A Molecular Approach to Apoptosis in the Human Heart During ... : Transplantation (original) (raw)
Brain dead (BD) patients are the main source of organs for transplant. However, in approximately 10%–30% of BD donors, severe myocardial ventricular dysfunction prevents the use of hearts for transplant (1), 19% of which are rejected because of contractility failure (2). The reason for this situation is related to the hemodynamic and neuroendocrine alterations that appear during cerebral herniation (3). Since initial studies performed in experimental models (4, 5), it has been reported that brain death leads to changes that compromise cardiac function. Recently, BD has also been reported to induce apoptosis in animals including the rat (6, 7) and rabbit (8) and in several human tissues such as kidney (9–11) and heart (12). Cardiac apoptosis reduces the contractile mass available, which has been linked to heart failure (13). Apoptosis is a cell function necessary for the removal of unwanted cells during normal development or aging. The mechanism is controlled through two major pathways, an extrinsic and intrinsic pathway (14), regulated by aspartate-specific cysteine proteases designated caspases (15). Activation of the extrinsic pathway makes surface cell death receptors such as Fas bind to their specific ligands (e.g., FasL). The intrinsic pathway is activated by various intracellular stimuli such anoxia or DNA damage, all of which affect mitochondria, where they promote mitochondrial membrane permeabilization and release of caspase activating factors like cytochrome c into the cytosol (16, 17). Members of the Bcl-2 family (proapoptotic Bax and antiapoptotic Bcl-2) control activation of the intrinsic pathway and play an important role in cardiomyocyte apoptosis in rats (18). Briefly, for each stimulus, the balance between death and survival seems to be determined by the ratio of apoptosis-stimulating to apoptosis-suppressing Bcl-2 family members (19, 20). Activation of both apoptotic pathways (extrinsic and intrinsic) culminates in the activation of effector caspases (caspase-3, -6, and -7). Effector caspases have many substrates, whose cleavage mediates chromatin condensation and DNA fragmentation as well as the cell morphology changes characteristic of apoptosis (21). Activation of the terminal stage of the apoptotic pathway has also been suggested to play an important role (8, 22) in the myocardial dysfunction that appears in grafted hearts from brain-dead donors (23, 24). Given that the process of apoptosis can be pharmaceutically manipulated, the noninvasive identification of apoptosis is critical (25). Because apoptosis is a controlled process with different targets for therapeutic intervention (26), antiapoptotic treatments could serve to prevent cardiac injury and avoid posttransplant complications. However, little is known about the molecular mechanisms that give rise to cardiac apoptotic alterations induced by BD in humans (9, 22). The aim of this study performed on cardiac tissue from brain-dead donors was to examine through real time-polymerase chain reaction (RT-PCR) the expression of several genes involved in the apoptotic cascade (BAX, BCL2, CASPASE 3, CYTOCHROME C, and FAS) to determine whether it is activated during BD. To exclude the possibility that our results could be attributable to hypoxia, _HIF1_α gene expression was also determined.
MATERIALS AND METHODS
Sample Collection
Tissue samples were obtained from the left ventricles of 50 heart beating donors (28 men, 22 women; mean age, 61 years; range, 30–80 years). Control samples were heart tissue specimens obtained from the left ventricles of 12 patients (6 men, 6 women; mean age, 65 years; range, 49–83 years) undergoing mitral valve replacement for double mitral lesion. In these patients, left ventricular end-diastolic pressures were moderately elevated over normal levels ≈8±2.2 mm Hg. Samples were obtained over a year, from April 2006 to May 2007. The heart-beating donors had died in a multidisciplinary intensive care unit of a tertiary university hospital. Brain death was attributed to several causes (28 deaths to subarachnoid or intracerebral hemorrhage, 3 to cerebrovascular anoxia, 16 to traumatic brain injury, and 3 to anoxic brain injury). Mean intensive care unit stay before donation was 93 hr (range, 7–624 hr). No donor had previous heart pathology. Consent for organ donation for transplant and clinical research was obtained from the relatives of each donor and control patient.
The donor hearts had been rejected for transplant because of donor age or weight, or recipient blood group incompatibility. The hearts from the 30- to 40-year-old donors that were incompatible with recipients on the Spanish waiting list, had not been accepted as competitive donors elsewhere in Europe. Left ventricular specimens were obtained by the Cavalieri principle of sectioning. After delivering cardioplegia, all cardiac tissues (from donors and controls) were freshly harvested, immediately embedded in O.C.T Compound (Tissue Tek, Sakura, Netherlands) and then stored at −80°C.
RNA Extraction and cDNA Synthesis
Total RNA was isolated using the Nucleo Spin RNA II Isolation Kit (Macherey-Nagel Gmb H & Co. KG) according to the manufacturer's instructions.
The quality of the RNA was assessed through absorbance measurements made using a NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDropTechnologies, Wilmington, DE). All the samples used in the PCR procedures showed a 260/280 nm absorbance ratio between 1.8 and 2.2. A ratio of approximately 2 is generally accepted as pure for RNA.
Each RNA sample was reverse transcribed using the oligo-pdT and random primers provided in the first-strand complementary DNA (cDNA) synthesis kit for RT-PCR (AMV; Roche Diagnostic, Switzerland) following the manufacturer's instructions. The cDNA samples were then cooled at 4°C and stored at −20°C until use. To confirm the total absence of residual RNA, we performed a conventional PCR on β-actin (GenBank: E00829) (β-actin forward primer 5′-ACTACCTCATGAAGATCCTC-3′; β-actin reverse primer 5′-CGGATGTCCACGTCACACTTC-3′). The PCR protocol consisted of an initial denaturation step at 94°C for 5 min, followed by 35 amplification cycles. Each cycle involved a denaturation step of 30 sec at 94°C, followed by 30 sec of primer annealing at 55°C and 30 sec of primer extension at 72°C, followed by a final extension of 10 min. Amplicon size was confirmed by electrophoresis using an ethidium bromide-stained 2% agarose gel in 1× TBE buffer.
Real-Time Quantitative Polymerase Chain Reaction
The primers for the genes examined were designed using Beacon Designer software (Premier Biosoft International, CA). Table 1 provides the primer sequences and annealing temperatures used.
Genbank accession numbers, annealing temperatures, and primer sequences for the human genes tested by RT-PCR
Quantitative PCR was performed using an i-Cycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). SYBR Green PCR Supermix (2×) (Bio-Rad), which detects amplification using Sybr Green as a double-stranded DNA-specific fluorescent dye, was used as a mastermix. Assays were performed in duplicate and a blank included in every assay. The reaction mixture for amplification comprised 2 μL of cDNA, 0.4 μL of each primer (forward and reverse primers; final concentration 0.1 μM), 10 μL of Sybr Green mix, and sterile water in a final volume of 20 μL. The RT-PCR protocol included an initial step of 95°C (3 min), followed by 40 cycles of 30 sec at 95°C for DNA denaturation, 40 sec for primer annealing at different temperatures depending on the gene (Table 1), and 40 sec at 72°C for primer extension. Fluorescence data were acquired at 72°C. Melting-curve analysis to confirm product specificity was performed immediately after amplification following 1 min of denaturation at 95°C, 1 min annealing at 65°C, and 60 cycles of 0.5°C increments (10 sec each) beginning at 65°C while monitoring fluorescence. Product identity was confirmed by electrophoresis using an ethidium bromide-stained 2% agarose gel in 1× TBE buffer.
Polymerase chain reaction efficiencies were calculated from a relative standard curve derived from a pool of heart tissue specimens from the same organ donors that was serially diluted (a 10-fold dilution series with at least six measuring points). Peptidylprolyl isomerase A (cyclophilin A) was used as the endogenous control (Human Endogenous Control Gene Panel, TATAA Biocenter, Goteborg, Sweden) according to the manufacturer's instructions.
Statistical Analysis
Data were statistically analyzed using the Proc MIXED procedure of the SAS/STAT v8.2 software package (SAS Institute, Inc., Cary, NC). _C_t values (number of cycles needed to generate a fluorescent signal above a predefined threshold) and Δ_C_t values (difference between the _C_t of the target gene and that of the housekeeping gene) for each gene and repetition were used as dependent variables. The relative standard method was used to quantify gene expression levels.
The model fitted included the type of sample (two levels: control vs. donor) as the fixed effect, and the sample as a random effect to account for the nonindependency of repetitions. Least-square means and their corresponding standard errors were computed for each sample level. For descriptive purposes, a Student's t test was also carried out on raw _C_t means computed for control and donor samples using Proc GLM of SAS/STAT, assuming that the sample type effect included two independent groups of normally distributed observations. Correlation coefficients between levels of expression were computed using the Proc CORR implemented in SAS/STAT.
RESULTS
Product identity was confirmed by electrophoresis on ethidium bromide-stained 2% agarose gels in 1× TBE buffer, which resulted in a single product of the desired length. In addition an i-Cycler iQ melting curve analysis was performed, which rendered single product specific melting temperatures. No primer-dimers were generated during the 40 real-time PCR cycles conducted (see Table 2).
Amplicon size and melting temperatures of the six genes analyzed by RT-PCR
Polymerase chain reaction efficiencies were 91.7% for BAX, 101.5% for BCL2, 94.3% for CASPASE 3, 101.1% for CYTOCHROME C, 94.7% for FAS, and 103.2% for _HIF1_α. Linearity was high, with correlation coefficients (_R_2) above 0.989.
To confirm the accuracy and reproducibility of real-time PCR, intra-assay precision was determined in two replicates within a single i-Cycler iQ run. Interassay variation was determined in two experimental runs performed on different days. The reproducibility of the data ranged from 95% for FAS to 98.8% for CASPASE 3 and CYTOCHROME C.
Gene Expression
The mixed linear model indicated a significant influence of the type of sample (donor vs. control) on _C_t values at P less than 0.0001. The Student's t test computed on raw means also revealed that differences between the type of sample were significant at the level P less than 0.05 for all the genes examined. Least-square _C_t means and their corresponding standard errors for each gene according to the sample type (control or donor) are given in Table 3.
C t (threshold cycle) values for the two study groups (brain-dead donors vs. control patients) represented as Lsmean and SEM
To quantify gene expression, the relative standard method (relative fold changes) was used and expression levels determined for the donor and control groups normalized to the level for peptidylprolyl isomerase A (PPIA). A marked increase was noted in the relative expression of three of the genes in the donor compared with control heart tissue specimens. These increases are plotted in Figure 1. In the heart-beating donor group, the genes FAS, CASPASE 3, and BAX were up-regulated 12.14-, 7.85-, and 4.89-fold, respectively, with respect to the basal levels recorded for the endogenous control (PPIA). For the same three genes increases in control group specimens were 3.57-, 2.64-, and 2.31-fold, respectively, over basal levels. Differences in expression level increases were significant between the donor and control groups.
Relative quantification of the studied genes. mRNA levels for apoptosis-related genes recorded in the heart tissue of brain-dead (BD) donors (n=50) and control patients (n=12) relative to levels for the endogenous control gene peptidylprolyl isomerase A. Values are expressed as mean±SEM. * indicates a significant difference compared with controls at P less than 0.05. ** indicates a significant difference between the two groups at P less than 0.01. CYT C, cytochrome C; Casp3, Caspase 3.
The remaining genes showed less pronounced increases in expression levels. In descending order, these increments were 3.76-fold for the BCL2 gene, 2.78-fold for _HIF1_α, and 2.56-fold for CYTOCHROME C compared with basal levels. Increases for the control group were 3.36-, 2.80-, and 2.45-fold, respectively. This time, differences between the control and donor groups were not significant.
Correlation coefficients among expression levels of the six genes in all the samples examined are provided in Table 4. All the computed correlation coefficients were significant at a P<0.0001 and ranged from moderate (0.395 for HIF1α-FAS) to high (0.852 for BAX-CYTC).
Intergene correlation analysis of q-PCR expression levels
DISCUSSION
Current estimates of the proportion of hearts rejected for transplant because of ventricular dysfunction range from 10% (27) to 30% (1). Of these, 19% have contractility problems (1) and are therefore not suitable for transplant purposes. Several authors have recently observed an active apoptotic process during BD (9, 10). The time taken from the initiation of the apoptotic process to phagocytosis of the dying cell is unknown, but estimates range from hours to days (28), a time interval that basically coincides with the death of brain-dead donors. Cardiac myocyte apoptosis occurs in several human heart diseases such as congestive heart failure (29, 30) or arrhythmogenic ventricular dysplasia (31) or myocarditis (32). If the apoptotic process is really activated during BD, this could lead to a substantial loss of contractile mass possibly accounting for the percentage of donor hearts with compromised contractility. In this study, we used a molecular approach to assess how the BD phenomenon affects human cardiac tissue in the damage sustained through apoptosis by detecting differences in the levels of expression of six different genes (BAX, BCL2, CASPASE 3, CYTOCHROME C, FAS, and _HIF1_α) using the PPIA gene to normalize the data (33) in a relative RT-PCR quantification procedure.
To establish whether apoptosis is indeed in an active mechanism in heart tissue during brain death, we examined several genes involved in the intrinsic and extrinsic apoptotic pathway. CASPASE 3 represents the final common pathway of the apoptotic cascade (34) and has been identified in neonatal (35) and adult rat cardiomyocytes (36). In our cardiac specimens from brain-dead donors, CASPASE 3 expression levels were 7.85-fold higher than basal levels and, remarkably, this increase differed significantly (P<0.05) from that recorded for the control cardiac specimens. We also examined expression levels of the FAS gene. FAS is a cell death receptor that belongs to the TNFR gene superfamily (37) with a role in activation of the extrinsic pathway. Our results indicate elevated expression levels of this gene by as much as 12.14-fold basal levels in heart tissue from heart beating donors, this increase being significantly greater than that detected in controls (P<0.01). Further apoptotic molecules determined here were BCL2 and BAX (members of the apoptosis-stimulating and suppressing Bcl-2 family). These molecules underwent 3.76- and 4.89-fold increases, respectively, over basal levels such that the ratio of BAX (proapoptotic)-to-BCL2 (antiapoptotic) expression was clearly in favor of the apoptotic molecule. These two molecules have been described to indicate the balance between cell death and survival (38). Finally, to complete our molecular approach to apoptosis we analyzed expression of the CYTOCHROME C gene. The release of this molecule and other mitochondrial proapoptotic factors into the cytosol (39) is induced by proapoptotic family members such as BAX as part of the intrinsic pathway. We observed no significant difference in the expression of this gene between the two groups of cardiac specimens. Because hypoxia can stimulate components of cell death receptor and mitochondrial pathways (40) and cardiac myocyte apoptosis has been induced by hypoxia in rats (41), we also examined the gene (_HIF1_α) to rule out the possibility that our results could be caused by hypoxia. Hypoxia-inducible factor 1 (HIF1α) is a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. HIF1α protein has been detected in distinct cell types in vivo and its expression is increased in response to hypoxia (42). We observed low-_HIF1_α expression levels in control and donor specimens such that we can preclude any major hypoxic damage to the heart tissue immediately after harvesting and, surprisingly, neither did there seem to be a marked difference in the hypoxia suffered by the two sources of heart tissue. Our results are in agreement with a report (43) that hypoxia seems not to be one of the starting points for the inflammatory cascade in brain-dead donor kidneys. In a molecular study performed on the liver of heart beating rats (6), apoptotic genes such as FAS, BAX, or CASPASE 3 increased their expression levels versus controls as a consequence of BD. Our data indicate a marked increase in the expression of three of the genes examined (BAX, CASPASE 3, and FAS) compared with controls. If we consider that each of these genes play a different role in the apoptotic process (BAX participates in the intrinsic pathway, FAS in the extrinsic pathway, and CASPASE 3 is an effector caspase), we could say that our results suggest that although both apoptotic pathways were active, the intrinsic pathway was activated to a much lesser extent than the extrinsic. Given that one of the main stimuli that activates the intrinsic pathway is anoxia, the lack of substantial increases in the expression levels of the gene related to hypoxia (_HIF1_α) and those involved in the intrinsic pathway (BAX or CYTOCHROME C) are consistent with our findings. In conclusion, our results suggest that the apoptotic process is initiated during brain death in cardiac tissue from heart beating donors. If these results are confirmed, they suggest the possible use of antiapoptotic treatments to prevent organ damage before transplantation.
ACKNOWLEDGMENTS
The authors thank Dr. Valle Castro for his help in obtaining the control tissue specimens and Benjamin Otero and Pilar Alonso for help with the donor tissue specimens.
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Keywords:
Brain death; Apoptosis genes; Heart transplant; RT-PCR; Organ donor
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