Effects of doxorubicin on cardiac muscle subsarcolemmal and intermyofibrillar mitochondria

Abstract

Doxorubicin (DOX) is a highly effective chemotherapeutic used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is highly limited by cumulative and irreversible cardiomyopathy that occurs following DOX treatment. The pathogenesis of DOX-induced cardiac muscle dysfunction is complex. However, it has been proposed that the etiology of this myopathy is related to mitochondrial dysfunction, as a result of the dose-dependent increase in the mitochondrial accumulation of DOX. In this regard, cardiac muscle possesses two morphologically distinct populations of mitochondria. Subsarcolemmal (SS) mitochondria are localized just below the sarcolemma, whereas intermyofibrillar (IMF) mitochondria are found between myofibrils. Mitochondria in both regions exhibit subtle differences in biochemical properties, giving rise to differences in respiration, lipid composition, enzyme activities and protein synthesis rates. Based on the heterogeneity of SS and IMF mitochondria, we hypothesized that acute DOX administration would have distinct effects on each cardiac mitochondrial subfraction. Therefore, we isolated SS and IMF mitochondria from the hearts of female Sprague-Dawley rats 48h after administration of DOX. Our results demonstrate that while SS mitochondria appear to accumulate greater amounts of DOX, IMF mitochondria demonstrate a greater apoptotic and autophagic response to DOX exposure. Thus, the divergent protein composition and function of the SS and IMF cardiac mitochondria result in differential responses to DOX, with IMF mitochondria appearing more susceptible to damage after DOX treatment.

Keywords: Anthracycline; Apoptosis; Heart.

Fig. 1

Mitochondrial DOX accumulation. Cardiac DOX concentration from cardiac cytosolic fraction (non-mitochondrial fraction) (Cytosolic), subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions from animals treated with DOX. Values are mean ± SEM. § Significantly different versus Cytosolic and IMF (p < 0.05). ^ Significantly different versus Cytosolic (p < 0.05).

Fig. 2

Mitochondrial respiration and protein content from isolated SS and IMF mitochondria. A) state 3 respiration; B) state 4 respiration; C) respiratory control ratio; D) citrate synthase protein expression and; E) mitochondrial respiratory chain protein content (complex I (CI), complex II (CII), complex III (CIII), complex IV(CIV) and complex V (CV)) in isolated SS and IMF mitochondria from CON and DOX treated animals. Pyruvate/malate was used as a complex I substrate to measure mitochondrial respiration. Representative western blot images are shown with the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05). # Significantly different versus SS mitochondria (p < 0.05).

Fig. 3

Mitochondrial ROS emission and pore opening. A) mitochondrial hydrogen peroxide emission; B) maximal rate of pore opening (V_max) and; C) time to reach V_max during exposure to both calcium and reactive oxygen species in isolated SS and IMF mitochondria from CON and DOX treated animals. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05). # Significantly different versus SS mitochondria (p < 0.05).

Fig. 4

Mitochondrial pro-apoptotic factors. A) Cytochrome c and B) AIF protein expression in isolated SS and IMF mitochondria from CON and DOX treated animals. Representative western blot images are shown to the right of the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05). # Significantly different versus SS mitochondria (p < 0.05).

Fig. 5

Markers of mitochondrial antioxidant capacity. A) GPX1; B) SOD1; C) SOD2; and D) catalase protein expression in isolated SS and IMF mitochondria from CON and DOX treated animals. Representative western blot images are shown below the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05). # Significantly different versus SS mitochondria (p < 0.05).

Fig. 6

Markers of mitochondrial dynamics. Mitochondrial fusion protein expression: A) OPA1 and B) Mfn2; and mitochondrial fission protein expression: C) Fis1 and D) DRP1 in isolated SS and IMF mitochondria from CON and DOX treated animals. Representative western blot images are shown below the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05). # Significantly different versus SS mitochondria (p < 0.05).

Fig. 7

Markers of mitophagy. A) p62; B) PINK1 and; C) Parkin protein expression in isolated SS and IMF mitochondria from CON and DOX treated animals. Representative western blot images are shown to the right of the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05).

Fig. 8

Markers of mitochondrial biogenesis. A) TFAM and B) Nrf1 protein expression in isolated SS and IMF mitochondria from CON and DOX treated animals. Representative western blot images are shown to the right of the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05).

Fig. 9

Markers of protein quality control. A) pUB; B) HSP70; C) HSP10; and D) Lon protein expression in isolated SS and IMF mitochondria from CON and DOX treated animals. Representative western blot images are shown below the graph. Values are mean ± SEM. * Significantly different versus control (CON) (p < 0.05). # Significantly different versus SS mitochondria (p < 0.05).