Global gene expression profiling of end-stage dilated cardiomyopathy using a human cardiovascular-based cDNA microarray

Abstract

To obtain a genomic portrait of heart failure derived from end-stage dilated cardiomyopathy (DCM), we explored expression analysis using the CardioChip, a nonredundant 10,848-element human cardiovascular-based expressed sequence tag glass slide cDNA microarray constructed in-house. RNA was extracted from the left ventricular free wall of seven patients undergoing transplantation, and five nonfailing heart samples. Cy3- and Cy5-labeled (and reverse dye-labeled) cDNA probes were synthesized from individual diseased or nonfailing adult heart RNA, and hybridized to the array. More than 100 transcripts were consistently differentially expressed in DCM >1.5-fold (versus pooled nonfailing heart, P < 0.05). Atrial natriuretic peptide was found to be up-regulated in DCM (19-fold compared to nonfailing, P < 0.05), as well as numerous sarcomeric and cytoskeletal proteins (eg, cardiac troponin, tropomyosin), stress response proteins (eg, HSP 40, HSP 70), and transcription/translation regulators (eg, CCAAT box binding factor, eIF-1AY). Down-regulation was most prominently observed with cell-signaling channels and mediators, particularly those involved in Ca(2+) pathways (Ca(2+)/calmodulin-dependent kinase, inositol 1,4,5-trisphosphate receptor, SERCA). Most intriguing was the co-expression of several novel, cardiac-enriched expressed sequence tags. Quantitative real-time reverse transcriptase-polymerase chain reaction of a selection of these clones verified expression. Our study provides a preliminary molecular profile of DCM using the largest human heart-specific cDNA microarray to date.

Figure 1.

Hierarchical cluster analysis of seven DCM and five nonfailing (NF) heart patient samples against human universal reference RNA. Six of seven DCM patient samples and four of the five nonfailing heart clustered together.

Figure 2.

Hierarchical cluster analysis of DCM versus pooled nonfailing (NF) adult heart samples. Six slides representing three individual DCM patients (three reverse-labeled) were clustered, ie, slides 1, 11, and 12 were hybridized with DCM-Cy5-dUTP, whereas slides 3, 9, and 13 were hybridized with DCM-Cy3-dUTP. Colors represent Cy3/Cy5 ratio; red = positive ratio, green = negative ratio. A selection of genes corresponding to regions of the cluster are shown.

Figure 3.

Real-time RT-PCR results of selected genes identified by the microarray analysis. a: Fold difference expression of each gene in DCM heart samples relative to nonfailing (NF) samples. ANP, atrial natriuretic peptide (positive control); CNN3870 and HA6788, novel heart transcripts; EIF1AY, elongation initiation factor 1A, Y isoform; CaCKK, Ca²⁺/calmodulin kinase kinase; InosK, inositol 1,4,5-triphosphate-3-kinase; InosTR, inositol 1,4,5-triphosphate receptor; CCaATPase, cardiac Ca²⁺ ATPase. b: Mean relative mRNA population of each gene from a versus GAPDH in DCM (n = 7) and NF (n = 5) samples. ^†, P < 0.01; *, P < 0.05 versus NF.