Autophagy, an evolutionarily conserved intracellular lysosomal degradative pathway, is constitutively active in the myocardium, and removes damaged organelles and proteins to ensure cardiomyocyte survival. Rapid upregulation of autophagy is essential for maintaining cardiomyocyte viability under stress, such as during starvation and ischemia, by generating nutrients as a source of energy and removing potentially deleterious proteins and organelles. Autophagy is also critical for removal of abnormal desmin aggregates that cause cardiomyopathy with the R120G mutation in chaperone protein, ?-B crystallin. It is therefore paradoxical that an upregulation of autophagy has been implicated in causing cardiomyocyte death in reperfusion injury, following an ischemic insult. With reperfusion, we have observed an impairment in autophagosome processing, triggered by a burst in reactive oxygen species generation, as a cause of autophagosome accumulation;which implies that flux through autophagy is impaired with ischemia-reperfusion injury. Indeed, we have demonstrated that autophagosome formation is induced in cardiomyocytes by expression of BNIP3, a pro-death protein that is transcriptionally induced by hypoxia and mediates cardiomyocyte death in ischemia- reperfusion injury;but autophagosome processing is impaired as the autophagy-lysosome machinery gets overwhelmed and autophagosomes enclosing damaged mitochondria accumulate. Exogenous expression of transcription factor EB (TFEB), a master regulator of autophagy-lysosome pathway biogenesis, re-established autophagosome processing, with removal of BNIP3-damaged mitochondria and attenuated cardiomyocyte death. We have also observed that starvation, a potent inducer of cardiomyocyte autophagy, provokes transcriptional upregulation of multiple components of the autophagy-lysosome machinery in the mouse heart; and repetitive starvation by intermittently fasting (depriving mice of food for 24 hours every other day for 6 weeks) results in protection against ischemia-reperfusion (IR) injury, with >50% reduction in infarct size as compared with ad-lib fed mice. This is associated with a rapid fasting-induced nuclear translocation of TFEB from the cytoplasm, suggesting that adequate priming of the autophagy-lysosome machinery by TFEB promotes sufficient autophagy, which is then beneficial in the setting of ischemia-reperfusion injury. In this proposal, we will test the hypothesis that intermittent fasting and exogenous expression of TFEB protect against ischemia-reperfusion induced cardiomyocyte death and protein-aggregate-induced cardiomyopathy and heart failure, by enhancing beneficial autophagy.
In specific aim 1, studies will be performed to determine the role of autophagy in intermittent fasting-induced cardioprotection against post-infarction remodeling, by subjecting mice deficient for LAMP2, (which are also autophagy-deficient as LAMP2 is critical for autophagosome-lysosome fusion) to IR injury followed by serial echocardiography and terminal invasive pressure-volume loop studies, to assess changes in left ventricular size and function and development of heart failure, as a function of LAMP2 expression.
In specific aim 2, mice with conditional adult onset cardiomyocyte specific overexpression of TFEB will be subjected to IR injury to determine whether exogenous TFEB confers protection against cell death, post-infarction remodeling and heart failure, by enhancing autophagy.
In specific aim 3, mice with cardiomyocyte-specific expression of mutant crystallin will be subjected to intermittent fasting or crossed with TFEB overexpressors, to determine whether induction of autophagy prevents protein- aggregate induced heart failure. These studies will evaluate the paradigm that transcriptional induction of autophagy-lysosome machinery enhances stress-induced autophagy, and protects against cardiomyocyte death and heart failure in ischemia-reperfusion injury and desmin-induced cardiomyopathy.

Public Health Relevance

Heart failure is a very common disease of the aging population, with the highest incidence and prevalence in the age group above 65, which is also the largest Veteran population group (more than 40%). Ischemic heart disease is the primary cause or a major contributor in 2/3rds of all cases of heart failure in Veterans. Cardiac muscle cell death plays a central role in causing cardiomyopathy, or impaired contractile function of the heart muscle;which is observed in more than half of all patients with heart failure. Autophagy is an intracellular process that breaks dow damaged cellular constituents, and is critical for cell survival. In this proposal, we will evaluat whether activation of autophagy can prevent cardiac muscle cell death in ischemia-reperfusion injury which causes ischemic heart disease;and prevent cardiomyopathy caused by aggregates of a mutant protein, B-crystallin;to develop new strategies to prevent and treat heart failure.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
1I01BX001969-01A2
Application #
8633520
Study Section
Cardiovascular Studies A (CARA)
Project Start
2013-10-01
Project End
2017-09-30
Budget Start
2013-10-01
Budget End
2014-09-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
St. Louis VA Medical Center
Department
Type
DUNS #
033986766
City
St. Louis
State
MO
Country
United States
Zip Code
63106