CaMKIIdelta-mediated SR Ca leak contributes to long-term Doxorubicin-induced cardiotoxicity

Abstract

Abstract Background Cardiotoxicity represents a major complication of cancer therapy with anthracyclines, whose main known compound is doxorubicine (DOX). Although mechanisms of DOX-induced toxic cardiomyopathy (DICM) are not entirely understood, CaMKIIδ-activity is known to be increased upon acute DOX exposure, contributing to impaired Calcium (Ca) handling. However, currently no studies have addressed the question, whether CaMKIIδ is involved in DOX-induced long-term cardiotoxicity. Purpose We aimed to investigate cardiac function and intracellular Ca handling using an in-vivo DOX treated mouse model, examining whether CaMKIIδ is involved in DOX-induced long-term cardiotoxicity. Methods Cardiac function and excitation-contraction coupling of CaMKIIdWT and CaMKIId-/- (global CaMKIId knockout) mice were tested in an in-vivo DOX treated mouse model (cumulative dose 12 mg/kg, intraperitoneal injection). Cardiac function was determined by echocardiography after 12 weeks. Ca handling of freshly isolated cardiomyocytes was assessed by epifluorescence microscopy (Fura2-AM) and confocal microscopy (Fluo4-AM). Histological analyses regarding cardiac fibrosis and cell dimensions were examined. Results Long-term DOX exposure reduced left ventricular ejection fraction (EF) by ∼13% and markedly increased cardiac fibrosis in CaMKIIδWT mice. Moreover, CaMKIIdWT hearts displayed decreased cell volume indicating atrophy following DOX treatment. Coinciding with the reduction in EF, CaMKIIdWT mice showed depressed Ca transient amplitudes as well as slowed Ca transient decay indicating depressed Ca uptake into the sarcoplasmic reticulum (SR). In addition, spontaneous diastolic Ca leakage from the SR was dramatically increased. In contrast, CaMKIId-/- mice showed preserved EF following 12 weeks of DOX treatment as compared to CaMKIIdWT in line with maintained Ca transient amplitudes. As an underlying cause, SR Ca leakage was not increased in CaMKIId-/- myocytes despite DOX-treatment. Pharmacological CaMKII inhibition using AIP (1µM) prevented SR Ca leak and restored Ca transient amplitudes in DOX-treated CaMKIIdWT myocytes. Conclusion Our study shows that CaMKIId contributes to long-term DOX-induced cardiotoxicity. We found that chronic DOX treatment induced depressed cardiac function and myocardial fibrosis, associated with impaired calcium handling as a consequence of CaMKIId-mediated SR Ca leak, which was absent in our CaMKIId-/- mouse model. In line, pharmacological inhibition of CaMKII rescued impaired Ca handling in DOX-treated CaMKIIdWT myocytes.