an effect that could reduce free intracellular calcium. On the other hand, NO has also been reported to modulate calcium release from the sarcoplasmic reticulum in skeletal muscle and papillary muscles,32 39 which would be expected to promote an increase in intracellular calcium. Thus, the net effect of NO on the intracellular calcium transient is difficult to predict.
In the present study, we observed that neither the NO donor SNP nor 8-bromo-cGMP caused steady-state depression of the amplitude of the intracellular calcium transient. In contrast, both SNP and 8-bromo-cGMP decreased the contractile response to calcium in normal adult myocytes. These observations are consistent with the findings of Shah et al,8 who have observed that in intact isolated rat myocytes loaded with the fluorescence indicator indo 1, exposure to 8-bromo-cGMP causes a very transient initial positive inotropic effect and an increase in the calcium transient. These initial effects are followed by a sustained, steady, stable negative inotropic effect that is not associated with a reduction in the amplitude of the calcium transient. Using analyses of steady-state tetanic contracture and the response to KT5823, a relatively specific inhibitor of protein kinase G, they concluded that 8-bromo-cGMP depresses the myofilament response to calcium via activation of protein kinase G, but they did not elucidate the underlying mechanism.8
NO and Intracellular Acidification
Our novel observations provide support for the theory that the predominant mechanism of the negative inotropic effect of NO in adult myocytes is the induction of intracellular acidification. Intracellular acidification is well established as a mechanism that depresses myofilament sensitivity to calcium and causes reductions in tension development and contraction amplitude and an increase in cell length.25 40 41 42 Using washout of an NH4Cl pulse to cause abrupt intracellular acidification, we showed that a similar degree of intracellular acidification caused by an intervention other than SNP depressed contractility to a similar extent in normal adult myocytes. These data lend support to a cause-and-effect relationship between the intracellular acidification caused by SNP and the depression in contractility. Our findings do not exclude the possibility that other factors, such as the presence of inorganic phosphate or the effects of protein kinase G on the calcium sensitivity of troponin C,43 44 may contribute to the modulation of myofilament sensitivity by NO. We observed that inhibition of the sodium-proton exchange with EIPA prevented SNP-induced intracellular acidification in normal myocytes.
In addition, NO markedly impaired recovery from the acute intracellular acidification induced by an NH4Cl pulse in control compared with hypertrophied myocytes, which depends on the stimulation of the forward sodium-proton exchange. These findings suggest that NO, via cGMP, may acutely impair contractility by disabling the forward sodium-proton exchange. In the present study, the direct inhibition of forward Na+-H+ exchange by exposure to a solution containing 0 Na+–0 Ca2+ also caused a more rapid and severe intracellular acidification in control than hypertrophied cells. Since extracellular Ca2+ was excluded, this difference is not related to Ca2+ entry via the Na+-Ca2+ exchange, which can modify pHi.45 Nor is this difference due to differences in intrinsic buffering power (Bi), since we have shown that Bi is comparable in control and hypertrophied cells from this model.26 The source of intracellular proton accumulation in response to disabling the forward Na+-H+ exchange is likely to be related to metabolic acid production, although a small contribution from transient reverse Na+-H+ exchange and passive proton influx due to the membrane potential cannot be excluded.
This study has several potential limitations. The changes in contraction amplitude and the earlier onset of relaxation are similar to changes observed in isolated papillary muscles and isolated hearts in response to “physiologically” derived NO from endothelial and endocardial cells. Nonetheless, it is important to recognize that the actual concentration of biologically active NO delivered to the myocyte target in response to exposure to SNP is difficult to estimate and to correlate with in vivo concentrations.46 Second, the effects of exogenous NO donors on contractility may be modulated or blunted by activation of the constitutive calcium-sensitive nitrate oxide synthase that is present in adult rat myocytes.35 In this regard, Kaye et al47 report that high pacing frequencies (3 Hz) increase constitutive NOS (NOS3) activity, NO production, and intracellular cGMP content in isolated adult rat myocytes, whereas this induction is not observed at lower pacing rates (0.5 Hz). For this reason, the present study was performed at a pacing frequency of 0.5 Hz to avoid the confounding variable of pacing-induced activation of constitutive NOS.
Differing Effects in Hypertrophied Versus Normal Myocytes
Our observation that the NO donor SNP did not modify contractility or promote intracellular acidification in hypertrophied rat myocytes is novel and unsuspected. This model of pressure-overload hypertrophy due to ascending aortic banding has been extensively characterized by our laboratory. At this stage of chronic pressure overload, animals are characterized by concentric LV hypertrophy with preserved systolic shortening indices, the absence of chamber dilation, and a predominant increase in myocyte width with a slight increase in myocyte length.9 10 11 12 26 Consistent with these characteristics, fractional cell shortening was similar in the control and hypertrophied myocytes in the present study. Thus, the failure to observe a negative inotropic effect of SNP in the hypertrophied myocytes is unlikely to be due to severe depression of contractile function at baseline. The different effects of SNP on intracellular acidification in control and hypertrophied cells could also potentially be related to differences in intrinsic Bi in hypertrophied and normal myocytes. However, we have demonstrated26 that Bi is comparable in control and hypertrophied myocytes from this aortic-banded model. In addition, we have shown that the effects of intracellular acidification per se on contractility are similar in normal and hypertrophied myocytes. In the present study, the observation that the cGMP analogue 8-bromo-cGMP simulated the effects of SNP on pHi in the normal but not the hypertrophied cells suggests that downstream signaling effects of cGMP on the sodium-proton exchange may be blunted in pressure-overload hypertrophy, which could be related to increased constitutive NOS activity.47 In this regard, Kelm et al48 have shown an increased basal production of NO in hypertrophic hearts from spontaneously hypertensive rats. A limitation of the present study is the possibility that our observations may apply only to hypertrophied adult rat cardiac myocytes. Thus, additional studies will be needed to confirm our observations in other species and models of pressure-overload hypertrophy.
Integrated Cardiac Paracrine Signaling: Role of pHi
The potential importance of the modulation of pHi in the regulation of the contractile response in well-oxygenated myocardium is not unique to the actions of NO. Several studies have shown that the positive inotropic effects of both endothelin and angiotensin II in adult myocytes are predominantly mediated by an increase in myofilament calcium sensitivity due to intracellular alkalization rather than a net increase in intracellular calcium.17 23 24 26 Relevant to the potential role of sodium-proton exchange in the signaling of NO, the positive inotropic effects of endothelin and angiotensin II appear to be related to activation of the sodium-proton exchanger. Taken together, these observations suggest the presence of complex and opposing paracrine signaling pathways of NO and endothelin in the heart, the acute effects of which on contractile function, and potentially cell growth, are mediated by opposing effects on pHi. Our observations support the hypothesis that the coupling of NO signaling with the Na+-H+ exchange differs in adult rat normal and hypertrophied myocytes.
Selected Abbreviations and Acronyms
|Bi||=||intracellular buffering power|
|[Ca2+]i||=||intracellular calcium concentration|
|LVH||=||left ventricular hypertrophy|
|NOS||=||nitric oxide synthase|
This work was supported in part by National Heart, Lung, and Blood Institute grants HL-38189 (B.H.L. and N.I.) and HL-42873 (K.S.) and a US Fogarty Fellowship (J.B.). We greatly appreciate the suggestions of Dr William Barry regarding the analysis of pHi. We also appreciate the help of Tony L. Baptista in the preparation of the manuscript.
- Received October 9, 1996.
- Revision received November 22, 1996.
- Accepted November 27, 1996.
- Copyright © 1997 by American Heart Association
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