Systemic inflammatory responses shape clinical outcomes in acute cardiovascular disease. Because of their functional plasticity and rapid turnover, neutrophils have emerged as dynamic indicators of inflammatory stress. Here we profile the appearance of distinct neutrophil maturation stages in patients with ST-elevation myocardial infarction, heart failure and stroke. Our data reveal the mobilization of immature neutrophils in all groups; however, patients with ST-elevation myocardial infarction exhibited the most pronounced engagement of this response, including the appearance of CD16lowCD10neg preneutrophils (preNeus), the final mitotic neutrophil progenitor, which was associated with disease outcome. Plasma cytokine profiling identified a coordinated inflammatory signature associated with preNeu mobilization, consistent with emergency granulopoiesis. PreNeus were identifiable as immature granulocytes in routine blood count analysis, where they predicted 30-day mortality better than established biomarkers in two clinical cohorts. Immature granulocytes also remained independently associated with survival in multivariate models incorporating risk factors, enabling the immediate identification of vulnerable patients with ST-elevation myocardial infarction upon hospital admission.

Acute hypoxia triggers multiple physiological and immune responses, yet the immediate systemic effects on circulating complement proteins remain insufficiently characterized. The complement cascade plays a central role in inflammation, host defense, and ischemia-related tissue injury, but its regulation during transient oxygen deprivation and reoxygenation in humans is poorly understood. Sixteen healthy volunteers were exposed to stepwise normobaric hypoxia simulating altitudes of 0, 2, 4, and 6 km (pO₂ = 9.64 kPa) followed by reoxygenation under normoxic conditions. Blood samples were collected at baseline, peak hypoxia (6 km), and after reoxygenation. Quantitative plasma proteomics was performed using targeted multiple-reaction-monitoring mass spectrometry to quantify key complement components (C1 complex, C3–C9, factor B) in 16 participants with complete datasets. Hematological parameters were analyzed in parallel. Hypoxia transiently increased leukocyte and platelet count, whereas hematocrit and mean corpuscular volume slightly decreased. While only slightly increasing during hypoxia, most complement peptides - including C1S, C1R, C3, C5, C7, C9, and CFAB - showed a coordinated reduction in relative abundance upon reoxygenation compared to both baseline and hypoxia (median fold-change ≈ 0.6–0.8; p < 0.05). Correlation analysis revealed coherent clustering among complement components but only weak associations with hematological indices. Acute hypoxia elicits rapid and reversible changes in the circulating complement peptide pool in healthy humans. Targeted plasma proteomics demonstrates clear oxygen-phase–dependent dynamics, with a coordinated decrease after reoxygenation. This pattern is consistent with reduced circulating availability of complement components, activation-associated consumption, and/or redistribution within the intravascular compartment. Future validation of these findings in certain patient cohorts may define translational relevance and functional consequences. Sixteen healthy volunteers were exposed to stepwise normobaric hypoxia simulating altitudes of 0 km, 2 km, 4 km, and 6 km. Hypoxia increased plasma concentrations of several proteins of the complement system as analyzed by quantitative mass spectrometry. Upon reoxygenation, most complement peptides decreased to or below baseline.

BACKGROUND:Inflammation orchestrates an outcome after acute myocardial infarction (AMI). Thromboinflammation, via the CD40- and CD40 ligand (CD40L)-mediated platelet-leukocyte interaction, is involved in post-AMI inflammation. OBJECTIVES:This study hypothesized that acetylsalicylic acid (ASA) exerts pleiotropic cardioprotective effects beyond prevention of reinfarction by reducing thromboinflammation and infarct size. METHODS:A murine AMI model was used to investigate the effects of low-dose ASA, which is applied preischemia or after induction of ischemia (intraischemia), on post-AMI thromboinflammation and the outcome. To investigate the underlying mechanisms, platelet and neutrophil depletion and genetically induced and antibody-induced CD40L deficiency were applied. Thromboinflammation markers were analyzed. Translationally, the outcome after ST-elevation myocardial infarction (STEMI) was measured in ASA-pretreated vs ASA-naive patients (ClinicalTrials.gov ID: NCT03539133). RESULTS:Both ASA treatment preischemia and intraischemia reduced infarct size and thromboinflammation and improved cardiac function and remodeling. The scar size was smaller with ASA preischemia 21 days after AMI but not with ASA intraischemia. This cardioprotection was blunted in the absence of (a) platelets or (b) neutrophils. Both pharmacologic inhibition or genetic deficiency of CD40L abrogated ASA's protective effect. Accordingly, ASA-pretreated patients with STEMI had improved outcome (12.5% vs 23.8%; hazard ratio, 0.50; 95% CI, 0.31-0.80; P < .001). This was driven by reduced mortality without differences in recurrent AMI. CONCLUSION:Existing ASA therapy shows pleiotropic effects in the reduction of thromboinflammation and improvement of outcome after AMI, independent of its effects on the occurrence of ischemia itself. This should be considered while choosing timing of initiation and the optimal antithrombotic regime post-AMI in patients with coronary artery disease.

Endothelial dysfunction (ED) is a hallmark of cardiovascular disease (CVD). We recently showed that anemia is associated with the progression of ED after acute myocardial infarction (AMI), which is partly mediated by red blood cells (RBCs). Extracellular vesicles (EVs) are efficient communicators between cells and can functionally contribute to various CVDs, including AMI. The potential role of RBC-derived large extracellular vesicles (REVs) in anemia-associated ED in stable coronary artery disease (CAD) patients has not yet been investigated. We hypothesize that REVs, but not plasma-derived EVs (PLEVs), mediate ED in anemic CAD patients. In this study, we demonstrated an increased release of REVs, but not PLEVs, in anemic patients compared to non-anemic patients. These REVs showed enhanced nitric oxide (NO) consumption in anemic patients. REVs and PLEVs were dose-dependently taken up by endothelial cells (ECs) in vitro. Aortic rings co-incubated with REVs, but not PLEVs, from anemic patients showed an attenuated endothelial NO-dependent (EDNO) relaxation. Mice injected with REVs from anemic patients showed impaired flow-mediated dilation responses in vivo, accompanied by reduced NO bioavailability. Proteomic analysis of REVs from anemic patients revealed increased myeloperoxidase (MPO) abundance. Co-incubation of ECs with REVs but not PLEVs from anemic patients increased reactive oxygen species (ROS) production. Pre-treatment of anemic REVs with an MPO inhibitor AZD-5904, followed by co-incubation with aortic rings, improved EDNO relaxation. These findings suggest that anemia increases the release of REVs with enhanced NO consumption. Additionally, anemic REVs promote ED in ECs by delivering oxidative stress-promoting MPO and increasing ROS production.

BACKGROUND:Cardiogenic shock (CS) is a severe complication of acute myocardial infarction (AMI) leading to poor outcomes. Specific biomarkers, with subsequent validation of their prognostic relevance in CS, are urgently needed to improve therapies and outcomes. Accordingly, the present study investigated the plasma proteome using proximity extension assay technology to identify novel specific biomarkers with subsequent validation of their prognostic relevance in CS. METHODS:Using proximity extension assay (Olink Explore, 2942 proteins), the proteomic signature in the plasma of 9 AMI patients without shock and 8 AMI patients with CS (AMICS; exploration cohort) at admission was analyzed. Candidate biomarkers were measured in the plasma of 421 patients with AMICS from the CULPRIT-SHOCK cohort (Culprit Lesion Only PCI Versus Multivessel PCI in Cardiogenic Shock; REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01927549, validation cohort). Their prognostic relevance was assessed for 180-day survival as the primary end point. RESULTS:Proteome profiling was successful for 2925 proteins and identified VEGFR1 (vascular endothelial growth factor receptor 1, also known as Flt1) as elevated in AMICS compared with nonshock AMI in the exploration cohort (P<0.001). In patients from the independent validation cohort, nonsurvivors had markedly higher VEGFR1 levels (6.8 versus 3.8 ng/L; P<0.001). In Cox regression, VEGFR1 levels were independently associated with a higher 180-day mortality risk even after adjusting for the Simplified Acute Physiology Score II (per ng/L; adjusted hazard ratio, 1.06 [95% CI, 1.03-1.09]; P<0.001) and yielded incremental prognostic information in addition to serum lactate levels (P<0.001). The levels of VEGFR1 in surviving (30 days; n=29) and nonsurviving (n=21) patients with AMICS were determined at different time points (days 0, 1, and 5) in a third cohort, showing continuously higher levels in nonsurvivors. CONCLUSIONS:Plasma proteomic screening identified VEGFR1 as an early biomarker in patients with AMICS that provided independent prognostic information in a large cohort of well-defined patients with AMICS.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson’s (r = 0.9920) and Gomori’s Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Background/Objectives: Acute myocardial infarction (AMI), characterized by irreversible heart muscle damage and impaired cardiac function caused by myocardial ischemia, is a leading cause of global mortality. The damage associated with reperfusion, particularly mitochondrial dysfunction and reactive oxygen species (ROS) formation, has emerged as a crucial factor in the pathogenesis of cardiac diseases, leading to the recognition of mitochondrial proteins as potential markers for myocardial damage. This study aimed to identify differentially expressed proteins based on the type of cardiac injury, in particular those with and without reperfusion. Methods: Male C57Bl/6J mice were either left untreated, sham-operated, received non-reperfused AMI, or reperfused AMI. Twenty-four hours after the procedures, left ventricular (LV) function and morphological changes including infarct size were determined using echocardiography and triphenyl tetrazolium chloride (TTC) staining, respectively. In addition, plasma was isolated and subjected to untargeted mass spectrometry and, further on, the ELISA-based validation of candidate proteins. Results: We identified mitochondrial creatine kinase 2 (Ckmt2) as a differentially regulated protein in plasma of mice with reperfused but not non-reperfused AMI. Elevated levels of Ckmt2 were significantly associated with infarct size and impaired LV function following reperfused AMI, suggesting a specific involvement in reperfusion damage. Conclusions: Our study highlights the potential of plasma Ckmt2 as a biomarker for assessing reperfusion injury and its impact on cardiac function and morphology in the acute phase of MI.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling β-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine was protective against obesity and insulin resistance but clinical development was halted by the emergence of CNTF-antibodies. The chimeric cytokine IC7 used the framework of Interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.

Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.

In Interleukin (IL)-6 signalling, IL-6 site I binds to the IL-6 receptor (IL-6R) first, following by IL-6 site II interaction to domain 2/3 of gp130 to form premature trimeric IL-6:IL-6R:gp130 receptor complexes. Formation of the mature hexameric receptor complex is then facilitated by the inter-trimeric interaction of IL-6 site III with domain 1 of the opposing gp130. The two gp130-associated Janus kinases (JAKs) trans-phosphorylate when their spatiotemporal pairing is correct, which causes the activation of STAT, ERK, and AKT pathways in a balanced manner. Since the intracellular domain (ICD) of IL-6R is not needed for STAT/ERK/AKT phosphorylation, we investigated the conditions under which a chimeric IL-6RECD-gp130TMD/ICD receptor protein confers biological activity. For IL-6RECD-gp130TMD/ICD, the extracellular domain (ECD) of IL-6R was fused to the transmembrane domain (TMD) and ICD of gp130. Co-expression of IL-6RECD-gp130TMD/ICD with signalling-deficient gp130 variants did not induce IL-6 signalling, suggesting that the assembly of hexameric complexes failed to dimerize the IL-6R-associated JAKs correctly. By mimicking the premature trimeric receptor complex, IL-6-mediated dimerization of IL-6RECD-gp130TMD/ICD with the single-cytokine-binding variant gp130ΔD1 induced signalling. Of note, IL-6 signalling via these synthetic gp130ΔD1:IL-6RECD-gp130TMD/ICD complexes resulted predominantly in STAT3 phosphorylation. A STAT3-dominated profile was also observed after IL-6-induced signalling mediated by a JAK-deficient IL-6RECD-gp130TMD/ICDΔJAK variant in complex with the JAK-proficient but STAT/ERK/AKT-deficient gp130JAKΔICD variant. Our data showed that effective ERK/AKT signalling could not be executed after intracellular domain swapping from gp130 to the IL-6R. Taken together, the chimeric IL-6R/gp130 receptor may be helpful in the creation of customized synthetic IL-6 signalling.

Gravitational changes between micro- and hypergravity cause several adaptations and alterations in the human body. Besides muscular atrophy and immune system impairment, effects on the circulatory system have been described, which can be associated with a wide range of blood biomarker changes. This study examined nine individuals (seven males, two females) during a parabolic flight campaign (PFC). Thirty-one parabolas were performed in one flight day, resulting in ~22 s of microgravity during each parabola. Each participant was subjected to a single flight day with a total of 31 parabolas, totaling 11 min of microgravity during one parabolic flight. Before and after (1 hour (h) and 24 h), the flights blood was sampled to examine potential gravity-induced changes of circulating plasma proteins. Proximity Extension Assay (PEA) offers a proteomic solution, enabling the simultaneous analysis of a wide variety of plasma proteins. From 2925 unique proteins analyzed, 251 (8.58%) proteins demonstrated a differential regulation between baseline, 1 h and 24 h post flight. Pathway analysis indicated that parabolic flights led to altered levels of proteins associated with vesicle organization and apoptosis up to 24 h post microgravity exposure. Varying gravity conditions are associated with poorly understood physiological changes, including stress responses and fluid shifts. We provide a publicly available library of gravity-modulated circulating protein levels illustrating numerous changes in cellular pathways relevant for inter-organ function and communication.

AimTo investigate the associations of the Dietary Approaches to Stop Hypertension (DASH) score with subcutaneous (SAT) and visceral (VAT) adipose tissue volume and hepatic lipid content (HLC) in people with diabetes and to examine whether changes in the DASH diet were associated with changes in these outcomes.MethodsIn total, 335 participants with recent-onset type 1 diabetes (T1D) and type 2 diabetes (T2D) from the German Diabetes Study were included in the cross-sectional analysis, and 111 participants in the analysis of changes during the 5-year follow-up. Associations between the DASH score and VAT, SAT and HLC and their changes were investigated using multivariable linear regression models by diabetes type. The proportion mediated by changes in potential mediators was determined using mediation analysis.ResultsA higher baseline DASH score was associated with lower HLC, especially in people with T2D (per 5 points: -1.5% [-2.7%; -0.3%]). Over 5 years, a 5-point increase in the DASH score was associated with decreased VAT in people with T2D (-514 [-800; -228] cm3). Similar, but imprecise, associations were observed for VAT changes in people with T1D (-403 [-861; 55] cm3) and for HLC in people with T2D (-1.3% [-2.8%; 0.3%]). Body mass index and waist circumference changes explained 8%-48% of the associations between DASH and VAT changes in both groups. In people with T2D, adipose tissue insulin resistance index (Adipo-IR) changes explained 47% of the association between DASH and HLC changes.ConclusionsA shift to a DASH-like diet was associated with favourable VAT and HLC changes, which were partly explained by changes in anthropometric measures and Adipo-IR.

Background and aims: Differences of dietary pattern adherence across the novel diabetes endotypes are unknown. This study assessed adherence to pre-speci fied dietary patterns and their associations with cardiovascular risk factors, kidney function, and neuropathy among diabetes endotypes. Methods and results: The cross-sectional analysis included 765 individuals with recent-onset (67 %) and prevalent diabetes (33 %) from the German Diabetes Study (GDS) allocated into severe autoimmune diabetes (SAID, 35 %), severe insulin-de ficient diabetes (SIDD, 3 %), severe insulinresistant diabetes (SIRD, 5 %), mild obesity-related diabetes (MOD, 28 %), and mild age-related diabetes (MARD, 29 %). Adherence to a Mediterranean diet score (MDS), Dietary Approaches to Stop Hypertension (DASH) score, overall plant -based diet (PDI), healthful (hPDI) and unhealthful plant -based diet index (uPDI) was derived from a food frequency questionnaire and associated with cardiovascular risk factors, kidney function, and neuropathy using multivariable linear regression analysis. Differences in dietary pattern adherence between endotypes were assessed using generalized mixed models. People with MARD showed the highest, those with SIDD and MOD the lowest adherence to the hPDI. Adherence to the MDS, DASH, overall PDI, and hPDI was inversely associated with highsensitivity C -reactive protein (hsCRP) among people with MARD ( b (95%CI): -9.18 % (-15.61; -2.26); -13.61 % (-24.17; -1.58); -19.15 % (-34.28; -0.53); -16.10 % (-28.81; -1.12), respectively). Adherence to the PDIs was associated with LDL cholesterol among people with SAID, SIRD, and MOD. Conclusions: Minor differences in dietary pattern adherence (in particular for hPDI) and associations with markers of diabetes -related complications (e.g. hsCRP) were observed between endotypes. So far, evidence is insufficient to derive endotype-specific dietary recommendations. Trial registration: Clinicaltrials.gov: NCT01055093. 2024 The Authors. Published by Elsevier B.V. on behalf of The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Objective Inflammation is a critical process during the progressive development and complication of abdominal aortic aneurysm. The co-stimulatory dyad CD40-CD40L is a major driver of inflammation and modulates immune responses. This study evaluates the potential of a small molecule inhibitor, which blocks the interaction between CD40 and tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6, referred to as TRAF-STOP, in the early and later phase during AAA progression. Methods and results AAAs were induced in C57BL/6J mice by infrarenal aortic porcine pancreatic elastase infusion for 7, 14 or 28 days. Inhibition of CD40 signaling by TRAF-STOP resulted in less severe AAA formation and reduced the incidence of AAA development. TRAF-STOP treatment attenuated aortic structural remodeling, characterized by a reduced elastic fiber degradation, lowered expression of matrix metalloproteinase (MMP)-2 and MMP9, as well as preserved collagen type IV content in aneurysmal tissue. Furthermore, this is accompanied by the reduction of key pro-inflammatory genes such as TNFα. Conclusion Pharmacological inhibition of CD40-TRAF6 signaling protects from adverse aortic structural remodeling during the early phase of AAA progression representing a translational strategy to limit progression of human AAA disease.

AbstractAll except one cytokine of the Interleukin (IL-)6 family share glycoprotein (gp) 130 as the common β receptor chain. Whereas Interleukin (IL-)11 signal via the non-signaling IL-11 receptor (IL-11R) and gp130 homodimers, leukemia inhibitory factor (LIF) recruits gp130:LIF receptor (LIFR) heterodimers. Using IL-11 as a framework, we exchange the gp130-binding site III of IL-11 with the LIFR binding site III of LIF. The resulting synthetic cytokimera GIL-11 efficiently recruits the non-natural receptor signaling complex consisting of gp130, IL-11R and LIFR resulting in signal transduction and proliferation of factor-depending Ba/F3 cells. Besides LIF and IL-11, GIL-11 does not activate receptor complexes consisting of gp130:LIFR or gp130:IL-11R, respectively. Human GIL-11 shows cross-reactivity to mouse and rescued IL-6R−/− mice following partial hepatectomy, demonstrating gp130:IL-11R:LIFR signaling efficiently induced liver regeneration. With the development of the cytokimera GIL-11, we devise the functional assembly of the non-natural cytokine receptor complex of gp130:IL-11R:LIFR.

SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD+‐dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2‐induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4‐eGFP decrease upon CoCl2 treatment of HEK293 cells. Co‐treatment with BafA1, an inhibitor of autophagosome–lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl2‐induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia‐induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild‐type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress‐induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.