Calcineurin Aß Gene Targeting Predisposes the Myocardium to Acute Ischemia-Induced Apoptosis and Dysfunction
From the Department of Pediatrics, Division of Molecular Cardiovascular Biology (O.F.B., R.A.K., B.J.W., Y.-S.D., R.K., T.E.H., J.D.M.), Children’s Hospital Medical Center, Ohio; Department of Cardiology (D.J.L., P.A.D.), Cardiovascular Research Institute Maastricht, University Hospital Maastricht, the Netherlands, and Department of Cardiology, Heart, Lung Center Utrecht, Utrecht, the Netherlands; Department of Pediatrics, Division of Cardiovascular Imaging (B.J.G., T.R.K.), Children’s Hospital Medical Center, Ohio; and the Department of Pediatrics, Division of Pediatric Informatics (B.J.A.), Children’s Hospital Medical Center, Ohio.y[+!;4, 百拇医药
Abstracty[+!;4, 百拇医药
Cardiovascular disease is the leading cause of mortality and morbidity within the industrialized nations of the world, with coronary heart disease (CHD) accounting for as much as 66% of these deaths. Acute myocardial infarction is a typical sequelae associated with long-standing coronary heart disease resulting in large scale loss of ventricular myocardium through both apoptotic and necrotic cell death. In this study, we investigated the role that the calcium calmodulin-activated protein phosphatase calcineurin (PP2B) plays in modulating cardiac apoptosis after acute ischemia-reperfusion injury to the heart. Calcineurin Aß gene–targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL, and a greater loss in functional performance compared with strain-matched wild-type control mice after ischemia-reperfusion injury. RNA expression profiling was performed to uncover potential mechanisms associated with this loss of cardioprotection. Interestingly, calcineurin Aß-/- hearts were characterized by a generalized downregulation in gene expression representing approximately 6% of all genes surveyed. Consistent with this observation, nuclear factor of activated T cells (NFAT)-luciferase reporter transgenic mice showed reduced expression in calcineurin Aß-/- hearts at baseline and after ischemia-reperfusion injury. Finally, expression of an activated NFAT mutant protected cardiac myocytes from apoptotic stimuli, whereas directed inhibition of NFAT augmented cell death. These results represent the first genetic loss-of-function data showing a prosurvival role for calcineurin-NFAT signaling in the heart.
Key Words: signaling calcineurin apoptosis heart failure transcriptionu0r^}!, 百拇医药
Introductionu0r^}!, 百拇医药
Cardiovascular disease is the leading cause of mortality in adults in the United States where it is thought to account for approximately 12 million deaths annually.1,2 Myocardial infarction, as well as nonischemic forms of heart failure, are thought to involve an irreversible loss of cardiac myocytes through apoptosis or programmed cell death.2–4In recent years, a great body of research has focused on identifying the signaling molecules responsible for myocardial cell death or those molecules that might protect the myocardium from ischemic insults.5 For example, numerous studies have identified signaling molecules that enhance cardiomyocyte apoptosis such as p38{alpha} , c-Jun N-terminal kinase, tumor necrosis factor-{alpha} , p53, ß-adrenergic receptors, and nitric oxide.5–7 In contrast, other signaling pathways have been shown to protect the heart from apoptosis such as cardiotrophin-1 through the gp130 receptor, p38ß, insulin-like growth factor-1 (IGF-1), Akt/protein kinase B, protein kinase C{epsilon} , nitric oxide, and extracellular signal-regulated kinases 1/2.5,6,8–12
Another potential regulator of cardiac apoptosis is the calcium-calmodulin–activated protein phosphatase calcineurin (or PP2B). Calcineurin was identified as an important regulator of cardiomyocyte hypertrophy in vivo and in vitro, while more recent investigation has suggested a role for calcineurin in the modulation of cardiomyocyte apoptosis.13,14 Studies conducted in neurons, lymphocytes, and tumor cell lines have demonstrated either pro- or antiapoptotic effects associated with calcineurin activation.15–22 Similarly, both pro- and antiapoptotic modulatory roles have been ascribed to calcineurin in cardiomyocytes. For example, calcineurin activation in cultured cardiac myocytes confers protection to H2O2- or 2-deoxyglucose–induced apoptosis, suggesting that calcineurin is cytoprotective.14,23 Moreover, transgenic mice expressing an activated form of calcineurin in the heart are largely protected from ischemia-reperfusion–induced DNA laddering, further suggesting that calcineurin activation antagonizes cardiomyocyte apoptosis in vivo.14 In contrast, isoproterenol stimulation of cardiac ß-adrenergic receptors promoted myocyte apoptosis, in part, by stimulating calcineurin activity.24 Part of this disparity potentially stems from the dichotomous actions of the often-employed inhibitory agent cyclosporine A. For example, cyclosporine A not only inhibits calcineurin enzymatic activity, but also inhibits mitochondrial permeability pore transition (MPTP) and apoptosis through its association with cyclophilin D in the inner mitochondrial membrane.25 However, use of cyclosporine-independent strategies have also revealed pro- and antiapoptotic regulatory roles for calcineurin, suggesting multiple levels of complexity underlying the biology of calcineurin and cell death. Given these issues, we pursued a genetic approach to evaluate the functional significance of calcineurin as a potential modulator of ischemia-reperfusion–induced apoptosis in vivo.
Materials and Methodszv(|, http://www.100md.com
Surgical Procedure and Animal Modelszv(|, http://www.100md.com
Mice were randomized to receive either ischemia-reperfusion injury or a sham procedure. Mice were anesthetized and placed on a warming pad maintained at 37°C. The trachea was cannulated with a polyethylene tube connected to a respirator (Harvard Apparatus) with a tidal volume of 0.6 mL (110/min). A left lateral thoracotomy was performed between the fourth and fifth ribs, pericardial tissue was removed, and the left anterior descending artery (LAD) was visualized and ligated with a slipknot of 8-0 silk. Reperfusion was initiated 60 minutes after the occlusion. The Calcineurin Aß (CnAß)-/- mouse and the NFAT-dependent luciferase reporter transgenic line (containing nine copies of an NFAT binding site upstream of a minimal promoter) were described previously.26,27 All procedures performed in animals were approved by the Institutional Animal Care and Use Committee.
Measurements of Ischemic Area at Risk and Infarct Size&.|m, 百拇医药
Twenty-four hours after reperfusion, mice were anesthetized, the LADs were reoccluded, and 1 mL of 1.0% Evans blue was injected into the apex of each heart to stain nonischemic tissue. The hearts were then excised, washed with PBS, embedded in agarose, and cut into five transverse slices for 15 minutes of incubation at room temperature with 1.5% 2,3,5-triphenyltetrazolium chloride (TTC) to measure viable myocardium (red staining). Slices were photographed (each side) under a microscope and left ventricular area, area at risk (AAR), and infarct area were determined by digital planimetry.&.|m, 百拇医药
Echocardiography and Working Heart Analyses&.|m, 百拇医药
CnAß-/- or wild-type mice were anesthetized with 2% isoflurane and hearts were visualized using a Hewlett Packard Sonos 5500 instrument and a 15-MHZ transducer. Cardiac ventricular dimensions were measured on M-mode three times for the number of animals indicated. The isolated ejecting mouse heart preparation has been described in detail previously.28
DNA Laddering and TUNEL+%s, http://www.100md.com
CnAß-/- mice or wild-type controls were subjected to 60 minutes LAD occlusion and 24 hours of reperfusion to induce cardiac apoptosis for DNA laddering and 3 weeks of reperfusion for terminal deoxyribonucleotide transferase (TdT)–mediated dUTP nick-end labeling (TUNEL) assessment as previously described.14+%s, http://www.100md.com
Assessment of Apoptosis in Cultured Cardiac Myocytes+%s, http://www.100md.com
Conditions for generating and culturing neonatal cardiac myocytes were described previously.14 The Ad{Delta} CnA and Ad{Delta} NFATc4 adenoviruses were described previously,14 whereas the AdVIVIT NFAT inhibitory adenovirus was composed of the green florescent protein (GFP) fused to the VIVIT sequence as described previously29 (gift of Dr Susan D. Kraner and Chris Norris, University of Kentucky, Lexington). Cardiac myocytes were treated with staurosporine (500 nmol/L) for 18 hours, and all manipulations were performed 24 hours after adenoviral infection to allow adequate protein expression.14 DNA laddering and measurement of TUNEL were described previously.14
Affymetrix Gene Expression Profiling and Bioinformaticsek, http://www.100md.com
Total RNA samples were prepared from four individual hearts from 8-week-old CnAß-/- or wild-type control mice. Biotin-labeled target cRNA was prepared from T7-transcribed cDNA made from 10 µg of the total RNA using the Affymetrix-recommended protocol30,31 and hybridized for expression analysis to the Affymetrix U74Av2 GeneChip using antibody-based fluorescence signal amplification. Data values used for filtering and clustering were Signal, Signal Confidence, Absolute Call (Absent/Present), and Change (Increase, Decrease, and Unchanged) as implemented in MicroArray Suite 5.0.ek, http://www.100md.com
Statistical Analysisek, http://www.100md.com
Statistical analyses between the experimental groups were performed using a Student’s t test or one-way ANOVA when comparing multiple groups. Data were reported as mean±SEM. Values of P=0.05 were considered significant.ek, http://www.100md.com
Results
Increased Myocardial Infarct Size in CnAß-/- Mice#, 百拇医药
Although conclusive evidence has emerged implicating calcineurin as a pivotal regulator of cardiac myocyte hypertrophy, its role in regulating cardiac myocyte survival or apoptosis after ischemic or stress-related injury has engendered some controversy. To more thoroughly evaluate the association between calcineurin signal transduction and cardiac survival/apoptosis, we analyzed CnAß-/- mice for susceptibility to ischemia-reperfusion injury. Null mice and strain-matched wild-type (WT) controls were subjected to 60 minutes of LAD coronary artery occlusion followed by 24 hours of reperfusion. After this 24-hour reperfusion period, hearts were removed and perfused with Evans blue dye, sectioned, and incubated in 1.5% TTC to quantify area at risk and area of infarct normalized to total left ventricular area (AAR/LV). The area at risk normalized to ventricular area was not different between the two groups (WT, 79.8±3.3%; CnAß-/- mice, 76.5±2.9%; A). These results indicated that there were no genotype-dependent differences in the perfused areas between the two groups. In contrast, loss of viable myocardium was increased by approximately 30% in CnAß-/- mice compared with WT mice (WT, 31.9±3.6%; CnAß-/- mice, 46.19±3.6%, n=8; P<0.05, C).
fig.ommittedfi, http://www.100md.com
Figure 1. CnAß-/- mice show greater myocardial injury after ischemia-reperfusion. A, Area at risk (AAR) to total left ventricular area ratios. B, Infarcted area (IA) normalized to AAR (n=8; P<0.05). C, Representative injured hearts from wild-type (Wt) and CnAß-/- mice stained with Evans blue and TTC.fi, http://www.100md.com
fig.ommittedfi, http://www.100md.com
Figure 2. CnAß-/- mice show greater myocyte death after ischemia-reperfusion. A, Representative DNA ladder as an indication of apoptosis from wild-type (WT) and CnAß-/- hearts after ischemia-reperfusion injury (n=6). B, Representative DNA fragmentation detection assays by ligation-mediated PCR. C, Representative histological sections showing greater scarring in CnAß-/- hearts after ischemia-reperfusion injury (n=6). D, TUNEL quantification of cardiomyocytes in 2 CnAß-/- and wild-type control hearts 3 weeks after ischemic injury expressed as a percentage of total myocytes in the remaining viable myocardium. E, Hematoxylin and eosin– and chloroacetate esterase–stained cardiac histological sections from the indicated mice (24 hours of reperfusion; arrows show mast cells).
CnAß-/- Mice Show Greater Cardiac Cell Death After Ischemia-Reperfusion Injury-s69x5c, 百拇医药
DNA laddering was examined by agarose gel electrophoresis using hearts from sham operated animals and mice subjected to ischemia-reperfusion injury. CnAß-/- mice showed a qualitative increase in the degree of DNA laddering compared with hearts from WT mice, suggestive of more apoptotic damage (n=6 hearts in each group, although only two each are shown) (A). These data were extended through independent assessment of DNA fragmentation by ligation-mediated polymerase chain reaction, which also showed significantly greater signal in the CnAß-/- hearts after ischemia-reperfusion injury (B). Histological analysis was performed using Masson’s trichrome staining (blue color shows fibrosis and scar) on myocardial tissue from WT and CnAß-/- mice subjected to ischemia and 3 weeks of reperfusion. Analysis of six individual hearts in each group revealed greater myocardial damage and increased interstitial fibrosis in CnAß-/- mice compared with WT animals (two representative hearts are shown for each group) (C). As a more quantifiable measure of cell death, TUNEL was performed from histological sections, 3 weeks after ischemia-reperfusion injury. Rates of TUNEL in cardiac myocytes were assessed within the remaining region of viable left ventricle and septum, which demonstrated approximately twice the rate in CnAß-/- hearts compared with WT controls (D). Basal TUNEL rates did not vary between WT and CnAß-/- hearts from sham animals (data not shown). Collectively, these results indicate that CnAß-/- mice undergo enhanced cell death after ischemia-reperfusion injury, suggesting that calcineurin is normally cytoprotective in the heart.
CnAß-/- mice have a partially compromised immune response,26 an effect that might secondarily lessen the degree of myocardial injury, ventricular remodeling, and the inflammatory response after ischemia-reperfusion injury. In attempt to control for such variables, cardiac histological sections were subjected to immunohistochemical analysis for CD45 (total leukocytes), CD3 (T lymphocytes), and B220 (B lymphocytes), which showed no differences in cellular recruitment 24 hours after ischemia-reperfusion in either group (data not shown). Histological sections were also stained with hematoxylin and eosin (H&E) and chloroacetate esterase (for mast cells) (E). The data show similar degrees of cellularity augmentation in both groups 24 hours after ischemia-reperfusion injury (H&E sections), as well as equivalent levels of mast cells in the pericardium (E).j, http://www.100md.com
CnAß-/- Mice Display Worse Cardiac Function After Ischemia-Reperfusion
At baseline, hearts from CnAß-/- mice functioned equivalent to WT control hearts with respect to +dP/dt, maximal left ventricular pressure developed, and time to peak pressure, each assessed by working heart preparation (). These results indicate that loss of calcineurin Aß does not obviously alter cardiac function in the unstimulated state. To further evaluate the functional ramifications of enhanced cell death after ischemia-reperfusion injury, transthoracic echocardiography was performed in CnAß-/- and WT mice. Echocardiography was performed at baseline and 1, 2, and 3 weeks after ischemia-reperfusion injury to serially evaluate function noninvasively. As shown in B, left ventricular end-diastolic dimensions (LVED) and left ventricular end-systolic dimensions (LVES) increased to a larger extent in CnAß-/- mice compared with WT mice (P<0.05). These results indicate greater left ventricular dilation in the heart, presumably due to enhanced cell death in the absence of CnAß. More importantly, fractional shortening, which approximates cardiac contractile performance, was reduced to a greater extent in CnAß-/- mice compared with WT mice 2 and 3 weeks after the ischemic event (P<0.05, n=9; C). These results indicate that CnAß-/- mice had reduced cardiac function after ischemia-reperfusion injury compared with WT controls, consistent with the notion that CnAß-/- hearts are more susceptible to a cell death–promoting stimulus. However, it is possible that CnAß-/- mice could also show alterations in function due to differences in ventricular remodeling that typifies postinfarction injury. To address this possibility, heart weight to body weight ratio assessments were performed after the final 3-week echocardiographic measurement in both groups. Neither WT nor CnAß-/- mice underwent significant cardiac hypertrophy over this time period under the conditions used, suggesting that the difference in functional performance likely reflects the degree of injury (data not shown).
fig.ommitted3y4h9vs, 百拇医药
Table 1. Cardiac Functional Analysis Using an Ex Vivo Working Heart Preparation in CnAß-/- and Wild-Type Controls3y4h9vs, 百拇医药
fig.ommitted3y4h9vs, 百拇医药
Figure 3. Cardiac morphology and function as determined by echocardiography. A and B, Left ventricular end-diastolic dimensions (LVED) and left ventricular end-systolic dimensions (LVES) in CnAß-/- mice compared with wild-type (WT) mice (*P<0.05 vs baseline; #P<0.05 vs WT after ischemia-reperfusion). Data are expressed in millimeters, average±SEM, n=9. C, Fractional shortening assessed by echocardiography in CnAß-/- and WT mice (*P<0.05 vs baseline; #P<0.05 vs WT after ischemia-reperfusion, n=9). Fractional shortening was calculated as (LVED-LVES)/LVEDx100 and expressed as percentage, average±SEM, n=9.3y4h9vs, 百拇医药
Altered Gene Expression in CnAß-/- Mice3y4h9vs, 百拇医药
The enhanced myocardial damage observed after ischemia-reperfusion injury in CnAß-/- mice suggested an alteration in one or more molecular pathway(s) that influences cell survival. Given the vast number of effectors that might potentially influence the survival versus apoptotic decision of myocardial cells after ischemia-reperfusion injury, we performed a large scale, unbiased screen for altered gene expression. Specifically, hearts from 8-week-old CnAß-/- and strain-matched WT mice (2 each) were harvested and RNA was purified for gene expression profiling using the Affymetrix U74Av2 array. This array contains all genes in the murine Unigene database that have been functionally characterized (approximately 6000) as well as an additional 6000 expressed sequence tags (ESTs). Duplicate heart samples were cross-compared between WT and CnAß-/- mice resulting in 5735 genes being significantly detected in one or more groups after internal normalization. Of these genes, 437 were significantly altered in expression between CnAß-/- and WT hearts (P<0.05). The raw data shown in represent the 2n relationship of expression between Wt and CnAß-/- hearts. For example, a WT value of 1.0 and a null value of 0.33 or 3.0 translates into an 8-fold change in gene expression either way. The data are also depicted on a colorimetric scale for simplicity (A). Remarkably, 383 genes showed downregulated expression in CnAß-/- hearts, whereas only 54 genes showed upregulated expression. This general profile suggests that the loss of CnAß reduces the expression of a subset of cardiac genes, implicating calcineurin as an important positive transcriptional effector pathway in the heart.
fig.ommitted#y+7o/, 百拇医药
Figure 4. A, Relative expression ratios of selected functional gene groups assessed from microarray screening. Cardiac RNA was collected from 2 CnAß-/- mice (KO) and 2 wild-type mice (WT) and subjected to expression profiling using the Affymetrix U74Av2 array. Left panels show a color scale representation of gene expression levels, with yellow equal to 1 (no change), blue equal to 0.2 (reduced expression), and red equal to 5 (increased expression). Absolute normalized expression data are shown for each sample in the right-hand columns along with the GenBank accession number. B, Select group of genes showing altered expression in the array screen was subject to a confirmatory RT-PCR analysis of expression levels at varying cycles (to show the most linear point in amplification).#y+7o/, 百拇医药
Expression signatures were further analyzed in CnAß-/- hearts to potentially implicate individual genes as phenotypic modulators. The most significantly altered genes within seven functional categories were assembled and annotated; any one of which might affect the viability of cardiac myocytes after injury. An interesting alteration was identified in a select group of genes that regulate sterol/cholesterol metabolism such as ATP-binding cassette transporter ABC1, the low-density lipoprotein receptor-related protein 10, and the stearoyl-coenzyme A desaturase 1 (A). Also of interest, four cell cycle regulatory genes were significantly altered in CnAß-/- hearts, as well as five genes encoding mitochondrial-localized proteins (A). Within the transcription category, the DNA binding factors Nkx2.5, Hand2, YB-1, and ATF-3 were each downregulated in CnAß-/- hearts, whereas ATF4 and DBP were upregulated. The observed reduction in Nkx2.5 and Hand2 expression, which themselves are important regulators of cardiac gene expression, is consistent with the proposed paradigm whereby calcineurin-NFAT signaling directly and indirectly augments expression of a subset of cardiac genes. Another intriguing pathway alteration was observed in structural genes that facilitate cellular adhesion and/or intracellular support. For example, four and a half LIM domains 1, {gamma} -sarcoglycan, {alpha} -sarcoglycan, desmoglein-2, {alpha} -6 integrin, and {alpha} -4 tubulin each showed altered expression in the CnAß-/- heart (A). A large number of genes that participate in cellular signaling, apoptosis signaling, or stress-responsive signaling were also identified as significantly altered in the absence of CnAß. Finally, a group of genes showing altered expression in the array screen were selected for reverse transcriptase polymerase chain reaction (RT-PCR) analysis at varying cycles to confirm the observed relationships (B). The data obtained by RT-PCR showed the same profile of increased or decrease expression, confirming the reliability of the array screen (normalized to L7 expression) (B).
The large number of candidate genes showing altered expression made it difficult to mechanistically establish which factors or pathways might ultimately underlie the observed profile of greater stress-induced cell death in the absence of CnAß. Despite this qualification, the overall observation that large subsets of genes are reduced in expression suggests that calcineurin functions as a general transcriptional regulator in the heart, which might predispose the heart to cell death. Indeed, NFAT transcription factors are primary effectors of calcineurin signaling that could fulfill such a role (see next section).6^, 百拇医药
NFAT Mediates Cardioprotection6^, 百拇医药
To determine the potential involvement of NFAT factors as regulators of cardiac myocyte cell survival, a neonatal cardiac myocyte culture–based model was employed. Whereas we have previously shown that calcineurin activation protects cultured myocytes from staurosporine- or 2-deoxyglucose–induced apoptosis, the necessity of NFAT factors as downstream mediators was not determined.14 To this end an NFAT-specific inhibitory adenovirus was used that expresses the VIVIT peptide as a GFP fusion.29 The specificity of this inhibitory virus was demonstrated by coinfection with an NFAT-dependent reporter adenovirus in cultured neonatal myocytes (A). AdVIVIT dramatically inhibited Ad{Delta} CnA-induced activation of the NFAT-dependent luciferase reporter. More importantly, AdVIVIT infection in neonatal cardiac myocytes augmented staurosporine-induced TUNEL, whereas expression of an activated NFATc4 truncation mutant antagonized TUNEL (P<0.05; B). Moreover, AdNFATc4 partially reduced enhanced TUNEL associated with calcineurin inhibition through Adcain infection (also see De Windt et al14) (P<0.05, B). Expression of Ad{Delta} NFATc4 infection also antagonized staurosporine-induced DNA laddering, further implicating NFAT factors as modulators of apoptosis in cardiac myocytes (C).
fig.ommitted\]+oyz, 百拇医药
Figure 5. NFAT mediates cytoprotection in the heart. A, Relative luciferase activity (RLU) derived from cultured neonatal cardiomyocytes infected with an NFAT-dependent reporter adenovirus (AdNFAT-luc) alone or in combination with AdCnA and/or AdVIVIT. B, TUNEL quantitation in neonatal cardiomyocytes infected with the indicated adenoviruses for 24 hours, followed by staurosporine treatment for 18 hours (*P<0.05 vs Adßgal with staurosporine; n=3). C, Representative DNA ladder analysis after adenoviral infection then straurosporine treatment (n=4 each).\]+oyz, 百拇医药
The results discussed above indicate that NFAT factors can function as regulators of apoptosis in cultured cardiac myocytes. However, it was uncertain whether NFAT factors might play a similar role within the intact adult heart, downstream of calcineurin signaling. To address this issue, NFAT-luciferase reporter transgenic mice were crossed into the CnAß-/- background and subjected to ischemia-reperfusion injury. NFAT-luciferase transgenic mice, WT for CnAß, were also generated from the same parental cross for direct comparison. Six CnAß-/- and five WT controls were subjected to 60 minutes of ischemia followed by 48 hours of reperfusion injury, at which time the hearts were removed and parsed into three components for measurement of luciferase activity: right ventricle (RV), nonischemic left ventricle and septum (LV-non), and the injured area of the LV (LV-injured). The RV was analyzed to measure NFAT activity as a secondary consequence of ischemia-reperfusion injury–induced remodeling/hypertrophy. Ischemia-reperfusion injury dramatically enhanced NFAT luciferase activity in each of the assayed ventricular compartments in WT controls (). More importantly, CnAß-/- mice consistently showed less NFAT activation after injury (). These results suggest an association between reduced NFAT transcriptional activity and diminished cardioprotection in CnAß-/- mice.
fig.ommittede, 百拇医药
Figure 6. Relative luciferase activity from the hearts of NFAT-luciferase reporter transgenic mice at baseline or after ischemia-reperfusion injury. These reporter mice were crossed into the CnAß-/- or CnAß+/+ background (n=6 and 5, respectively). Activity is shown from the whole heart (control), right ventricle (RV), nonischemic region of the LV and septum (LV-non), and the LAD-perfused area of the LV subjected to ischemia (LV-injured).e, 百拇医药
Discussione, 百拇医药
In this study, we investigated the association between the calcineurin-NFAT signaling pathway and cell death in the myocardium after stress stimulation. Loss of CnAß rendered the myocardium more susceptible to ischemia-reperfusion injury, suggesting that calcineurin signaling helps maintain myocyte viability in vivo. Part of the mechanism responsible for increased ischemic damage in CnAß-/- mice is likely a secondary consequence of altered gene expression due to reduced NFAT activity.
Role of Calcineurin as a Modulator of Cellular Apoptosisat, 百拇医药
Although calcineurin has been recognized as a central regulator of the hypertrophic growth of the myocardium, its role as an effector of myocardial cell death is more controversial. In other tissues or cell-types, calcineurin has been shown to either agonize or antagonize apoptosis after stress stimulation. Studies conducted in neurons, lymphocytes, and tumor cell lines have shown both pro- or antiapoptotic effects of calcineurin activation.15–22 The exact decision of cytoprotection versus apoptosis is likely regulated by coordinated signals from other costimulated signaling pathways or depends on cell-type specific calcineurin effector/docking proteins. Indeed, calcineurin activation was shown to either induce apoptosis or to antagonize apoptosis depending on the status of p38 activation.32 More recently, calcineurin was shown to localize to the mitochondria in fibroblasts through docking with the inhibitory protein FKBP38, resulting in Bcl-2 and Bcl-xl redistribution.33 Calcineurin has also been implicated as a direct inducer of apoptosis in primary hippocampal neurons through dephosphorylating the proapoptotic factor Bad.15 In addition to cell-type specific regulatory considerations, the pro- or antiapoptotic effects attributed to calcineurin have been obscured by the use of cyclosporine A, which can modulate apoptosis independent of calcineurin through direct effects on mitochondrial permeability transition pore (MPTP) formation within the inner mitochondrial membrane.25 Given the calcineurin-independent effects associated with cyclosporine A and FK506, the use of calcineurin gene-targeted mice and/or null cells derived from these mice should help elucidate the direct role that calcineurin plays in modulating cellular apoptosis in diverse tissues.
In the present study, we demonstrated that genetic disruption of the CnAß gene in the mouse enhanced cardiac damage induced by ischemia-reperfusion injury. This result indicates that calcineurin signaling imparts a degree of protection against cell death in the heart. Consistent with this notion, we have previously shown that transgenic mice expressing a constitutively active mutant of calcineurin in the heart are significantly protected from ischemia-reperfusion-induced cell death.14 In cultured cardiomyocytes, adenoviral-mediated gene transfer of activated calcineurin reduced 2-deoxyglucose–induced TUNEL, whereas calcineurin inhibition with a Cain-expressing adenovirus increased TUNEL.14 In contrast, a subsequent study reported that isoproterenol stimulation of neonatal cardiomyocytes promoted apoptosis, in part, by stimulating calcineurin activity.24 These authors demonstrated that cyclosporine A and FK506 blocked the increase in cardiomyocyte apoptosis induced by isoproterenol stimulation and, more significantly, that transgenic mice expressing dominant-negative calcineurin in the heart were refractory to isoproterenol-induced TUNEL reactivity in vivo. These results suggest that calcineurin activation is associated with enhanced apoptosis in cardiomyocytes, in contrast to our original and subsequent observations. It is likely that the nature of the stimulus (isoproterenol) partially underlies the disparity between the two studies discussed above. For example, isoproterenol induces a prominent elevation in cAMP, which induces protein kinase A signaling and secondary alterations in inotropy, events not typically associated with ischemic injury or straurosporine stimulation.
By comparison, Kakita et al23 recently identified an antiapoptotic role for calcineurin activation in cardiomyocytes after endothelin-1 stimulation. Specifically, endothelin-1 stimulation protected cardiac myocytes in culture from H2O2-induced TUNEL reactivity, DNA laddering, caspase-3 cleavage, and loss of mitochondrial membrane potential. This endothelin-1–mediated cytoprotection from H2O2-induced apoptosis was blocked by inhibition of calcineurin with either cyclosporine A or FK506. Collectively, these disparate accounts underscore the complexity of intracellular signaling networks within mammalian cells, such that seemingly related stress stimuli can elicit fundamentally different responses depending on the nature of the stimulus (mitochondrial- versus death receptor–mediated), the status of other parallel signaling pathways, and the context of cell-type specific calcineurin modulatory factors.pu-&n#7, http://www.100md.com
Mechanism Whereby Calcineurin Signaling Protects the Myocardium
Although a large number of studies have implicated calcineurin as a modulator of cell death in varied cell types, only a few downstream mechanisms responsible for death or protection have been identified. As discussed above, calcineurin was reported to directly dephosphorylate Bad in neurons, thus enhancing apoptosis.15 Careful evaluation of Bad phosphorylation at serine 112, 136, and 128 from the hearts of calcineurin transgenic mice or CnAß-/- mice failed to show any difference from controls (data not shown). Kakita et al23 reported that calcineurin-mediated protection from H2O2-induced apoptosis was associated with an increase in Bcl-2 expression in cultured neonatal cardiac myocytes. However, expression levels of Bcl-2 did not change in the hearts of either calcineurin TG mice or CnAß-/- mice, nor was expression altered for Bcl-xl, Bad, caspase-1, -3, -8, -9, or FKBP38 (data not shown). Previously, we identified a minor but significant increase in Akt phosphorylation in the hearts of calcineurin transgenic mice.14 However, CnAß-/- mice showed no difference in basal or stimulated Akt phosphorylation in the heart, ruling out this potential mechanism (data not shown). Collectively, these negative findings suggest that the classical effectors of the apoptotic response are not directly linked to calcineurin signaling in the heart.
To investigate other potential mechanisms, an unbiased array screen was performed from the hearts of CnAß-/- and matched WT mice. Although a number of potential regulatory proteins were altered in expression, the most significant observation was the dramatic downregulation in expression of large subsets of unrelated genes (about 6% of detectable genes in the heart). This observation suggested a defect in the transcriptional potency underlying a group of genes in the hearts of CnAß-/- mice. This interpretation is consistent with known role of NFAT transcription factors as important effectors of calcineurin-regulated gene expression in most cell types.34 Indeed, the full potency of calcineurin-induced hypertrophy in the heart was shown to require NFATc3 using gene-targeted mice.35t\v}#, 百拇医药
With respect to apoptosis signaling, we previously observed that overexpression of an activated NFATc4 in cultured neonatal cardiomyocytes partially antagonized 2-deoxyglucose-induced apoptosis.14 Furthermore, Kakita et al23 showed that endothelin-1–mediated protection from H2O2-induced apoptosis also promoted NFAT dephosphorylation. In this study, we showed that specific inhibition of NFAT with AdVIVIT augmented cardiomyocyte cell death after staurosporine treatment. More importantly, CnAß-/- mice showed reduced NFAT transcriptional activity in vivo after ischemia-reperfusion injury. These results are also consistent with a recent report by Izumo and colleagues36 in which NFAT inhibition augmented cardiac myocyte apoptosis after phenylephrine stimulation in culture.
Collectively, these results discussed above suggest that the homeostatic transcriptional activity of NFAT provides the necessary framework of basal gene expression that affords cardiac "health" and resistance to apoptotic stimuli. However, the exact array of downstream effectors that are regulated by NFAT factors in providing protection is not known. Despite this, the results of this study provide the first genetic data indicating that calcineurin signaling is necessary for cardiac protection and suggests that strategies to acutely agonize calcineurin might be of therapeutic benefit during or after myocardial infarction. However, this notion is in dramatic contrast to the function of calcineurin as a mediator of cardiac hypertrophy, which itself can lead to cardiomyopathy and heart failure if constitutively activated (although it is apoptosis-independent). These observations suggest that calcineurin may function as a "double-edge sword," such that transient activation antagonizes myocyte apoptosis, but long-standing activation induces cardiac hypertrophy and deleterious ventricular remodeling associated with heart failure.
Acknowledgments$, 百拇医药
This work was supported by the NIH (to J.D.M. and B.J.A.) and the Pew Charitable Trusts (J.D.M). O.F.B was supported by Post-Doctoral Fellowships from the NIH (HL10336). B.J.W. was supported by an MD/PhD scholar award from the University of Cincinnati Physician Scientist Training Program and the Albert J. Ryan Foundation.$, 百拇医药
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Abstracty[+!;4, 百拇医药
Cardiovascular disease is the leading cause of mortality and morbidity within the industrialized nations of the world, with coronary heart disease (CHD) accounting for as much as 66% of these deaths. Acute myocardial infarction is a typical sequelae associated with long-standing coronary heart disease resulting in large scale loss of ventricular myocardium through both apoptotic and necrotic cell death. In this study, we investigated the role that the calcium calmodulin-activated protein phosphatase calcineurin (PP2B) plays in modulating cardiac apoptosis after acute ischemia-reperfusion injury to the heart. Calcineurin Aß gene–targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL, and a greater loss in functional performance compared with strain-matched wild-type control mice after ischemia-reperfusion injury. RNA expression profiling was performed to uncover potential mechanisms associated with this loss of cardioprotection. Interestingly, calcineurin Aß-/- hearts were characterized by a generalized downregulation in gene expression representing approximately 6% of all genes surveyed. Consistent with this observation, nuclear factor of activated T cells (NFAT)-luciferase reporter transgenic mice showed reduced expression in calcineurin Aß-/- hearts at baseline and after ischemia-reperfusion injury. Finally, expression of an activated NFAT mutant protected cardiac myocytes from apoptotic stimuli, whereas directed inhibition of NFAT augmented cell death. These results represent the first genetic loss-of-function data showing a prosurvival role for calcineurin-NFAT signaling in the heart.
Key Words: signaling calcineurin apoptosis heart failure transcriptionu0r^}!, 百拇医药
Introductionu0r^}!, 百拇医药
Cardiovascular disease is the leading cause of mortality in adults in the United States where it is thought to account for approximately 12 million deaths annually.1,2 Myocardial infarction, as well as nonischemic forms of heart failure, are thought to involve an irreversible loss of cardiac myocytes through apoptosis or programmed cell death.2–4In recent years, a great body of research has focused on identifying the signaling molecules responsible for myocardial cell death or those molecules that might protect the myocardium from ischemic insults.5 For example, numerous studies have identified signaling molecules that enhance cardiomyocyte apoptosis such as p38{alpha} , c-Jun N-terminal kinase, tumor necrosis factor-{alpha} , p53, ß-adrenergic receptors, and nitric oxide.5–7 In contrast, other signaling pathways have been shown to protect the heart from apoptosis such as cardiotrophin-1 through the gp130 receptor, p38ß, insulin-like growth factor-1 (IGF-1), Akt/protein kinase B, protein kinase C{epsilon} , nitric oxide, and extracellular signal-regulated kinases 1/2.5,6,8–12
Another potential regulator of cardiac apoptosis is the calcium-calmodulin–activated protein phosphatase calcineurin (or PP2B). Calcineurin was identified as an important regulator of cardiomyocyte hypertrophy in vivo and in vitro, while more recent investigation has suggested a role for calcineurin in the modulation of cardiomyocyte apoptosis.13,14 Studies conducted in neurons, lymphocytes, and tumor cell lines have demonstrated either pro- or antiapoptotic effects associated with calcineurin activation.15–22 Similarly, both pro- and antiapoptotic modulatory roles have been ascribed to calcineurin in cardiomyocytes. For example, calcineurin activation in cultured cardiac myocytes confers protection to H2O2- or 2-deoxyglucose–induced apoptosis, suggesting that calcineurin is cytoprotective.14,23 Moreover, transgenic mice expressing an activated form of calcineurin in the heart are largely protected from ischemia-reperfusion–induced DNA laddering, further suggesting that calcineurin activation antagonizes cardiomyocyte apoptosis in vivo.14 In contrast, isoproterenol stimulation of cardiac ß-adrenergic receptors promoted myocyte apoptosis, in part, by stimulating calcineurin activity.24 Part of this disparity potentially stems from the dichotomous actions of the often-employed inhibitory agent cyclosporine A. For example, cyclosporine A not only inhibits calcineurin enzymatic activity, but also inhibits mitochondrial permeability pore transition (MPTP) and apoptosis through its association with cyclophilin D in the inner mitochondrial membrane.25 However, use of cyclosporine-independent strategies have also revealed pro- and antiapoptotic regulatory roles for calcineurin, suggesting multiple levels of complexity underlying the biology of calcineurin and cell death. Given these issues, we pursued a genetic approach to evaluate the functional significance of calcineurin as a potential modulator of ischemia-reperfusion–induced apoptosis in vivo.
Materials and Methodszv(|, http://www.100md.com
Surgical Procedure and Animal Modelszv(|, http://www.100md.com
Mice were randomized to receive either ischemia-reperfusion injury or a sham procedure. Mice were anesthetized and placed on a warming pad maintained at 37°C. The trachea was cannulated with a polyethylene tube connected to a respirator (Harvard Apparatus) with a tidal volume of 0.6 mL (110/min). A left lateral thoracotomy was performed between the fourth and fifth ribs, pericardial tissue was removed, and the left anterior descending artery (LAD) was visualized and ligated with a slipknot of 8-0 silk. Reperfusion was initiated 60 minutes after the occlusion. The Calcineurin Aß (CnAß)-/- mouse and the NFAT-dependent luciferase reporter transgenic line (containing nine copies of an NFAT binding site upstream of a minimal promoter) were described previously.26,27 All procedures performed in animals were approved by the Institutional Animal Care and Use Committee.
Measurements of Ischemic Area at Risk and Infarct Size&.|m, 百拇医药
Twenty-four hours after reperfusion, mice were anesthetized, the LADs were reoccluded, and 1 mL of 1.0% Evans blue was injected into the apex of each heart to stain nonischemic tissue. The hearts were then excised, washed with PBS, embedded in agarose, and cut into five transverse slices for 15 minutes of incubation at room temperature with 1.5% 2,3,5-triphenyltetrazolium chloride (TTC) to measure viable myocardium (red staining). Slices were photographed (each side) under a microscope and left ventricular area, area at risk (AAR), and infarct area were determined by digital planimetry.&.|m, 百拇医药
Echocardiography and Working Heart Analyses&.|m, 百拇医药
CnAß-/- or wild-type mice were anesthetized with 2% isoflurane and hearts were visualized using a Hewlett Packard Sonos 5500 instrument and a 15-MHZ transducer. Cardiac ventricular dimensions were measured on M-mode three times for the number of animals indicated. The isolated ejecting mouse heart preparation has been described in detail previously.28
DNA Laddering and TUNEL+%s, http://www.100md.com
CnAß-/- mice or wild-type controls were subjected to 60 minutes LAD occlusion and 24 hours of reperfusion to induce cardiac apoptosis for DNA laddering and 3 weeks of reperfusion for terminal deoxyribonucleotide transferase (TdT)–mediated dUTP nick-end labeling (TUNEL) assessment as previously described.14+%s, http://www.100md.com
Assessment of Apoptosis in Cultured Cardiac Myocytes+%s, http://www.100md.com
Conditions for generating and culturing neonatal cardiac myocytes were described previously.14 The Ad{Delta} CnA and Ad{Delta} NFATc4 adenoviruses were described previously,14 whereas the AdVIVIT NFAT inhibitory adenovirus was composed of the green florescent protein (GFP) fused to the VIVIT sequence as described previously29 (gift of Dr Susan D. Kraner and Chris Norris, University of Kentucky, Lexington). Cardiac myocytes were treated with staurosporine (500 nmol/L) for 18 hours, and all manipulations were performed 24 hours after adenoviral infection to allow adequate protein expression.14 DNA laddering and measurement of TUNEL were described previously.14
Affymetrix Gene Expression Profiling and Bioinformaticsek, http://www.100md.com
Total RNA samples were prepared from four individual hearts from 8-week-old CnAß-/- or wild-type control mice. Biotin-labeled target cRNA was prepared from T7-transcribed cDNA made from 10 µg of the total RNA using the Affymetrix-recommended protocol30,31 and hybridized for expression analysis to the Affymetrix U74Av2 GeneChip using antibody-based fluorescence signal amplification. Data values used for filtering and clustering were Signal, Signal Confidence, Absolute Call (Absent/Present), and Change (Increase, Decrease, and Unchanged) as implemented in MicroArray Suite 5.0.ek, http://www.100md.com
Statistical Analysisek, http://www.100md.com
Statistical analyses between the experimental groups were performed using a Student’s t test or one-way ANOVA when comparing multiple groups. Data were reported as mean±SEM. Values of P=0.05 were considered significant.ek, http://www.100md.com
Results
Increased Myocardial Infarct Size in CnAß-/- Mice#, 百拇医药
Although conclusive evidence has emerged implicating calcineurin as a pivotal regulator of cardiac myocyte hypertrophy, its role in regulating cardiac myocyte survival or apoptosis after ischemic or stress-related injury has engendered some controversy. To more thoroughly evaluate the association between calcineurin signal transduction and cardiac survival/apoptosis, we analyzed CnAß-/- mice for susceptibility to ischemia-reperfusion injury. Null mice and strain-matched wild-type (WT) controls were subjected to 60 minutes of LAD coronary artery occlusion followed by 24 hours of reperfusion. After this 24-hour reperfusion period, hearts were removed and perfused with Evans blue dye, sectioned, and incubated in 1.5% TTC to quantify area at risk and area of infarct normalized to total left ventricular area (AAR/LV). The area at risk normalized to ventricular area was not different between the two groups (WT, 79.8±3.3%; CnAß-/- mice, 76.5±2.9%; A). These results indicated that there were no genotype-dependent differences in the perfused areas between the two groups. In contrast, loss of viable myocardium was increased by approximately 30% in CnAß-/- mice compared with WT mice (WT, 31.9±3.6%; CnAß-/- mice, 46.19±3.6%, n=8; P<0.05, C).
fig.ommittedfi, http://www.100md.com
Figure 1. CnAß-/- mice show greater myocardial injury after ischemia-reperfusion. A, Area at risk (AAR) to total left ventricular area ratios. B, Infarcted area (IA) normalized to AAR (n=8; P<0.05). C, Representative injured hearts from wild-type (Wt) and CnAß-/- mice stained with Evans blue and TTC.fi, http://www.100md.com
fig.ommittedfi, http://www.100md.com
Figure 2. CnAß-/- mice show greater myocyte death after ischemia-reperfusion. A, Representative DNA ladder as an indication of apoptosis from wild-type (WT) and CnAß-/- hearts after ischemia-reperfusion injury (n=6). B, Representative DNA fragmentation detection assays by ligation-mediated PCR. C, Representative histological sections showing greater scarring in CnAß-/- hearts after ischemia-reperfusion injury (n=6). D, TUNEL quantification of cardiomyocytes in 2 CnAß-/- and wild-type control hearts 3 weeks after ischemic injury expressed as a percentage of total myocytes in the remaining viable myocardium. E, Hematoxylin and eosin– and chloroacetate esterase–stained cardiac histological sections from the indicated mice (24 hours of reperfusion; arrows show mast cells).
CnAß-/- Mice Show Greater Cardiac Cell Death After Ischemia-Reperfusion Injury-s69x5c, 百拇医药
DNA laddering was examined by agarose gel electrophoresis using hearts from sham operated animals and mice subjected to ischemia-reperfusion injury. CnAß-/- mice showed a qualitative increase in the degree of DNA laddering compared with hearts from WT mice, suggestive of more apoptotic damage (n=6 hearts in each group, although only two each are shown) (A). These data were extended through independent assessment of DNA fragmentation by ligation-mediated polymerase chain reaction, which also showed significantly greater signal in the CnAß-/- hearts after ischemia-reperfusion injury (B). Histological analysis was performed using Masson’s trichrome staining (blue color shows fibrosis and scar) on myocardial tissue from WT and CnAß-/- mice subjected to ischemia and 3 weeks of reperfusion. Analysis of six individual hearts in each group revealed greater myocardial damage and increased interstitial fibrosis in CnAß-/- mice compared with WT animals (two representative hearts are shown for each group) (C). As a more quantifiable measure of cell death, TUNEL was performed from histological sections, 3 weeks after ischemia-reperfusion injury. Rates of TUNEL in cardiac myocytes were assessed within the remaining region of viable left ventricle and septum, which demonstrated approximately twice the rate in CnAß-/- hearts compared with WT controls (D). Basal TUNEL rates did not vary between WT and CnAß-/- hearts from sham animals (data not shown). Collectively, these results indicate that CnAß-/- mice undergo enhanced cell death after ischemia-reperfusion injury, suggesting that calcineurin is normally cytoprotective in the heart.
CnAß-/- mice have a partially compromised immune response,26 an effect that might secondarily lessen the degree of myocardial injury, ventricular remodeling, and the inflammatory response after ischemia-reperfusion injury. In attempt to control for such variables, cardiac histological sections were subjected to immunohistochemical analysis for CD45 (total leukocytes), CD3 (T lymphocytes), and B220 (B lymphocytes), which showed no differences in cellular recruitment 24 hours after ischemia-reperfusion in either group (data not shown). Histological sections were also stained with hematoxylin and eosin (H&E) and chloroacetate esterase (for mast cells) (E). The data show similar degrees of cellularity augmentation in both groups 24 hours after ischemia-reperfusion injury (H&E sections), as well as equivalent levels of mast cells in the pericardium (E).j, http://www.100md.com
CnAß-/- Mice Display Worse Cardiac Function After Ischemia-Reperfusion
At baseline, hearts from CnAß-/- mice functioned equivalent to WT control hearts with respect to +dP/dt, maximal left ventricular pressure developed, and time to peak pressure, each assessed by working heart preparation (). These results indicate that loss of calcineurin Aß does not obviously alter cardiac function in the unstimulated state. To further evaluate the functional ramifications of enhanced cell death after ischemia-reperfusion injury, transthoracic echocardiography was performed in CnAß-/- and WT mice. Echocardiography was performed at baseline and 1, 2, and 3 weeks after ischemia-reperfusion injury to serially evaluate function noninvasively. As shown in B, left ventricular end-diastolic dimensions (LVED) and left ventricular end-systolic dimensions (LVES) increased to a larger extent in CnAß-/- mice compared with WT mice (P<0.05). These results indicate greater left ventricular dilation in the heart, presumably due to enhanced cell death in the absence of CnAß. More importantly, fractional shortening, which approximates cardiac contractile performance, was reduced to a greater extent in CnAß-/- mice compared with WT mice 2 and 3 weeks after the ischemic event (P<0.05, n=9; C). These results indicate that CnAß-/- mice had reduced cardiac function after ischemia-reperfusion injury compared with WT controls, consistent with the notion that CnAß-/- hearts are more susceptible to a cell death–promoting stimulus. However, it is possible that CnAß-/- mice could also show alterations in function due to differences in ventricular remodeling that typifies postinfarction injury. To address this possibility, heart weight to body weight ratio assessments were performed after the final 3-week echocardiographic measurement in both groups. Neither WT nor CnAß-/- mice underwent significant cardiac hypertrophy over this time period under the conditions used, suggesting that the difference in functional performance likely reflects the degree of injury (data not shown).
fig.ommitted3y4h9vs, 百拇医药
Table 1. Cardiac Functional Analysis Using an Ex Vivo Working Heart Preparation in CnAß-/- and Wild-Type Controls3y4h9vs, 百拇医药
fig.ommitted3y4h9vs, 百拇医药
Figure 3. Cardiac morphology and function as determined by echocardiography. A and B, Left ventricular end-diastolic dimensions (LVED) and left ventricular end-systolic dimensions (LVES) in CnAß-/- mice compared with wild-type (WT) mice (*P<0.05 vs baseline; #P<0.05 vs WT after ischemia-reperfusion). Data are expressed in millimeters, average±SEM, n=9. C, Fractional shortening assessed by echocardiography in CnAß-/- and WT mice (*P<0.05 vs baseline; #P<0.05 vs WT after ischemia-reperfusion, n=9). Fractional shortening was calculated as (LVED-LVES)/LVEDx100 and expressed as percentage, average±SEM, n=9.3y4h9vs, 百拇医药
Altered Gene Expression in CnAß-/- Mice3y4h9vs, 百拇医药
The enhanced myocardial damage observed after ischemia-reperfusion injury in CnAß-/- mice suggested an alteration in one or more molecular pathway(s) that influences cell survival. Given the vast number of effectors that might potentially influence the survival versus apoptotic decision of myocardial cells after ischemia-reperfusion injury, we performed a large scale, unbiased screen for altered gene expression. Specifically, hearts from 8-week-old CnAß-/- and strain-matched WT mice (2 each) were harvested and RNA was purified for gene expression profiling using the Affymetrix U74Av2 array. This array contains all genes in the murine Unigene database that have been functionally characterized (approximately 6000) as well as an additional 6000 expressed sequence tags (ESTs). Duplicate heart samples were cross-compared between WT and CnAß-/- mice resulting in 5735 genes being significantly detected in one or more groups after internal normalization. Of these genes, 437 were significantly altered in expression between CnAß-/- and WT hearts (P<0.05). The raw data shown in represent the 2n relationship of expression between Wt and CnAß-/- hearts. For example, a WT value of 1.0 and a null value of 0.33 or 3.0 translates into an 8-fold change in gene expression either way. The data are also depicted on a colorimetric scale for simplicity (A). Remarkably, 383 genes showed downregulated expression in CnAß-/- hearts, whereas only 54 genes showed upregulated expression. This general profile suggests that the loss of CnAß reduces the expression of a subset of cardiac genes, implicating calcineurin as an important positive transcriptional effector pathway in the heart.
fig.ommitted#y+7o/, 百拇医药
Figure 4. A, Relative expression ratios of selected functional gene groups assessed from microarray screening. Cardiac RNA was collected from 2 CnAß-/- mice (KO) and 2 wild-type mice (WT) and subjected to expression profiling using the Affymetrix U74Av2 array. Left panels show a color scale representation of gene expression levels, with yellow equal to 1 (no change), blue equal to 0.2 (reduced expression), and red equal to 5 (increased expression). Absolute normalized expression data are shown for each sample in the right-hand columns along with the GenBank accession number. B, Select group of genes showing altered expression in the array screen was subject to a confirmatory RT-PCR analysis of expression levels at varying cycles (to show the most linear point in amplification).#y+7o/, 百拇医药
Expression signatures were further analyzed in CnAß-/- hearts to potentially implicate individual genes as phenotypic modulators. The most significantly altered genes within seven functional categories were assembled and annotated; any one of which might affect the viability of cardiac myocytes after injury. An interesting alteration was identified in a select group of genes that regulate sterol/cholesterol metabolism such as ATP-binding cassette transporter ABC1, the low-density lipoprotein receptor-related protein 10, and the stearoyl-coenzyme A desaturase 1 (A). Also of interest, four cell cycle regulatory genes were significantly altered in CnAß-/- hearts, as well as five genes encoding mitochondrial-localized proteins (A). Within the transcription category, the DNA binding factors Nkx2.5, Hand2, YB-1, and ATF-3 were each downregulated in CnAß-/- hearts, whereas ATF4 and DBP were upregulated. The observed reduction in Nkx2.5 and Hand2 expression, which themselves are important regulators of cardiac gene expression, is consistent with the proposed paradigm whereby calcineurin-NFAT signaling directly and indirectly augments expression of a subset of cardiac genes. Another intriguing pathway alteration was observed in structural genes that facilitate cellular adhesion and/or intracellular support. For example, four and a half LIM domains 1, {gamma} -sarcoglycan, {alpha} -sarcoglycan, desmoglein-2, {alpha} -6 integrin, and {alpha} -4 tubulin each showed altered expression in the CnAß-/- heart (A). A large number of genes that participate in cellular signaling, apoptosis signaling, or stress-responsive signaling were also identified as significantly altered in the absence of CnAß. Finally, a group of genes showing altered expression in the array screen were selected for reverse transcriptase polymerase chain reaction (RT-PCR) analysis at varying cycles to confirm the observed relationships (B). The data obtained by RT-PCR showed the same profile of increased or decrease expression, confirming the reliability of the array screen (normalized to L7 expression) (B).
The large number of candidate genes showing altered expression made it difficult to mechanistically establish which factors or pathways might ultimately underlie the observed profile of greater stress-induced cell death in the absence of CnAß. Despite this qualification, the overall observation that large subsets of genes are reduced in expression suggests that calcineurin functions as a general transcriptional regulator in the heart, which might predispose the heart to cell death. Indeed, NFAT transcription factors are primary effectors of calcineurin signaling that could fulfill such a role (see next section).6^, 百拇医药
NFAT Mediates Cardioprotection6^, 百拇医药
To determine the potential involvement of NFAT factors as regulators of cardiac myocyte cell survival, a neonatal cardiac myocyte culture–based model was employed. Whereas we have previously shown that calcineurin activation protects cultured myocytes from staurosporine- or 2-deoxyglucose–induced apoptosis, the necessity of NFAT factors as downstream mediators was not determined.14 To this end an NFAT-specific inhibitory adenovirus was used that expresses the VIVIT peptide as a GFP fusion.29 The specificity of this inhibitory virus was demonstrated by coinfection with an NFAT-dependent reporter adenovirus in cultured neonatal myocytes (A). AdVIVIT dramatically inhibited Ad{Delta} CnA-induced activation of the NFAT-dependent luciferase reporter. More importantly, AdVIVIT infection in neonatal cardiac myocytes augmented staurosporine-induced TUNEL, whereas expression of an activated NFATc4 truncation mutant antagonized TUNEL (P<0.05; B). Moreover, AdNFATc4 partially reduced enhanced TUNEL associated with calcineurin inhibition through Adcain infection (also see De Windt et al14) (P<0.05, B). Expression of Ad{Delta} NFATc4 infection also antagonized staurosporine-induced DNA laddering, further implicating NFAT factors as modulators of apoptosis in cardiac myocytes (C).
fig.ommitted\]+oyz, 百拇医药
Figure 5. NFAT mediates cytoprotection in the heart. A, Relative luciferase activity (RLU) derived from cultured neonatal cardiomyocytes infected with an NFAT-dependent reporter adenovirus (AdNFAT-luc) alone or in combination with AdCnA and/or AdVIVIT. B, TUNEL quantitation in neonatal cardiomyocytes infected with the indicated adenoviruses for 24 hours, followed by staurosporine treatment for 18 hours (*P<0.05 vs Adßgal with staurosporine; n=3). C, Representative DNA ladder analysis after adenoviral infection then straurosporine treatment (n=4 each).\]+oyz, 百拇医药
The results discussed above indicate that NFAT factors can function as regulators of apoptosis in cultured cardiac myocytes. However, it was uncertain whether NFAT factors might play a similar role within the intact adult heart, downstream of calcineurin signaling. To address this issue, NFAT-luciferase reporter transgenic mice were crossed into the CnAß-/- background and subjected to ischemia-reperfusion injury. NFAT-luciferase transgenic mice, WT for CnAß, were also generated from the same parental cross for direct comparison. Six CnAß-/- and five WT controls were subjected to 60 minutes of ischemia followed by 48 hours of reperfusion injury, at which time the hearts were removed and parsed into three components for measurement of luciferase activity: right ventricle (RV), nonischemic left ventricle and septum (LV-non), and the injured area of the LV (LV-injured). The RV was analyzed to measure NFAT activity as a secondary consequence of ischemia-reperfusion injury–induced remodeling/hypertrophy. Ischemia-reperfusion injury dramatically enhanced NFAT luciferase activity in each of the assayed ventricular compartments in WT controls (). More importantly, CnAß-/- mice consistently showed less NFAT activation after injury (). These results suggest an association between reduced NFAT transcriptional activity and diminished cardioprotection in CnAß-/- mice.
fig.ommittede, 百拇医药
Figure 6. Relative luciferase activity from the hearts of NFAT-luciferase reporter transgenic mice at baseline or after ischemia-reperfusion injury. These reporter mice were crossed into the CnAß-/- or CnAß+/+ background (n=6 and 5, respectively). Activity is shown from the whole heart (control), right ventricle (RV), nonischemic region of the LV and septum (LV-non), and the LAD-perfused area of the LV subjected to ischemia (LV-injured).e, 百拇医药
Discussione, 百拇医药
In this study, we investigated the association between the calcineurin-NFAT signaling pathway and cell death in the myocardium after stress stimulation. Loss of CnAß rendered the myocardium more susceptible to ischemia-reperfusion injury, suggesting that calcineurin signaling helps maintain myocyte viability in vivo. Part of the mechanism responsible for increased ischemic damage in CnAß-/- mice is likely a secondary consequence of altered gene expression due to reduced NFAT activity.
Role of Calcineurin as a Modulator of Cellular Apoptosisat, 百拇医药
Although calcineurin has been recognized as a central regulator of the hypertrophic growth of the myocardium, its role as an effector of myocardial cell death is more controversial. In other tissues or cell-types, calcineurin has been shown to either agonize or antagonize apoptosis after stress stimulation. Studies conducted in neurons, lymphocytes, and tumor cell lines have shown both pro- or antiapoptotic effects of calcineurin activation.15–22 The exact decision of cytoprotection versus apoptosis is likely regulated by coordinated signals from other costimulated signaling pathways or depends on cell-type specific calcineurin effector/docking proteins. Indeed, calcineurin activation was shown to either induce apoptosis or to antagonize apoptosis depending on the status of p38 activation.32 More recently, calcineurin was shown to localize to the mitochondria in fibroblasts through docking with the inhibitory protein FKBP38, resulting in Bcl-2 and Bcl-xl redistribution.33 Calcineurin has also been implicated as a direct inducer of apoptosis in primary hippocampal neurons through dephosphorylating the proapoptotic factor Bad.15 In addition to cell-type specific regulatory considerations, the pro- or antiapoptotic effects attributed to calcineurin have been obscured by the use of cyclosporine A, which can modulate apoptosis independent of calcineurin through direct effects on mitochondrial permeability transition pore (MPTP) formation within the inner mitochondrial membrane.25 Given the calcineurin-independent effects associated with cyclosporine A and FK506, the use of calcineurin gene-targeted mice and/or null cells derived from these mice should help elucidate the direct role that calcineurin plays in modulating cellular apoptosis in diverse tissues.
In the present study, we demonstrated that genetic disruption of the CnAß gene in the mouse enhanced cardiac damage induced by ischemia-reperfusion injury. This result indicates that calcineurin signaling imparts a degree of protection against cell death in the heart. Consistent with this notion, we have previously shown that transgenic mice expressing a constitutively active mutant of calcineurin in the heart are significantly protected from ischemia-reperfusion-induced cell death.14 In cultured cardiomyocytes, adenoviral-mediated gene transfer of activated calcineurin reduced 2-deoxyglucose–induced TUNEL, whereas calcineurin inhibition with a Cain-expressing adenovirus increased TUNEL.14 In contrast, a subsequent study reported that isoproterenol stimulation of neonatal cardiomyocytes promoted apoptosis, in part, by stimulating calcineurin activity.24 These authors demonstrated that cyclosporine A and FK506 blocked the increase in cardiomyocyte apoptosis induced by isoproterenol stimulation and, more significantly, that transgenic mice expressing dominant-negative calcineurin in the heart were refractory to isoproterenol-induced TUNEL reactivity in vivo. These results suggest that calcineurin activation is associated with enhanced apoptosis in cardiomyocytes, in contrast to our original and subsequent observations. It is likely that the nature of the stimulus (isoproterenol) partially underlies the disparity between the two studies discussed above. For example, isoproterenol induces a prominent elevation in cAMP, which induces protein kinase A signaling and secondary alterations in inotropy, events not typically associated with ischemic injury or straurosporine stimulation.
By comparison, Kakita et al23 recently identified an antiapoptotic role for calcineurin activation in cardiomyocytes after endothelin-1 stimulation. Specifically, endothelin-1 stimulation protected cardiac myocytes in culture from H2O2-induced TUNEL reactivity, DNA laddering, caspase-3 cleavage, and loss of mitochondrial membrane potential. This endothelin-1–mediated cytoprotection from H2O2-induced apoptosis was blocked by inhibition of calcineurin with either cyclosporine A or FK506. Collectively, these disparate accounts underscore the complexity of intracellular signaling networks within mammalian cells, such that seemingly related stress stimuli can elicit fundamentally different responses depending on the nature of the stimulus (mitochondrial- versus death receptor–mediated), the status of other parallel signaling pathways, and the context of cell-type specific calcineurin modulatory factors.pu-&n#7, http://www.100md.com
Mechanism Whereby Calcineurin Signaling Protects the Myocardium
Although a large number of studies have implicated calcineurin as a modulator of cell death in varied cell types, only a few downstream mechanisms responsible for death or protection have been identified. As discussed above, calcineurin was reported to directly dephosphorylate Bad in neurons, thus enhancing apoptosis.15 Careful evaluation of Bad phosphorylation at serine 112, 136, and 128 from the hearts of calcineurin transgenic mice or CnAß-/- mice failed to show any difference from controls (data not shown). Kakita et al23 reported that calcineurin-mediated protection from H2O2-induced apoptosis was associated with an increase in Bcl-2 expression in cultured neonatal cardiac myocytes. However, expression levels of Bcl-2 did not change in the hearts of either calcineurin TG mice or CnAß-/- mice, nor was expression altered for Bcl-xl, Bad, caspase-1, -3, -8, -9, or FKBP38 (data not shown). Previously, we identified a minor but significant increase in Akt phosphorylation in the hearts of calcineurin transgenic mice.14 However, CnAß-/- mice showed no difference in basal or stimulated Akt phosphorylation in the heart, ruling out this potential mechanism (data not shown). Collectively, these negative findings suggest that the classical effectors of the apoptotic response are not directly linked to calcineurin signaling in the heart.
To investigate other potential mechanisms, an unbiased array screen was performed from the hearts of CnAß-/- and matched WT mice. Although a number of potential regulatory proteins were altered in expression, the most significant observation was the dramatic downregulation in expression of large subsets of unrelated genes (about 6% of detectable genes in the heart). This observation suggested a defect in the transcriptional potency underlying a group of genes in the hearts of CnAß-/- mice. This interpretation is consistent with known role of NFAT transcription factors as important effectors of calcineurin-regulated gene expression in most cell types.34 Indeed, the full potency of calcineurin-induced hypertrophy in the heart was shown to require NFATc3 using gene-targeted mice.35t\v}#, 百拇医药
With respect to apoptosis signaling, we previously observed that overexpression of an activated NFATc4 in cultured neonatal cardiomyocytes partially antagonized 2-deoxyglucose-induced apoptosis.14 Furthermore, Kakita et al23 showed that endothelin-1–mediated protection from H2O2-induced apoptosis also promoted NFAT dephosphorylation. In this study, we showed that specific inhibition of NFAT with AdVIVIT augmented cardiomyocyte cell death after staurosporine treatment. More importantly, CnAß-/- mice showed reduced NFAT transcriptional activity in vivo after ischemia-reperfusion injury. These results are also consistent with a recent report by Izumo and colleagues36 in which NFAT inhibition augmented cardiac myocyte apoptosis after phenylephrine stimulation in culture.
Collectively, these results discussed above suggest that the homeostatic transcriptional activity of NFAT provides the necessary framework of basal gene expression that affords cardiac "health" and resistance to apoptotic stimuli. However, the exact array of downstream effectors that are regulated by NFAT factors in providing protection is not known. Despite this, the results of this study provide the first genetic data indicating that calcineurin signaling is necessary for cardiac protection and suggests that strategies to acutely agonize calcineurin might be of therapeutic benefit during or after myocardial infarction. However, this notion is in dramatic contrast to the function of calcineurin as a mediator of cardiac hypertrophy, which itself can lead to cardiomyopathy and heart failure if constitutively activated (although it is apoptosis-independent). These observations suggest that calcineurin may function as a "double-edge sword," such that transient activation antagonizes myocyte apoptosis, but long-standing activation induces cardiac hypertrophy and deleterious ventricular remodeling associated with heart failure.
Acknowledgments$, 百拇医药
This work was supported by the NIH (to J.D.M. and B.J.A.) and the Pew Charitable Trusts (J.D.M). O.F.B was supported by Post-Doctoral Fellowships from the NIH (HL10336). B.J.W. was supported by an MD/PhD scholar award from the University of Cincinnati Physician Scientist Training Program and the Albert J. Ryan Foundation.$, 百拇医药
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