Nemutlu, Emirhan

Background: Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by recurrent sinopulmonary infections due to motile cilia defects. The disease is genetically heterogeneous, with abnormalities in structural ciliary proteins. Zinc finger MYND-type containing 10 (ZMYND10) is essential for the assembly of outer dynein arms (ODA), with chaperones like Glucose-regulated protein 78 (GRP78) facilitating protein folding. This study investigates ZMYND10 and Dynein axonemal heavy chain 5 (DNAH5) mutations in individuals with PCD. Methods: Eight individuals aged 14–22 with clinical PCD symptoms and confirmed DNAH5 mutations were included. We analyzed the correlation between DNAH5 abnormalities and preassembly/chaperone proteins using immunofluorescence labeling. Nasal swabs were double-labeled (DNAH5–β-tubulin, β-tubulin–ZMYND10, β-tubulin–GRP78) and examined via fluorescence microscopy. Serum metabolomics and proteomics were also assessed. Results: The corrected total cell fluorescence (CTCF) levels of DNAH5, ZMYND10, and GRP78 were significantly different between PCD individuals and controls. Metabolomic analysis showed reduced valine, leucine, and isoleucine biosynthesis, with increased malate and triacylglycerol biosynthesis, malate-aspartate and glycerol phosphate shuttles, and arginine/proline metabolism, suggesting mitochondrial and ER stress. Conclusions: The altered expression of DNAH5, ZMYND10, and GRP78, along with metabolic shifts, points to a complex link between ciliary dysfunction and cellular stress in PCD. Further studies are needed to clarify the underlying mechanisms.

The mammalian heart loses its regenerative potential within a week after birth. Although under certain circumstances adult cardiomyocytes can re-enter the cell cycle, initiate DNA synthesis and progress towards mitosis, however, they do not complete a conventional mitosis cycle and proceed towards endomitosis resulting in polyploidy. This suggests that high-energy dependent steps in cell cycle and myofibrillar disassembly are malfunctioning in adult cardiomyocytes. It is known that nucleotide-based energetic signaling and metabolic environment are critical for initiation of tissue regeneration after injury. The regulatory mechanisms and energetic-metabolic signaling circuits that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Using mouse neonatal (2 and 10 days) hearts, with high and low regenerative capacity, respectively, we have determined metabolomic profiles and dynamics of phosphotransfer circuits and AMP-signaling using 18O-phosphoryl labeling mass-spectrometric and 18O-assisted 31P NMR techniques. The results demonstrate that loss of heart regenerative capacity 10 days after birth is associated with marked changes in heart metabolome and adenylate kinase (AK)-catalyzed phosphotransfer flux. Among most altered metabolites were putrescine and myo-inositol, involved in tissue regeneration, Krebs cycle and amino acid metabolites, creatinine, glucose-6-phosphate and lactate indicating rearrangements in energy and substrate metabolic systems. AK flux was the only phosphotransfer flux significantly upregulated in hearts at 10 days of age along with increased in mitochondrial and glycolytic capacities and mitochondrial AK2 expression. Increased AK2 expression was associated with augmented AMP phosphorylation, measured by generation of β-ATP[18O], and reduction of AMPK activity. Thus, increased channeling of AMP into mitochondria and reduction of AK-AMP-AMPK signaling axis could contribute to the withdrawal from completion of cell cycle in terminally differentiated adult cardiomyocytes. The uncovered metabolic signaling mechanism opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.

Introduction: Cardiac remodeling in heart failure (HF) is associated with conduction abnormalities and dyssynchrony which results in poorly coordinated contractions precipitating metabolic heterogeneity and energetic inefficiency. Cardiac resynchronization therapy (CRT) improves HF symptoms, ventricular function and overall survival. The aim of this study was to determine the changes in metabolomic profiles and substrate utilization after CRT. Methods: 18 patients with advanced HF and 7 healthy controls were included in this study. Blood samples were collected from peripheral vein, femoral artery (FA) and coronary sinus (CS) before and 10-min after CRT. Metabolomic profiling of plasma samples were performed using GC/MS and 1 H NMR spectroscopy. Partial Least Squares Discriminant Analysis (PLS-DA) score plots and regression coefficients were used to examine metabolomic profiles. Results: HF patients have altered plasma metabolomic profile (Fig. A) with distinct panel of metabolites including free fatty acids (FFA), glycerol, 2-hydroxybutyrate, glycine, succinate and uric acid important in group classification compared to controls. Increased release of fumarate and glycerol-3-phosphate from myocardium in HF indicate mitochondrial and substrate shuttle dysfunction. Immediately after CRT, transcardiac metabolomic profile of plasma from CS shifted to a different state with increased myocardial uptake and metabolism of FFA and decreased release of creatinine indicating improved energetic conditions (Fig. B). Conclusion: Metabolomic profiling indicates that CRT improves myocardial substrate metabolism and utilization in patients with advanced HF.

Evaluation of disease phenotypes and drug effects requires knowledge not only metabolite levels but also their turnover rates from which metabolic fluxes and status of the whole energetic system can be determined. Oxygen-18-assited 31 P NMR and mass spectrometric technique uniquely allow simultaneous measurements of phosphorus-containing metabolite levels and their turnover rates. This includes simultaneous recordings of 18 O-labeling rates reflecting ATP synthesis (γ-ATP) and ATP utilization (Pi), phosphotransfer fluxes through adenylate kinase (β-ATP/ADP), creatine kinase (CrP) and glycolytic pathways (G-6-P) as well as Krebs cycle associated mitochondrial nucleotide turnover (γ/β-GTP), substrate shuttle (G-3-P) and glycogen metabolism (G-1-P). Metabolomic and fluxomic profiling of hearts and body fluids of phosphotransfer enzyme deficient transgenic animals using 18 O-assisted GC/MS, 1 H and 18 O-assisted 31 P NMR indicate selective metabolic perturbations and adaptations in the whole energetic system. Adenylate kinase and creatine kinase deficiencies were associated with altered metabolomic profiles, increased mitochondrial capacities and redistribution of phosphotransfer flux through glycolytic and guanine nucleotide systems. These phosphometabolomic alterations were more expressed when animals were subjected to treadmill exercise, mitochondria-targeting drugs such as metformin and high-fat diet. Metabolite turnover rates are correlated with heart performance unveiling robustness and dynamics rearrangements in cell bioenergetics infrastructure. Glycerophosphate, G-3-P, metabolic dynamics is altered in transgenic animal models indicating defects in substrate shuttle and supply of reducing equivalents to mitochondria. This is of importance since G-3-P metabolic abnormalities and metabolic arrest are linked to human diseases such as sudden death syndrome. Thus, our study demonstrates that phosphometabolomic and fluxomic profiling is a valuable tool for metabolic phenotyping of transgenic animal models of human diseases as well as for plasma biomarker identification and monitoring of treatment efficacy and drug toxicity.

Adenylate kinase 2 (AK2) isoform in mitochondrial intermembrane/intracristal space provides a conduit facilitating ATP export and delivery to cellular ATPases. AK2+/- transgenic mice is viable while homozygous AK2-/- deficiency results in midgestation embryonic lethality. AK2-/- mouse embryonic fibroblasts (MEFs) have severely disrupted mitochondrial cristae structure and display low growth and proliferation potential. Protein knockdown using siRNA indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Transgenic AK2+/- hearts display greater changes in AMP/ATP ratio and AMPK activation during ischemia compared to wild type suggesting deficient tuning of AMP signals. Metabolomic profile of AK2+/- hearts subjected to myocardial ischemia indicate lower ability to maintain adenine and guanine nucleotide pools. AK2+/- mice have abnormal metabolomic response to treadmill exercise compared to wild type. Metabolomic profiling revealed that most significant plasma metabolites critical in the discrimination between wild type and AK2+/- phenotypes were adenosine, tyrosine, tryptophan and hypoxanthine indicating lower energetic potential of AK2+/- mice. AK2+/- hearts have increased mitochondrial creatine kinase CKmit content which could be viewed as a compensatory mechanism. Thus, AK2 is the first phosphotransfer enzyme which deficiency causes embryonic lethality indicating critical significance of catalyzed nucleotide exchange and conductive transfer in the narrow and crowded mitochondrial intermembrane/intracristal space.

Metabolic drugs frequently target mitochondrial functions and AMP signaling. Metformin, one of the most prescribed anti-diabetic medications, exerts its glucose lowering effect through mild interference with mitochondrial energy metabolism triggering homeostatic adenylate kinase (AK)-catalyzed AMP and downstream AMPK signaling. However, metabolomic signature of drug-induced “therapeutic mitochondrial toxicity" and energetic imbalance-induced AMP signaling are poorly understood. This work was aimed to determine metabolomic representation of metformin-induced mitochondrial and AMP signaling stress by comparing wild type and AK1-deficient mice with compromised AMP-signaling. Metabolomic profiling of plasma and urine samples from wild type mice treated with metformin revealed distinct metabolic signature suggesting altered energy, Krebs cycle, lipid and ketone body metabolism, with higher levels of creatinine, malate, α-ketoglutarate, FFA, glycerol, glycerol-3-phosphate, 3-hydroxybutyrate and hypoxanthine. Contrary to expectations glucose lowering effect of metformin was greater in energetically and AMP-signaling compromised AK1-/- mice. Metformin action on AK1 deficient mice was characterized by increased levels of creatinine, lactate, alanine, FFA, phosphoserine, acetoacetate, 3-hydroxybutyrate, malate and α-ketoglutarate indicating broader alterations in energy, glycolytic, Krebs cycle, ketone body, lipid and amino acid metabolism. At baseline, livers from AK1-/- mice had higher AMP/ADP and pAMPKα/AMPKα ratios compared to wild type. There were no significant sustained changes in global nucleotide ratios after metformin treatment. Overall metabolomic profile of AK1-/- mice indicate larger disturbances in energetic and substrate metabolic networks after metformin treatment compared to wild type. Thus, this study revealed specific metabolomic signatures of metformin therapeutic action and mitochondrial toxicity indicating that genetic and metabolic background modulate individual response to metformin treatment.

The mammalian heart loses its regenerative potential within a week after birth. Although under certain circumstances adult cardiomyocytes can re-enter the cell cycle, initiate DNA synthesis and progress towards mitosis, however, they do not complete a conventional mitosis cycle and proceed towards endomitosis resulting in polyploidy. This suggests that high-energy dependent steps in cell cycle and myofibrillar disassembly are malfunctioning in adult cardiomyocytes. It is known that nucleotide-based energetic signaling and metabolic environment are critical for initiation of tissue regeneration after injury. The regulatory mechanisms and energetic-metabolic signaling circuits that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Using mouse neonatal (2 and 10 days) hearts, with high and low regenerative capacity, respectively, we have determined metabolomic profiles and dynamics of phosphotransfer circuits and AMP-signaling using 18O-phosphoryl labeling mass-spectrometric and 18O-assisted 31P NMR techniques. The results demonstrate that loss of heart regenerative capacity 10 days after birth is associated with marked changes in heart metabolome and adenylate kinase (AK)-catalyzed phosphotransfer flux. Among most altered metabolites were putrescine and myo-inositol, involved in tissue regeneration, Krebs cycle and amino acid metabolites, creatinine, glucose-6-phosphate and lactate indicating rearrangements in energy and substrate metabolic systems. AK flux was the only phosphotransfer flux significantly upregulated in hearts at 10 days of age along with increased in mitochondrial and glycolytic capacities and mitochondrial AK2 expression. Increased AK2 expression was associated with augmented AMP phosphorylation, measured by generation of β-ATP[18O], and reduction of AMPK activity. Thus, increased channeling of AMP into mitochondria and reduction of AK-AMP-AMPK signaling axis could contribute to the withdrawal from completion of cell cycle in terminally differentiated adult cardiomyocytes. The uncovered metabolic signaling mechanism opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope18O-assisted31P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The18O labeling procedure is based on the incorporation of one18O into Pifrom [18O]H2O with each act of ATP hydrolysis and the distribution of18O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded18O-labeling procedures has revealed significant differences in the dynamics of G-6-P[18O] (glycolysis), G-3-P[18O] (substrate shuttle), and G-1-P[18O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[18O], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1−/−), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[18O] and decreased G-1-P[18O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit−/−-deficient hearts is characterized by a higher G-6-[18O]P turnover rate, G-6-P levels, glycolytic capacity, γ/β-phosphoryl of GTP[18O] turnover, as well as β-[18O]ATP and β-[18O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus,18O-assisted gas chromatography-mass spectrometry and31P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.