Abstract Introduction Transthyretin amyloidosis (ATTR) is a prevalent form of cardiac amyloidosis (CA), a progressive and life-threatening disease. In ATTR, the precursor protein is transthyretin (TTR), a homotetrameric transporter, which forms amyloid due to its dissociation into monomers and/or proteolysis. TTR protein entities or oligomers have been associated with cytotoxicity, but their specific cardiotoxic effects remain elusive. Purpose We aimed to develop an in vitro model to 1) identify the cardiotoxic effects of wtTTR and cardiac-related TTR variants: V122I, L11M, and V30M, on primary murine ventricular cardiomyocytes (pAVMCs) and 2) investigate the underlying mechanisms of cardiotoxicity in vitro. Methods Recombinant wtTTR, TTRV122I, TTRL111M and TTRV30M were biotechnologically produced in E. coli BL23 (DE3) cells and purified, as confirmed by SDS-PAGE. PAVMCs were isolated from C57BL6/J mice and exposed to clinically relevant TTR concentrations over short (24 hours, 0-16 μM of TTR tetramer) and extended (48 and 72 hours, 0-8 μΜ of TTR tetramer) time periods for the evaluation of cell death and investigation of the cardiotoxicity mechanisms via protein expression. After identifying the toxic proteins and concentrations, co-treatment with TTR stabilizers, tafamidis and acoramidis, was performed in therapeutic concentrations (8-32 μΜ). Cell viability was assessed via MTT and LDH assays, while TTR polymerization and protein expression were examined with Western blot. Results None of the TTR proteins exhibited cytotoxicity at 24 or 48 hours. In pAVMCs, 72-hour exposure to 8 μΜ of wtTTR, TTRV122I and TTRV30M resulted in a statistically significant reduction in mitochondrial MTT reaction. Notably, only wtTTR and TTRV122I increased LDH release, suggesting TTR-induced cardiomyocyte damage. PAVMC supernatants contained TTR monomers and oligomers, reflecting TTR instability. TTR cardiotoxicity corroborated with decreased protein levels of OXPHOS complexes II–V and activation of apoptosis via increase of BAX and cleaved caspase-3. Tafamidis (≥16 μΜ) and acoramidis (≥8 μΜ) restored pAVMC damage, providing proof-of-concept that TTR instability or misfolding is causative for cytotoxicity. Conclusion Prolonged exposure to TTR proteins at clinically relevant concentrations induces cardiotoxicity, an effect mitigated by TTR stabilizers. The underlying mechanism involves the induction of apoptosis and the impairment of mitochondrial function, enhancing understanding of ATTR-CA pathophysiology.
Choustoulaki et al. (Fri,) studied this question.