A detailed and accurate real-time analysis of mitochondrial malfunctioning in organs of living human patients in a hospital setting is impractical. Usually, mechanisms must be inferred from tissue specimens obtained from non-vital organs (e.g., blood and skeletal muscle), or from postmortem tissues examinations. Such specimens should carry out relevant biological signatures that mirror whether a mechanistic target is related to the severity of sepsis and to clinical outcomes. Whereas alterations in several dimensions of mitochondrial function in sepsis were already reported using preclinical tools, there is no recommended use for the hospital routine. Figure 2 presents an overview of the different ways to measure mitochondrial damage, activity and recovery in sepsis.

Mechanisms of mitochondrial damage, functional impairment and recovery. BCE: biochemical coupling efficiency; PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1α; PPAR-γ: peroxisome proliferator-activated receptor gamma; NRF-1: nuclear respiratory factor 1

Mitochondrial biogenesis refers to the process by which cells generate new mitochondria, and depending on the demands also increases the number. This process requires the synthesis of mitochondrial proteins, which are encoded by nuclear DNA and subsequently imported and integrated into mitochondria [7]. Biogenesis further relies on the transcription of mtDNA, which encodes 13 genes that translate proteins to the respiratory complexes [37]. Biogenesis along with mitophagy serves to replace damaged/dysfunctional mitochondria, thus enhancing the ability to generate ATP in response to increased energy demands over time. In postmortem studies, septic patients exhibit mild-to-moderate mitochondrial swelling and increased markers of mitophagy in the kidney, with minimal cell death or indications of permanent damage, such as tissue fibrosis [36]. In addition, postmortem studies using liver and rectus abdominis muscle samples from critically ill patients have revealed an increase expression of transcriptional factors linked to mitochondrial biogenesis [37]. Similarly, it was found in biopsies of skeletal muscle of septic patients a partial activation of the mitochondrial biogenesis pathway involving NRF2α/GABP and its target genes, which does not parallel the benefit for mitochondrial function. However, research has also shown a decrease in mitochondrial content in the muscles of critically ill patients with sepsis-induced multiple organ failure [38]. Although these studies contribute to a better understanding of the mitochondrial signatures of the disease, they demanded muscle biopsy samples, which bring into light important practical limitations. However, these data suggest that biogenesis activation may play a role in the recovery phase of critical illness [39], albeit not yet correlating with improved mitochondrial function [17]. Hence, compromised mitochondrial biogenesis in critically ill patients, and activation of the biogenesis pathway may represent a key prognostic factor in critically ill patients associated with recovery of the initial injury [39].

Proteins involved in the regulation of mitochondrial morphology and maintenance of the fission–fusion balance are necessary for overall cellular health. Mitochondrial fission is necessary to preserve an ideal number of mitochondria during cellular growth and division processes, while fusion enables the unification of two mitochondria forming a more elongated one. In a homeostatic environment both fission and fusion processes result in healthy mitochondria [40]. It is tentative to assume that mitochondrial fusion and fission undergo alterations during the acute inflammatory response observed in sepsis. These changes can be assessed by measuring the levels of fusion proteins, such as mitofusins 1 and 2 and optic atrophy 1 protein, as well as fission proteins, including dynamin-related protein 1 and fission 1 protein [39]. Despite these proteins being promising biomarkers of mitochondrial dysfunction caused by sepsis, the extent of mitochondrial dynamics (fusion and fission) in sepsis patients remains largely speculative. In this context, new clinical studies may provide insights related to the association between dynamics and specific clinical outcomes in sepsis.

Mitochondria undergo various morphological changes during events such as fusion and fission, which help maintain a healthy mitochondrial population by facilitating the exchange of mtDNA, preserving mtDNA integrity, and regulating the size, quantity, distribution, thereby sustaining OXPHOS capacity [8]. These changes are also crucial for cell division and proliferation, as well as in the selective elimination of damaged or excess mitochondria through a process known as mitophagy [7]. Proteins involved in fusion and fission events, such as mitofusin-2 and dynamin-related protein-1, have been linked to changes in mitochondrial membrane potential and reduced oxygen consumption [41]. Fission and fusion processes become more prevalent under stressful conditions and play critical roles in eliminating damaged mitochondria and enhancing repair mechanisms. However, there is limited data regarding these mitochondrial dynamics in patients with sepsis, and the data may vary depending on the tissue type. From a theoretical standpoint, mitochondrial dynamics in patients with sepsis is a plasticity phenomenon, aimed at improving mitochondrial bioenergetic function, and decreasing oxidative stress in favor of cell survival [42].

However, mtDNA levels in the serum should be interpreted as a potential damage-associated molecular pattern that propagates an inflammatory response through interactions with the immune system [12, 43]. Therefore, mtDNA damage can lead to a pathological cycle, resulting in metabolic dysfunction, particularly in white blood cells [44]. The reduction in mtDNA content in the peripheral blood observed in the acute phase of sepsis could be only due to an increased concentration of neutrophils in the peripheral blood [45]. Also, it remains uncertain whether there is an interaction between mDNA concentration and changes in mitochondrial bioenergetics, particularly in immune cells. The assessment of serum levels of mtDNA is easy to perform and applicable, however, the role of these limitations and their prognostic impact must be better elucidated in new studies. Mitochondria possess their own DNA, and the depletion of mtDNA in an injury process may, hypothetically, cause defects in the ETC compromising OXPHOS [45, 46]. Another alternative is the organ-specific evaluation, through tissue biopsies, both in vivo and postmortem [47]. Although it is not a clinically applicable assessment, it allows for an organ-specific assessment regarding quantitative mtDNA polymerase chain reaction (qPCR) and mtDNA damage. Moreover, tissue utilization allows the evaluation of the interaction between mtRNA and mtDNA expression of mitochondrial biomarkers.

In addition to alterations in mitochondrial mass, various hormones, enzymes, and regulatory pathways within cells are responsible for regulating the quality of mitochondrial function and promoting a shift towards glycolytic pathways, alternatively promoting the hierarchization of oxidative e non-oxidative metabolic pathways depending on the required pro- or anti-inflammatory response. Reactive derivatives of nitric oxide and superoxide anions, such as peroxynitrite, which cause oxidative stress, stimulate glycolysis by activating the rate-limiting step of the PPP, glucose-6-phosphate dehydrogenase. This pathway leads to the formation of NADPH in relation to NADH. NADH is a substrate for mitochondrial OXPHOS of high-energy phosphates, while NADPH is essential for the formation and repair of proteins, DNA, and lipids. By diverting glycolytic intermediates from the Krebs cycle and suppressing aerobic mitochondrial respiration, cells and tissues transition into a state of reduced oxygen consumption and ATP production. This phenomenon is commonly referred to as the “Warburg effect”, particularly in the context of cancer [48]: cells are less reliant on oxidative metabolism, thereby decreasing oxidative stress and promoting the formation of reducing equivalents (e.g., lactic acid and NADPH) that induce cell repair [49]. The Warburg effect and related mediators, such as hypoxia-inducible factor 1-alpha (HIF-1α), are induced in sepsis models, potentially providing cytoprotection and modulating inflammation in response to acute cell stress [50]. However, HIF-1α activation in immune cells can perpetuate the activation of the pro-inflammatory signaling through the stimulation of glycolytic pathway [51]. An estimation of PPP components, NADH:NAD+ ratio, and HIF-1α can be easily determined in immune cells through biochemistry assays, reactive polymerase chain reaction, and Western blotting.

Proton pumps of the ETC, in conjunction with F1Fo-ATP synthase, establish a proton gradient across the inner membrane, generating both an electrochemical potential [proton motive force (pmf), in mV] and a flux of protons (proton current in nmol of protons/min). Mitochondrial membrane potential is a critical component of healthy mitochondrial metabolism and contributes to determining the pmf [10]. Thus, measuring the pmf serves as a surrogate marker of mitochondrial functionality. This methodology, however, is currently feasible only in isolated mitochondria and cells, is relatively time-efficient, and, although valuable for understanding disease mechanisms, has yet to be integrated into clinical practice.

Reductions in both the expression and activity of Complexes I, II, III, and IV, as well as of cytochrome c (Cyt c) have been reported in critically ill patients [15, 39]. However, it is still doubtful whether these alterations are determinants of the patient's prognosis, or if they are just epiphenomena in the acute context of critical illness. However, they are useful and commonly used to assess mitochondrial activity [52,53,54,55], especially when normalized by enzymatic activity, protein, or DNA concentration. This type of measurement can be done on circulating cells (mononuclear cells, platelets) or on biopsy-derived cells (muscle cells, for example). Relative content of mitochondrial proteins are determined by Western blotting, using primary antibodies for mitochondrial complexes subunits. Also, Cyt c content can also be quantified in immunoassay of isolated cells.

Quantifying the efficiency of mitochondrial ATP production is a logical method for evaluating their functional integrity. Measuring mitochondrial respiration is a cost-effective and time-efficient approach compared to conventional methods of assessing mitochondrial function through biopsies, making it widely accessible. High-resolution measurements of mitochondrial respiration can be obtained using advanced instruments equipped with highly sensitive microcathode oxygen electrodes, which can be utilized in acute care settings. The measurement of mitochondrial respiration can be performed using the substrate–uncoupler–inhibitor titration (SUIT) protocol, which is a commonly used method for quantifying this process. This protocol entails the titration of various combinations of substrates, uncouplers, and inhibitors in order to evaluate mitochondrial respiratory function [11]. The SUIT protocol permits the investigation of intricate interactions between coupling and substrate control in a single assay, thereby measuring multiple aspects of mitochondrial physiology [56].

Respirometry allows for real-time measurement of mitochondrial respiration, providing crucial parameters through the application of established inhibitors and uncouplers, which serve as sensitive indicators of the response to mitochondrial stress. Figure 3 depicts the mitochondrial respiration trace derived from the SUIT protocol, which was employed in the following procedures [8, 56]: (1) routine respiration: routine respiration, also known as basal respiration, measures the oxygen consumption resulting from ATP production and proton leak. This represents energy demand under steady-state conditions. Changes in routine respiration in patients with disease compared to controls may indicate altered mitochondrial function and should be interpreted in the context of the following mitochondrial parameters: (2) proton leak: after measuring routine respiration, cells are exposed to oligomycin, an inhibitor of Complex V. The remaining mitochondrial respiration after the addition of oligomycin was attributable to proton leak. Although some proton leaks are expected under physiological conditions, significant proton leak may indicate damage to the mitochondrial membrane and/or complex damage. The use of oligomycin also allows for the estimation of oxygen consumption secondary to ATP production, often referred to as ATP-linked respiration. (3) Maximal respiration: the addition of a mitochondrial uncoupler, such as dinitrophenol or carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, stimulates maximal respiration by mimicking the physiological energy demand, leading to an increase in oxygen consumption. The difference between maximal respiration and routine respiration represents the spare respiratory capacity (SRC) of the cell. SRC indicates the ability of a cell to respond to energetic stress and is a measure of a cell’s fitness. A decrease in SRC may limit the cell's ability to handle stressors, resulting in mitochondrial dysfunction. (4) Residual oxygen consumption; the addition of mitochondrial inhibitors, such as the combination of rotenone (Complex I) and antimycin (Complex III), completely inhibits the electron transport system. The remaining oxygen is consumed by non-mitochondrial respiration in the form of oxidases and other cellular enzymes that utilize oxygen. Residual oxygen consumption may increase in the presence of a stress response.

Example of a respirometry assay. ATP: adenosine triphosphate

Under normal conditions with excess ADP and oxygen, mitochondrial respiration, known as state 3 respiration, occurs rapidly. Conversely, when ADP was fully consumed, state 4 respiration occurred, which was significantly slower. State 4 respiration can be induced by “uncoupling” oxygen consumption from OXPHOS, leading to proton leakage back into the mitochondrial matrix without the production of cellular energy. One of the most promising indicators of mitochondrial function is biochemical coupling efficiency (BCE), which is calculated as the quotient between OXPHOS and proton leak. BCE reflects the true effectiveness of mitochondria in utilizing oxygen for ATP production [8, 75]. This is a useful way to gain more insight into the site of the dysfunction, namely, respiratory control decreases because of dysfunction in localized sites of substrate oxidation, ATP synthesis, proton conductance, or F1Fo-ATP synthase [10].

Although several centers may not have access to equipment for real-time measurement of mitochondrial respiratory rates, this limitation can be easily overcome by utilizing simpler biochemical colorimetric methods that maintain a reasonable level of sensitivity and offer short assay times, which can be easily integrated into the hospital laboratory routine. For example, measurements of succinate dehydrogenase enzyme activity for Complex II (succinate: DCIP-oxidoreductase), Complex IV, and Complex V are routinely performed in research laboratories [76, 77]. However, these aforementioned colorimetric methods only allow for the evaluation of metabolic activity in a single complex at a time, while mitochondrial respirometry assays enable the simultaneous assessment of the metabolic activity of multiple complexes. This approach has the potential for clinical and large-scale applicability, but further studies are necessary for its validation.