Profile of MicroRNAs Differentially Produced in Hearts from Patients with Hypertrophic Cardiomyopathy and Sarcomeric Mutations

To the Editor: MicroRNAs (miRNAs)1 regulate cardiac growth and conduction and play an important role in cardiac diseases (1). Several miRNAs are differentially produced in cardiac hypertrophic tissue, compared with normal tissue, and may contribute to the development of cardiomyocyte hypertrophy (2, 3). Hypertrophic cardiomyopathy (HCM) is frequently familial and caused by mutations in sarcomeric genes. To our knowledge, no study has reported the miRNA production profile in HCM tissues with sarcomeric gene mutations. To better define the molecular changes in HCM, we defined the production of well-characterized miRNAs in left ventricular (LV) heart tissue from 5 patients who underwent a cardiac transplantation and a control heart tissue (human LV tissue, Ambion/Applied Biosystems), and we compared their production profiles. Two of the patients were familial HCM patients who were carriers of missense mutations in the MYH72 (myosin, heavy chain 7, cardiac muscle, beta) gene (Val822Met and Arg453Cys). Three patients were cases of sporadic LV hypertrophy secondary to heart valve disease. The study was approved by the Ethical Committee of Hospital Universitario Central Asturias (HUCA), and all of the patients provided written informed consent. We isolated total RNA with TRIzol (Invitrogen) and used the TaqMan MicroRNA Reverse Transcription Kit, Megaplex RT Human Pool A primers, and TaqMan human MicroRNA TLDA plate A (all from Applied Biosystems) to determine the production profile of 377 human miRNAs in the healthy LV tissues and in a pool of the 2 tissues with MYH7 mutations. Each sample was analyzed in triplicate, and the mean threshold cycle (CT) value for each miRNA was normalized by using mammalian U6 as the reference gene. A P value <0.05 with respect to the -fold difference in miRNA production (HCM pool vs healthy LV tissue) was considered statistically significant. The detailed experimental procedure is available upon request to the corresponding author. Compared with the healthy LV tissue, the HCM tissue showed an overall downregulation of miRNAs. Although the 2 tissues were not significantly different with respect to most of the miRNAs, with a CT difference between the 2 tissues of <4 (data not shown), the 2 tissues were significantly different with respect to the production of 19 of the miRNAs (Table 1). These miRNAs were individually assayed in triplicate in the LV control and the 5 pathologic tissues via real-time TaqMan miRNA assays (Applied Biosystems). Ten miRNAs were underproduced (miR-1, miR-133b, miR-191, miR-208b, miR-218, miR-30b, miR-374, miR-454, and miR-495) in the 5 pathologic tissues, and 2 miRNAs were overproduced (miR-590–5p and miR-92a). miR-495 was the only miRNA that differentiated hearts with and without sarcomeric mutations. Compared with the healthy tissue, miR-495 was underproduced in the 2 samples with MYH7 mutations and overproduced in the 3 samples without sarcomeric mutations. This miRNA is deregulated in primary muscular disorders but not in cardiac diseases. miR-590–5p and miR-92a were overproduced in all of the pathologic tissues. Neither of these 2 miRNAs had previously been reported as deregulated in cardiac hypertrophy and other heart diseases. H1 and H9 correspond to the patients with MYH7 mutations, and H2, H3, and H5 correspond to the patients with disease secondary to cardiac valvular disease. The mean CT values for the healthy tissue are also indicated. HC, healthy control. H1 and H9 correspond to the patients with MYH7 mutations, and H2, H3, and H5 correspond to the patients with disease secondary to cardiac valvular disease. The mean CT values for the healthy tissue are also indicated. HC, healthy control. miR-1 and miR-133 were underproduced in the hypertrophic tissues and have previously been implicated in cardiac development. They are significantly downregulated in hearts from patients with idiopathic and ischemic cardiomyopathies (4, 5). miR-208a and miR-208b are encoded by introns in the MYH6 (myosin, heavy chain 6, cardiac muscle, alpha) and MYH7 genes, respectively. In mice, the reexpression of Myh7 {myosin, heavy polypeptide 7, cardiac muscle, beta [Mus musculus]} and the production of miR-208b is a characteristic of cardiac hypertrophy in response to pressure overload. In agreement with a role for these miRNAs in the development of HCM, miR-208a was also overproduced in the 2 patients with MYH7 mutations. Interestingly, the -fold change in miR-208a in one of the HCM patients was the highest among all the miRNAs analyzed in our study (Table 1). The downregulation of miR-208b was lower in the 3 patients with cardiac hypertrophy secondary to valve disease, suggesting that the changes in production of this miRNA could differ between hypertrophic hearts with and without sarcomeric gene mutations. Compared with other studies of samples from patients with heart failure, we analyzed pathologic tissues with a recognized sarcomeric mutation that would be the primary cause of the hypertrophy in these patients. Changes in miRNA production might differ between hypertrophic hearts with sarcomeric mutations and hearts in which the disease was secondary to another condition causing the hypertrophy. The difference between these cases and those without sarcomeric mutations should be replicated with other patients, including cases with mutations in different sarcomeric genes. We also studied failing explanted hearts that represented advanced stages of the disease; thus, we cannot exclude the possibility that some of the deregulated miRNAs were not representative of the changes at the initial stages of the disease. microRNA hypertrophic cardiomyopathy left ventricular Hospital Universitario Central Asturias threshold cycle. myosin, heavy chain 7, cardiac muscle, beta myosin, heavy chain 6, cardiac muscle, alpha myosin, heavy polypeptide 7, cardiac muscle, beta [Mus musculus].