A new unique form of microRNA from human heart, microRNA-499c, promotes myofibril formation and rescues cardiac development in mutant axolotl embryos

Abstract

Background: A recessive mutation "c" in the Mexican axolotl, Ambystoma mexicanum, results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development.

Results: We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper.

Conclusions: This new form, microRNA-499c, plays an important role in cardiac development.

Figure 1

Successful cloning of human RNAs. Gel electrophoresis of (A) individual plasmid digestion fragments with BsrGI. A uniform 2.5 kb band represents the unchangeable vector portion, and variable bands are different size cloned RNAs. (B) Gel electrophoresis of cloned DNAs from PCR. (C) Gel electrophoresis of cloned RNAs from reverse transcription reaction.

Figure 2

Tropomysin expression in RNA-treated mutant axolotl hearts revealed by immunofluorescent staining with confocal microscopy. (A) Normal heart. (B) Mutant untreated heart. (C) Mutant heart treated with cloned RNA (clone #291) after 5 days of incubation in organ culture. Magnification is the same in all images.

Figure 3

(A) DNA sequence of cloned RNA from the clone #291 (microRNA-499c). Underlined area is 22 bp microRNA-499. (B) Alignment of cloned RNA with microRNA-499 generated in Mirbase [8].

Figure 4

Alignment of cloned RNA (microRNA-499c) with (A) microRNA-499a and (B) microRNA-499b.

Figure 5

Time-course study of the relative quantities of tropomyosin expression by immunofluorescence staining in RNA-treated (7 ng/μl of microRNA499c) axolotl hearts. A. The immunofluorescent images show mutant hearts that were fixed and stained for tropomyosin after incubation with 7 ng/μl of microRNA499c for 0, 1, 2, 3, 4 and 5 days; the 0- control was without treatment. B. The average levels of fluorescence were quantified with ImageJ software as a percentage of that expressed in normal hearts, which was set to 100%. Significance (*p-value) between treated on the second day following days hearts and untreated hearts is p < 0.05, n=3.

Figure 6

Relative expression of cardiac markers by RT-PCR in mutant hearts (c/c), mutant hearts transfected with the microRNA-499c (c/c RNA) and normal hearts (+/+): (A) tropomyosin, (B) cardiac Troponin T and (C) α-syntrophin. Expression was calculated as percentage of that expressed in normal hearts (expression in normal hearts was assumed as 100%). Significance between treated and untreated mutant hearts is for tropomyosin, p < 0.05, cardiac Troponin T p < 0.02 and α-syntrophin p < 0.03, n=10.