Abstract Body: Background: Hexokinase II (HKII) binds to the mitochondrial (MITO) outer membrane, directing glucose for energy production. Cyclophilin D (CypD) regulates the MITO permeability transition pore (MPTP) and interacts with HKII. Increased CypD in failing hearts of obese patients exacerbates MITO dysfunction, contributing to heart failure. CypD regulation involves post-translational modifications. We previously showed that failing hearts show reduced MITO function and increased protein acetylation. Purpose: This study investigates the differential expression of HKII and CypD in the mitochondria of left ventricular (LV) myocardium in dogs with chronic heart failure (CHF). Methods: Frozen LV tissues from 7 normal (NL) dogs and 7 dogs with CHF (LV ejection fraction≤35%) induced by intracoronary microembolization were used. Protein levels of HXII (102 kDa), CypD (37 kDa), and porin (33 kDa, unchanged in failing hearts) were determined by Western blotting with chemiluminescence, using commercially available antibodies. The MITO fraction lysate was analyzed, and band intensities were quantified in densitometric units (du) and normalized to porin. Results: Porin protein levels were similar in the MITO fractions of NL and HF dogs (2.12 ± 0.15 vs. 2.16 ± 0.12 du). HXII levels remained unchanged (0.47 ± 0.07 vs. 0.48 ± 0.08 du), while CypD levels were upregulated (2.95 ± 0.49 vs. 0.47 ± 0.05 du) in HF dogs compared to NL dogs. In HF dogs, HXII levels normalized to porin remained unchanged (0.22 ± 0.03 vs. 0.24 ± 0.05), but CypD levels were significantly upregulated (1.40 ± 0.23 vs. 0.23 ± 0.04, p<0.05) compared to NL dogs. Conclusions: Unchanged HXII but upregulated CypD protein levels in LV mitochondria from HF dogs may contribute to MITO dysfunction via enhanced MPTP opening, triggering cardiomyocyte apoptosis. Pharmacologic interventions modulating CypD levels could help prevent or slow MITO dysfunction and cardiomyocyte loss in failing hearts.