CaMKII-dependent SR Ca leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac myocytes

Abstract

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca](i)) handling. Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ(-/-), KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 μmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS](i)) and [Ca](i) handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 μmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 μmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. DOX increased [ROS](i) and led to significant diastolic [Ca](i) overload in rat myocytes. This was associated with reduced [Ca](i) transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca](i) overload after DOX superfusion and led to partially restored [Ca](i) transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca](i) overload and Ca spark frequency. DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca](i) homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca](i). In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.

Fig. 1. ROS concentration is elevated upon DOX treatment

Using the fluorescent dye CM-H₂DCFDA, oxidation-sensitive fluorescence was significantly elevated upon DOX exposition at a concentration of 10 µmol/L (n=6, black squares) compared to Ctrl group (n=5, white squares). ^#indicates significance vs. Ctrl. using Student-Newman-Keuls post hoc test.

Fig. 2. DOX exposure decreases Ca transient amplitudes and induces Ca overload

Fig. 2A shows decreased Ca transient amplitudes upon DOX exposition (10 µmol/L) in line with elevated diastolic Ca over a time period of 15 min. Average data for B: Ca transient amplitudes and C: diastolic Ca. * indicates significance using paired t-test, ^#indicates significance vs. Ctrl. using Student-Newman-Keuls post hoc test.

Fig. 2. DOX exposure decreases Ca transient amplitudes and induces Ca overload

Fig. 2A shows decreased Ca transient amplitudes upon DOX exposition (10 µmol/L) in line with elevated diastolic Ca over a time period of 15 min. Average data for B: Ca transient amplitudes and C: diastolic Ca. * indicates significance using paired t-test, ^#indicates significance vs. Ctrl. using Student-Newman-Keuls post hoc test.

Fig. 3. DOX exposure induces diastolic SR Ca leakage

A: Diastolic spark frequency is elevated by ~5.8 fold as compared to Ctrl during DOX treatment (10 µmol/L). B: Average data for A. *indicates significance using student‘s t-test.

Fig. 4. DOX treatment decreases SR Ca content

Fig. 4A demonstrates reduced SR Ca content after 30 min of DOX exposure. Average data for B: fractional release and C: SR Ca content. *indicates significance using student‘s t-test.

Fig. 5. ROS scavenging attenuates SR Ca loss upon DOX

Fig. 5A shows original line scans demonstrating decreased SR Ca loss in case of ROS scavenging using 100µmol/L Melatonin (right panel). Average data for B: Ca spark frequency, C: SR Ca content and D: diastolic Ca overloading after 30 min of DOX exposure. * indicates significance using student‘s t-test.

Fig. 6. DOX activates CaMKII

Fig. 6A shows original Western Blots demonstrating increased CaMKII phosphorylation status after 30 min DOX exposure (10 µmol/L). B: Average data for A. C depicts slightly increased ox-CaMKII upon DOX exposition. D: Average data for C. * indicates significance using student‘s t-test.

Fig. 7. CaMKII inhibition prevents diastolic Ca overload and maintains Ca transient amplitudes upon DOX treatment

Fig. 7A depicts original Ca recordings showing decreased Ca transient amplitudes and diastolic Ca overload upon DOX exposure (KN-92, upper panel), but not in case of CaMKII-inhibition (KN-93, lower panel). Fig. 7B shows expanded original Ca transients upon DOX exposure (upper panel) and upon simultaneous CaMKII inhibition (KN-93, lower panel). Average data for C: Ca transient amplitudes and D: Diastolic Ca levels. * indicates significance using paired t-test. ^#indicates significance vs. Ctrl. using Student-Newman-Keuls post hoc test.

Fig. 7. CaMKII inhibition prevents diastolic Ca overload and maintains Ca transient amplitudes upon DOX treatment

Fig. 7A depicts original Ca recordings showing decreased Ca transient amplitudes and diastolic Ca overload upon DOX exposure (KN-92, upper panel), but not in case of CaMKII-inhibition (KN-93, lower panel). Fig. 7B shows expanded original Ca transients upon DOX exposure (upper panel) and upon simultaneous CaMKII inhibition (KN-93, lower panel). Average data for C: Ca transient amplitudes and D: Diastolic Ca levels. * indicates significance using paired t-test. ^#indicates significance vs. Ctrl. using Student-Newman-Keuls post hoc test.

Fig. 7. CaMKII inhibition prevents diastolic Ca overload and maintains Ca transient amplitudes upon DOX treatment

Fig. 7A depicts original Ca recordings showing decreased Ca transient amplitudes and diastolic Ca overload upon DOX exposure (KN-92, upper panel), but not in case of CaMKII-inhibition (KN-93, lower panel). Fig. 7B shows expanded original Ca transients upon DOX exposure (upper panel) and upon simultaneous CaMKII inhibition (KN-93, lower panel). Average data for C: Ca transient amplitudes and D: Diastolic Ca levels. * indicates significance using paired t-test. ^#indicates significance vs. Ctrl. using Student-Newman-Keuls post hoc test.

Fig. 8. CaMKII inhibition reduces diastolic SR Ca loss upon DOX exposure

Fig. 8A shows reduced Ca spark frequency in case of CaMKII inhibition (AIP, lower panel) despite DOX exposure. Fig. 8B: Average data for A. *indicates significance using student‘s t-test.

Fig. 9. CaMKII inhibition restores SR Ca content despite DOX exposure

Fig. 9A shows increased SR Ca content in case of CaMKII inhibition (KN-93, lower panel) despite DOX exposure. Fig. 9B: Average data for A. *indicates significance using student‘s t-test.

Fig. 10. Genetic CaMKIIδ-KO decreases SR Ca loss

Fig. 10 A shows decreased SR Ca spark frequency in cardiac myocytes from CaMKIIδ-KO mouse. Average data for B: Ca spark frequency and C: diastolic Ca upon 12 min DOX exposure in KO myocytes (light grey) vs WT control cells. *indicates significance using student‘s t-test.