Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy

Abstract

Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart.

Methods: H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction.

Results: LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality.

Conclusions: Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.

Keywords: Drp1; Heart failure; Mitochondrial fission; Sepsis.

Figure 1:

Sepsis-induced mitochondrial fission is associated with mitochondrial dysfunction and increased mitochondrial oxidative stress. (A) Immunofluorescent microscopy co-stained with Mitotracker (purple) and Hoechst (blue) demonstrating mitochondrial fragmentation in H9C2 cardiomyocytes following 12 hrs. of LPS treatment. Mitochondrial fragmentation associated with mitochondrial dysfunction represented by: 1) decreased tetramethylrhodamine methyl ester (TMRM™) fluorescence (n=3 biological replicates). (B) as a surrogate for decreased membrane potential, 2) increased mitochondrial-specific ROS production (MitoSox™) (n=3 biological replicates). (C) and total cell ROS production (CellRos™). (D) (n=3 biological replicates) and 3) decreased cellular respiration, measured by changes in oxygen consumption rate on seahorse XF-24e at different time points following LPS treatment (n=5 biological replicates) (E).

Figure 2:

(A) Western blot showing Drp1 phosphorylation levels in total lysate by immunoblotting using anti-phosporylated-S616-Drp1 antibodies at different time points following LPS treatment (n=3 biological replicates). (B) Western blot demonstrating Drp1 levels in mitochondrial-enriched fractions 4 hrs after LPS treatment as compared to control treatment (n=4 biological replicates). VDAC was used as a loading control and protein levels were quantified and represented as a ratio. (C) Mitochondrial content was evaluated using western blot analyses. VDAC and Tim22 were used as markers of outer and inner mitochondrial membrane and were normalized to tubulin (n=4 biological replicates). (D) Lysosomal accumulation quantified by fluorophore (Lyso-ID™) quantification normalized to Hoechst (n=4 biological replicates). (E) LDH release in the cell supernatant was utilized as a surrogate for cell death and quantified using Cytotoxicity Detection Kit (n=4 biological replicates).

Figure 3:

(A) Western blot analysis demonstrating Drp1 levels in mitochondrial-enriched fractions in 3 conditions (control, LPS treated, LPS + P110 treated) after 24 hrs. (n=3 biological replicates). VDAC was used as a loading control and protein levels were quantified and represented as a ratio. (B) Cellular respiration measured by changes in oxygen consumption rate on seahorse XF-24e in control, LPS treatment and LPS+P110 treatment (n=6 biological replicates). Quantification for basal oxygen consumption, maximum oxygen consumption, spare respiratory capacity, ATP-dependent oxygen consumption presented in pmol/min. (C) Tetramethylrhodamine methyl ester (TMRM™) fluorescence utilized as a surrogate for mitochondrial membrane potential across the three groups. Quantification was normalized to Hoechst (n=3 biological replicates). (D) Increased mitochondrial-specific oxidative stress quantified by fluorescence assay (MitoSox™) and quantification normalized to Hoechst(n=4 biological replicates).

Figure 4:

Evaluation of mitochondrial function, oxidative stress, lysosomal accumulation and cell death in H9C2 cardiomyocytes stratified to 4 different conditions (control, LPS treatment, LPS along with P110 treatment, LPS along with P259 treatment). (A) Mitochondrial-specific ROS (MitoSox™) (n=4 biological replicates). and (B) total cell ROS (CellRos™) (n=4 biological replicates) were quantified and normalized to Hoechst. (C) Tetramethylrhodamine methyl ester (TMRM™) fluorescence utilized as a surrogate for mitochondrial membrane potential across the 4 groups (n=3 biological replicates). (D) ATP production was quantified by ATP colorimetric/fluorometric assay kit (Biovision, Milpitas, CA) (n=3 biological replicates). (E) Lysosomal accumulation was quantified by fluorophore (Lyso-ID™) quantification normalized to Hoechst (n=5 biological replicates). (F) LDH release in the cell supernatant was utilized as a surrogate for cell death and quantified using Cytotoxicity Detection Kit (n=3 biological replicates).

Figure 5:

Balb/c mice were treated with LPS (8mg/kg IP) to induce murine model of sepsis. Peptide P110 (0.5mg/kg IP) was administered in a subset of septic animals 3hrs following LPS treatment. (A) Illness severity and outcome quantified by change in temperature, weight, mouse sepsis score (previously published illness severity score for murine sepsis) as well as mortality. Variables evaluated every 3 hrs following LPS treatment. Temperature and weight displayed as a change from baseline (n=6 mice for control, 12 for LPS-treated mice, and 12 for LPS+P110-treated mice). (B) Change in cardiac function was measured by change in ejection fraction (EF), fractional shortening (FS) and change in velocity time integral (VTI) across the Left ventricular outflow tract. (C) Mouse sepsis score is displayed as a cumulative score per animal (n=6 control, 12=LPS, 12=LPS+P110).

Figure 6:

(A) Electron microscopy of cardiac tissue from mice treated with control, LPS or LPS followed by P110 (n=4 per condition). Images quantified for changes in: (B) Percent altered mitochondria, (C) form factor (FF) [(perimeter²)/(4π·surface area)], which reflects the complexity and branching aspect of mitochondria, (D) aspect ratio (AR) [(major axis)/(minor axis)], which reflects mitochondrial “length-to-width ratio”, and (E) mitochondrial cross-sectional area. (F) Cardiac tissue oxidative stress was quantified by the level of oxidative post-translational modification (s-nytrosylation) proteins in the total cardiac lysates. (G) Mitochondrial content was evaluated by quantifying mitochondrial DNA as a ratio to total DNA across the three groups. Quantification and scoring done by scientist who was blinded to conditions (n=3 control mice, n=5 of LPS-treated mice and n= 5 for LPS+P110-treated mice).