Defining Chaperone Mediated Autophagy in Ischemic and Proteotoxic Models of Cardiac Pathology

Background Heart failure (HF) is a leading cause of death worldwide and is estimated to increase 46% more by 2030. Accumulation of aberrant and misfolded proteins contributes to HF pathology including ischemic and hypertrophic cardiomyopathies, and desmin‐related myopathy. Normal physiological conditions require protein degradation pathways for removing damaged and superfluous proteins. Chaperone mediated autophagy (CMA) is a protein degradation pathway unique to mammalian cells and selectively degrades substrate proteins. In CMA, chaperone bound proteins are translocated to a lysosome membrane protein type 2a (LAMP2a) receptor for lysosomal degradation. Several cardiac regulatory proteins like RyR2, RCAN1, and MEF2D are substrates of CMA. Although CMA is active in the heart, the causal role of CMA in cardiac physiology and pathology has not been defined. Objective To show that CMA plays both a necessary and a sufficient role in ischemic and proteotoxic cardiomyocyte stress. Methods CMA activity, measured by LAMP2a protein levels, was determined in two different models of cardiomyocyte pathology: 1) an ischemic stress model where CoCl 2 , a hypoxia mimetic agent, was added to neonatal rat ventricular cardiomyocytes (NRVMs), and 2) a proteotoxic model where the mutant CryAB R120G is expressed in NRVMs. A missense mutation in aB crystallin causes the accumulation of insoluble protein aggregates. To determine CMA activity in vivo , LAMP2a levels were measured in mouse hearts following myocardial infarction (MI) or transverse aortic constriction (TAC) and in transgenic mice expressing the mutant CryAB R120G . Gain and loss of CMA function studies were done in NRVMs using a LAMP2a‐expressing adenovirus and a LAMP2a silencing siRNA. Results LAMP2a levels were significantly increased in the hearts of the mice 4 weeks post‐MI (1.81 fold) and 1 week post‐TAC surgery (3.5 fold). Consistently, hypoxic stress induced by CoCl 2 treatment of NRVMs also increased LAMP2a levels. Loss of CMA function studies indicated that the CoCl 2 ‐induced increase in LAMP2a (26%) was blunted by LAMP2a siRNA. Gain of CMA function studies demonstrated that adenoviral expression of LAMP2a in NRVMs treated with CoCl 2 further augmented LAMP2a levels (55%). These data suggest a crucial role of CMA in cardiac cell pathophysiology subjected to ischemic stress. In hearts from mutant CryAB R120G transgenic mice, LAMP2a levels were increased 3‐fold compared to control hearts. Co‐infection of NRVMs with CryAB R120G and LAMP2a decreased the mutant CryAB R120G protein levels, consistent with CMA degradation. Similarly, LAMP2a overexpression significantly reduced CryAB R120G aggregate pathology and soluble ubiquitinated protein levels. Conversely, LAMP2a knock‐down did not affect CryAB R120G aggregate pathology. While gain of LAMP2a increased cytotoxicity in the hypoxia‐stressed cells, CryAB R120G pathology was significantly attenuated by LAMP2a overexpression. Additional studies are underway to determine the differential role of CMA in cardiac cells under these pathophysiological conditions. Conclusion Targeting the CMA pathway may offer a new avenue for treating and preventing certain forms of heart failure. Support or Funding Information This work was supported by the American Heart Association Post‐Doctoral Fellowship (Grant No. 17POST33670412) and a Protein Quality Control grant through the Sanford School of Medicine. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .