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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - RAMBO (Mitochondrial DNA degradation and sterile inflammation in the heart)

Teaser

Summary

Heart failure occurs when the heart is unable to pump enough blood to the other organs to satisfy their need for oxygen and nutrients. It usually manifests as tiredness and weakness, breathlessness and swelling of the legs and abdomen. Less than 50 percent of patients are living five years after their initial diagnosis and less than 25 percent are alive at 10 years. Poor prognosis can be attributed to a limited understanding of how the heart weakens. Many studies indicate that inflammation play an important role when people have got heart failure. However, a drug which inhibits one inflammation mediator (cytokine) worsened heart failure, suggesting that targeting a single component of the inflammation is not sufficient to cure the disease. If we understand how the inflammation is regulated, we can know useful information to mechanisms of heart failure and to identify therapeutic targets against heart failure.
Mitochondria are small compartments in cell that generate energy needed to power the cell\'s biochemical reactions. Mitochondria have striking similarities to bacteria cells and have their own DNA containing unmethylated motif as bacteria. Damaged mitochondria are degraded by a system called as autophagy, by which mitochondria are engulfed in membrane and fused with lysosomes for degradation. Mitochondrial autophagy is called mitophagy. Lysosomes contain many enzymes such as DNase II, which digest DNA. We have previously reported that incomplete digestion of damaged mitochondria by DNase II in mitophagy-lysosome system results in inflammation and heart failure. In addition, we have reported a protein Bcl2-L-13 is involved in mitophagy, which is a mammalian counterpart of Atg32, an essential mitophagy receptor in yeast. In this study, we are attempting how Bcl2-L-13 induces mitophagy, how mitochondrial DNA is methylated, and how DNase activity is regulated in heart failure.
Heart failure has become a global health problem affecting 26 million worldwide. The prevalence of heart failure in developed countries is estimated to be between 0.4 and 2.2%. Thus, we should develop novel and effective drug for heart failure treatment. In heart failure patients, inflammatory cytokines are increased in blood and the levels of cytokines are related to the severity and prognosis of the disease, yet the mechanisms of inflammation are not well defined. If we know the precise overall mechanism by which regulate inflammation in heart failure, we can develop a new therapy for heart failure.
Overall objectives in this study are 1) elucidate molecular mechanisms underlying inflammation in heart failure, focusing on mitochondrial DNA and 2) develop novel and effective therapeutics to treat patients with heart failure.

Work performed

Many kinds of proteins are required for mitophagy-mediated mitochondrial degradation. We screened a series of yeast autophagy mutants and found a different set of autophagy genes is used for Bcl2-L-13- and Atg32-mediated mitophagy in yeast. The components of the Atg1 complex essential for starvation-induced autophagy were necessary for Bcl2-L-13-, but not Atg32-mediated, mitophagy. The ULK1 complex, a counterpart of the Atg1 complex, is necessary for Bcl2-L-13-mediated mitophagy in mammalian cells. We propose a model where, upon mitophagy induction, Bcl2-L-13 recruits the ULK1 complex to process mitophagy. We published these discoveries in a journal.
We performed a large-scale study to identify the binding proteins which are necessary for Bcl2-L-13 function. We have identified possible candidates to bind Bcl2-L-13. However, the candidate proteins had no effects on mitochondrial fragmentation. We repeated the screening. We are now examining the role of their knockdown in Bcl2-L-13-induced mitochondrial fragmentation.
To know the role of Bcl2-L-13 in hearts, we examined animals which has no Bcl2-L-13. The animals showed normal heart function. When the animals have high blood pressure, their hearts did not work well. Thus, Bcl2-L-13 is very important to protect hearts against high pressure. Under pressure overload, mitochondria become smaller. Our data suggest that Bcl2-L-13 is necessary to generate smaller mitochondria and to produce enough energy for hearts.
We have reported that phosphorylation of Bcl-L-13 is important for mitophagy. We inhibited all enzymes which phosphorylate proteins each by each and found some proteins can phosphorylate Bcl2-L13. We found that one protein can phosphorylates Bcl2-L-13. In order to know the role of the phosphorylation of Bcl2-L-13, we have generated the animals, in which Bcl2-L-13 is not phosphorylated. The mice showed normal heart function and their hearts did not work well under high blood pressure.
To demonstrate that the level of mitochondrial DNA methylation is important for inflammation, we analysed the level of mitochondrial DNA methylation in disease hearts. DNA methylation level was determined using very high-speed machine. We could not detect any changes in DNA methylation levels in hearts. We are examining the methylation in mice which cannot degrade the DNA.
DNase II activity increased when animals show a big heart but not in heart failure. Increases in DNase II inhibited heart failure. By screening whole genes, we identified molecules which inhibited DNase II. The molecule, called microRNA (miRNA), can bind DNase II mRNA. Once the miRNA binds to DNase II mRNA, DNase II mRNA is degraded and cannot be translated to DNase II protein. The miRNA is increased in mouse and human failing hearts.

Final results

We will find how mitochondria are fragmented and degraded in heart failure and how we can regulate the degradation of mitochondria and inflammation. We also find whether mitochondrial DNA is modified or not and we can change the step. In addition, we will know why mitochondrial DNA is accumulated in heart failure. These results tell us how inflammation and heart failure occur and give us hints to develop new and effective drugs to cure patients with heart failure.