Effects of iron overload on apoptosis and titin proteolysis in cardiomyocytes

Abstract

Iron is one of the essential elements involved in various fundamental biological activities. However, excess iron may bypass the negative feedback regulatory systems, leading to the formation of iron overload. The increase of iron deposition generates cellular toxicity and subsequently damages vital organs. Primary and secondary iron overload are affecting patients worldwide. Iron overload cardiomyopathy is the primary cause of cardiac dysfunction and cardiovascular mortality in β-thalassaemia major patients. Current effective therapy includes chelation treatment with conventional and new iron chelators, while potential new therapies are currently under development.

The pathophysiology of iron overload cardiomyopathy remains unclear. Controversial findings on the mechanism of excessive iron entry into cardiomyocytes exist. Using novel real-time approach to trace iron entry into HL-1 cardiomyocytes, the only beating cardiac cell line with mature cardiac phenotype available currently, we visualized the patterns of iron entry following ferric iron incubation with and without ascorbate. Iron entry could be partly blocked by pretreatment with L-type calcium channel blockers but not T-type calcium channel blocker. Such blockage effect by L-type calcium channel blockers occurred in ferric iron overload. This finding suggested a role of L-type calcium channels for ferric iron uptake into cardiomyocytes under iron overload condition.

For the pathophysiology of iron cardiac toxicity, we assessed the iron overload induced apoptosis using both in vitro and in vivo approaches. The results demonstrated that iron-overloaded mouse HL-1 atrial cardiomyocytes and human embryonic stem cell derived ventricular cardiomyocytes underwent apoptosis via the mitochondria-mediated caspase-3 dependent pathway. Supportive data was found in iron-overloaded mouse myocardium by an increase in DNA fragmentation. However, despite the blockage of iron entry, L-type calcium channel blockers did not significantly prevent iron induced apoptosis in vitro.

The mechanism of cardiac contractile dysfunction caused by iron overload on cardiomyopathy has not yet been fully characterized. Given the central role of titin, the giant myofilament protein, as the main determinant in myocardial passive tension, stiffness, diastolic and systolic cardiac function, as well as myocardial twisting and untwisting motion, we investigated its expression in iron-overloaded cardiomyocytes in vitro and in vivo. Our results indicated that significant degradation of cardiomyocytes titin was induced by iron overload. This was associated with the cleavage at the elastic domain. Its potential upstream protease, calpain, was further identified to be activated under iron overload. The specific role of titin proteolysis in iron-overloaded cardiomyocytes merited further investigation.

The findings in this project provided new insights to the pathophysiology of iron overload cardiomyopathy, in terms of the route for iron entry, iron induced cardiac apoptosis, and titin proteolysis. Novel therapeutic approaches for prevention and treatment of iron overload cardiomyopathy can focus on inhibiting excessive iron uptake, as well as by targeting pathways involved in cardiac apoptosis and titin proteolysis.