Mitophagy Regulators as Novel Targets in Sepsis-Induced Myocardial Dysfunction

Corresponding Author

Sepsis, a life-threatening condition defined as a dysregulated immune response to infection, is characterized by multiorgan failure, including, eg, severe cardiovascular dysfunction.¹ It has also been described that microcirculation undergoes severe alterations during sepsis, including maldistribution/slowing of capillary blood flow, decreased oxygen delivery, endothelial dysfunction, and increased vascular permeability.^2,3 Particularly in relation to high-energy-demanding organs, such as the heart, the critical role of microcirculation in facilitating the transport of oxygen and nutrients to tissue cells, which is essential for maintaining organ function, becomes especially evident.⁴ Essential elements of microcirculatory autoregulatory mechanisms, specifically the regulated production of vasodilatory nitric oxide and the regulatory functions of endothelial cells, are severely compromised. This impairment plays a crucial role in the pathophysiology of sepsis.^3,4

Sepsis-induced myocardial dysfunction (SIMD) is characterized by both systolic and diastolic dysfunction of the myocardium affecting both sides of the heart as a consequence of sepsis.⁵ It is estimated that approximately up to 50% of patients with sepsis will develop SIMD.⁵ Furthermore, patients with SIMD have a higher mortality rate than sepsis patients who do not develop SIMD.⁵

In this issue of JACC: Basic to Translational Science, Gao et al⁶ used an established SIMD model that induces bacterial peritonitis followed by sepsis and SIMD via a cecal ligation and puncture surgery. The rat model used in this study is characterized by cardiac dysfunction, the up-regulation of sepsis-associated biomarkers, dysmorphic microvessel anatomy, and an altered erythrocyte arrangement as an indicator of impaired blood flow (Figures 1, 4 and 5 in Gao et al⁶). Furthermore, this SIMD-induced model involves a disrupted endothelial barrier, as indicated by altered vascular endothelial-cadherin expression, increased infiltration of albumin and neutrophils into the myocardium, decreased cardiac function parameters, and a myocardial injury phenotype (Figure 5 in Gao et al⁶).

Early during sepsis, an imbalance of oxygen delivery and consumption leads to microcirculatory and mitochondrial distress/dysfunction syndrome (MMDS), which is a hallmark of sepsis and a major driver of multiorgan failure.^4,7 Understanding the molecular regulatory mechanisms between mitochondrial dysfunction and microcirculatory alterations in the early stages of sepsis is of pivotal interest in the development of new sepsis therapies.

In MMDS, mitochondrial dysfunction seems to occur downstream of and is presumably caused by microcirculatory changes occurring in the early stages of sepsis.^4,7 During sepsis, the release of proinflammatory cytokines and generation of reactive oxygen species cause excessive inflammation and oxidative stress, which leads to further mitochondrial damage and promotes cell death.^3,4,8 Sepsis-induced nitric oxide overproduction might also contribute to mitochondrial impairment.⁴

Using the SIMD model, Gao et al⁶ reported that microcirculatory dysfunction as part of MMDS is a key feature of SIMD characterized by increased apoptotic markers Caspase-3 and -9 and a decrease in the expression of the endothelial marker CD34 (Figures 2 and 4 in Gao et al⁶). Using a lipopolysaccharide (LPS)-based inflammation induction cell culture model in cardiac microvascular endothelial cells, the effects of inflammation on mitochondrial function were further assessed. Here, acute mitochondrial damage occurred after LPS induction, followed by the activation of mitochondrial degradation via autophagy and, later in time, apoptosis (Figures 3 and 6 to 8 in Gao et al⁶).

Mitochondrial autophagy, commonly referred to as mitophagy, is a selective degradation mechanism that facilitates the removal of aged, damaged, or dysfunctional mitochondria from the cell, thus ensuring the maintenance of mitochondrial function.⁹ This process is crucial for cellular energy homeostasis, and its proper function is essential for maintaining the integrity of the microcirculation. The accumulation of defective mitochondria results in increased reactive oxygen species production,⁸ which in the context of sepsis, may contribute to endothelial dysfunction and impaired microcirculation, thereby playing a pathophysiological role in the initiation and propagation of sepsis-related organ failure.^4,8 In the cell culture model of Gao et al,⁶ LPS-induced acute up-regulation of mitophagy was observed via an increase in autophagy markers and detection of autophagosomes via electron microscopy. This was followed by the suppression/loss of mitophagy and the accumulation of mitochondrial membrane proteins later in time, resulting in cell death (Figures 3 and 6 to 8 in Gao et al⁶).

Several studies have indicated that the induction of mitophagy can mitigate sepsis-induced damage to mitochondria by removing dysfunctional mitochondria, thereby restoring endothelial function and microcirculatory homeostasis.⁸ The molecular mechanisms that regulate mitophagy are potential therapeutic targets for sepsis. The best known regulatory pathway of mitophagy is the PTEN-induced putative protein kinase 1 (PINK1)-mediated ubiquitination of mitochondrial proteins by the E3 ligase Parkin.⁸ PINK1, located at the outer mitochondrial membrane, senses mitochondrial damage and recruits cytosolic Parkin, whereupon the autophagosome receptor is recruited and the formation of the autophagosome membrane is facilitated.^8,9 Another regulatory pathway of mitophagy that occurs under hypoxic conditions, termed hypoxia-induced mitophagy, involves the outer mitochondrial membrane proteins FUNDC1, NIX, and BNIP3, which promote Beclin-mediated initiation of the Atg5-dependent autophagy pathway.⁹ The selective degradation of mitochondria is mediated by LC3 (microtubule-associated proteins 1A/1B light chain 3B)-binding motifs of the proteins, which thereby facilitate the engulfment of mitochondria by autophagosomes.⁹ Hypoxia-induced mitophagy might be altered during sepsis-induced MMDS.

Understanding the role of hypoxia-induced mitophagy in sepsis-induced cardiac microcirculatory dysfunction is needed for the development of targeted therapies. Therefore, insights into the molecular mechanisms that regulate this pathway are essential.

Gao et al⁶ studied the regulatory mechanism of mitophagy occurring during SIMD.⁶ They identified cellular FLICE-inhibitory protein (c-FLIP) as a regulator of FUNDC1-mediated mitophagy during SIMD. c-FLIP is located at the interface of apoptosis and autophagy and has antiapoptotic and antiautophagic effects under physiological conditions but can induce autophagy after pathological triggers.¹⁰ In both models of this study, the SIMD rat model and the LPS-induced inflammatory cell culture model, the c-FLIP expression levels were reduced (Figures 2 to 4 in Gao et al⁶). Manipulation of c-FLIP levels in the SIMD model attenuated MMDS, as shown by the restoration of increased apoptosis, endothelial cell loss, altered microvessel morphology, altered blood flow, defective endothelial barrier function, increased inflammation, impaired cardiac function, and myocardial damage to control levels (Figures 4 and 5 in Gao et al⁶).

In the LPS cell culture model, the overexpression of c-FLIP restored early mitochondrial damage by ameliorating the LPS-induced overshooting of FUNDC1-mediated mitophagy (Figure 6 in Gao et al⁶). Thus, in early disease stages, the inhibition of excessive autophagy plays a protective role. In contrast, the overexpression of c-FLIP at later stages induced FUNDC1-mediated mitophagy, thereby clearing accumulated damaged mitochondria and restoring the protective functions of mitophagy (Figure 6 in Gao et al⁶). Consequently, the overexpression of c-FLIP in the late stages of the LPS model resulted in the restoration of mitochondrial function, apoptosis levels and endothelial function through FUNDC1-mediated mitophagy (Figures 7 and 8 in Gao et al⁶). Furthermore, Gao et al⁶ studied the regulation of c-FLIP/FUNDC1-mediated mitophagy via JNK. Inhibiting JNK activity in the late LPS-induced cell culture model phenocopied c-Flip overexpression, as observed by restoring the JNK/FUNDC1 interaction and thereby activating FUNDC1-mediated mitophagy (Figure 9 in Gao et al⁶). In summary, the findings of this study identify c-FLIP as a potential candidate for targeted therapies for sepsis/SIMD (Figure 1).

Figure 1.

Graphical Illustration of Dysregulated Mitophagy During Sepsis-Induced Myocardial Dysfunction and a Possible Strategy for Targeted Therapy

(A) Early during sepsis-induced myocardial dysfunction, impaired microcirculation leads to hypoxia and oxidative stress (reactive oxygen species [ROS]) in cardiac microvascular endothelial cells, resulting in mitochondrial damage and the induction of hypoxia-induced autophagy of mitochondria (mitophagy). This process is mediated by mitophagy receptors (FUNDC1/BNIP3/NIX), which are disinhibited at this stage because of the loss of cellular FLICE-inhibitory protein (c-FLIP). This disinhibition causes excessive mitophagy, accelerating cell death. Later, during SIMD, FUNDC1/BNIP3/NIX-mediated mitophagy is inhibited because of the absence of regulation by c-FLIP, leading to the accumulation of damaged mitochondria and subsequent apoptosis. (B) The reintroduction of c-FLIP ameliorates hyperactivated mitophagy at early stages and activates down-regulated mitophagy at later stages, thereby restoring mitophagy homeostasis. The modulation of the activity or expression of regulators or receptors involved in mitophagy represents a promising strategy for targeted therapy in sepsis-induced myocardial dysfunction and warrants further validation in additional sepsis models.

Mitophagy is modulated through multiple regulatory mechanisms, and the intricacies of its cross-regulatory mechanisms under both physiological and pathophysiological conditions remain to be comprehensively elucidated. Although this study presents a highly promising candidate for targeted sepsis therapy, numerous questions remain unresolved: How do impairments in alternative mitophagy pathways, such as the PINK1/Parkin pathway, contribute to the early and late stages of SIMD, and how is their regulation affected by modulating c-FLIP overexpression? What is the role of the c-FLIP/FUNDC1-mediated mitophagy pathway in other organ dysfunctions induced by sepsis, including those affecting the kidneys, liver, and lungs? Does the modulation of c-FLIP levels have the potential to mitigate MMDS in other sepsis-induced organ models and thus could be applied systemically? Finally, does the restoration of microcirculation and mitochondrial function facilitate the clinical improvement of sepsis?

Taken together, Gao et al⁶ identified a novel molecular target for the treatment of sepsis and sepsis-induced MMDS, which has the potential to restore mitophagy and microcirculation by modifying its activity and/or expression levels.⁶ However, future research should develop new molecular tools that specifically alter the expression or activity of c-FLIP. In addition, their potential to alleviate MMDS in SIMD and in a broader spectrum of sepsis-induced MMDS disease models should be validated.

Funding Support and Author Disclosures

Dr Friedrich and Dr Kappert are supported by the Investitionsbank Berlin (Pro FIT - Program for the Promotion of Research, Innovation and Technology, 10209032 and 10210792). Dr Kappert is supported by the German Research Association (Deutsche Forschungsgemeinschaft, KA 1820/9-1 and KA 1820/10-1). The authors have reported that they have no relationships relevant to contents of this paper to disclose.