Desmin Amyloid-like Oligomers Generated by Mono-Phospho Mimetic Mutants Decrease Respiration in Isolated Cardiac Sub-Mitochondrial Particles

BACKGROUND: Despite its prevalence the molecular bases of heart failure (HF) remain obscure. Our group recently reported a role for amyloid-like oligomers (ALOs) generated by mono-phosphorylated desmin as a potential mechanism underlying HF development and progression. We reported that differential desmin phosphorylation at Ser 27 and 31 may drive the formation of 600 kDa desmin ALOs. We first optimized a way to isolate these ALOs and tested their potential toxicity on cardiac sub-mitochondrial particles. METHODS: We implemented an electrophoretic method to separate and identify the ALOs in-gel. The procedure was optimized using a well-established murine model of cardiac proteotoxicity (R120G CryAB mice). We alternatively generated desmin ALOs by transducing cultured cardiac myocytes with a lentivirus carrying desmin double mutants that mimic or prevent desmin phosphorylation at Ser27/31, by substitution to Asp or Ala, respectively. ALOs at both 600 kDa and 1200 kDa, serving as a negative control, were purified using gel electrophoresis, followed by electroelution of the ALOs. We incubated cardiac sub-mitochondrial particles (SMPs) with 600 and 1200 kDa ALOs from double mutants and measured O2 consumption by polarographic analysis. RESULTS & CONCLUSIONS: Only the 600kDa oligomers showed amyloid-like properties as specified by Thioflavin T binding using our new optimized separation. Furthermore, only the mono-phosphomimetic desmin ALOs decreased O2 consumption in SMPs in a dose- (and sequence-) dependent fashion when SMPs were energized by NADH. These data point to a novel mechanism of mitochondrial toxicity for ALOs generated by mono- hosphorylated desmin, which is increased in both human and experimental HF. This work is supported by the American Heart Association (12SDG9210000), the NIH Proteomic innovation contract P01HL081427 and PPG on cardiac resynchronization therapy PO1-HL077180, Fondazione del Monte, and RFO University of Bologna (Bologna, Italy) to GA