Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy
Background— Hspa1a and Hspa1b genes encode stress-inducible 70-kDa heat shock proteins (Hsp70) that protect cells from insults such as ischemia. Mice with null mutations of both genes (KO) were generated, and their cardiac phenotype was explored.
Methods and Results— Heart rate and blood pressures were normal in the KO mice. Hearts from KO mice were more susceptible to both functional and cellular damage by ischemia/reperfusion. Cardiac hypertrophy developed in Hsp70-KO mice. Ca2+ transients in cardiomyocytes of KO mice showed a delayed (120%) calcium decline and decreased sarcoplasmic reticulum calcium content. Cell shortening was decreased by 35%, and rates of contraction and relaxation were slower by 40%. These alterations can be attributed to the absence of Hsp70 because viral expression of Hsp70 in KO cultured cardiomyocytes restored these parameters. One mechanism underlying myocyte dysfunction could be decreased SERCA2a expression. This hypothesis was supported by a prolonged calcium decline and decreased SERCA2a protein. Viral SERCA2a expression restored contractility and Ca2+ transients. We examined the involvement of Jun N-terminal kinase (JNK), p38-mitogen–activated protein kinase (p38-MAPK), Raf-1, and extracellular signal–regulated kinase (ERK) in SERCA2a downregulation and the cardiac phenotype of KO mice. Levels of phosphorylated JNK, p38-MAPK, Raf-1, and ERK were elevated in KO hearts. Activation of the Raf-1–ERK pathway in normal cardiomyocytes resulted in decreased SERCA2a.
Conclusions— Absence of Hsp70 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp70-KO hearts against ischemia/reperfusion. In addition, deletion of Hsp70 genes might induce cardiac dysfunction and development of cardiac hypertrophy through the activation of JNK, p38-MAPK, Raf-1, and ERK.
Received October 29, 2005; revision received March 15, 2006; accepted April 7, 2006.
Heat shock proteins (HSP) are a family of proteins induced by an increase in temperature as well as other environmental stresses and are well known to play a role in protein folding, translocation, and the assembly of intracellular protein, which may protect against various environmental challenges.1–3 The 70-kDa family of HSP (Hsp70) is one of the most extensively studied groups. In the mouse, there are at least 7 different subgroups of Hsp70 (now referred to as the Hspa family of genes and proteins), including 5 constitutively expressed forms.4,5 Ischemia, heat shock, and exercise can induce two additional forms of Hsp70: Hsp70-1 (now Hspa1b) and Hsp70-3 (now Hspa1a). However, induction of Hsp70 can also lead to changes in the expression of a wide spectrum of other proteins related to cellular defense mechanisms such as oxygen–free radical scavenging enzymes and several other members of the heat shock protein family.6,7 Therefore, in the experimental setting of heat shock or other stresses, it is hard to distinguish the protective effect of one specific heat shock protein from other induced proteins.1,8
Clinical Perspective p 2597
Several studies have shown that overexpression of exogenous copies of Hsp70 confers protection against simulated ischemia and metabolic stress.8–10 Adenovirus-mediated transfer of Hsp70 was also highly effective in providing protection against simulated ischemic injury.11,12 Recently, a knockout mouse strain has been developed that lacks functional Hspa1a and Hspa1b genes.13,14 The Hspa1a gene and the Hspa1b gene encode the Hsp70 proteins, which only differ by one amino acid. Both are expressed in a wide range of tissues, constitutively as well as in response to a wide range of environmental stress.14–16
Initial observations of these Hspa1a−/−/Hspa1b−/− double-knockout (KO) mice indicated the presence of mild cardiac hypertrophy. The detailed cardiac phenotype and stress-induced responses of these KO mice have not yet been explored. Whether this hypertrophy is associated with cardiac dysfunction is unknown.
Stress-activated protein kinases such as Jun N-terminal kinase (JNK), p38-mitogen–activated protein kinase (p38-MAPK), Raf-1, and extracellular signal–regulated kinase (ERK) play key roles in regulating cardiac hypertrophy by activation of different transcription factors.17,18 JNK activation appears to be necessary in the cardiac hypertrophic response both in vitro and in vivo. It has been previously shown that an acute elevation of Hsp70 inhibits activity of JNK, p38-MAPK, and Raf-1.19,20 These observations led us to hypothesize that the absence of Hsp70 might activate the JNK, p38-MAPK, and/or Raf-1/ERK signaling pathways and subsequently induce cardiac hypertrophy and may lead to cardiac dysfunction in the KO animals. In the present study, we sought to investigate the consequences of Hsp70 KO on contractile properties and calcium handling of the heart. In addition, we studied changes in cardiac hypertrophy–related kinases and stress-induced response specifically against ischemia-related injury. Our findings showed impaired cardiac function and calcium handling associated with hypertrophy in the Hsp70-KO mice.
Additional method details can be found at the online-only Data Supplement.
Generation of the Hspa1a−/−/Hspa1b−/− KO Mice
The targeted deletions, or KO of Hspa1a and Hspa1b, were carried out at the US Environmental Protection Agency (Research Triangle Park, NC), as described previously.14 Homozygous nulls (KO) and wild-type (WT) lines were generated. These mice are available for distribution to the scientific community through the Mutant Mouse Regional Resource Centers (MMRRC) network (http://www.mmrrc.org/). The KO mice and alleles are referenced in Mouse Genome Informatics (http://www.informatics.jax.org/searches/allele.cgi?26337) as Hspa1a/Hspa1btm1Dix.
Male mice (16 to 24 months old) were anesthetized with ketamine (100 mg/kg) and heparinized (7.5 U/30 g body wt). We used an isovolumic heart preparation described previously.21
Global ischemia was initiated by stopping perfusion to the heart and submerging it into the perfusate maintained at 37°C. Pacing was ceased during the ischemic period (12 minutes). After ischemia, perfusion and pacing were reinitiated and maintained for 1 hour. The time frames for ischemia and reperfusion were chosen on the basis of results from preliminary studies, in which the ischemic duration was sufficient to elicit mechanical and hemodynamic changes during reperfusion but not long enough to induce detrimental effects on hearts.
Assessment of Left Ventricular Function and Cellular Damage
Left ventricular function was monitored continuously throughout each experiment as previously described.21
Assessment of Cardiac Hypertrophy
Hearts were isolated from the body and rinsed with 4°C perfusate to remove blood; atria and aortas were then removed. The left ventricle was isolated from the right ventricle, and both were weighed. Heart weight–to–body weight ratios were calculated as an index of cardiac hypertrophy.22 Isolated ventricular myocytes were plated on a cover-glass, and cell lengths and widths were measured under the microscope.
Isolation and Adenoviral Infection of Cardiomyocytes
Ca2+-tolerant adult cardiomyocytes were isolated from ventricular tissue by a standard enzymatic digestion procedure described previously.23 Myocytes were cultured on glass coverslips treated with laminin. Cells were infected with either an empty adenovirus (Adv-Ctr) or adenovirus containing the rat Hsp70-2 gene24 (homolog of mouse Hspa1a) (Adv-Hsp70) 2 hours after isolation with a multiplicity of infection of 20 pfu/cell. To test the expression efficiency, myocytes were infected with an adenoviral vector carrying a marker gene, β-galactosidase, at a multiplicity of 20 pfu/cell. After 48 hours, infection efficiency determined under these conditions was 94%.
Measurements were performed 48 hours after infection. Adenoviruses were constructed as described previously.12 In a separate set of experiments, an adenovirus containing the catalytic domain of Raf-1 was used to express an activated form of Raf.
Calcium transients were recorded in either fresh plated or cultured myocytes, with the use of Indo-1 or Fluo-3. The method has been described previously.23 Calcium transients were recorded from at least 20 cells per heart and for at least 3 hearts per treatment. Diastolic and systolic intracellular Ca2+ levels were inferred from the basal and maximal indo-1 ratio per cycle, respectively. Diastolic decay time (Tdecay) was calculated from the normalized Ca2+ transient.
Sarcoplasmic Reticulum Ca2+ Load
Experiments were performed at room temperature, as described by Shannon et al.25 In brief, cells were superfused with normal Krebs solution and paced at 0.3 Hz at least 20 times; solution then was rapidly switched to 0 Na, 0 Ca2+ normal Tyrode solution. After 30 seconds, 10 mmol/L caffeine was added to cause sarcoplasmic reticulum (SR) calcium release. The difference between the basal and peak total systolic [Ca2+]i in the presence of caffeine is therefore SR calcium content.
Measurement of Myocyte Contractility by Edge Detection
Contractile properties of single myocytes were measured by using edge detection, as described.23 Myocyte fractional shortening, maximal shortening rate (+dL/dt), and relengthening rate (−dL/dt) were analyzed with the use of Felix32 software (Photon Technology International Inc, Birmingham, NJ). Data were collected from at least 20 cells per heart and 3 hearts per group.
Papillary muscles were prepared as previously described.26 Post–rest potentiation protocol was performed to determine maximal force (Fmax) by stopping stimulation ranging up to 20 seconds and then resuming regular stimulation. Force-frequency protocol was performed from 0.5 to 7 Hz. Assessment of rapid cooling contractures was adapted from a previously described method.27
Western Blot Analyses
Western blot analyses were performed as previously described.9,21 Membranes were probed with antibodies for Hsp70, Hsc70, SERCA2a, and α-actin antibodies (Stressgen, Victoria, BC, and Affinity Bioreaents Inc, Golden, Colo). Raf-1, JNK, p38-MAPK, and ERK protein level were measured by using phosphorylation state-specific as well as nonspecific antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, Calif, and Upstate Inc, Waltham, Mass). Immunoprecipitation was performed with the use of the Raf-1 antibody.
Data are given as mean±SEM. Statistical comparison was performed with the use of 1-way ANOVA in conjunction with the Fisher protected least-squares-difference test, independent t tests, and repeated-measures ANOVA.
The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.
Targeted deletion of the Hspa1a and Hspa1b genes did not generate obvious effects on the natural development of these mice. Initial phenotype analysis during the first 8 weeks after birth revealed no differences in body weight, gross physical appearance, or behavioral vigor between littermates of all genotypes. However, in the adult stage (16 to 24 weeks), KO mice exhibited significantly greater left ventricular weight–to–body weight ratio (Figure 1A, P<0.01), whereas right ventricular weight–to–body weight ratio was unchanged, which suggests the specific development of left ventricular hypertrophy in Hsp70-KO mice. Isolated cardiac myocyte dimensions also show a significant increase in parallel with heart weight–to–body weight ratio in the KO mice compared with WT (Figure 1B, P<0.01). However, liver weight–to–body weight ratio was not different in both WT and KO groups (data not shown). Heart rate and blood pressure were similar between groups (WT, 404.43±5.92 and KO, 379.45±31.59 bpm; WT, 76.25±0.6/44.75±1.4 and KO, 78.00±2.88/44.50±2.0 mm Hg, respectively, n=4 for each group).
Expression of Hsp70 Protein in WT and Hspa1a−/−/Hspa1b−/− KO Hearts
To ensure the absence of Hsp70 proteins in KO mice, Hsp70 protein was assessed by Western blot after ischemia/reperfusion (I/R) challenge. Western blot analysis indicated the complete absence of expression of Hsp70 protein in the KO group after 12 minutes of ischemia and 60 minutes of reperfusion, whereas there was an obvious induction of Hsp70 in the WT group after ischemia compared with WT (normoxic) or KO (Figure 1C). However, Hsc70 expression was not altered between groups.
Calcium Handling and Contractility in Cardiac Myocyte of KO Mice
We studied calcium transients in myocytes isolated from control and KO mice plated for 1 hour on coverslips. Calcium transients of cardiac myocytes from KO mice were prolonged (Figure 2A). Calcium decline (half-time, t1/2) was prolonged by 120% compared with WT (Table), suggesting lower SERCA2a activity. Time to peak (Tmax) was also prolonged in KO mice. Diastolic calcium (Rdia) was decreased in the KO mice (Table). Systolic Ca2+ peak was diminished by &10% in the myocytes from KO mice (Table), although the magnitude of the calcium transient was similar in both groups. Contractility, assessed by edge detection, was also impaired in cardiac myocytes from KO mice (Figure 2B). Cell shortening was decreased by 35% in the KO (Figure 2C). Rate of contraction (+dL/dt) and rate of relaxation (−dL/dt) were diminished by &40% in myocytes from KO mouse hearts (Figure 2, D and E). Western blot analysis revealed an &20% decrease in SERCA2a expression in the KO hearts (KO, 1.07±0.06 versus WT, 1.37±0.01 arbitrary units normalized by α-actin; P<0.05; n=8 for each group; Figure 1C). Diminished SERCA2a expression can lead to lower SR calcium loading. Myocytes from KO mice presented a decreased caffeine-induced Ca2+ transient (Figure 3, A and B). Ca2+ content was &20% lower in the KO mice (Figure 3C).
In addition to these studies, we measured rapid cooling contracture in isolated papillary muscle to evaluate SR calcium loading.27 Figure 3D shows that the magnitude of developed force after rapid cooling of the cardiac muscle was lower in KO than in WT mice (2.6±0.3 versus 6.6±2.6 mN/mm2, respectively; P<0.01, n=5).
Effects of I/R on Cardiac Mechanical Function
We sought to investigate if myocyte dysfunction was reflected in global cardiac function for KO mice. We did not see alterations on baseline parameters of perfused mouse hearts from KO mice compared with control (Figure 4). However, I/R induced a significant decrease in cardiac mechanical function in both WT and KO hearts. Before ischemia, left ventricular developed pressure was similar in both WT and KO groups (72.5±2.0 versus 80.2±6.3 mm Hg, respectively; Figure 4A; n=5). However, developed pressure was significantly decreased in both KO and WT groups throughout reperfusion. As expected, the recovery of left ventricular developed pressure was significantly lower in the KO group compared with the WT group (33.89±3.70 versus 44.69±1.71 mm Hg at 60 minutes of reperfusion, P<0.05). Other indexes of contractility such as dp/dtmax and dp/dtmin followed a similar trend. During the preischemic period, dp/dtmax and dp/dtmin were similar between both the WT and the KO groups; however, after ischemia, the KO group showed significantly poorer recoveries in the dp/dtmax and dp/dtmin compared with the WT group throughout the reperfusion period (1242.4±129.6 versus 1848.5±18.0, P<0.01, and −810.3±83.1 versus −1169.8±64.3 at the 60 minutes of reperfusion, P<0.03; n=5 for each group).
End-diastolic pressure was set at 10 mm Hg during the preischemic period. After ischemia, end-diastolic pressure increased significantly in the KO group, which was prominent at the early reperfusion period and was significantly higher compared with the WT group (29.65±3.50 versus 13.78±3.61 mm Hg at 15 minutes of reperfusion, P<0.01; Figure 3D; n=5). The WT group did not show significant changes in end-diastolic pressure compared with the preischemic level throughout the reperfusion period. Also, T1/2 was significantly increased in the KO group compared with its preischemic level, and it was significantly higher compared with the WT group throughout reperfusion (P<0.05, n=5, not shown).
Creatine kinase activity in the perfusion effluent was significantly increased after I/R, especially at the early phase of reperfusion in the KO hearts compared with WT control hearts (Figure 4C).
Rescue of Myocyte Contractile Dysfunction by Adenoviral-Induced Expression of Hsp70 or SERCA2a
To investigate if lack of Hsp70 was responsible for the observed alterations on contractile properties of KO myocytes, we infected cardiac myocytes from KO mice with an adenovirus carrying the Hspa1a gene. Hsp70 protein expression was confirmed by Western blot (not shown). Figure 5 shows the recovery of contractile properties toward control in the KO cardiac myocytes expressing Hsp70. Hsp70 expression increased cell shortening by 120% in the myocytes from KO mice compared with KO myocytes infected with empty adenovirus (KO+Adv-Ctr, Figure 5B). The function of rescued myocytes was not statistically different from WT myocytes. The rate of contraction and relaxation was also improved (Figure 5C). In addition, we measured the expression of SERCA2a protein in these myocytes. Cultured KO myocytes presented a 15% decrease in SERCA2a expression, which was returned toward WT after Hsp70 adenoviral expression (Figure 5D). Furthermore, replacement of SERCA2a protein by adenoviral expression, restored contractile function, and calcium transients toward control (see Figure in the online-only Data Supplement).
Effect on the Active Form of MAPK
The level of 46-kDa phosphorylated JNK (p-JNK) was significantly greater in KO hearts compared with WT hearts (Figure 6A). Total JNK protein levels were similar among all groups. Phosphorylated p38-MAPK (p-p38-MAPK) protein level was also significantly higher in KO hearts, with the (nonphosphorylated) p38-MAPK (p38-MAPK) level remaining similar in KO group compared with the WT hearts. There was also an increase in p-ERK level in the KO group, and the total ERK level was similar in both the WT and the KO group. α-Actin level was similar between the WT and KO groups. When these bands were normalized to α-actin, p-JNK, p-p38-MAPK, and p-ERK levels were significantly higher in the KO group (Figure 6B, P<0.01, n=4). Figure 6C shows phospho-active kinase levels normalized to total kinase level. p-JNK, p-p38-MAPK, and p-ERK were significantly higher in the KO group (P<0.01, n=4).
Role of Raf-1 Activation on the KO Cardiac Phenotype
Phosphorylated Raf-1 (p-Raf-1) was 65% higher in the KO mice compared with WT (Figure 7A). In contrast, transgenic mice overexpressing Hsp70 presented 42% decreased p-Raf-1. Immunocoprecipitation experiments indicated a direct interaction between Raf-1 and Hsp70 (Figure 7B). Adenoviral expression of activated Raf produced a 2-fold increase in phosphorylated ERK (Figure 7C). Interestingly, SERCA2a expression was decreased by 54% after Raf activation.
The present study was undertaken to investigate the effects of a genetic ablation of the Hspa1a and Hspa1b genes on the heart. The null mutation of Hspa1a/ Hspa1b did not produce obvious abnormalities in the physical appearance. However, as shown here for the first time, absence of Hsp70 leads to a cardiac phenotype characterized by impaired contractile function and altered calcium handling associated with mild hypertrophy.
After we found cardiac hypertrophy in the KO mice, we searched at the myocyte level for contractile dysfunction. We found alterations of the calcium transient characterized by a prolonged time to peak and time of calcium decline. These alterations may explain the impaired contractility in the myocyte, as measured by edge detection, with diminished cell shortening and slowed rate of contraction and relaxation. These alterations were specific for the Hsp70 ablation because adenoviral-induced expression of Hsp70 reverted the contractile dysfunction.
One of the possible mechanisms to explain the cardiac alterations observed here is a diminished SERCA2a expression. We observed a discrete but consistent SERCA2a protein downregulation in the hearts of KO mice. The prolonged calcium transient and the slowed cell relaxation support a diminished SERCA2a activity. It is well accepted that diminished SERCA2a activity can lead to lower calcium loading and an impaired contraction. Diminished calcium loading was demonstrated by our findings, with an increased time to peak in the calcium transient, a decreased caffeine-induced calcium release, and a decreased cooling contracture in the papillary muscle. In addition, adenoviral SERCA2a expression reestablished Ca2+ transients and contractility of KO cardiomyocytes. The mechanism involved in the decrease of SERCA2a activity/expression can be related to absence of the inhibitory effect of Hsp70 on p38-MAPKs and/or Raf-1/ERK pathway activity. It has been demonstrated that expression of active Raf-1 prolongs calcium transients in neonatal rat cardiomyocytes.28 Similarly, activation of MKK6/p38 MAPK pathway prolongs the calcium transient and downregulates SERCA2a.29 Here, we found that p-Raf-1 is increased in KO mice, which explains the activation of ERK and decreased SERCA2a observed. In support of this idea, we were able to induce SERCA2a decrease in cardiomyocytes by activation of the Raf-1/ERK pathway. In the immunocoprecipitation experiment, we found that Hsp70 interacts directly with Raf-1, possibly modulating its activity.
Challenging Hspa1a−/−/ Hspa1b−/− KO hearts with global I/R revealed impaired responses in KO hearts compared with WT, indicating the involvement of Hsp70 in protection against I/R injury. Hsp70 appears to protect cells through multiple mechanisms by targeting key cellular components and regulatory processes. Our study demonstrated that the protection conferred by Hsp70 occurs during the early phase of protection and is evident within 1 to 2 hours after I/R-mediated injury. A role for Hsp70 protecting the late phase of I/R injury has been demonstrated by Nakano et al30 and Hampton et al.13 In the same model used in the present work, it was demonstrated that targeted deletion of the Hspa1a/1b genes abolished the late infarct-sparing effect of ischemic preconditioning after I/R injury.13 It has been suggested that cardiac resistance against I/R insults after heat conditioning is a multifactorial phenomenon and includes more than one member of heat shock protein induction.5,31–33 Our study and the study of Hampton et al,13 which use a genetic ablation of both Hsp70 genes, provide more direct evidence for a role of Hsp70 in its protection of the heart against I/R-related injury.
The hypertrophic response of the myocardium is an important pathophysiological adaptation in which specific members of the MAPK play a role. Accumulating evidence suggests that especially JNK, ERK, and p38-MAPKs play key roles in regulating hypertrophy in cardiac myocytes.34,35 Particularly, JNK activation has been implicated as a necessary molecular event in the cardiac hypertrophic response, both in vitro and in vivo.36–40 Other factors are also involved, as demonstrated by the increased activity of p38-MAPK after pressure-overload hypertrophy in aortic-banded mice and in human hearts with heart failure.34,41 Thus, the activation of intracellular signaling pathways, including JNK and p38-MAPKs, is associated with the development of cardiac hypertrophy. Gaia et al42 showed that whereas hypertrophy gradually develops in spontaneously hypertensive rats, heat stress induces a more pronounced stimulation of Hsp70 proteins. More interestingly, it has also been reported that the acute elevation of Hsp70 inhibits JNK and p38-MAPKs and subsequently inhibits a signal transduction pathway leading to programmed cell death.20 Our study clearly demonstrates that the active forms of JNK and p38-MAPKs were significantly elevated in KO hearts, and these KO mice had significant left ventricular hypertrophy compared with the WT littermates. Thus, the cardiac hypertrophy seen in KO mice could result from removing the inhibiting effects of Hsp70 on the JNK pathway.
This is the first study that defines a role for Hsp70 in maintaining cardiac contractility and calcium handling. In addition, our findings are consistent with previous observations that overexpression of Hsp70 confers cardioprotection against myocardial ischemia. Thus, these findings strongly support the hypothesis that Hsp70 not only plays an important role in cardiac protection against I/R insults but also is necessary for the basal cardiac function. We also observed that a genetic knockout of Hsp70 genes results in the development of cardiac hypertrophy. The subcellular mechanism for the development of cardiac hypertrophy in Hsp70-KO mice appears to be related to the inductions in the MAPK pathway, including JNK, p38-MAPK, and Raf-1/ERK.
This work was subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Adenovirus encoding the activated Raf was a gift from Dr Kevin M. Pumiglia, Albany Medical College, Albany, NY.
Sources of Funding
The National Institutes of Health (grant HL52946, to W.H.D.) contributed to the funding of these studies. This work was also funded in part by the US Environmental Protection Agency.
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This report demonstrates that deletion of the inducible heat shock protein 70 in mouse hearts results in cardiac hypertrophy, decreased contractile function, and abnormal calcium handling. These findings indicate that heat shock protein 70, which participates in the refolding of proteins during ischemic injury, has the additional function of inhibiting a pathological type of cardiac hypertrophy. Therefore, not only does increased expression of the heat shock protein protect against ischemic injury, but it may have additional beneficial functions that result from its inhibiting influence on specific kinases that mediate cardiac hypertrophy. In this report, we did show that deletion of the heat shock protein 70 leads to the activation of kinase signaling cascades, which induces cardiac hypertrophy. One may speculate that increased expression of heat shock protein 70 could also modify other forms of cardiac hypertrophy that lead to abnormal calcium handling in the myocyte, resulting in delayed diastolic relaxation and decreased contractile function. In summary, therapies designed to increase expression of heat shock protein 70 may be of benefit by decreasing ischemic heart injury resulting from protein malfolding, and, in addition, it may counteract pathological remodeling leading to cardiac hypertrophy and the accompanying abnormalities in calcium handling.
↵*Drs Kim and Suarez contributed equally to this work.
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.598409/DC1.