Published online before print June 16, 2005, doi: 10.1161/01.RES.0000173849.68636.1e
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© 2005 American Heart Association, Inc.
Integrative Physiology |
Expression of Slow Skeletal Troponin I in Adult Mouse Heart Helps to Maintain the Left Ventricular Systolic Function During Respiratory Hypercapnia
From the Center for Cardiovascular Research, Department of Physiology and Biophysics (D.U., L.A.W., R.J.S., B.M.W.) and Department of Medicine (F.A.L.D., J.R.P., L.A.W., B.M.W.), Section of Cardiology, University of Illinois at Chicago.
Correspondence to Dr Beata M. Wolska, University of Illinois at Chicago, Dept of Medicine, Section of Cardiology, 840 S Wood St (M/C 715), Chicago, IL 60612. E-mail bwolska{at}uic.edu
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Abstract |
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Compared with the adult, neonatal heart muscle is less sensitive to deactivation by acidic pH. We hypothesized that expression of slow skeletal troponin I (ssTnI), the embryonic isoform, in adult heart would help maintain left ventricular (LV) systolic function during respiratory hypercapnia. We assessed LV function by transthoracic 2D-targeted M-mode and pulsed Doppler echocardiography in transgenic (TG) mice in which cardiac TnI was replaced with ssTnI and in nontransgenic (NTG) littermates. Anesthetized mice were ventilated with either 100% oxygen or 35% CO2 balanced with oxygen. Arterial blood pH with 35% CO2 decreased to the same levels in both groups of animals. In the absence of propranolol, the LV fractional shortening was higher in TG compared with NTG mice throughout most of the experimental protocol. LV diastolic function was impaired in TG compared with NTG mice both at 100% oxygen and 35% CO2 because E-to-A wave ratio of mitral flow was significantly lower, and E-wave deceleration time and LV isovolumic relaxation time were longer in TG compared with NTG mice. When compensatory mechanisms that occur through stimulation of ß-adrenergic receptors during hypercapnia were blocked by continuous perfusion with propranolol, we found that NTG mice died within 3 to 4 minutes after switching to 35% CO2, whereas TG mice survived. Our experiments demonstrate the first evidence that specific replacement of cardiac TnI with ssTnI has a protective effect on the LV systolic function during hypercapnic acidosis in situ.
Key Words: troponin I • hypercapnia • acidosis
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Introduction |
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It is known that acidosis alters cardiac contractility to a smaller degree in neonatal than adult heart.1,2 The lower sensitivity to deactivation by acidic pH during development may be attributable to differences in (1) isoforms of myofilament proteins expressed in neonatal and adult heart, (2) effects of acidosis on Ca2+ fluxes, and/or (3) intracellular pH and/or Ca2+ buffering. Based on experiments comparing adult and neonatal rat ventricular muscles, we have hypothesized that slow skeletal troponin I (ssTnI) is the major determinant of the differential response to acidic pH between adult and neonatal hearts.2 The ssTnI isoform is expressed during embryonic and early postnatal life, whereas cardiac TnI (cTnI) is expressed during adult life.3,4 The role of TnI isoforms and the role of specific molecular differences between these two isoforms have been extensively studied.1,2,5–10 We have reported that expression of ssTnI in adult cardiac papillary muscles is sufficient to prevent the force decline during hypercapnic acidosis in isolated papillary muscles.8 These data provide direct evidence for the important protective role of ssTnI during acidosis and most likely during ischemia/reperfusion. However, it is still unclear whether ssTnI has the same protective effect in the in situ ejecting heart during acidic conditions. In the present study, our objective was to test the protective effect of ssTnI on cardiac function during acidic conditions in vivo. An important rationale for our experiments comes from our recent data indicating that the role of TnI on cardiac function is best measured in ejecting, afterloaded hearts.11–13 Our experiments provide the first evidence that specific replacement of cTnI with ssTnI has a protective effect on the left ventricular (LV) systolic function during hypercapnic acidosis in the intact animal.
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Materials and Methods |
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Animal Preparations
Adult 8- to 10-month old male and female nontransgenic (NTG) and transgenic (TG) mice that express ssTnI in the heart14 were used from colonies bred and maintained at the Biologic Resources Laboratory at the University of Illinois at Chicago. All experiments were performed in compliance with animal care policies of the Animal Care Committee of the University of Illinois at Chicago. Mice were anesthetized with 1.0% to 1.5% isoflurane mixed with the 100% oxygen delivered through a vaporizer connected to a rodent ventilator. Stroke volume was set at 0.25 to 0.30 mL, and a respiration rate was set at 136 breaths per minute.15 After 20 minutes of baseline conditions, 100% oxygen was switched to 35% CO2 balanced with oxygen for 40 minutes and back to 100% oxygen for 40 minutes. In some mice, the right femoral vein was isolated and catheterized as previously described,12 and propranolol (34 ng/g body weight/min) was continuously infused using a pump.
Echocardiography
Transthoracic 2D-targeted M-mode and pulsed Doppler echocardiography (ECHO) were performed with a 15-MHz linear array transducer (Acuson Sequoia C256 system). The transducer was placed on a layer of acoustic coupling gel that was applied to the left hemithorax; adequate contact was maintained while avoiding excessive pressure on the chest. Mice were imaged in a shallow left lateral decubitus position. M-mode images of the left ventricle were obtained from the parasternal short axis view at the level of the papillary muscles. Interventricular septal and LV posterior wall thicknesses and LV internal dimensions at the end of diastole and systole were measured by the American Society of Echocardiography leading-edge method on the M-mode tracings.16 Fractional shortening of the left ventricle, a measure of LV systolic function, was calculated from digital images as: LV fractional shortening (FS) (FS) (%)=(LVIDd–LVISd)/LVIDdx100, where LVIDd is the internal diastolic dimension of the LV, and LVISd is the internal systolic dimension of the LV.17
Diastolic transmitral inflow recordings were acquired from apical four-chamber view using 7 MHz pulsed Doppler ECHO. The probe was positioned substernally at the xyphoid applying minimal pressure. The Doppler range gate depth was set at 4 mm to obtain optimal signals from the LV inflow and outflow tracks. The sample volume was positioned along the long axis in the middle of the mitral ring at the tips of the opened cusps of the mitral valve. Three parameters of the LV diastolic function were evaluated: (1) E/A ratio–ratio of the maximal velocity of E (early LV filling) and A (atrial contraction) waves; (2) E-wave deceleration time (DT)–the time from the peak of the E wave to the intersection of the deceleration slope of the E wave with the baseline; and (3) LV isovolumic relaxation time (IVRT), which was measured from the aortic valve closure to the mitral valve opening.17,18 The M-mode and Doppler tracings were conducted with a paper speed of 200 mm/sec.
Blood Gas Analysis
Blood gas analysis was performed using IL Synthesis Systems (Instrumentation Laboratory, 1998, Milano, Italy). The right carotid artery or left femoral artery was dissected, and a polyethylene catheter (PE-10) was inserted into the artery up to the aortic arch. Four mice in each group were anesthetized as described above and exposed to 100% O2, and 3 mice in each group were ventilated with 35% CO2. Approximately 0.3 mL of blood was drawn into a syringe for measurement of arterial blood pH after 40 minutes of ventilation with either 100% oxygen or 35% CO2 balanced with oxygen. During the exposure to either gas, LV function was monitored by ECHO.
Electrophoresis and Western Blot Analysis
TnI Phosphorylation
The apex of the left ventricle was placed directly into 10% TCA in acetone (–70°C) and placed in the –70°C freezer for a minimum of 16 hours. Samples were slowly warmed to room temperature (1 hour at –20°C, 1 hour at 4°C, and 1 hour at room temperature) to allow acetone substitution of the tissue, preserving the phosphorylation status of the proteins. TnI phosphorylation was analyzed by nonequilibrium 1D isoelectric focusing as described previously.19 Percentage phosphorylation was expressed as TnI (phosphorylated)/(TnI [dephosphorylated]+TnI [phosphorylated])x100%.
Expression of SERCA2 and Phospholamban Phosphorylation
Hearts were frozen in liquid nitrogen and stored at –80°C. Samples were homogenized in modified radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors (Upstate). SDS-PAGE was performed using 8% acrylamide gels (SERCA2a) and 4% to 20% acrylamide gradient gels (phospholamban [PLB]). Proteins were transferred to nitrocellulose membranes, blocked with 5% nonfat milk, and incubated with anti-PLB (1:2000; Upstate), anti–phospho-PLB Ser16 (Upstate; 1:1000), anti–phospho-PLB Thr17 (1:2500; Badrilla-UK) or anti-SERCA2a (1:2000; ABR) antibodies. Following incubation with primary antibody, membranes were incubated in horseradish peroxidase–coupled secondary antibodies (Amersham) (1:5000 for anti–phospho-PLB and 1:10000 for anti-PLB and anti-SERCA2a) followed by ECL detection (Pierce). Membranes incubated initially in anti–phospho-PLB antibody were stripped using a commercial stripping buffer (Pierce), and the same membranes were probed using an anti-PLB antibody. To assess the levels of expression of PLB and SERCA2a, densitometric analysis (Personal densitometer; Amershan Biosciences) was performed using the DataQuaNT software.
Statistical Analysis
All results are presented as mean±SE. The statistical significance of differences between groups of NTG and TG mice was determined by one-way or one-way repeated-measures ANOVA followed by the Student–Newman–Keuls test, as appropriate. A value of P<0.05 was considered significant.
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Results |
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Effect of Hypercapnic Acidosis on LV Systolic and Diastolic Function in the Absence of Propranolol
Figure 1A shows a summary of the changes in FS in NTG (n=5) and TG (n=5) mice during the baseline conditions (100% O2), hypercapnic acidosis (35% CO2), and recovery (100% O2) assessed by ECHO. FS was significantly higher in TG compared with NTG mice throughout most of the experimental protocol. In NTG mice during hypercapnic acidosis, FS dropped from 47.3±2.1% at baseline (20 minutes of 100% O2) to 27.8±1.0% at 5 minutes of CO2 and returned to the baseline level at 20 minutes of CO2 (43.4±3.0%). During recovery, there was a transient increase in FS that reached 63.0±2.2% at 5 minutes of recovery time. In TG mice, hypercapnic acidosis resulted in an increase in FS throughout the exposure to CO2. During baseline conditions, FS was 51.0±2.4% (20 minutes of 100% O2) and increased to 74.2±1.0% at 20 minutes of hypercapnic acidosis. Moreover, FS stayed significantly elevated during the recovery time in TG mice. At 20 minutes of recovery, FS was 70.4±4.6% in TG and 55.6±4.0% in NTG mice. Figure 1B shows representative M-mode ECHO recordings of the LV in NTG and TG mice at 20 minutes of 100% oxygen (baseline conditions) and at 5 minutes of 35% CO2. Under baseline conditions, FS was 50% in NTG and 48% in TG hearts; however, after 5 minutes exposure to CO2, FS dropped to 29% in NTG and increased to 72% in TG hearts in this experiment.
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Diastolic function was assessed using the pulsed Doppler ECHO. Figure 2 shows the E-to-A wave ratios (E/A) (Figure 2A), E-wave DTs (Figure 2B) and LV isovolumic relaxation times (IVRT) (Figure 2C) at 20 minutes of baseline conditions, 20 minutes of hypercapnic acidosis, and 20 minutes of recovery. At all time points during the experiments, the E/A ratios were significantly lower in TG compared with NTG hearts. In TG mice, the E-wave DT was prolonged at 20 minutes of baseline conditions and 20 minutes of hypercapnic acidosis (Figure 2B). LV IVRT was the same in NTG and TG mice during baseline conditions but was longer in TG compared with NTG at 20 minutes of 35% CO2 (Figure 2C). In TG hearts, LV IVRT was longer at 20 minutes of CO2 than in baseline and recovery conditions. Figure 2D shows representative examples of pulsed Doppler mitral flow recordings in NTG and TG mice at baseline level and at 20 minutes of 35% CO2. At baseline the E/A ratios were 1.3 in NTG and 1.0 in TG hearts and at 20 minutes of CO2, decreased to 1.2 in NTG and 0.9 in TG hearts, respectively. E-wave DT was 25 ms in NTG and 31 ms in TG hearts at baseline and decreased to 21 ms in NTG and 29 ms in TG hearts at 20 minutes of CO2. LV IVRTs were the same at baseline (15 ms in NTG and 15 ms in TG hearts) but at 20 minutes of CO2, increased only in TG heart to 23 ms.
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Effect of Hypercapnic Acidosis on LV Systolic and Diastolic Functions in the Presence of Propranolol
To determine whether the response from the sympathetic nervous system is critical for the survival of NTG and TG mice during the hypercapnic conditions, we blocked the ß-adrenergic receptors by continuous infusion of propranolol. As illustrated in Figure 3A, in the presence of propranolol (at 20 minutes of propranolol), FS decreased from a baseline (20 minutes of O2) value 42.0±1.9% to 25.2±1.9% in NTG mice (n=5) and from 51.6±3.4% to 27.6±2.8% in TG mice (n=5). In NTG mice, switching to 35% CO2 resulted in a precipitous, sudden decrease in FS, and by 4 minutes all 5 NTG mice had died. However, in TG mice, FS decreased only slightly initially, and during 15 to 20 minutes of hypercapnic acidosis, they recovered fully to the baseline level. After switching back to 100% oxygen, FS transiently increased to 68.0±1.5% (5 minutes of recovery) and then returned to a level slightly below FS in baseline conditions. Figure 3B presents M-mode ECHO recordings of the LV in NTG and TG mice during exposure to hypercapnic acidosis in the presence of propranolol. The image of the LV in the NTG mouse was taken at the moment of death, 4 minutes after switching to 35% CO2. Under the same conditions, however, there was normal LV systolic function in TG mouse. The M-mode ECHO recordings of LV in TG mouse is presented 7 minutes after the onset of ventilation with 35% CO2.
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The effects of propranolol on LV diastolic function of TG mice are presented in Figure 4. There was no difference in the E/A ratio between the TG mice with and without propranolol (Figure 4A). In the presence of propranolol at 20 minutes of CO2, E-wave DT was significantly prolonged compared with E-wave DT at 20 minutes of CO2 without propranolol and with baseline and recovery (with and without propranolol) (Figure 4B). Infusion with propranolol also resulted in prolongation of LV IVRT (Figure 4C).
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Table 1 summarizes the effect of hypercapnic acidosis on heart rate (HR) in the presence and absence of propranolol. In surviving mice, HR was not significantly different between TG and NTG groups under any experimental condition. Infusion of propranolol resulted in significant decreases in HRs in NTG and TG mice.
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Blood Gas Analysis During Hypercapnic Acidosis
Under baseline conditions blood pH, PCO2, and PO2 were not different between NTG and TG animals (Table 2). Hypercapnic acidosis resulted in a significant decrease in blood pH, an increase in PCO2 and a decrease in PO2 in both groups of animals (Table 2), but there was no significant difference between NTG and TG animals.
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Effect of Hypercapnic Acidosis on TnI and PLB Phosphorylation
Because hypercapnia is associated with stimulation of the sympathetic nervous system and activation of protein kinases, in the next series of experiments we determined the states of TnI and PLB phosphorylation in NTG and TG groups of mice during the baseline condition and during hypercapnia (4 minutes of CO2) in the presence and absence of propranolol. The results shown in Figure 5 demonstrate that cTnI appears in an unphosphorylated and three phosphorylated (P1, P2, and P3) species. NTG mice subjected to 35% CO2 show a significantly increased level of cTnI phosphorylation that is prevented by propranolol. However, as expected, there were no changes in ssTnI phosphorylation in any of the experimental conditions (data not shown).
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Figure 6A and 6B show phosphorylation of Ser16 and Thr17 of PLB in TG (lanes 1 to 4) and NTG (lanes 5 to 8) hearts at baseline (20 minutes of O2) (lanes 1 and 5), 20 minutes of propranolol (lanes 2 and 6), 20 minutes of hypercapnic acidosis (lanes 3 and 7), and at 4 minutes of hypercapnic acidosis in the presence of propranolol (lanes 4 and 8). In the baseline condition, the phosphorylation of Ser16 was low (2 of 3 samples) or not detectable (1 sample). Phosphorylation of Thr17 was not detectable in any of the three baseline samples. In the absence of propranolol, hypercapnia resulted in an increase in phosphorylation of Ser16 and Thr17 in both groups of animals. The hypercapnia-induced increase in phosphorylation of Ser16 and Thr17 was completely prevented by propranolol. Figure 6C shows that the level of expression of PLB was not different between TG (lanes 1 to 4) and NTG (lanes 5 to 8) mice. There was no change in the expression levels of SERCA2a between NTG and TG groups (data not shown).
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Discussion |
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These data are the first to demonstrate that hearts expressing ssTnI in cardiac muscle show a remarkable resistance to acidosis in vivo and that the compensatory response from the sympathetic nervous system is critical for survival of NTG mice expressing cTnI. To the best of our knowledge, our work is also the first to show that effects of hypercapnic acidosis can be studied directly in vivo.
Differences in the response to acidic pH among various types of muscles lie in the isoforms of myofilament proteins. Donaldson et al20 reported the first evidence that effects of acidosis on Ca2+-force relations of isolated myofilaments differ among fast, slow, and cardiac muscle. Early studies also demonstrated that Ca2+ activation of neonatal heart myofilaments is relatively insensitive to deactivation by acidic pH, when compared with adult myofilaments,2 and that this differential effect is localized in thin filament proteins.1 With identification of ssTnI as the neonatal isoform,21 we set out to determine whether this isoform switch is responsible for the differential effect of acidosis.7 Definitive evidence that this is indeed the case came from investigations of myofilaments from TG mice in which ssTnI completely replaced cTnI.8 Wolska et al8 reported that Ca2+-force relations of skinned fiber bundles from hearts of the ssTnI-TG mice were less right shifted in acidosis than the NTG controls. With acidosis, there was also a significant and sustained fall in tension of electrically stimulated NTG papillary muscles but no effect in the TG muscles.
Various regions of TnI have been proposed to be responsible for the differential response to acidic pH between ssTnI and cTnI. Studies using different chimeras composed of regions of cTnI and ssTnI suggest that a pH-sensitive domain may reside in the carboxyl terminus9 or in the inhibitory portion of TnI.10 However, more recent studies have localized a specific amino acid (Ala162) of cTnI as largely responsible for the differential response to acidosis.5 When Ala162 was replaced with His (the corresponding ssTnI amino acid), the Ca2+ sensitivity of ATPase activity of reconstituted myofilament preparations at pH 6.5 was restored to that at pH 7.0. An important question, which we have addressed here, is the relative significance of these changes in hearts in vivo. Apart from the differences between ssTnI and cTnI in their response to acidosis, there are other differences between the two isoforms that may play a role in the ability of ssTnI to protect the myocardium from deactivation by acidic pH. One major difference between cTnI and ssTnI is the presence of a 32-amino acid N-terminal extension in cTnI.22 This extension contains 2 serines at positions 22 and 23, which are phosphorylated by protein kinase A.23 Phosphorylation of these sites results in a decrease in myofilament sensitivity to Ca2+ and contributes to the enhanced rate of relaxation in situ during ß-adrenergic stimulation.12,14,24
During acidosis, in the absence of the ß-adrenergic blocker propranolol, we observed the complete recovery of FS in NTG hearts and an increase in FS in TG hearts. The mechanical recovery during acidosis has been previously reported by us8 and others6,25,26 and can be explained by partial recovery of intracellular pH and/or an increase in Ca2+ transient amplitude attributable to increased sarcoplamic reticulum (SR) Ca2+ load as a consequence of increased activity of Na+/H+ exchanger, inhibition of Na+/K+ pump, and phosphorylation of PLB. Our current data demonstrate that acidosis in the absence of propranolol significantly increases the level of phosphorylation of PLB (Figure 6). The phosphorylation of PLB releases SERCA2a inhibition and results in an increased SR Ca2+ uptake and increased Ca2+ transient. However, at the same time, acidic pH directly suppresses the SR CaATPase activity; therefore, the net effect of SERCA2a activity depends on multiple factors. It has been reported in isolated cells that the Ca2+ transient is significantly increased not only during acidosis but also stays elevated after switching to solutions with normal pH.6,27 Small transient increases above the baseline value (before acidosis) in intracellular pH have also been reported during the first few minutes after switching to control solution.6,27,28 These alterations in pH and Ca2+ transients can significantly influence the recovery kinetics of FS and may have more profound effects on TG mice because their myofilaments are more sensitive to Ca2+.8 During recovery, intracellular pH comes to a baseline value faster than dephosphorylation of PLB and myofilament regulatory proteins that were phosphorylated during hypercapnic acidosis. In Langendorff perfused hearts stimulated with isoproterenol, cTnI stayed phosphorylated after 15 minutes perfusion with drug-free solution, whereas, at the same time, PLB was almost completely dephosphorylated.29
Our data indicate that during hypercapnic conditions in situ, reflex activation of the sympathetic nervous system is critical for survival of NTG mice. Because we used propranolol, a specific ß-adrenergic receptor blocker, the increase in the FS during hypercapnic acidosis in TG mice is most likely attributable to stimulation of 
-adrenergic receptors, an increase in Ca2+ transient amplitude, and partial recovery of intracellular pH. Stimulation of -adrenergic receptors by phenylephine in Langendorff perfused mouse hearts in the presence of a ß-blocker results in positive inotropic effect.30,31 Moreover, it has been demonstrated in swine that acute hypercapnia was associated with release of both norepinephrine and epinephrine.32 Plasma norepinephrine concentration increased 3.4-fold and epinephrine increased 1.8-fold compared with basal concentrations. A similar increase in catecholamine levels was most likely present in our experimental conditions. Our functional (ECHO) and biochemical (TnI and PLB phosphorylation) data indicate that during hypercapnic conditions, there is a significant release of catecholamines that stimulate both adrenergic receptors in the absence of propranolol and -adrenergic receptors in the presence of propranolol. In NTG hearts, which demonstrate a more significant reduction of myofilament sensitivity to Ca2+ during acidosis than TG hearts, the contribution of -receptors is apparently too small to prevent mice from death. Interestingly, in the presence of propranolol, acidosis decreases HR in TG mice, despite an increase in FS. In these conditions, HR is a net result of at least two opposite effects: stimulation of -adrenergic receptors and acidification, which is known to decrease the frequency of spontaneous beating of the sino-atrial node.33
The rate of cardiac relaxation is influenced by both myofilament properties and Cai2+ decay. We have previously shown in vitro that myofilaments from TG mice expressing ssTnI demonstrate higher myofilament sensitivity to Ca2+ that resulted in an impairment of diastolic function in TG mice.8,14 This was confirmed in the experiments reported here and manifested by a smaller E/A ratio and longer E-wave DT in TG mice compared with NTG mice. Acidosis in situ is associated with release of catecholamines and stimulation of adrenergic receptors. Stimulation of ß-adrenergic receptors results in phosphorylation of multiple proteins in the sarcolemma, SR, and myofilaments.13,34 Phosphorylation of PLB appears to be the most dominant among the proteins responsible for the enhanced rate of relaxation; however, the role of cTnI phosphorylation cannot be excluded.12 Our data show that in the absence of propranolol, hypercapnic acidosis induced a significant increase in cTnI phosphorylation in NTG hearts and increased phosphorylation of Ser16 and Thr17 of PLB in both groups. Additionally in NTG hearts, phosphorylation of cTnI contributes to enhanced rate of relaxation. At the same time, acidic pH directly inhibits SERCA2a activity. Therefore, the overall effect of hypercapnic acidosis on the rate of relaxation in situ is the net result of multiple factors. Based on ECHO relaxation parameters during hypercapnia, the relaxation was significantly impaired in TG compared with NTG hearts. This is most likely attributable to smaller desensitization of myofilaments in TG compared with NTG hearts. Our previous studies have indicated that ventricular myocytes isolated from TG hearts exhibit no major differences in Ca2+ transients from myocytes isolated from NTG hearts, both in control conditions and during isoproterenol stimulation.14 We have also reported13 data that show that the expression and distribution of ß1 and ß2 receptors are not different between NTG and TG hearts. Therefore, we do not anticipate a significantly different response of the sympathetic nervous system to acidosis between NTG and TG mice. Moreover, there were no differences between NTG and TG hearts in isoform population of myosin or thin filament proteins other than TnI that could account for the differential response to hypercapnic conditions.
In summary, data presented here strongly support the hypothesis that myofilament deactivation by acidosis is the major factor responsible for depression of contractility in acidosis. These data indicate that ssTnI, which is expressed in fetal and neonatal myocardium, plays a critical function in protecting immature hearts in acidic conditions from significant decreases in contractility caused by myofilament desensitization. Our data also support the more general hypothesis that alteration in myofilament response to Ca2+ is a major variable affecting cardiac function.
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Acknowledgments |
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This research was supported by NIH research grants RO1 HL-64209 (to B.M.W.), R37 HL-22231 (to R.J.S.), and PO1 HL-62426 (to R.J.S.). B.M.W. is an Established Investigator of the American Heart Association.
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Footnotes |
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Original received January 26, 2005; revision received June 3, 2005; accepted June 6, 2005.
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References |
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