Author + information
- Received January 31, 2001
- Revision received June 11, 2001
- Accepted June 26, 2001
- Published online October 1, 2001.
- ↵*Reprint requests and correspondence:
Dr. A. J. Marian, Section of Cardiology, Baylor College of Medicine, One Baylor Plaza, 543E, Houston, Texas 77030
The goal of this study was to identify genes upregulated in the heart in human patients with hypertrophic cardiomyopathy (HCM).
Hypertrophic cardiomyopathy is a genetic disease caused by mutations in contractile sarcomeric proteins. The molecular basis of diverse clinical and pathologic phenotypes in HCM remains unknown.
We performed polymerase chain reaction-select complementary DNA subtraction between normal hearts and hearts with HCM and screened subtracted libraries by Southern blotting. We sequenced the differentially expressed clones and performed Northern blotting to detect increased expression levels.
We screened 288 independent clones, and 76 clones had less than twofold increase in the signal intensity and were considered upregulated. Sequence analysis identified 36 genes including those encoding the markers of pressure overload-induced (“secondary”) cardiac hypertrophy, cytoskeletal proteins, protein synthesis, redox system, ion channels and those with unknown function. Northern blotting confirmed increased expression of skeletal muscle alpha-actin (ACTA1), myosin light chain 2a (MLC2a), GTP-binding protein Gs-alpha subunit (GNAS1), NADH ubiquinone oxidoreductase (NDUFB10), voltage-dependent anion channel 1 (VDAC1), four-and-a-half LIM domain protein 1 (FHL1) (also known as SLIM1), sarcosin (SARCOSIN) and heat shock 70kD protein 8 (HSPA8) by less than twofold. Expression levels of ACTA1, MLC2aand GNAS1were increased in six additional and FHL1in four additional hearts with HCM.
A diverse array of genes is upregulated in the heart in human patients with HCM, which could account for the diversity of clinical and pathologic phenotypes. Markers of secondary hypertrophy are also upregulated, suggesting commonality of pathways involved in HCM and the acquired forms of cardiac hypertrophy. Elucidation of the role of differentially expressed genes in HCM could provide for new therapeutic targets.
Hypertrophic cardiomyopathy (HCM) is a genetic disease caused by mutations in the contractile sarcomeric proteins (1). Hypertrophic cardiomyopathy is diagnosed clinically by the presence of unexplained left ventricular hypertrophy and pathologically by myocyte hypertrophy, disarray and interstitial fibrosis (2). While the causality of mutant contractile proteins in the pathogenesis of HCM is well established, the molecular bases of diverse cardiac phenotypes in HCM remain unknown.
We and others have proposed cardiac hypertrophy in HCM is a “compensatory” phenotype due to increased cardiac myocyte stress or altered Ca+2sensitivity of the contractile apparatus imparted by the mutant contractile proteins (3). Accordingly, increased myocyte stress leads to expression of a variety of cardiac genes that activate the transcription machinery leading to hypertrophy and other phenotypes of HCM. Thus, the pathogenesis of hypertrophy, a common programmed response of the myocardium to any form of stress, whether caused by a genetic defect or by an acquired condition, involves common pathways. Similarly, pathogenesis of diverse cardiac phenotypes also results from upregulation of expression of a variety of genes in response to the primary impetus provided by the mutant contractile protein (3). To identify genes that are upregulated in the heart in HCM, as the initial step for delineating their role in induction of cardiac phenotypes, we performed subtraction hybridization between normal hearts and hearts with HCM followed by Northern blotting to confirm upregulation of expression of the differentially expressed genes in HCM.
Subtraction hybridization and differential screening
Each patient signed an informed consent, and the institutional review board approved the protocols. Subtraction hybridization was performed between two messenger ribonucleic acid (mRNA) pools extracted from a heart with HCM and a normal heart, using a polymerase chain reaction (PCR)-select complementary deoxyribonucleic acid (cDNA) subtraction kit (CLONETECH, Palo Alto, California). In brief, left ventricular septal tissue was obtained by myomectomy from a patient who had a long-standing history of symptomatic HCM with left ventricular outflow tract obstruction. A corresponding segment of the interventricular septum was excised from an age- and gender-matched normal donor heart that was not used for cardiac transplantation. Tissues were frozen in liquid nitrogen and stored at −135°C until mRNA extraction. Total RNA and poly A+ RNA were extracted from hearts with HCM and control hearts side-by-side using the same reagents and protocols and quantified by measuring absorption at 260 nm. To select genes that were differentially expressed in the heart in HCM, 2 μg of polyA+ mRNAs extracted were used to generate subtraction cDNA libraries. The cDNA library generated from the normal heart was subtracted from that of the heart with HCM (forward subtraction) through two sets of hybridization, which resulted in a library enriched for genes that were upregulated in the heart with HCM. A reverse subtraction cDNA library was generated by subtracting the cDNA library generated from the heart with HCM from that of the normal heart. To reduce the background, two rounds of suppression PCR were performed to amplify the differentially expressed genes. The enriched cDNA library was subcloned into a T/A cloning plasmid vector, and the resulting products were transformed into Escherichia coliand plated on agar plates.
To confirm that the subtracted clones represented differentially expressed genes and to reduce the number of false positives, colonies were initially screened by PCR-select differential screening (CLONETECH). In brief, the cloned inserts were amplified by PCR using flanking primers, and 288 randomly selected clones in the forward-subtracted cDNA library were arrayed on three sets of 96-spot dot blot membranes in duplicates for subsequent hybridization. Probes were prepared by PCR amplification of the forward and reverse subtraction cDNA libraries, restriction enzyme digestion to remove the adapter sequences in order to reduce the background and radiolabeling with [32P] dCTP a specific activity of more than 109cpm/μg. Each set of membranes was hybridized either with forward or reverse subtracted probe in ExpressHyb solution (CLONETECH). After washing in high stringency conditions, the membranes were exposed to X-ray films in the presence of intensifying screens for 24 h.
Differentially expressed clones were sequenced using Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Genetic Analyzer 310 (PE Biosystem, Foster City, California). Sequences were analyzed using BLAST search of gene bank database.
To confirm differential expression of the putative candidate genes in HCM, Northern blotting was performed on mRNA extracts from hearts with HCM and control hearts. In brief, 10 μg aliquots of total RNA extracts were loaded onto a formaldehyde-agarose gel, subjected to electrophoresis and were transferred to a nylon membrane (Bio-Rad Laboratories, Cambridge, Massachusetts). The forward-subtracted cDNA probes were radiolabeled with [32P] dCTP and hybridized to mRNAs on the membranes in the presence of Denhardt’s reagent (0.1% ficol, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 100 μg/ml of denatured fragmented salmon sperm DNA) in hybridization solution (6 × sodium chloride-sodium citrate, 0.5% sodium dodecylsulfate). After washing, the membrane was exposed to an X-ray film for 24 to 48 h.
To determine whether the differentially expressed clones were also upregulated in additional hearts with HCM, Northern blotting was performed on mRNA extracts from six additional hearts with HCM and two control hearts.
Genes upregulated in HCM
A total of 288 clones from the subtracted library were screened by duplicate dot-blot hybridization. A twofold or greater difference in the intensity of signals detected from colonies hybridized with the forward and reverse-subtraction probes (forward less than reverse) was considered evidence of differential expression. We identified 76 differentially expressed clones (Fig. 1)that were sequenced. Blast analyses of the sequences identified 36 differentially expressed genes (Table 1)that code for a diverse array of proteins with a variety of functions.
Northern blot analyses
Northern blot analyses were performed to compare expression of skeletal muscle alpha-actin (ACTA1), myosin light chain 2a (MLC2a), guanosine 5′-triphosphate (GTP)-binding protein Gs-alpha subunit (GNAS1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase (NDUFB10), voltage-dependent anion channel 1 (VDAC1), four-and-a-half LIM domain protein 1 (FHL1) (also known as SLIM1), sarcosin (SARCOSIN) and heat shock 70kD protein 8 (HSPA8) between hearts with HCM and control hearts. The results, shown in Figure 2, confirmed upregulation of expression of ACTA1(fourfold), MLC2a(fivefold), GNAS1(threefold), NDUFB10(eightfold), VDAC1(twofold), FHL1(twofold), SARCOSIN(twofold) and HSPA8(ninefold).
Expression levels of ACTA1, GNAS1, MLC2aand FHL1genes were determined by Northern blotting in six additional left ventricular tissues from patients with HCM and two normal hearts. The results are shown in Figure 3. As shown, expression levels of ACTA1, GNAS1and MLC2awere increased in all six hearts by less than twofold, while expression levels of FHL1were increased by more than twofold in four hearts.
A diverse array of genes encoding for proteins with variety of functions, including markers of cardiac hypertrophy due to pressure overload (secondary hypertrophy), is upregulated in the human heart in HCM. The diversity of molecular phenotype in HCM is in accord with the diversity of pathologic and clinical phenotypes that encompass not only myocyte hypertrophy and disarray but also interstitial fibrosis, thickening of the media of intramural coronary arteries and arrhythmias (2). Increased expression of the markers of “secondary” cardiac hypertrophy supports the hypothesis that hypertrophy in HCM is also a “secondary” phenotype (3), and common pathways are involved in the induction of cardiac hypertrophy in genetic and nongenetic forms.
We have confirmed increased expression of selected differentially expressed genes (eight genes) by Northern blotting in the myomectomy sample that was used for subtraction subsequently and subsequently for four genes in multiple hearts with HCM, further validating the observed results. We used PCR-select cDNA subtraction hybridization, which is considered a robust technique for identification of differentially expressed genes (4,5). Unlike microarray DNA chips, which are restricted to known sequences and are subject to bias for the selection of putative genes, subtraction hybridization affords the opportunity to screen all differentially expressed genes without a selection bias or a priori knowledge of their sequences. However, it is a tedious method, and, in each set of subtraction experiments, a fraction of total differentially expressed genes are identified, and the yield decreases with each additional subtraction. We performed three sets of colony screening from a single cDNA subtraction experiment and selected genes that showed at least a twofold increase in the expression levels. Therefore, the total number of differentially expressed genes in the heart in HCM could be greater. In addition, expression of many genes could be decreased in HCM, and, since we did not screen the reverse subtracted library, they were not detected. Furthermore, cDNA subtraction hybridization detects gene expression at the mRNA stage, which may not correspond to protein expression. Moreover, expression profiling detects only expression of the genes and not the activity of their proteins, which, for many proteins, such as the intracellular signaling kinases, is the primary determinant of protein function. Therefore, it not surprising that upregulation of expression of intracellular signaling kinases, which play fundamental roles in induction of acquired forms of cardiac hypertrophy (6)and, likewise, are expected to modulate cardiac hypertrophy in HCM, were not detected. Furthermore, expression profiling is an initial step that provides a basis for future studies to delineate the role of differentially expressed genes in the pathogenesis of cardiac phenotype.
A variety of genes including those encoding for contractile sarcomeric proteins, cytoskeletal proteins, ion channels, intracellular signal tranducers, protein maintaining the redox state of the myocardium, along with transcriptional and translation machinery were upregulated in HCM. The most common upregulated genes were the markers of “secondary” cardiac hypertrophy, such as skeletal alpha-actin, isoforms of myosin light chain and, less commonly, brain natriuretic factor, also known to be activated in pressure-overload induced (secondary) cardiac hypertrophy. Many of the upregulated genes have not been previously implicated in cardiac hypertrophy, and function of a few, such as sarcosin (7)and SLIM1 (8), is unknown. In addition, structure and function of proteins encoded by HSPC040(9)and C1ORF8(10), which have been identified by expressed sequence tags analysis and comparative proteomics, respectively, are unknown. Among the genes with the highest upregulation were HSPA8, also known as HSP73, which is a member of HSP70 multigene family with chaperoning roles for nascent polypeptide facilitating their correct folding, translocation and degradation (11). Expression of NDUFB10encoding for the first enzyme complex in the electron transport chain of mitochondria (12)was also upregulated significantly. Upregulation of the expression of HSPA8and NDUFB10(12), along with increased expression of many ribosomal proteins is in accord with the increased protein synthesis and mitochondria function in hypertrophic states and signifies their role as potential modulators of cardiac phenotype in HCM. Other notable genes included GNAS1, encodes for the GTP-binding protein Gs and couples the hormone-receptor binding to adenylyl cyclase activation (13); VDAC1, encodes for VDAC1 that forms the major pathway for movement of adenine nucleotides through the outer membrane of mitochondria (14)and sodium hydrogen exchanger, which is a ubiquitous membrane-bound enzyme involved in pH regulation and activated by a variety of signals including growth factors (15).
In summary, we have identified a variety of genes that are upregulated in human patients with HCM that could contribute to the diversity of clinical and pathologic phenotypes of HCM. Increased expression of genes encoding for the markers of “secondary” cardiac hypertrophy supports the hypothesis that hypertrophy in HCM, a genetic disease, is also a “secondary” phenotype and involves pathways that are similar to those implicated in the acquired forms. Characterization of the role of upregulated gene in HCM could provide clues to the diversity of clinical and pathologic phenotypes and for new therapeutic and preventive targets.
☆ Supported by grants from the National Heart, Lung, and Blood Institute, Specialized Centers of Research (P50-HL42267-01) and an Established Investigator Award (9640133N) from the AHA, National Center, Dallas, Texas.
- skeletal muscle alpha-actin
- complementary deoxyribonucleic acid
- four-and-a-half LIM domain protein 1
- guanosine 5′-triphosphate (GTP)-binding protein Gs-alpha subunit
- hypertrophic cardiomyopathy
- heat shock 70kD protein 8
- myosin light chain 2A
- messenger ribonucleic acid
- reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase
- polymerase chain reaction
- voltage-dependent anion channel 1
- Received January 31, 2001.
- Revision received June 11, 2001.
- Accepted June 26, 2001.
- American College of Cardiology
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