Uncompensated upregulation of ryanodine receptor 2 with unchanged other calcium regulating proteins expression plays a significant role in early development of anthracycline cardiomyopathy

J Klimas1, D Kucerova1,2, G Doka1, J Kmecova1, P Musil3, K Turcekova1, P Ochodnicky4, P Krenek1, P Babal5, P Boknik2, J Kyselovic1. 1Faculty of Pharmacy, Comenius University in Bratislava, Dept. of Pharmacology and Toxicology 832 32, Slovakia, 2Universitätsklinikum Münster, Institut für Pharmakologie und Toxikologie D-48149, Germany, 3International Laser Centre, 841 04, Slovakia, 4Academic Medical Center, University of Amsterdam, Dpt. of Pharmacology and Pharmacotherapy 1105 AZ, The Netherlands, 5Faculty of Medicine, Comenius University in Bratislava, Dpt. of Pathology 813 72, Slovakia

Calcium release channel on the sarcoplasmic reticulum of cardiomyocytes (also known as ryanodine receptor type 2, RyR2), and its regulatory protein complex consisting of junctin, triadin, calsequestrin (JCN, TRD, CSQ) and FKBP12.6, plays a critical role in the regulation of calcium. Consequently, dysregulation inside this complex was identified as a crucial factor for development of cardiac failure. Recently, importance of RyR2 has been suggested in the pathomechanism of anthracycline cardiotoxicity in vitro. However, little is known about its influence in the early stages of such cardiac injury in vivo.

To study this, 10-12 week old male Wistar rats were treated for two weeks with daunorubicin (DAU, 3 mg/kg, i.p., in 48 h intervals), controls (CON) received vehicle. Left ventricular function (left ventricular pressure – LVP, rate of pressure development – dP/dtmax and decline – dP/dtmin) were measured using left ventricular catheterization and twelve lead electrocardiograms were recorded under tribromethanol anaesthesia (15 ml/kg b.w.; n=8-10 per group). Cell shortening and calcium were measured in enzymatically isolated cardiomyocytes. The expressions of RyR2 and associated intracellular calcium regulating proteins (including SERCA2a, phospholamban, and L-type calcium channel) and cardiac failure defining markers (atrial natriuretic peptide, ANP; muscle myosin regulating genes, MYH6 and MYH7) were detected in ventricular tissue samples using SDS PAGE and immunoblotting and/or by RT-PCR. Histopathological evaluation was used to characterize cardiomyopathy.

Application of daunorubicin led to a decreased function of the left ventricle (LVP by 14%, dP/dtmax by 36% and dP/dtmin by 30%; all p<0.05). On the other side, we observed increased cell shortening of isolated cardiac cells by 31% (p<0.05). Moreover, DAU cells showed two-fold increase in the incidence of resting spontaneous calcium releases and increase of the magnitude of calcium release (by 39%, p<0.05). Following DAU administration, ANP expression was increased 5-fold (p<0.05), MYH6 was decreased (by 15%), and MYH7 was 5-fold increased (p<0.05). Interestingly, RyR2 was significantly upregulated by 94% (P<0.05) but all other Ca2+ regulating proteins remained stable. Histopathological evaluation revealed cardiomyocytes hypotrophy (reduction of cell width by 30%, p<0.05) with presence of edematous swelling.

In conclusion, overexpression of RyR overexpression of RyR may be responsible for modulated calcium handling, which contributes, at least in part, to the development of anthracycline induced cardiomyopathy. Moreover, the missing conterregulatory changes in expression of other Ca2+ regulatory proteins might be co-responsible for the cardiac damage.

This project was supported by the grants VEGA 1/0981/12 and 1/1253/12.