(left to right, back row) Kathy He, research teaching specialist; Jacquelin Lypowy, research teaching specialist; Shweta Rane, graduate student. (front row) Maha Abdellatif, PhD, associate professor, UMDNJ-New Jersey Medical School; Danish Sayed, graduate student; Andy Chen, research teaching specialist
Newly discovered regulators
in the heart
The recently discovered tiny RNA (microRNA) molecules that exist in a wide variety of species managed to elude scientists for decades. Now, these molecules are providing us with new insights into the mechanisms underlying the development of diseases such as cancer. We recently reported that microRNAs also play a major role in the development of cardiac enlargement and failure. We found that one microRNA in particular was capable of inhibiting the enlargement of heart cells, while others delayed programmed cell death. These molecules have the potential to serve as predictive markers in disease, and more importantly, as promising drug targets.
MicroRNAs have changed our perception of how cellular functions are regulated. The central dogma is that the genomic DNA is transcribed into message RNA that is translated into protein. In turn, proteins are the main regulators of these transcription and translation steps. MicroRNAs have now added a new dimension to our understanding. These molecules have the capacity to specifically bind to message RNA and prevent their translation, which explains the discrepancies observed between the levels of some message RNA and their translated proteins. This also explains why message RNA profiling is not a reliable diagnostic or prognostic marker of disease. It is important to note that while one message RNA molecule translates into one protein (and sometimes splice variants), one microRNA regulates multiple message RNA that may collaborate in a specific cellular function. Since, mainly, a single cellular function is executed by a consortium of genes it becomes clear that microRNA levels must be more tightly regulated compared to a single gene’s message RNA levels, which in many cases can be offset by redundant isoforms. Thus, it was no surprise to find that microRNA profiling offered a more precise diagnostic and prognostic parameter in cancer.
||Figure 1: Progressive deregulation of microRNA
expression during pressure-overload cardiac hypertrophy. Differentially expressed microRNA were analyzed by hierarchical clustering of the log2 value of each aortic constriction/sham operated pair of microRNA micro
array signal at 1d, 7d, and 14d post-aortic constriction, using Cluster v3.0 software. The results are displayed in a Heatmap generated by Java TreeView v1.0.13
software. Red represents up-regulation; green,
down-regulation; black, no change. The legend on the right indicates the microRNA represented in the corres -
ponding row, all of which are mus musculus specific. The barcode on the bottom, right, represents the color scale of the log2 values. Each column represents the data from a given time point indicated at the top of the Heatmap.
We questioned whether microRNA expression is deregulated in the heart after an increase in its workload, imposed by pressure overload. The model we used for this experiment was the mouse, in which constriction of the aorta imposes pressure on the heart causing it to enlarge. This mimics cardiac enlargement in humans due to high blood pressure, which may lead to failure in severe cases. After a detailed time course we found progressive changes in the microRNA levels, with unique patterns characterizing each stage in the heart’s dysfunction. We predicted that those microRNAs that change acutely upon imposition of pressure might be a cause rather than an effect of enlargement. One microRNA, in particular, microRNA-1, which is muscle-specific, and has the highest levels of microRNA in the heart, was reduced by ~50% within 24 hours of pressure overload, before any increase in heart weight. As mentioned earlier, microRNAs bind to their target message RNA and inhibit its translation. Thus, if the level of a microRNA is reduced, we expect an increase in the translation of those targets.
MicroRNA-1 has several predicted targets that we have previously shown to be involved in heart growth and enlargement. Some of those genes include Ras GTPase-activating protein and cyclin dependent kinase 9, which regulate translation and transcription, and fibronectin, which enhances cell attachment and promotes growth. When cultured myocytes are stimulated with growth-inducing factors, the cells enlarge and the expression of these genes increases, concomitant with the decrease in microRNA-1 expression, similar to what we observe in the mouse heart in vivo. By replenishing the levels of microRNA in these cells we were able to inhibit the increase in the levels of Ras GTPase-activating protein, cyclin dependent kinase 9, and fibronectin, as well as, the myocytes’ growth. This led us to conclude that reduction of microRNA-1 in the heart during increased workload is necessary for the increase in heart mass.
We also found many other microRNA changes in the heart and are still pursuing their functions. Of particular interest are those that change dramatically preceding or during the transition of cardiac enlargement to failure. Preceding cardiac failure, we found that microRNA-21 is increased about 8-fold. Interestingly, this microRNA was also reported to increase in many forms of cancer. It was shown that if it is inhibited, the cancer cells die or become more sensitive to chemotherapy, concluding that microRNA-21 is pro-survival. In agreement, we found that this molecule plays a similar role in heart cells. We know that the heart copes with pressure overload by a compensatory mechanism that involves cell enlargement and an increase in pro-survival genes. But chronic pressure eventually results in heart failure. One reason may be attributed to exhaustion that results in activation of programmed death pathways and down-regulation of the pro-survival genes, which results in further weakening of the heart walls, initiating a vicious cycle. Thus pro-survival and anti-survival genes such as microRNA-21 are therapeutic candidates.
Targeting specific microRNA molecules by therapeutic agents is not as simple as targeting specific protein molecules, due to the nature of their structure. One approach that has recently emerged is to target those molecules via systemically delivering the complimentary sequence to a specific microRNA. The limitations of this method, which include poor cell permeability and rapid hydrolysis, were overcome by adding a cholestryl moiety and non-hydrolysable sulfur bonds, respectively, to these molecules. This product was tested in animals with great success and is currently being tested in cancer clinical trials. But in the cardiovascular field, the research of these micoRNA molecules has just begun.
In short, the deregulation of microRNA is proving to be an underlying cause of cellular dysfunction and disease. This is especially true in many forms of cancer, where these regulatory molecules have provided better prognostic and diagnostic tools, as well as new therapeutic targets. Our data suggest that microRNA play an essential role during cardiac hypertrophy and failure and have the potential for providing us with new diagnostic, prognostic, and therapeutic targets for cardiovascular diseases.
Maha Abdellatif was born in Alexandria, Egypt, and received her medical degree from the University of Alexandria. She earned a PhD in biochemistry from the University of Maryland, and did postdoctoral training with Dr. Michael Schneider at Baylor College of Medicine, in Houston, Texas, where she joined the faculty in 1995. She was recruited to UMDNJ-New Jersey Medical School by Dr. Stephen F. Vatner in 2001.