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The synergistic effects of aging and heart failure on myocardial insulin signaling in SHHF rats
The 7th Annual Scientific Meeting
•
HFSA
S15
048
049
Sarcomere Length-Tension Relationship in Failing Human Myocardium Eric I. Rossman,1 Remus M. Berretta,1 Rebecca E. Petre,1 Khuram W. Chaudhary,1 John P. Gaughan,1 Steven R. Houser,1 Kenneth B. Margulies1—1Cardiovascular Research Group, Temple University School of Medicine, Philadelphia, PA
MMP-7 Deletion Improves Survival Post-MI G. Patricia Escobar,1 Jennifer W. Hendrick,1 Jennifer S. Leiser,1 Jeffrey A. Sample,1 Kathryn B. Dowdy,1 Sarah E. Sweterlitsch,1 Joseph T. Mingoia,1 Lynn M. Matrisian,2 Merry L. Lindsey1—1Division of Cardiothoracic Surgery Research, Medical University of South Carolina, Charleston, SC; 2Department of Cancer Biology, Vanderbilt University, Nashville, TN
The Starling relationship is a fundamental component of cardiac muscle physiology. Although many researchers have studied the length-tension relationship in human myocardium, none have done it at the sarcomere level. The present study examined the effects of sarcomere length (SL) on contractile parameters in cardiac trabeculae from ischemic and non-ischemic failing human hearts, as well as those mechanically unloaded by a left ventricular assist device (LVAD). Methods: Using laser diffraction techniques, resting SLs were measured in right ventricular cardiac trabeculae (n ⫽ 10: Average dimensions (mm): 0.33 ⫾ 0.05 Width, 0.19 ⫾ 0.01 Thickness, 2.94 ⫾ 0.32 Length) from failing human hearts. As muscle length was incremented in a stepwise manner (25.0 um/step), resting SL and isometric contractile tension were assessed. Based on the SL-tension relationship, we determined the SLs that produced a developed tension of 20, 50, and 80% of maximal developed tension (Tmax) for each trabeculae. Results: For all groups, developed tension, rate of maximal tension rise, and rate of maximal tension decline all increased linearly as SL was increased. Diastolic tension increased minimally between SLs of 1.7 um to 2.0 um, and in some cases at SLs beyond 2.0 um, it increased in a nonlinear fashion. The SL associated with a particular percent of Tmax was remarkably consistent across all trabeculae: 1.92 ⫾ 0.02 um -⬎ 20%(Tmax-To); 2.09 ⫾ 0.02 um -⬎ 50%(Tmax-To); and 2.25 ± 0.01 um -⬎ 80%(Tmax-To); (To represents the minimal developed tension). These relationships did not seem to be affected by etiology or prior LVAD support. Conclusion: SL can be reliably measured in very thin cardiac trabeculae from human hearts. Although the shapes of the active SL-tension relationships are quite similar, there is substantial heterogeneity in the shapes of the passive SL-tension relationships. Our study also shows that Tmax, often used as a starting point during in vitro analysis, is achieved at non-physiological SLs (ⱖ2.35 um). Therefore, in studies where SL cannot be measured, using a percent of the tension between Tmax and To one can reliably set the preload at a more physiologic SL.
Matrix metalloproteinases (MMPs) are proteolytic enzymes that actively participate in tissue remodeling events. MMP levels increase in response to myocardial infarction (MI); and left ventricular (LV) remodeling is improved in animals treated with MMP inhibitors or in mice with MMP gene deletions. MMP-7, also known as matrilysin1, is present in macrophages; and MMP-7 levels increase in the myocardium of older mice, unoperated MMP-9 null mice, or in rabbits treated with MMPi post-MI. MMP7 roles in the myocardium have not been examined. To test the hypothesis that MMP-7 gene deletion would attenuate LV remodeling post-MI, MMP-7 null mice (n ⫽ 16; 5 female and 11 male) and C57/BL6 wild type mice (n ⫽ 24; 11 female and 13 male) underwent coronary artery ligation for 7 days. There was no difference in baseline echocardiographic parameters between the two groups. Surprisingly, mortality rates were different. Five wild type (n ⫽ 3 female and 2 male) and 3 MMP-7 null (n ⫽ 2 female and 1 male) died perioperatively. Of the remaining 19 wild type and 13 MMP7 null mice, survival rates were 31.6% for wild type and 92.3% for MMP-7 null mice (p ⬍ 0.001). Males accounted for the disparity: 9 of 11 male wt mice did not survive 7 days post-MI, whereas 1 of 10 male MMP-7 null mice did not survive. There was no difference in mortality rates in the females. LV rupture occurred in 4 of the 8 male and 0 of the 2 female wild type mice that underwent autopsy. In the surviving mice, heart weight to body weight ratios and infarct sizes were similar between the two groups (see table below). By echocardiography, wild type mice had greater infarct wall thinning, however, than the MMP-7 null mice. Wall thickness was not significantly decreased in the MMP-7 null mice at day 7 post-MI, indicating preservation of myocardial structure. End-systolic volumes (ESV) were also different between the two groups. The increase in wild type mice ESV was significantly greater than the increase in MMP-7 null mice (p ⬍ 0.05), indicating that wild type mice had a greater change in chamber geometry. Macrophage levels (16.7 ⫾ 2.3% area, n ⫽ 5 vs 16.2 ⫾ 0.9% area, n ⫽ 9 for MMP7 null, p ⫽ 0.80) were also similar between the two groups. Differences in post-MI remodeling may contribute to the improved mortality seen in the MMP-7 group. Together, these data suggest a role for MMP-7 in early survival post-MI that is not compensated by other MMPs and is not explained by differences in infarct size or macrophage infiltration.
050
051
The Synergistic Effects of Aging and Heart Failure on Myocardial Insulin Signaling in SHHF Rats Lazaros A. Nikolaidis,1 Indu Poornima,1 George Sokos,1 Carol Stolarski,1 Richard P. Shannon1—1Medicine, Division of Cardiology, Drexel University College of Medicine, Allegheny General Hospital, Pittsburgh, PA
Mitogen-Activated Protein Kinases Are Differentially Activated in Connection with Cardiomyocyte Apoptosis and Cardiac Dysfucntion in Heart Failure after Myocardial Infarction Fuzhong Qin,1 Shuji Fukuoka,1 Chikao Iwai,1 Michelle Liang,1 Chang-seng Liang1—1Cardiology Unit, University of Rochester Medical Center, Rochester, NY
Background: Clinical and experimental studies have suggested that advanced heart failure (HF) is associated with the development of whole body and myocardial insulin resistance. Myocardial insulin resistance has been implicated in abnormal myocardial substrate metabolism and energetic inefficiency in advanced HF. The precise insulin signaling defects in the failing myocardium are not well characterized. Because the development of both HF and insulin resistance become more prevalent with age, it is also plausible that age associated increases in body weight may contribute to altered myocardial insulin signaling. Methods: We studied 7 spontaneously hypertensive heart failure prone (SHHF) rats (12 months), 6 old controls (OC, 16 months) and 10 younger controls (YC, 5 month old). LV function was assessed by 2-D Echo under brief (5 min) isoflurane 1% anesthesia. Plasma was obtained to measure insulin, glucose, and NEFA. The cellular insulin signaling cascade was investigated on LV myocardial samples to measure GLUT-4 translocation and Akt-1 protein abundance and the insulin induced PI-3 kinase dependent Akt-1 phosphorylation at Ser-473 and Thr-308 using Western blots. Results: OC rats were more obese (612 ⫾ 37g*) than YC (490 ⫾ 5g) and had higher plasma insulin levels (225 ⫾ 38 pmol/L vs 148 ⫾ 23 pmol/L*) and NEFA (312 ⫾ 30 mmol/L vs 253 ⫾ 25mmol/L), despite normal glucose, consistent with insulin resistance. SHHF were more obese (797 ⫾ 12 g*) had higher insulin levels (1221 ⫾ 166 pmol/L*) and NEFA (398 ⫾ 70mmol/L*) and normal plasma glucose compared to OC. SHHF hearts were more dilated (LVEDD (mm): 8.5 ⫾ 0.2 vs OC: 7.5 ⫾ 0.7, YC: 7.5 ⫾ 0.1), and hypertrophied (LV mass (g): 1.6 ⫾ 0.1, OC: 1.3 ⫾ 0.1, YC: 1.2 ⫾ 0.1). LV systolic function was similar between SHHF and OC, but significantly impaired compared to YC (Fractional Shortening: SHHF: 37 ⫾ 2%, OC: 39 ⫾ 2%, YC: 50 ⫾ 2%, p ⬍ 0.05). Protein levels of Akt-1 were reduced by 40% (p ⬍ 0.05) in myocardium from SHHF compared to OC or YC. Insulin stimulated Ser 473/Thr 308 phosphorylation of Akt-1 was reduced in both OC and SHHF, but insulin stimulated Glut-4 translocation was impaired to a greater extent in SHHF (5 ⫾ 4%) compared to OC (12 ⫾ 4%) or YC (30 ⫾ 5%). Conclusions: Aging and HF are associated with greater plasma insulin levels, NEFA, and increased body weight compared to aging alone. Aging alone is associated with impaired insulin stimulated Akt-1 phosphorylation and Glut-4 translocation. However, the combined effects of aging and HF are synergistic with respect to myocardial insulin signaling abnormalities. This may contribute to the higher incidence and morbidity of HF with advanced age.
Mitogen-activated protein kinases (MAPKs) have been shown to be activated in congestive heart failure in which oxidative stress and cell apoptosis also have been observed. However, little is known of the time course changes of MAPKs and cardiomyocyte apoptosis after myocardial infarction (MI). In this study, we proposed to determine whether the changes of MAPKs are associated with progression of cardiac dysfunction in heart failure after MI. Methods: Rabbits were randomly assigned to ligation of the circumflex coronary artery to produce MI or sham operation. The animals were sacrificed at 1, 4, 8, 12 weeks after MI. Sham rabbits were sacrificed at 12 weeks after surgery. The remote non-infarcted myocardial tissue was used for the measurements. We measured cardiac function by echocardiography weekly, hemodynamics, infarct size, the activities and the protein levels of extracellular-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 MAPK (p38) by Western blot (arbitrary units), cardiac total oxidative stress by examining the ratio of reduced to oxidized glutathione (GSH/GSSG), and cardiomyocyte apoptosis (apoptotic nuclei per 10,000 cardiomyocytes) by monoclonal antibody against single stranded DNA. Results: There were no significant differences in the ratio of left ventricle to body weight between the sham and MI rabbits. MI rabbits exhibited progressive increases of left ventricular end-diastolic pressure and end-diastolic dimension, and progressive decreases of left ventricular fractional shortening and dP/dt over the 12week period compared with sham animals. This decrease of left ventricular systolic function was associated with a time-dependent decrease of GSH/GSSG and increase of cardiomyocyte apoptosis. Simultaneously, we found that the ERK and JNK activities decreased with age of MI, while the p38 activity was increased. There was, however, no difference in the protein levels of ERK, JNK and p38 between the sham and MI animals. Conclusion: MAPKs are activated differentially in heart failure after MI. These changes are not only time-dependent, but also correlated with oxidative stress and cardiomyocyte apoptosis. The findings suggest that activation of the MAPKs may play a role in left ventricular systolic dysfunction after MI. Group
DP/dt (mmHg) GSH/GSSG Apoptotic cells ERK activity JNK activity p38 activity
Sham MI-1W MI-4W MI-8W MI-12W
4470 ⫾ 205 3958 ⫾ 299* 3871 ⫾ 188* 3753 ⫾ 260* 3080 ⫾ 220*
117 ⫾ 23 75 ⫾ 18* 55 ⫾ 12* 33 ⫾ 9* 13 ⫾ 4*
6.0 ⫾ 1.4 29.1 ⫾ 10.0* 43.5 ⫾ 7.8* 60.3 ⫾ 14.8* 89.4 ⫾ 24.8*
0.99 ⫾ 0.05 0.88 ⫾ 0.08* 0.73 ⫾ 0.06* 0.58 ⫾ 0.08* 0.52 ⫾ 0.03*
n ⫽ 5–8. Values are mean ⫾ SEM. *P ⬍ 0.05 vs Sham. MI: myocardial infarction. W: week.
0.99 ⫾ 0.03 0.74 ⫾ 0.01* 0.71 ⫾ 0.11* 0.77 ⫾ 0.01* 0.60 ⫾ 0.16*
1.00 ⫾ 0.03 1.19 ⫾ 0.07* 1.25 ⫾ 0.13* 1.28 ⫾ 0.04* 1.36 ⫾ 0.11*
•
HFSA
S15
048
049
Sarcomere Length-Tension Relationship in Failing Human Myocardium Eric I. Rossman,1 Remus M. Berretta,1 Rebecca E. Petre,1 Khuram W. Chaudhary,1 John P. Gaughan,1 Steven R. Houser,1 Kenneth B. Margulies1—1Cardiovascular Research Group, Temple University School of Medicine, Philadelphia, PA
MMP-7 Deletion Improves Survival Post-MI G. Patricia Escobar,1 Jennifer W. Hendrick,1 Jennifer S. Leiser,1 Jeffrey A. Sample,1 Kathryn B. Dowdy,1 Sarah E. Sweterlitsch,1 Joseph T. Mingoia,1 Lynn M. Matrisian,2 Merry L. Lindsey1—1Division of Cardiothoracic Surgery Research, Medical University of South Carolina, Charleston, SC; 2Department of Cancer Biology, Vanderbilt University, Nashville, TN
The Starling relationship is a fundamental component of cardiac muscle physiology. Although many researchers have studied the length-tension relationship in human myocardium, none have done it at the sarcomere level. The present study examined the effects of sarcomere length (SL) on contractile parameters in cardiac trabeculae from ischemic and non-ischemic failing human hearts, as well as those mechanically unloaded by a left ventricular assist device (LVAD). Methods: Using laser diffraction techniques, resting SLs were measured in right ventricular cardiac trabeculae (n ⫽ 10: Average dimensions (mm): 0.33 ⫾ 0.05 Width, 0.19 ⫾ 0.01 Thickness, 2.94 ⫾ 0.32 Length) from failing human hearts. As muscle length was incremented in a stepwise manner (25.0 um/step), resting SL and isometric contractile tension were assessed. Based on the SL-tension relationship, we determined the SLs that produced a developed tension of 20, 50, and 80% of maximal developed tension (Tmax) for each trabeculae. Results: For all groups, developed tension, rate of maximal tension rise, and rate of maximal tension decline all increased linearly as SL was increased. Diastolic tension increased minimally between SLs of 1.7 um to 2.0 um, and in some cases at SLs beyond 2.0 um, it increased in a nonlinear fashion. The SL associated with a particular percent of Tmax was remarkably consistent across all trabeculae: 1.92 ⫾ 0.02 um -⬎ 20%(Tmax-To); 2.09 ⫾ 0.02 um -⬎ 50%(Tmax-To); and 2.25 ± 0.01 um -⬎ 80%(Tmax-To); (To represents the minimal developed tension). These relationships did not seem to be affected by etiology or prior LVAD support. Conclusion: SL can be reliably measured in very thin cardiac trabeculae from human hearts. Although the shapes of the active SL-tension relationships are quite similar, there is substantial heterogeneity in the shapes of the passive SL-tension relationships. Our study also shows that Tmax, often used as a starting point during in vitro analysis, is achieved at non-physiological SLs (ⱖ2.35 um). Therefore, in studies where SL cannot be measured, using a percent of the tension between Tmax and To one can reliably set the preload at a more physiologic SL.
Matrix metalloproteinases (MMPs) are proteolytic enzymes that actively participate in tissue remodeling events. MMP levels increase in response to myocardial infarction (MI); and left ventricular (LV) remodeling is improved in animals treated with MMP inhibitors or in mice with MMP gene deletions. MMP-7, also known as matrilysin1, is present in macrophages; and MMP-7 levels increase in the myocardium of older mice, unoperated MMP-9 null mice, or in rabbits treated with MMPi post-MI. MMP7 roles in the myocardium have not been examined. To test the hypothesis that MMP-7 gene deletion would attenuate LV remodeling post-MI, MMP-7 null mice (n ⫽ 16; 5 female and 11 male) and C57/BL6 wild type mice (n ⫽ 24; 11 female and 13 male) underwent coronary artery ligation for 7 days. There was no difference in baseline echocardiographic parameters between the two groups. Surprisingly, mortality rates were different. Five wild type (n ⫽ 3 female and 2 male) and 3 MMP-7 null (n ⫽ 2 female and 1 male) died perioperatively. Of the remaining 19 wild type and 13 MMP7 null mice, survival rates were 31.6% for wild type and 92.3% for MMP-7 null mice (p ⬍ 0.001). Males accounted for the disparity: 9 of 11 male wt mice did not survive 7 days post-MI, whereas 1 of 10 male MMP-7 null mice did not survive. There was no difference in mortality rates in the females. LV rupture occurred in 4 of the 8 male and 0 of the 2 female wild type mice that underwent autopsy. In the surviving mice, heart weight to body weight ratios and infarct sizes were similar between the two groups (see table below). By echocardiography, wild type mice had greater infarct wall thinning, however, than the MMP-7 null mice. Wall thickness was not significantly decreased in the MMP-7 null mice at day 7 post-MI, indicating preservation of myocardial structure. End-systolic volumes (ESV) were also different between the two groups. The increase in wild type mice ESV was significantly greater than the increase in MMP-7 null mice (p ⬍ 0.05), indicating that wild type mice had a greater change in chamber geometry. Macrophage levels (16.7 ⫾ 2.3% area, n ⫽ 5 vs 16.2 ⫾ 0.9% area, n ⫽ 9 for MMP7 null, p ⫽ 0.80) were also similar between the two groups. Differences in post-MI remodeling may contribute to the improved mortality seen in the MMP-7 group. Together, these data suggest a role for MMP-7 in early survival post-MI that is not compensated by other MMPs and is not explained by differences in infarct size or macrophage infiltration.
050
051
The Synergistic Effects of Aging and Heart Failure on Myocardial Insulin Signaling in SHHF Rats Lazaros A. Nikolaidis,1 Indu Poornima,1 George Sokos,1 Carol Stolarski,1 Richard P. Shannon1—1Medicine, Division of Cardiology, Drexel University College of Medicine, Allegheny General Hospital, Pittsburgh, PA
Mitogen-Activated Protein Kinases Are Differentially Activated in Connection with Cardiomyocyte Apoptosis and Cardiac Dysfucntion in Heart Failure after Myocardial Infarction Fuzhong Qin,1 Shuji Fukuoka,1 Chikao Iwai,1 Michelle Liang,1 Chang-seng Liang1—1Cardiology Unit, University of Rochester Medical Center, Rochester, NY
Background: Clinical and experimental studies have suggested that advanced heart failure (HF) is associated with the development of whole body and myocardial insulin resistance. Myocardial insulin resistance has been implicated in abnormal myocardial substrate metabolism and energetic inefficiency in advanced HF. The precise insulin signaling defects in the failing myocardium are not well characterized. Because the development of both HF and insulin resistance become more prevalent with age, it is also plausible that age associated increases in body weight may contribute to altered myocardial insulin signaling. Methods: We studied 7 spontaneously hypertensive heart failure prone (SHHF) rats (12 months), 6 old controls (OC, 16 months) and 10 younger controls (YC, 5 month old). LV function was assessed by 2-D Echo under brief (5 min) isoflurane 1% anesthesia. Plasma was obtained to measure insulin, glucose, and NEFA. The cellular insulin signaling cascade was investigated on LV myocardial samples to measure GLUT-4 translocation and Akt-1 protein abundance and the insulin induced PI-3 kinase dependent Akt-1 phosphorylation at Ser-473 and Thr-308 using Western blots. Results: OC rats were more obese (612 ⫾ 37g*) than YC (490 ⫾ 5g) and had higher plasma insulin levels (225 ⫾ 38 pmol/L vs 148 ⫾ 23 pmol/L*) and NEFA (312 ⫾ 30 mmol/L vs 253 ⫾ 25mmol/L), despite normal glucose, consistent with insulin resistance. SHHF were more obese (797 ⫾ 12 g*) had higher insulin levels (1221 ⫾ 166 pmol/L*) and NEFA (398 ⫾ 70mmol/L*) and normal plasma glucose compared to OC. SHHF hearts were more dilated (LVEDD (mm): 8.5 ⫾ 0.2 vs OC: 7.5 ⫾ 0.7, YC: 7.5 ⫾ 0.1), and hypertrophied (LV mass (g): 1.6 ⫾ 0.1, OC: 1.3 ⫾ 0.1, YC: 1.2 ⫾ 0.1). LV systolic function was similar between SHHF and OC, but significantly impaired compared to YC (Fractional Shortening: SHHF: 37 ⫾ 2%, OC: 39 ⫾ 2%, YC: 50 ⫾ 2%, p ⬍ 0.05). Protein levels of Akt-1 were reduced by 40% (p ⬍ 0.05) in myocardium from SHHF compared to OC or YC. Insulin stimulated Ser 473/Thr 308 phosphorylation of Akt-1 was reduced in both OC and SHHF, but insulin stimulated Glut-4 translocation was impaired to a greater extent in SHHF (5 ⫾ 4%) compared to OC (12 ⫾ 4%) or YC (30 ⫾ 5%). Conclusions: Aging and HF are associated with greater plasma insulin levels, NEFA, and increased body weight compared to aging alone. Aging alone is associated with impaired insulin stimulated Akt-1 phosphorylation and Glut-4 translocation. However, the combined effects of aging and HF are synergistic with respect to myocardial insulin signaling abnormalities. This may contribute to the higher incidence and morbidity of HF with advanced age.
Mitogen-activated protein kinases (MAPKs) have been shown to be activated in congestive heart failure in which oxidative stress and cell apoptosis also have been observed. However, little is known of the time course changes of MAPKs and cardiomyocyte apoptosis after myocardial infarction (MI). In this study, we proposed to determine whether the changes of MAPKs are associated with progression of cardiac dysfunction in heart failure after MI. Methods: Rabbits were randomly assigned to ligation of the circumflex coronary artery to produce MI or sham operation. The animals were sacrificed at 1, 4, 8, 12 weeks after MI. Sham rabbits were sacrificed at 12 weeks after surgery. The remote non-infarcted myocardial tissue was used for the measurements. We measured cardiac function by echocardiography weekly, hemodynamics, infarct size, the activities and the protein levels of extracellular-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 MAPK (p38) by Western blot (arbitrary units), cardiac total oxidative stress by examining the ratio of reduced to oxidized glutathione (GSH/GSSG), and cardiomyocyte apoptosis (apoptotic nuclei per 10,000 cardiomyocytes) by monoclonal antibody against single stranded DNA. Results: There were no significant differences in the ratio of left ventricle to body weight between the sham and MI rabbits. MI rabbits exhibited progressive increases of left ventricular end-diastolic pressure and end-diastolic dimension, and progressive decreases of left ventricular fractional shortening and dP/dt over the 12week period compared with sham animals. This decrease of left ventricular systolic function was associated with a time-dependent decrease of GSH/GSSG and increase of cardiomyocyte apoptosis. Simultaneously, we found that the ERK and JNK activities decreased with age of MI, while the p38 activity was increased. There was, however, no difference in the protein levels of ERK, JNK and p38 between the sham and MI animals. Conclusion: MAPKs are activated differentially in heart failure after MI. These changes are not only time-dependent, but also correlated with oxidative stress and cardiomyocyte apoptosis. The findings suggest that activation of the MAPKs may play a role in left ventricular systolic dysfunction after MI. Group
DP/dt (mmHg) GSH/GSSG Apoptotic cells ERK activity JNK activity p38 activity
Sham MI-1W MI-4W MI-8W MI-12W
4470 ⫾ 205 3958 ⫾ 299* 3871 ⫾ 188* 3753 ⫾ 260* 3080 ⫾ 220*
117 ⫾ 23 75 ⫾ 18* 55 ⫾ 12* 33 ⫾ 9* 13 ⫾ 4*
6.0 ⫾ 1.4 29.1 ⫾ 10.0* 43.5 ⫾ 7.8* 60.3 ⫾ 14.8* 89.4 ⫾ 24.8*
0.99 ⫾ 0.05 0.88 ⫾ 0.08* 0.73 ⫾ 0.06* 0.58 ⫾ 0.08* 0.52 ⫾ 0.03*
n ⫽ 5–8. Values are mean ⫾ SEM. *P ⬍ 0.05 vs Sham. MI: myocardial infarction. W: week.
0.99 ⫾ 0.03 0.74 ⫾ 0.01* 0.71 ⫾ 0.11* 0.77 ⫾ 0.01* 0.60 ⫾ 0.16*
1.00 ⫾ 0.03 1.19 ⫾ 0.07* 1.25 ⫾ 0.13* 1.28 ⫾ 0.04* 1.36 ⫾ 0.11*