Cardioprotective Roles of β-Hydroxybutyrate Against Doxorubicin Induced Cardiotoxicity

Abstract

Background: β-Hydroxybutyrate (BHB) is produced by fatty acid oxidation in the liver under the fasting state and confirmed to play a cardioprotective role in ischemia and hypertensive settings. Doxorubicin (DOX) is an effective chemotherapeutic drug, but limited by serious irreversible cardiotoxicity. However, whether BHB can protect from DOX-induced cardiotoxicity remains unknown. Methods and Results: C57BL/6 mice were intraperitoneally injected with DOX to induce cardiac toxicity and intragastrically administered into BHB for treatment. They were randomly divided into three groups, namely a sham group (Sham), a doxorubicin group (DOX), and a doxorubicin+β-Hydroxybutyrate group (DOX + BHB). Echocardiography and pathological staining were performed to evaluate cardiac function and fibrosis. H9c2 cardiomyocyte was treated with DOX or BHB for in vitro experiments. Cell apoptosis and ROS were determined by flow cytometry. BHB significantly restored DOX-induced cardiac function decline and partially prevented cardiac reverse remodeling, characterized by increased cell size and decreased fibrosis. In vitro, BHB treatment decreased cellular injury and apoptosis. Also, BHB alleviated oxidative stress level and increased mitochondrial membrane potential. Conclusion: Our results suggested that BHB could protected from DOX-induced cardiotoxicity by inhibiting cell apoptosis and oxidative stress and maintaining mitochondrial membrane integrity.

Keywords: cardiotoxicity; doxorubicin; heart failure; metobolites; β-hydroxybutyrate.

FIGURE 1

BHB prevents doxorubicin-induced left ventricular dysfunction. (A) Ejection fraction (EF); (B) fractional shortening (FS); (C) left ventricular diastolic internal dimension (LVIDd); (D) and left ventricular systolic internal dimension (LVIDs) among sham, DOX and DOX + BHB groups; (F) the presentative echocardiography images among groups. Each group had 5 mice. ##p < 0.01, ###p < 0.001 vs. Sham. **p < 0.01 vs. DOX group. n. s, not significant.

FIGURE 2

BHB protects from DOX-induced cardiac remodeling. (A) HE staining; (B) Sirius red of tissues from sham, DOX and DOX + BHB group. Each group had 5 mice. ###p < 0.001 vs. Sham; *p < 0.05, **p < 0.01 vs. DOX.

FIGURE 3

BHB prevents DOX-induced cardiac cell apoptosis. Blue indicates nucleus, green indicates TUNEL positive cells, and Merge for apoptotic cells. Each group had 3 samples. ###p < 0.001 vs. Sham; ***p < 0.001 vs. DOX.

FIGURE 4

BHB alleviates oxidative stress induced by doxorubicin. (A) The MDA level among groups. (B) The ROS detection of cardiomyocytes among groups. Each group had 3 samples. ##p < 0.01 vs. Sham; *p < 0.05 vs. DOX.

FIGURE 5

BHB counteracts mitochondrial membrane potential decrease induced by doxorubicin (A) The LDH release level among groups. (B) The JC-1 staining of different groups. Red fluorescent signal (aggregated dye) indicated a normal mitochondrial membrane potential while green fluorescent signal (monomeric dye) suggested a decreased mitochondrial membrane potential. Each group had 3 samples. ##p < 0.01 vs. Sham; *p < 0.05 vs. DOX.

FIGURE 6

BHB alleviated cardiac injury via modulating ERK1/2 phosphorylation and MMP9 expression. The up-left is immunoblotting bands while two else are quantitative results. Each group had 2 samples. #p < 0.05 vs. Sham; *p < 0.05 vs. DOX.