Abstract Introduction Transthyretin amyloidosis (ATTR) is one of the most common types of cardiac amyloidosis (CA), a progressive, 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. ATTR primarily presents with heart involvement, especially in the wild-type form (ATTRwt), while point mutations in the TTR gene are responsible for the manifestation of ATTR with cardiac, neurological and mixed phenotypes (ATTRv). In both ATTR-CA subtypes, cardiac involvement is the key determinant of prognosis, quality of life, and mortality. However, while TTR protein entities or oligomers have been associated with cytotoxicity, their specific cardiotoxic effects remain unclear. Purpose We aimed to develop an in vitro model to investigate the cardiotoxic effects of wtTTR and TTR variants: V122I and L11M, which affect the heart, and V30M, which has cardiac and neurological manifestations. Our study focused on evaluating these effects on distinct cardiac cell populations, particularly primary murine ventricular cardiomyocytes (pAVMCs) and fibroblasts (FBs). Methods Recombinant wtTTR, TTR V122I, TTR L111M, TTR V30M were biotechnologically produced in E.coli BL23(DE3) cells and purified, as confirmed by SDS-PAGE. pAVMCs and FBs were isolated from C57BL6 mice and exposed to various TTR concentrations over short (24 hours, 0-16 μM of TTR tetramer) and prolonged time periods (48 and 72 hours, 0-8 μΜ of TTR tetramer). The selected concentrations were based on plasma TTR tetramer levels of healthy individuals and ATTR patients. Cell viability was assessed by MTT and LDH assays. The polymerization of TTR in cell culture supernatant was examined via native PAGE and Western blot. Results None of the TTR proteins exhibited cytotoxicity in any cardiac cell population after 24 or 48 hours of treatment. However, in pAVMCs, exposure to wtTTR, TTR V122I, and TTR V30M for 72 hours at the highest concentration (8 μΜ) resulted in a statistically significant reduction in MTT absorbance, suggesting a mitochondrial impact of TTR proteins. Notably, only TTR V122I induced a corresponding increase in LDH release in the cell culture supernatant, indicating TTR V122I-induced necrosis. In FBs, treatment with wtTTR and TTR L111M at 8 μM for 72 hours resulted in a statistically significant increase in LDH suggesting that these cardiac related TTRs induce cell necrosis upon extended treatment in FBs. Both pAVMC and FB supernatants contained TTR monomers and oligomers reflecting TTR instability. Conclusions Our findings suggest that TTR proteins act as toxic species at clinically relevant concentrations following prolonged exposure, with notable differences observed among wild-type and variant TTRs. These results provide valuable insight into ATTR-CA pathophysiology, and ongoing research will enhance our understanding of the mechanisms of cardiac toxicity.
Choustoulaki et al. (Sat,) studied this question.