In this study, we investigated the function and molecular mechanisms of the miR-208 family in adult heart physiology. Our findings, which we believe are novel, include the following: (a) miR-208a and miR-208b are members of miR-208 family and are differentially expressed during heart development and heart pathology, paralleling the expression of their respective host genes, αMHC and βMHC. (b) Cardiac overexpression of miR-208a is sufficient to induce hypertrophy. (c) miR-208a–induced βMHC expression is restricted to a subset of cardiomyocytes associated with fibrosis. (d) βMHC expression is dramatically decreased in Mir208a–/– mouse hearts. (e) Both miR-208a and miR-208b target Thrap1 and myostatin, 2 important negative regulators of muscle growth and hypertrophy. (f) miR-208a is necessary for normal cardiac conduction. miR-208a gain- and loss-of-function are associated with arrhythmias. Together, the results of our study provide critical functional and mechanistic insights into how miRNAs participate in the regulatory cascade to modulate cardiac remodeling and normal physiology.
The βMHC and αMHC isoforms are major contractile proteins of cardiomyocytes and differ primarily in their ability to convert ATP to mechanical work at different rates, thus their relative protein expression ratio affects contractility of the cardiac sarcomeres (56
). Increased expression of βMHC is a common feature of cardiac hypertrophy and heart failure that reduces contractile performance and is thought to be a maladaptive response (28
). The shift towards βMHC is reversible under particular conditions that are associated with improved cardiac performance, including the regression of hypertrophy and human patients that respond favorably to β blocker therapy (58
). Furthermore, mutations in the Myh7
gene are commonly associated with hypertrophic cardiomyopathies (63
). Therapies that inhibit the maladaptive features of hypertrophy might be useful in improving the function of the diseased heart (20
). However, the molecular events that induce hypertrophy and govern hypertrophic gene expression are not well defined. More specifically, it is not fully understood how βMHC is regulated during cardiac remodeling and how its reactivation contributes to functional maladaptation. Our experimental results demonstrate that miR-208a is sufficient to induce cardiac hypertrophy in Tg hearts. Interestingly, the hypertrophic growth induced by miR-208a is only accompanied by increased βMHC expression in a subset of hypertrophied cardiomyocytes. It will be important to perform time course experiments in the future to determine the temporal regulation of βMHC by miR-208a.
In contrast, deletion of miR-208a resulted in decreased βMHC expression in the adult heart, providing genetic evidence that miR-208a is required for βMHC expression. Our results are consistent with a prior report showing that loss of miR-208a blunted stress-induced cardiac hypertrophy and βMHC reactivation (17
). Strikingly, upregulated βMHC expression in miR-208a Tg hearts is tightly associated with regional fibrosis, similar to what we previously found in renin-induced hypertrophy (31
). The strong correlation of re-expression of βMHC and fibrosis development may explain why such reactivation of fetal genes is associated with a maladaptive phenotype. Further studies to understand how induced βMHC expression is associated with fibrosis will likely shed light on the biology of cardiac hypertrophy and heart failure.
Our findings demonstrate that miR-208a posttranscriptionally represses the expression of Thrap1, a component of the thyroid hormone nuclear receptor complex. Thyroid hormone signaling has long been an established regulator of cardiac myosin heavy chain isoform expression: a surge of circulating thyroid hormone that occurs after birth transcriptionally represses βMHC expression while activating αMHC expression (34
). Studies have also shown that excessive administration of thyroid hormone leads to the development of cardiac hypertrophy, but the molecular mechanism was elusive (64
). Our findings, in which increasing the level of miR-208a in Tg hearts reduced Thrap1 levels and induced hypertrophic growth, provide a link between the action of miR-208a and thyroid hormone in cardiac hypertrophy. We find that miR-208a posttranscriptionally represses Thrap1, which is consistent with a previous report (17
). We extended our previous observations by uncovering an additional conserved miR-208 family binding site within the Thrap1 3′ UTR. Furthermore, we demonstrated that both miR-208a and miR-208b target those sites. In addition, we found that miR-208a and miR-208b target myostatin, a negative regulator of hypertrophic growth. The findings that both miR-208a and miR-208b repress Thrap1 and myostatin to a comparable level further suggest that miR-208 family members may target similar genes in vivo. A key disparity between our findings and that of an earlier report is the ability of miR-208a to induce cardiac hypertrophy. We believe the degree of miR-208a transgene expression accounts for this difference; we report higher miR-208a transgene expression levels, which indicate that surpassing a particular threshold of miR-208 family expression is important for facilitating hypertrophic growth.
In addition to the important role for miR-208a in cardiac hypertrophy, our studies also identify miR-208a as a key regulatory molecule necessary for proper cardiac conduction. Our experiments demonstrate that miR-208a is sufficient to induce cardiac arrhythmias, while miR-208a is also required to maintain proper cardiac conduction. Our results further indicate that miR-208a is required to maintain the expression of Cx40, whose misexpression is associated with cardiac arrhythmias (49
). We also find that GATA4 and Hop are upregulated and downregulated, respectively, in Mir208a–/–
hearts. While GATA4 is directly targeted by miR-208a, Hop is likely indirectly regulated by miR-208a. We found that both transcript and protein levels of Hop were lower in Mir208a–/–
hearts compared with wild-type hearts. Thus, altering miR-208a expression affects the delicate regulation of potent cardiac transcription factors. However, the cardiac conduction defects revealed in miR-208a Tg and Mir208a–/–
mice did not phenocopy Cx40 and Hop loss-of-function animal models. This is perhaps not entirely surprising, since miRNAs are proposed to fine-tune the expression of targeted mRNAs that produce protein products important for a particular tissue and to reduce transcriptional noise by helping to turn over misexpressed mRNAs (68
). By direct or indirect effects, a single miRNA may fine-tune the expression of thousands of genes (73
). In this sense, the precise molecular mechanisms underlying miR-208a–mediated regulation of cardiac conduction system components have yet to be defined. Identification of the cardiac transcription factors whose expressions are posttranscriptionally fine-tuned by miR-208a will likely be the key to understanding miR-208a function. Additional studies are needed to test and further define this intriguing possibility.
Our present finding that miR-208a is required to maintain Hop expression may also help to explain the blunted hypertrophic growth response of Mir208a–/–
). Unlike most homeobox transcription factors, Hop does not bind DNA directly. Instead, Hop recruits histone deacetylase 2 and inhibits the transcriptional activity of serum response factor in cardiomyocytes (74
). Interestingly, Hop overexpression was reported to induce cardiac hypertrophy and is proposed to inhibit an antihypertrophy gene program in the adult heart (76
). A potential explanation for the inability of Mir208a–/–
hearts to undergo hypertrophic growth may stem from the lack of Hop protein available to repress this antihypertrophy gene program. It will be important to understand how miR-208a regulates the expression of Hop and connexin proteins in the future.
Cardiac function and remodeling are intimately linked to the regulation of complex genetic pathways, and much effort has been expended in attempts to understand the molecular mechanisms underlying these pathways, with the ultimate goal of improving the prognosis of heart patients (77
). Much of our current understanding of how cardiac gene expression is controlled is at the level of transcriptional regulation, in which transcription factors associate with their regulatory DNA elements (enhancer/promoter sequences) to activate gene expression (78
). The regulation of cardiac gene expression is complex, with individual cardiac genes controlled by multiple independent enhancers that direct very restricted expression patterns in the heart. miRNAs have reshaped our view of how cardiac gene expression is regulated, increasing this complexity even further, by adding another layer of regulation at the posttranscriptional level. The results reported in this study clearly established a role for miR-208a in repressing antihypertrophy genes as part of the genetic program needed for hypertrophic growth. Although the arrhythmogenesis induced by perturbing miR-208a levels warrants much caution, we anticipate that inhibition of miR-208a may be a viable therapeutic strategy to repress βMHC expression and might remove some of the maladaptive features of hypertrophy.