Abstract P3033: The Pathogenic R120G Mutant Of Alpha-B-crystallin Accumulates Within Cardiac Mitochondria

Alpha-B-crystallin (CryAB) is an abundant molecular chaperone comprising nearly 5% of total cardiac mass. While predominately cytosolic, some studies note mitochondrial translocation of CryAB during stress. The autosomal dominant R120G mutation leads to CryAB misfolding and protein aggregation resulting in cardiomyopathy. Although mitochondrial dysfunction has been reported in cardiac-specific CryAB R120G transgenic mice, it is unclear whether these effects are secondary to the formation of cytotoxic aggregates or directly relate to mitochondrial damage. Using proteinase K protection assays in isolated mitochondria from mouse hearts and embryonic fibroblasts (MEFs), we have discovered that wild-type (WT) CryAB is present in the mitochondrial matrix indicating that this protein likely functions as a chaperone within the organelle. Interestingly, however, compared to the WT protein, CryAB R120G was significantly more enriched in MEF mitochondria. Furthermore, treatment of mice with adeno-associated virus serotype 9 (AAV9) encoding CryAB R120G under the control of a cardiac-specific promoter resulted in time-dependent intramitochondrial CryAB R120G accumulation across 2 and 4-weeks post injection. These data are the first to indicate that misfolded CryAB R120G resides specifically within the mitochondrial matrix. Knockdown of the mitochondrial matrix protease LonP1 significantly increased intramitochondrial as well as total CryAB R120G protein levels, and sensitized MEFs to cell death indicating that Lonp1 functions to proteolytically process imported CryAB R120G . While this response may acutely preserve mitochondrial function, sustained accumulation of intramitochondrial CryAB R120G likely exhausts proteolytic machinery within the organelle leading to induction of mitochondrial autophagy (mitophagy). Indeed, mitochondrial respiration was unchanged in both AAV9-treated and homozygous CryAB R120G knock-in mice at early time-points, whereas mitophagy-deficient Parkin -/- mice displayed significantly exacerbated mitochondrial CryAB R120G protein accumulation. Altogether, these data indicate aberrant accumulation of misfolded proteins within cardiac mitochondria underlies the pathogenicity of CryAB R120G .