Context-dependent roles for ubiquitous mitochondrial creatine kinase CKMT1 in breast cancer progression.

Metabolic reprogramming is a hallmark of cancer, enabling cancer cells to rapidly proliferate, invade, and metastasize. We show that creatine levels in metastatic breast cancer cell lines and secondary metastatic tumors are driven by the ubiquitous mitochondrial creatine kinase (CKMT1). We discover that, while CKMT1 is highly expressed in primary tumors and promotes cell viability, it is downregulated in metastasis. We further show that CKMT1 downregulation, as seen in breast cancer metastasis, drives up mitochondrial reactive oxygen species (ROS) levels. CKMT1 downregulation contributes to the migratory and invasive potential of cells by ROS-induced upregulation of adhesion and degradative factors, which can be reversed by antioxidant treatment. Our study thus reconciles conflicting evidence about the roles of metabolites in the creatine metabolic pathway in breast cancer progression and reveals that tight, context-dependent regulation of CKMT1 expression facilitates cell viability, cell migration, and cell invasion, which are hallmarks of metastatic spread.

Role of creatine shuttle in colorectal cancer cells.

The creatine shuttle translocates the energy generated by oxidative phosphorylation to the cytoplasm via mitochondrial creatine kinase (MTCK) and creatine kinase B (CKB) in the cytoplasm. It is not apparent how the creatine shuttle is related to cancer. Here, we analyzed the expression and function of CKB and MTCK in colorectal cancer (CRC) and investigated the role of the creatine shuttle in CRC. Compared with normal mucosa, 184 CRC tissues had higher levels of CKB and MTCK, and these levels were associated with histological grade, tumor invasion, and distant metastasis. CK inhibitor dinitrofluorobenzene (DNFB) on CRC cell lines HT29 and CT26 inhibited cell proliferation and stemness to less than 2/3 and 1/20 of their control levels, respectively. In this treatment, the production of reactive oxygen species increased, mitochondrial respiration decreased, and mitochondrial volume and membrane potential decreased. In a syngeneic BALB/c mouse model using CT26 cells pretreated with DNFB, peritoneal metastasis was suppressed to 70%. Phosphorylation of EGFR, AKT, and ERK1/2 was inhibited in DNFB-treated tumors. High ATP concentrations prevented EGFR phosphorylation in HT29 cells following DNFB treatment, CKB or MTCK knockdown, and cyclocreatine administration. Despite not being immunoprecipitated, CKB and EGFR were brought closer together by EGF stimulation. These findings imply that blocking the creatine shuttle decreases the energy supply, suppresses oxidative phosphorylation, and blocks ATP delivery to phosphorylation signals, preventing signal transduction. These findings highlight the critical role of the creatine shuttle in cancer cells and suggest a potential new cancer treatment target.

A Zeb1/MtCK1 metabolic axis controls osteoclast activation and skeletal remodeling.

Osteoclasts are bone-resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc-finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial-mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1-null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat-like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP-buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo.

Characterization and tissue expression analysis of mitochondrial creatine kinases (types I and II) from Pelodiscus sinensis.

The aim of this study was to characterize the functions of the mitochondrial creatine kinases in the Chinese soft-shelled turtle Pelodiscus sinensis (PSCK-MT1 and PSCK-MT2) to characterize function in relation to hibernation. Computational prediction via molecular dynamics simulations showed that PSCK-MT1 had stronger kinase- and creatine-binding affinity than PSCK-MT2. We measured PSCK-MT1 and PSCK-MT2 levels in the myocardium, liver, spleen, lung, kidney, and ovary of P. sinensis before and after hibernation and found that the expression of these enzymes was the most significantly upregulated in the ovary. We enumerated the ovarian follicles and evaluated the physiological indices of P. sinensis and discovered that fat was the main form of energy storage in P. sinensis. Moreover, both PSCK-MTs promoted follicular development during hibernation. Immunohistochemistry was used to study follicular development and revealed that both PSCK-MTs were expressed primarily in the follicular fluid and granulosa layer before and after hibernation. We found that PSCK-MT1 and PSCK-MT2 could play important roles in ovarian follicular development under hibernation. Hence, both PSCK-MTs probably function effectively under the conditions of low temperature and oxygen during hibernation. Communicated by Ramaswamy H. Sarma.

Ubiquitous mitochondrial creatine kinase promotes the progression of gastric cancer through a JNK-MAPK/JUN/HK2 axis regulated glycolysis.

BACKGROUND: Ubiquitous mitochondrial creatine kinase (uMtCK) transfers high-energy phosphates from mitochondrially generated ATP to creatine to generate phosphocreatine. uMtCK overexpression has been reported in several malignant tumors, however, the clinical significance and impact of uMtCK in gastric cancer (GC) has not been comprehensively studied. METHODS: We first examined uMtCK expression in GC by quantitative real-time PCR and western blot assays. Then the clinicopathological significance of aberrant uMtCK expression was determined by immunohistochemical staining in a GC tissue microarray. Kaplan-Meier analysis was used for survival analysis. The biological functions of uMtCK in GC cells were explored by wound-healing, transwell assays and glucose metabolism assays in vitro as well as a liver metastasis model by spleen injection in nude mice in vivo. RESULTS: We verified that the expression of uMtCK was substantially elevated in GC tissues, significantly associating with a poorer prognosis in GC patients, especially for those with advanced stage. In univariate and multivariate analyses, uMtCK expression emerged as an independent prognostic factor for both disease-free survival and overall survival. Functionally, we demonstrated that uMtCK promoted glycolysis in GC cells and facilitated their migration, invasion and liver metastasis in vitro and in vivo. Mechanistically, uMtCK enhanced GC progression in a HK2-dependent glycolysis via acting the JNK-MAPK/JUN signaling pathway. CONCLUSIONS: uMtCK could serve as a novel independent prognostic biomarker as well as potential therapeutic target for GC patients, particularly for GC patients with an advanced UICC stage and tumor recurrence.

Interaction between glycolysis‒cholesterol synthesis axis and tumor microenvironment reveal that gamma-glutamyl hydrolase suppresses glycolysis in colon cancer.

Background: Metabolic reprogramming is a feature of cancer. However, colon cancer subtypes based on the glycolysis‒cholesterol synthesis axis have not been identified, and little is known about connections between metabolic features and the tumor microenvironment. Methods: Data for 430 colon cancer cases were extracted from The Cancer Genome Atlas, including transcriptome data, clinical information, and survival outcomes. Glycolysis and cholesterol synthesis-related gene sets were obtained from the Molecular Signatures Database for a gene set variation analysis. The relationship between the genomic landscape and immune landscape were investigated among four metabolic subtypes. Hub genes were determined. The clinical significance of candidate hub gene was evaluated in 264 clinical samples and potential functions were validated in vitro and in vivo. Results: Colon cancer cases were clustered into four metabolic subtypes: quiescent, glycolytic, cholesterogenic, and mixed. The metabolic subtypes differed with respect to the immune score, stromal score, and estimate score using the ESTIMATE algorithm, cancer-immunity cycle, immunomodulator signatures, and signatures of immunotherapy responses. Patients in the cholesterogenic group had better survival outcomes than those for other subtypes, especially glycolytic. The glycolytic subtype was related to unfavorable clinical characteristics, including high mutation rates in TTN, APC, and TP53, high mutation burden, vascular invasion, right colon cancer, and low-frequency microsatellite instability. GGH, CACNG4, MME, SLC30A2, CKMT2, SYN3, and SLC22A31 were identified as differentially expressed both in glycolytic-cholesterogenic subgroups as well as between colon cancers and healthy samples, and were involved in glycolysis‒cholesterol synthesis. GGH was upregulated in colon cancer; its high expression was correlated with CD4+ T cell infiltration and longer overall survival and it was identified as a favorable independent prognostic factor. The overexpression of GGH in colon cancer-derived cell lines (SW48 and SW480) inhibited PKM, GLUT1, and LDHA expression and decreased the extracellular lactate content and intracellular ATP level. The opposite effects were obtained by GGH silencing. The phenotype associated with GGH was also validated in a xenograft nude mouse model. Conclusions: Our results provide insight into the connection between metabolism and the tumor microenvironment in colon cancer and provides preliminary evidence for the role of GGH, providing a basis for subsequent studies.

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Post-COVID Complications after Pressure Ulcer Surgery in Patients with Spinal Cord Injury Associate with Creatine Kinase Upregulation in Adipose Tissue.

The risk of complications following surgical procedures is significantly increased in patients with SARS-CoV-2 infection. However, the mechanisms underlying these correlations are not fully known. Spinal cord injury (SCI) patients who underwent reconstructive surgery for pressure ulcers (PUs) before and during the COVID-19 pandemic were included in this study. The patient's postoperative progression was registered, and the subcutaneous white adipose tissue (s-WAT) surrounding the ulcers was analyzed by proteomic and immunohistochemical assays to identify the molecular/cellular signatures of impaired recovery. Patients with SCI and a COVID-19-positive diagnosis showed worse recovery and severe postoperative complications, requiring reintervention. Several proteins were upregulated in the adipose tissue of these patients. Among them, CKMT2 and CKM stood out, and CKM increased for up to 60 days after the COVID-19 diagnosis. Moreover, CKMT2 and CKM were largely found in MGCs within the s-WAT of COVID patients. Some of these proteins presented post-translational modifications and were targeted by autoantibodies in the serum of COVID patients. Overall, our results indicate that CKMT2, CKM, and the presence of MGCs in the adipose tissue surrounding PUs in post-COVID patients could be predictive biomarkers of postsurgical complications. These results suggest that the inflammatory response in adipose tissue may underlie the defective repair seen after surgery.

Mitochondrial Creatine Kinase Attenuates Pathologic Remodeling in Heart Failure.

BACKGROUND: Abnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF. METHODS: First, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice. RESULTS: In people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling. CONCLUSIONS: In the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

System analysis of VEGFA in renal cell carcinoma: The expression, prognosis, gene regulation network and regulation targets.

BACKGROUND: VEGFA is one of the most important regulators of angiogenesis and plays a crucial role in cancer angiogenesis and progression. Recent studies have highlighted a relationship between VEGFA expression and renal cell carcinoma occurrence. However, the expression level, gene regulation network, prognostic value, and target prediction of VEGFA in renal cell carcinoma remain unclear. Therefore, system analysis of the expression, gene regulation network, prognostic value, and target prediction of VEGFA in patients with renal cell carcinoma is of great theoretical significance as there is a clinical demand for the discovery of new renal cell carcinoma treatment targets and strategies to further improve renal cell carcinoma treatment efficacy. METHODS: This study used multiple free online databases, including cBioPortal, TRRUST, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, Metascape, and TIMER for the abovementioned analysis. RESULTS: VEGFA was upregulated in patients with kidney renal clear cell carcinoma (KIRC) and kidney chromophobe (KICH), and downregulated in patients with kidney renal papillary cell carcinoma (KIRP). Moreover, genetic alterations of VEGFA were found in patients with renal cell carcinoma as follows: 4% (KIRC), 8% (KICH), and 4% (KIRP). The promoter methylation of VEGFA was lower and higher in patients with clinical stages of KIRC and stage 1 KIRP, respectively. VEGFA expression significantly correlated with KIRC and KIRP pathological stages. Furthermore, patients with KICH and KIRP having low VEGFA expression levels had a longer survival than those having high VEGFA expression levels. VEGFA and its neighboring genes functioned in the regulation of protein methylation and glycosylation, as well as muscle fiber growth and differentiation in patients with renal cell carcinoma. Gene Ontology enrichment analysis revealed that the functions of VEGFA and its neighboring genes in patients with renal cell carcinoma are mainly related to cell adhesion molecule binding, catalytic activity, acting on RNA, ATPase activity, actin filament binding, protease binding, transcription coactivator activity, cysteine-type peptidase activity, and calmodulin binding. Transcription factor targets of VEGFA and its neighboring genes in patients with renal cell carcinoma were found: HIF1A, TFAP2A, and ESR1 in KIRC; STAT3, NFKB1, and HIPK2 in KICH; and FOXO3, TFAP2A, and ETS1 in KIRP. We further explored the VEGFA-associated kinase (ATM in KICH as well as CDK1 and AURKB in KIRP) and VEGFA-associated microRNA (miRNA) targets (MIR-21 in KICH as well as MIR-213, MIR-383, and MIR-492 in KIRP). Furthermore, the following genes had the strongest correlation with VEGFA expression in patients with renal cell carcinoma: NOTCH4, GPR4, and TRIB2 in KIRC; CKMT2, RRAGD, and PPARGC1A in KICH; and FLT1, C6orf223, and ESM1 in KIRP. VEGFA expression in patients with renal cell carcinoma was positively associated with immune cell infiltration, including CD8+T cells, CD4+T cells, macrophages, neutrophils, and dendritic cells. CONCLUSIONS: This study revealed VEGFA expression and potential gene regulatory network in patients with renal cell carcinoma, thereby laying a foundation for further research on the role of VEGFA in renal cell carcinoma occurrence. Moreover, the study provides new renal cell carcinoma therapeutic targets and prognostic biomarkers as a reference for fundamental and clinical research.

CKMT1B is a potential prognostic biomarker and associated with immune infiltration in Lower-grade glioma.

BACKGROUND: Lower-grade glioma (LGG) is the most common histology identified in glioma. CKMT1B has not been investigated in glioma. The purpose of this research was to investigate the prognostic value of CKMT1B and its correlation with immune infiltration in LGG. METHODS: We used Gene Expression Profiling Interactive Analysis (GEPIA) to analyze the expression of CKMT1B in LGG. Univariate and multivariate Cox regression analyses were used to assess the effect of CKMT1B expression and screened variables on survival. The correlation between CKMT1B and immune infiltration was evaluated by TIMER and CIBERSORT. Moreover, the possible biological functions of CKMT1B were studied by GSEA. The statistical analysis was conducted by R software. RESULTS: The expression of CKMT1B was significantly lower than the normal samples in LGG. Low expression of CKMT1B predicts a worse prognosis. Multivariate Cox analyses revealed that CKMT1B might be an independent favorable prognostic indicator. TIMER analysis revealed that CKMT1B expression level was related to immune infiltration. CIBERSORT analysis showed that CKMT1B expression was positively related to the infiltration level of activated mast cells and negatively related to macrophage M2 in LGG. Moreover, GESA showed that multiple cancer-related and immune-related gene sets were enriched in the low-CKMT1B group in the top 5 of the most significant differences. CONCLUSION: CKMT1B is a prognostic biomarker with potential applications and associated with immune infiltration in Lower-grade glioma.

A Small-Molecule Approach to Restore a Slow-Oxidative Phenotype and Defective CaMKIIß Signaling in Limb Girdle Muscular Dystrophy.

Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIß signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII (Myl2) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo, including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIß signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1.

Zbtb20 deficiency causes cardiac contractile dysfunction in mice.

The zinc-finger protein ZBTB20 regulates development and metabolism in multiple systems, and is essential for postnatal survival in mice. However, its potential role in the cardiovascular system remains undefined. Here, we demonstrate that ZBTB20 is critically involved in the regulation of cardiac contractility and blood pressure in mice. At the age of 16 days, the relatively healthy Zbtb20-null mice exhibited hypotension without obvious change of heart rate or other evidence for heart failure. Moreover, Zbtb20 deletion led to a marked reduction in heart size, left ventricular wall thickness, and cell size of cardiomyocytes, which was largely proportional to the decreased body growth. Notably, echocardiographic and hemodynamic analyses showed that cardiac contractility was greatly impaired in the absence of ZBTB20. Mechanistically, ZBTB20 deficiency decreased cardiac ATP contents, and compromised the enzyme activity of mitochondrial complex I in heart as well as L-type calcium current density in cardiomyocytes. Furthermore, the developmental activation of some mitochondrial function-related genes was significantly attenuated in Zbtb20-null myocardium, which included Hspb8, Ckmt2, Cox7a1, Tfrc, and Ogdhl. Put together, these results suggest that ZBTB20 plays a crucial role in the regulation of heart development, energy metabolism, and contractility.

The relationship between skin ultrasound images and muscle damage using skin blotting in wheelchair basketball athletes.

STUDY DESIGN: Secondary analysis of a cross-sectional observation study. OBJECTIVES: To determine the relationship between skin ultrasound images and muscle damage in wheelchair basketball athletes, using skin blotting examinations of the ischial regions. SETTING: Community, Japan. METHODS: Fourteen elite wheelchair basketball athletes were recruited. We obtained data regarding participants' characteristics. We undertook ultrasonographic images and quantitative skin blotting of the ischial region before and after training, and after rest. RESULTS: We identified Category II and III pressure injuries in 2 of the 12 participants. Structural features were classified into four categories based on ultrasonographic features, namely, normal skin structure, unclear superficial and deep fascia, cloudy fat layer, and fat infiltration and low-echoic lesion/anechoic lesions. The muscle-type creatinine kinase (CK-M) level (median [interquartile range: IQR], 2.98 [2.80-3.47]) in the fat infiltration and low-echoic lesion/anechoic lesion group was significantly higher (1.43 [1.41-1.49]) than in a nonfat infiltration and low-echoic lesion/anechoic lesion group after training (p = 0.03). The interleukin-6 (IL-6) level (median [IQR], 23.5 [16.15-58.97]) in the fat infiltration and low-echoic lesion/anechoic lesion group was significantly higher (1.94 [1.74-4.44]) than in the nonfat infiltration and low-echoic lesion/anechoic lesion group after rest (mean difference = -25.4, 95% CI -61.1 to 10.7, p = 0.03). CONCLUSIONS: The combination of ultrasonographic images and skin blotting using CK-M and IL-6, could detect early deep tissue damage in wheelchair athletes. These techniques could be potentially useful in the treatment and prevention of pressure injuries. SPONSORSHIP: This study was supported in part by YAMAHA Motor Foundation for Sports.

Overexpression of mitochondrial creatine kinase preserves cardiac energetics without ameliorating murine chronic heart failure.

Mitochondrial creatine kinase (Mt-CK) is a major determinant of cardiac energetic status and is down-regulated in chronic heart failure, which may contribute to disease progression. We hypothesised that cardiomyocyte-specific overexpression of Mt-CK would mitigate against these changes and thereby preserve cardiac function. Male Mt-CK overexpressing mice (OE) and WT littermates were subjected to transverse aortic constriction (TAC) or sham surgery and assessed by echocardiography at 0, 3 and 6 weeks alongside a final LV haemodynamic assessment. Regardless of genotype, TAC mice developed progressive LV hypertrophy, dilatation and contractile dysfunction commensurate with pressure overload-induced chronic heart failure. There was a trend for improved survival in OE-TAC mice (90% vs 73%, P = 0.08), however, OE-TAC mice exhibited greater LV dilatation compared to WT and no functional parameters were significantly different under baseline conditions or during dobutamine stress test. CK activity was 37% higher in OE-sham versus WT-sham hearts and reduced in both TAC groups, but was maintained above normal values in the OE-TAC hearts. A separate cohort of mice received in vivo cardiac 31P-MRS to measure high-energy phosphates. There was no difference in the ratio of phosphocreatine-to-ATP in the sham mice, however, PCr/ATP was reduced in WT-TAC but preserved in OE-TAC (1.04 ± 0.10 vs 2.04 ± 0.22; P = 0.007). In conclusion, overexpression of Mt-CK activity prevented the changes in cardiac energetics that are considered hallmarks of a failing heart. This had a positive effect on early survival but was not associated with improved LV remodelling or function during the development of chronic heart failure.

Distribution and Activity of Mitochondrial Proteins in Vascular and Avascular Retinas: Implications for Retinal Metabolism.

Purpose: Understanding the energetics of retinal neurons and glia is crucial for developing therapies for diseases that feature deficits in nutrient or oxygen availability. Herein, we performed a detailed characterization of the distribution and activity of mitochondrial proteins in the vascularized retinas of rat and marmoset, and the avascular retinas of rabbit and guinea pig. Further, we delineated expression of ubiquitous mitochondrial creatine kinase (uMtCK). Methods: Expression of eight mitochondrial proteins was investigated using Western blotting, single- and double-labeling immunohistochemistry. Activities of cytochrome c oxidase, succinate dehydrgogenase, and isocitrate dehydrogenase were determined by enzyme histochemistry using unfixed tissue sections. Results: In vascularized retinas, immunoreactivities were characterized by strong, punctate labeling in the plexiform layers, photoreceptor inner segments, somas of various cell types, notably retinal ganglion cells (RGCs), and the basolateral surface of the retinal pigment epithelium. In avascular retinas, immunoreactivities featured intense labeling of inner segments, together with weak, but unambiguous, staining of both plexiform layers. RGCs were relatively enriched. In Müller cells of avascular retinas, mitochondria were restricted to scleral-end processes. For each species, enzyme activity assays yielded similar results to the protein distributions. Labeling for uMtCK in vascular and avascular retinas was fundamentally similar, being restricted to neuronal populations, most notably inner segments and RGCs. Of all of the mitochondrial proteins, uMtCK displayed the strongest labeling in avascular retinas. uMtCK was not detectable in Müller cells in any species. Conclusions: The current findings advance our understanding of the metabolic similarities and differences between vascular and avascular retinas.

Exercise-induced reductions in mitochondrial ADP sensitivity contribute to the induction of gene expression and mitochondrial biogenesis through enhanced mitochondrial H2O2 emission.

Acute exercise rapidly induces mitochondrial gene expression, however, the intracellular events regulating this process remain incompletely understood. The purpose of this study was to determine whether reductions in mitochondrial ADP sensitivity during exercise have a biological role in regulating mitochondrial-derived reactive oxygen species (ROS) production and the induction of mitochondrial biogenesis. Mitochondrial creatine kinase wildtype (WT) and knockout (KO) mice have divergent responses in ADP sensitivity during exercise, and we therefore used these mice to determine the relationship between mitochondrial ADP sensitivity, ROS production, and mitochondrial adaptations to exercise. In WT mice, acute exercise reduced mitochondrial ADP respiratory sensitivity and the ability of ADP to suppress ROS production, while increasing mitochondrial gene transcription (PGC-1α, PGC-1ß and PDK4). In stark contrast, in KO mice, exercise increased ADP sensitivity, reduced mitochondrial ROS emission, and did not induce gene transcription. Despite the divergence in mRNA responses, exercise similarly induced calcium/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK) phosphorylation in WT and KO mice, however only WT mice were associated with redox stress (4HNE). These data implicate acute changes in ADP sensitivity in mitochondrial adaptations to exercise. To further examine this we chronically exercise trained mice. While training increased mitochondrial content and reduced ADP sensitivity in WT mice, KO mice did not exhibit adaptations to exercise. Combined, these data suggest that exercise-induced attenuations in mitochondrial ADP sensitivity mediate redox signals that contribute to the induction of acute and chronic mitochondrial adaptations.

Involvement of the phosphoryl transfer network on cardiac energetic metabolism during Staphylococcus aureus infection and its association to disease pathophysiology.

Evidences have suggested that the phosphoryl transfer network by the enzymatic activities of creatine kinase (CK), adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), shows new perspectives to understand some disturbances in the energy metabolism during bacterial infections. Thus, the aim of this study was to evaluate whether Staphylococcus aureus infection in mice could alter serum and cardiac activities of these enzymes and their association to disease pathophysiology. For that, we measured total leukocytes, lymphocytes and neutrophils (just 48 h of infection) that were lower in infected animals after 48 and 72 h in infected mice compared with negative control, while total protein and globulin plasma levels were higher after 72 h of infection. The serum CK activity was higher in infected animals 48 and 72 h post-infection compared to the control group, as well as observed for mitochondrial cardiac CK activity. The serum PK activity was higher in infected animals after 72 h of infection compared to the control group, and lower in the cardiac tissue. The cardiac AK activity was lower in infected animals 48 h and 72 h post-infection compared to the control group, while serum and cardiac LDH activities were higher. Based on these evidences, it is possible to conclude that the stimulation of CK activity exerts a key role as an attempt to maintain the bioenergetic homeostasis by the production of phosphocreatine to avoid a rapid fall on the concentrations of total adenosine triphosphate. In summary, the phosphoryl transfer network can be considered a pathway involved in the improvement on tissue and cellular energy homeostasis of S. aureus-infected mice.

Listeria monocytogenes impairs enzymes of the phosphotransfer network and alters antioxidant/oxidant status in cattle brain structures.

Several evidences have suggested the involvement of enzymes belonging to the phosphotransfer network, formed by creatine kinase (CK), pyruvate kinase (PK) and adenylate kinase (AK), as well the oxidative stress on the pathogenesis of infectious diseases associated with the central nervous system (CNS). Thus, the aim of this study was to evaluate whether listeriosis alters the brain energy metabolism and/or causes oxidative stress in different brain structures of cattle experimentally infected by Listeria monocytogenes. The cytosolic CK activity was inhibited in the cerebral cortex, cerebellum, brainstem and hippocampus of infected animals compared to uninfected animals, while the mitochondrial CK activity was increased. The PK activity was inhibited in all brain structures of infected animals, while the AK activity was unchanged. Na+, K+-ATPase activity decreased in the cerebral cortex, cerebellum and hippocampus of animals infected by L. monocytogenes. Regarding the oxidative strees variables, the cerebellum and brainstem of infected animals showed increased thiobarbituric acid reactive substances, while the catalase activity was inhibited. Glutathione S-transferarase was inhibited in the cerebral cortex and brainstem of infected animals, and it was increased in the cerebellum. L. monocytogenes was quantified in the liver (n = 5/5) and cerebral cortex (n = 4/5) of the infected cattle. Based on these evidences, the nucleocytoplasmic communication between CK isoenzymes was insufficient to avoid an impairment of cerebral bioenergetics. Moreover, the inhibition on brain PK activity caused an impairment in the communication between sites of ATP generation and ATP utilization. The lipid peroxidation and alteration on antioxidant status observed in some brain structures were also involved during the disease. In summary, these alterations contribute to disease pathogenesis linked to CNS during cattle listeriosis.

[Effect of ubiquitous mitochondrial creatine kinase on cisplatin sensitivity of human nasopharyngeal carcinoma cell line CNE-1].

Objective: To investigate the effect of ubiquitous mitochondrial creatine kinase 1(CKMT1) on the sensitivity of human nasopharyngeal carcinoma cell line CNE-1 to DDP. Methods: CNE-1 cells were transiently transfected with CKMT1 overexpression (CKMT1) or empty vector (EV). The growth curve and DDP IC50 were developed by MTT assay, plate clone formation assay was performed by gradient concentration of DDP treatment, cell cycle and apoptosis were detected by flow cytometry, levels of apoptosis related protein Bax/Bcl-2/C-PARP and the transcription factor p-STAT3-Tyr705 were detected by Western Blot. Results: The transfection efficiencies of CKMT1 and EV were more than 90% with a higher proliferation rate in the CKMT1-transfected cells. However, the CKMT1-transfected cells had a DDP IC50 of 2.76 µmol/L, which was significantly lower than that of 4.60 µmol/L in the EV-transfected cells (P<0.01). With the treatment of certain concentration of DDP, the CKMT1-transfected cells had a lower clone formation rate, the cell cycle arrested more obviously in G2/M phase, and the apoptosis rate was higher (P<0.01), with higher levels of Bax/C-PARP (P<0.05 or P<0.01), but lower levels of Bcl-2 (P<0.01) and p-STAT3-Tyr705 (P<0.01), compare with the EV-transfected cells. Conclusions: CKMT1 may inhibit the activation of STAT3, increasing the sensitivity of CNE-1 to chemotherapeutic drug DDP.