Periodic Report Summary 1 - MRNA IN HEART (mRNA translational regulation in heart failure)
The aim of our work was to determine the mechanisms through which mTOR and its targets 4E-BP1 and 2 regulate translation and thereby cardiac function. Using genetically recombinant mice, we have previously determined that the absence of mTOR in cardiac cells induce heart failure rapidly; this event could be partially but significantly prevented through the deletion of a target of mTOR, the inhibitor of translation 4E-BP1. Deleting both 4E-BP1 and 2, heart failure and mortality could be prevented even further. In addition, the sole deletion of 4E-BP1 and 2 could improve cardiac function during stress. These results indicated the necessity to determine the "translatome" that is, the mRNA associated with polysomes, in normal conditions and during stress, and determine which mRNA molecules are directly regulated by the inhibitors of translation 4E-BPs and which are influenced by mTOR itself.
During this period of the study (six months), mRNA was collected from the myocardium of wild-type (WT) adult (2-month-old) sham-operated (sham) mice and from WT mice three days after transverse aortic constriction (TAC), a model in which blood pressure is greatly increased and myocardial stress induced. mRNA was also obtained from 4E-BP1-knock out (KO) mice before and three days after TAC. mRNA was also purified from ribosomes by immunoprecipitating them with an antibody specific for a protein component. cDNA libraries were generated and sequenced in our facility. Through this approach, we identified a pool of mRNAs which associated with ribosomes after stress and that therefore seems to be differentially regulated. In addition, we identified a pool of mRNAs which are regulated by 4E-BPs.
These results should be confirmed by further analyses, which will be carried out by other investigators. Eventually, we should be able to identify mRNAs intimately related to the control of cardiac function, a result which will lead to a better understanding of the molecular mechanisms underlying heart failure and to new molecular tools which could be employed to combat this disease, which is one of the major health care burdens in the Western world.
During this period of the study (six months), mRNA was collected from the myocardium of wild-type (WT) adult (2-month-old) sham-operated (sham) mice and from WT mice three days after transverse aortic constriction (TAC), a model in which blood pressure is greatly increased and myocardial stress induced. mRNA was also obtained from 4E-BP1-knock out (KO) mice before and three days after TAC. mRNA was also purified from ribosomes by immunoprecipitating them with an antibody specific for a protein component. cDNA libraries were generated and sequenced in our facility. Through this approach, we identified a pool of mRNAs which associated with ribosomes after stress and that therefore seems to be differentially regulated. In addition, we identified a pool of mRNAs which are regulated by 4E-BPs.
These results should be confirmed by further analyses, which will be carried out by other investigators. Eventually, we should be able to identify mRNAs intimately related to the control of cardiac function, a result which will lead to a better understanding of the molecular mechanisms underlying heart failure and to new molecular tools which could be employed to combat this disease, which is one of the major health care burdens in the Western world.