Mitophagy coordinates the mitochondrial unfolded protein response to attenuate inflammation-mediated myocardial injury

Abstract

Mitochondrial dysfunction is a fundamental challenge in septic cardiomyopathy. Mitophagy and the mitochondrial unfolded protein response (UPR^mt) are the predominant stress-responsive and protective mechanisms involved in repairing damaged mitochondria. Although mitochondrial homeostasis requires the coordinated actions of mitophagy and UPR^mt, their molecular basis and interactive actions are poorly understood in sepsis-induced myocardial injury. Our investigations showed that lipopolysaccharide (LPS)-induced sepsis contributed to cardiac dysfunction and mitochondrial damage. Although both mitophagy and UPR^mt were slightly activated by LPS in cardiomyocytes, their endogenous activation failed to prevent sepsis-mediated myocardial injury. However, administration of urolithin A, an inducer of mitophagy, obviously reduced sepsis-mediated cardiac depression by normalizing mitochondrial function. Interestingly, this beneficial action was undetectable in cardiomyocyte-specific FUNDC1 knockout (FUNDC1^CKO) mice. Notably, supplementation with a mitophagy inducer had no impact on UPR^mt, whereas genetic ablation of FUNDC1 significantly upregulated the expression of genes related to UPR^mt in LPS-treated hearts. In contrast, enhancement of endogenous UPR^mt through oligomycin administration reduced sepsis-mediated mitochondrial injury and myocardial dysfunction; this cardioprotective effect was imperceptible in FUNDC1^CKO mice. Lastly, once UPR^mt was inhibited, mitophagy-mediated protection of mitochondria and cardiomyocytes was partly blunted. Taken together, it is plausible that endogenous UPR^mt and mitophagy are slightly activated by myocardial stress and they work together to sustain mitochondrial performance and cardiac function. Endogenous UPR^mt, a downstream signal of mitophagy, played a compensatory role in maintaining mitochondrial homeostasis in the case of mitophagy inhibition. Although UPR^mt activation had no negative impact on mitophagy, UPR^mt inhibition compromised the partial cardioprotective actions of mitophagy. This study shows how mitophagy modulates UPR^mt to attenuate inflammation-related myocardial injury and suggests the potential application of mitophagy and UPR^mt targeting in the treatment of myocardial stress.

Keywords: FUN14 domain-containing 1; Inflammation; Mitochondrial unfolded protein response; Mitophagy; Septic cardiomyopathy.

Fig. 1

Activation of FUNDC1-associated mitophagy attenuates sepsis-induced myocardial injury. FUNDC1^f/f mice and cardiomyocyte-specific FUNDC1 knockout (FUNDC1^CKO) mice were injected intraperitoneally with a single dose of PBS or LPS (5 mg/kg) to induce septic cardiomyopathy. Blood and heart samples were isolated 48 h after LPS injection. To activate mitophagy, urolithin A (UA, 30 mg/kg) was injected intraperitoneally in sterile saline with 0.1% DMSO. A-C. ELISA analysis of the concentration of cardiac injury markers, including LDH, troponin T, and CK-MB. D-F. Echocardiographic data showing left ventricular ejection fraction (LVEF), left ventricular diastolic dimension (LVDd), and fractional shortening (FS) in FUNDC1^CKO and FUNDC1^f/f mice in the presence of LPS. G-H. The AC16 human ventricular cardiomyocyte cell line was treated with 10 μg/mL of LPS to induce sepsis-related cardiomyocyte damage. siRNA against FUNDC1 (si-FUNDC1) was transfected into AC15 to inhibit mitophagy activation. UA, an inducer of mitophagy, was used to culture AC16 cells. Next, mitophagy activity was observed using the mt-Keima assay. A yellow signal highlights increased mitophagic flux within cardiomyocytes. Data are presented as mean ± SEM, normalized per 1000 cardiomyocytes. n = 6 per group. *P<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Loss of FUNDC1-associated mitophagy promotes cardiomyocyte death and mitochondrial dysfunction. The AC16 human ventricular cardiomyocyte cell line was treated with 10 μg/mL of LPS to induce sepsis-related cardiomyocyte damage. UA, an inducer of mitophagy, was used to culture AC16 cells. siRNA against FUNDC1 (si-FUNDC1) was transfected into AC15 to inhibit mitophagy activation. A. Cell viability was determined using an MTT assay. B. ELISA analysis of caspase-3 activity. C-D. AC16 cells were stained with JC-1 to observe changes in the mitochondrial membrane potential. E-F. AC16 cells were stained with the MitoSOX red mitochondrial superoxide indicator to show changes in mitochondrial ROS. G. Total ATP production was determined using the Cell Titer-Glo Luminescent Viability assay. H-J. Mitochondrial morphology was revealed using confocal immunofluorescence. TOM20 was used to show the shape of mitochondria in response to LPS or FUNDC1 deletion. Data are presented as mean ± SEM, normalized per 1000 cardiomyocytes. *P<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

UPR^mtis activated in response to FUNDC1 deficiency. FUNDC1^f/f and cardiomyocyte-specific FUNDC1 knockout (FUNDC1^CKO) mice were injected intraperitoneally with a single dose of PBS or LPS (5 mg/kg) to induce septic cardiomyopathy. Blood and heart samples were isolated 48 h after LPS injection. To activate mitophagy, urolithin A (UA, 30 mg/kg) was injected intraperitoneally in sterile saline with 0.1% DMSO. A-G. Total RNA was isolated from the ventricular tissue of FUNDC1^CKO and FUNDC1^f/f mice. RT-PCR was conducted to assess genes involved in UPR^mt. Data are presented as mean ± SEM. n = 6 per group. *P<0.05.

Fig. 4

Activation of UPR^mtpartly reduces sepsis-induced myocardial injury and mitochondrial dysfunction in FUNDC1-knockout mice. FUNDC1^f/f and cardiomyocyte-specific FUNDC1 knockout (FUNDC1^CKO) mice were injected intraperitoneally with a single dose of PBS or LPS (5 mg/kg) to induce septic cardiomyopathy. Blood and heart samples were isolated 48 h after LPS injection. To induce UPR^mtin vivo, mice were injected with oligomycin (500 μg/kg) intraperitoneally in sterile saline with 0.1% DMSO. A-C. Echocardiographic data showing left ventricular ejection fraction (LVEF), left ventricular diastolic dimension (LVDd), and fractional shortening (FS) in FUNDC1^CKO and FUNDC1^f/f mice in the presence of LPS or oligomycin. D-F. ELISA analysis of the concentration of cardiac injury markers, including LDH, troponin T, and CK-MB. G-H. The AC16 human ventricular cardiomyocyte cell line was treated with 10 μg/mL of LPS to induce sepsis-related cardiomyocyte damage. siRNA against FUNDC1 (si-FUNDC1) was transfected into AC15 to inhibit mitophagy activation. Oligomycin, an inducer of UPR^mt, was used to culture AC16 cells. Next, cells were stained with JC-1 to observe changes in the mitochondrial membrane potential. I-J. AC16 cells were stained with the MitoSOX red mitochondrial superoxide indicator to show changes in mitochondrial ROS. K-L. Mitophagy activity was observed using the mt-Keima assay. A yellow signal highlights increased mitophagic flux within cardiomyocytes. Data are presented as mean ± SEM, normalized per 1000 cardiomyocytes. n = 6 per group. *P<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Inhibition of UPR^mtpartly abolishes the cardioprotective effect of mitophagy activation. The AC16 human ventricular cardiomyocyte cell line was treated with 10 μg/mL of LPS to induce sepsis-related cardiomyocyte damage. UA, an inducer of mitophagy, was used to culture AC16 cells. siRNA against FUNDC1 (si-FUNDC1) and Atf6 (si-Atf6) were transfected into AC15 to inhibit the activation of mitophagy and UPR^mt, respectively. A-B. Mitophagy activity was observed using the mt-Keima assay. A yellow signal highlights increased mitophagic flux within cardiomyocytes. C. Cell viability was determined using an MTT assay. D. ELISA analysis of caspase-3 activity. E-F. AC16 cells were stained with JC-1 to observe changes in the mitochondrial membrane potential. G-H. AC16 cells were stained with the MitoSOX red mitochondrial superoxide indicator to show changes in mitochondrial ROS. I. Total ATP production was determined using the Cell Titer-Glo Luminescent Viability assay. J-L. Mitochondrial morphology was revealed using confocal immunofluorescence. TOM20 was used to show the shape of mitochondria in response to LPS or FUNDC1 deletion. Data are presented as mean ± SEM, normalized per 1000 cardiomyocytes. *P<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)