View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Journal of the American College of Cardiology Vol. 38, No. 4, 2001 © 2001 by the American College of Cardiology ISSN 0735-1097/01/$20.00 Published by Elsevier Science Inc. PII S0735-1097(01)01509-1 Hypertrophic Cardiomyopathy Expression Profiling of Cardiac Genes in Human Hypertrophic Cardiomyopathy: Insight Into the Pathogenesis of Phenotypes Do-Sun Lim, MD, Robert Roberts, MD, FACC, Ali J. Marian, MD, FACC Houston, Texas OBJECTIVES The goal of this study was to identify genes upregulated in the heart in human patients with hypertrophic cardiomyopathy (HCM). BACKGROUND 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. METHODS 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. RESULTS 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, MLC2a and GNAS1 were increased in six additional and FHL1 in four additional hearts with HCM. CONCLUSIONS 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. (J Am Coll Cardiol 2001;38: 1175–80) © 2001 by the American College of Cardiology Hypertrophic cardiomyopathy (HCM) is a genetic disease transcription machinery leading to hypertrophy and other caused by mutations in the contractile sarcomeric proteins phenotypes of HCM. Thus, the pathogenesis of hypertro- (1). Hypertrophic cardiomyopathy is diagnosed clinically by phy, a common programmed response of the myocardium the presence of unexplained left ventricular hypertrophy and to any form of stress, whether caused by a genetic defect or pathologically by myocyte hypertrophy, disarray and inter- by an acquired condition, involves common pathways. stitial fibrosis (2). While the causality of mutant contractile Similarly, pathogenesis of diverse cardiac phenotypes also proteins in the pathogenesis of HCM is well established, results from upregulation of expression of a variety of genes the molecular bases of diverse cardiac phenotypes in HCM in response to the primary impetus provided by the mutant remain unknown. contractile protein (3). To identify genes that are upregu- We and others have proposed cardiac hypertrophy in lated in the heart in HCM, as the initial step for delineating HCM is a “compensatory” phenotype due to increased their role in induction of cardiac phenotypes, we performed ϩ cardiac myocyte stress or altered Ca 2 sensitivity of the subtraction hybridization between normal hearts and hearts contractile apparatus imparted by the mutant contractile with HCM followed by Northern blotting to confirm proteins (3). Accordingly, increased myocyte stress leads to upregulation of expression of the differentially expressed expression of a variety of cardiac genes that activate the genes in HCM. From the Section of Cardiology, Department of Medicine, Baylor College of METHODS Medicine, Houston, Texas. Supported by grants from the National Heart, Lung, and Blood Institute, Specialized Centers of Research (P50-HL42267-01) and an Estab- Subtraction hybridization and differential screening. lished Investigator Award (9640133N) from the AHA, National Center, Dallas, Each patient signed an informed consent, and the institu- Texas. Manuscript received January 31, 2001; revised manuscript received June 11, 2001, tional review board approved the protocols. Subtraction accepted June 26, 2001. hybridization was performed between two messenger ribo- 1176 Lim et al. JACC Vol. 38, No. 4, 2001 Gene Expression in HCM October 2001:1175–80 by subtracting the cDNA library generated from the heart Abbreviations and Acronyms with HCM from that of the normal heart. To reduce the ACTA1 ϭ skeletal muscle alpha-actin background, two rounds of suppression PCR were per- cDNA ϭ complementary deoxyribonucleic acid formed to amplify the differentially expressed genes. The ϭ FHL1 four-and-a-half LIM domain protein 1 enriched cDNA library was subcloned into a T/A cloning GNAS1 ϭ guanosine 5Ј-triphosphate (GTP)- binding protein Gs-alpha subunit plasmid vector, and the resulting products were transformed HCM ϭ hypertrophic cardiomyopathy into Escherichia coli and plated on agar plates. HSPA8 ϭ heat shock 70kD protein 8 To confirm that the subtracted clones represented differ- ϭ MLC2a myosin light chain 2A entially expressed genes and to reduce the number of false mRNA ϭ messenger ribonucleic acid NDUFB10 ϭ reduced nicotinamide adenine positives, colonies were initially screened by PCR-select dinucleotide ubiquinone oxidoreductase differential screening (CLONETECH). In brief, the cloned PCR ϭ polymerase chain reaction inserts were amplified by PCR using flanking primers, and SARCOSIN ϭ sarcosin 288 randomly selected clones in the forward-subtracted ϭ VDAC1 voltage-dependent anion channel 1 cDNA library were arrayed on three sets of 96-spot dot blot membranes in duplicates for subsequent hybridization. nucleic acid (mRNA) pools extracted from a heart with Probes were prepared by PCR amplification of the forward HCM and a normal heart, using a polymerase chain and reverse subtraction cDNA libraries, restriction enzyme reaction (PCR)-select complementary deoxyribonucleic acid digestion to remove the adapter sequences in order to reduce 32 (cDNA) subtraction kit (CLONETECH, Palo Alto, Cal- the background and radiolabeling with [ P] dCTP a 9 ␮ ifornia). In brief, left ventricular septal tissue was obtained specific activity of more than 10 cpm/ g. Each set of by myomectomy from a patient who had a long-standing membranes was hybridized either with forward or reverse history of symptomatic HCM with left ventricular outflow subtracted probe in ExpressHyb solution (CLONE- tract obstruction. A corresponding segment of the interven- TECH). After washing in high stringency conditions, the tricular septum was excised from an age- and gender- membranes were exposed to X-ray films in the presence of matched normal donor heart that was not used for cardiac intensifying screens for 24 h. transplantation. Tissues were frozen in liquid nitrogen and Sequencing. Differentially expressed clones were se- stored at Ϫ135°C until mRNA extraction. Total RNA and quenced using Big Dye Terminator Cycle Sequencing poly Aϩ RNA were extracted from hearts with HCM and Ready Reaction Kit on an ABI Genetic Analyzer 310 (PE control hearts side-by-side using the same reagents and Biosystem, Foster City, California). Sequences were ana- protocols and quantified by measuring absorption at lyzed using BLAST search of gene bank database. 260 nm. To select genes that were differentially expressed in Northern blotting. To confirm differential expression of the heart in HCM, 2 ␮g of polyAϩ mRNAs extracted were the putative candidate genes in HCM, Northern blotting used to generate subtraction cDNA libraries. The cDNA was performed on mRNA extracts from hearts with HCM library generated from the normal heart was subtracted from and control hearts. In brief, 10 ␮g aliquots of total RNA that of the heart with HCM (forward subtraction) through extracts were loaded onto a formaldehyde-agarose gel, two sets of hybridization, which resulted in a library subjected to electrophoresis and were transferred to a nylon enriched for genes that were upregulated in the heart with membrane (Bio-Rad Laboratories, Cambridge, Massachu- HCM. A reverse subtraction cDNA library was generated setts). The forward-subtracted cDNA probes were radiola- Figure 1. Subtracted clones dot blotted at 96-spot membranes. Membranes hybridized with radiolabeled forward and reverse subtracted complementary deoxyribonucleic acid libraries. Differentially expressed genes, identified by a twofold increase in signal intensity in the membrane hybridized with the forward subtraction probes, are shown by arrows. Clones hybridized with the reverse subtraction probes are considered background. JACC Vol. 38, No. 4, 2001 Lim et al. 1177 October 2001:1175–80 Gene Expression in HCM Table 1. Differentially Expressed Genes in the Heart in HCM Gene Symbol Accession # Locus # of Clone Sarcomeric Skeletal muscle ␣-actin ACTA1 J00068 1q42.13–q42.2 12 Myosin light chain 2a LOC58498