Regulate ATP Production Research Articles

Inhibition of mitochondrial cyclophilin D, a downstream target of glycogen synthase kinase 3α, improves sperm motility.

Cyclophilin D (CypD) negatively regulates ATP production by opening of the mitochondrial permeability transition pore. This study aimed to understand the role of CypD in sperm motility regulation. Changes in CypD during sperm capacitation and its interaction with glycogen synthase kinase 3α (GSK3α), a key kinase regulating sperm motility, were examined in mouse spermatozoa. The effects of CypD inhibitor cyclosporin A (CsA) and GSK3 inhibitor 6-bromo-indirubin-3'-oxime (BIO) on sperm motility, p-GSK3α(Ser21), mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), and ATP production were examined. The effect of proteasome inhibitor MG115 on the cellular levels of CypD was examined. In cauda epididymal spermatozoa, GSK3α was found in both cytosolic and mitochondrial fractions whereas CypD was primarily found in the mitochondrial fraction together with ATP synthase F1 subunit alpha (ATP5A), a mitochondrial marker. GSK3α and CypD were co-localized in the sperm midpiece. Interaction between GSK3α and CypD was identified in co-immunoprecipitation. CsA, a CypD inhibitor, significantly increased sperm motility, tyrosine phosphorylation, mPTP closing, MMP, and ATP levels in spermatozoa, suggesting that CypD acts as a negative regulator of sperm function. Under capacitation condition, both GSK3α and CypD were decreased in spermatozoa but ATP5A was not. The GSK3 inhibitor BIO markedly increased p-GSK3α(Ser21) and decreased CypD but significantly increased mPTP closing, MMP, ATP production, and motility of spermatozoa. This suggests that inhibitory phosphorylation of GSK3α is coupled with degradation of CypD, potentiating the mitochondrial function. Degradation of CypD was attenuated by MG115, indicative of involvement of the ubiquitin proteasome system. During sperm capacitation, CypD act as a downstream target of GSK3α can be degraded via the ubiquitin proteasome system, stimulating mitochondrial function and sperm motility.

Abstract 19126: Discovery of TMEM65 as a Positive Regulator of Mitochondrial Calcium Efflux

Introduction: The balance between mitochondrial calcium ( m Ca 2+ ) uptake and efflux regulates ATP production, but if perturbed causes m Ca 2+ depletion and energy starvation or m Ca 2+ overload and cell death. Disrupted m Ca 2+ homeostasis is implicated in ischemia-reperfusion injury, heart failure, and neurodegeneration. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of m Ca 2+ efflux in excitable tissues, supporting NCLX as a promising therapeutic target to limit pathogenic m Ca 2+ overload in organs like the heart and brain. However, a critical barrier to translation is that little is known about the mechanisms controlling NCLX activity. Goals: We used proximity biotinylation screening to identify the NCLX interactome and proteins that may regulate NCLX function. We identified TMEM65, a mitochondrial inner membrane protein of unknown function, as an NCLX-proximal protein. Hypothesis: Null mutation of TMEM65 causes mitochondrial encephalomyopathy and phenocopies genetic NCLX disruption. Therefore, we hypothesized that TMEM65 modulates m Ca 2+ efflux through NCLX. Approach: Here, we used measurements of m Ca 2+ exchange in AC16 cardiomyocytes with TMEM65 overexpression (OE) or CRISPR/Cas9 genetic disruption of TMEM65 to test TMEM65’s effect on m Ca 2+ flux and its functional dependence on NCLX. We used AAV-mediated Tmem65 knockdown (KD) and TMEM65 OE in mice to assess the effects of gain- or loss of TMEM65 expression on cardiac function. Results: TMEM65 OE potently enhanced m Ca 2+ efflux in vitro , and NCLX inhibition with CGP-37157 mitigated this effect. Knockout of TMEM65 attenuated m Ca 2+ efflux. In vivo Tmem65 KD reduced left ventricular fractional shortening and reduced S293 phosphorylation of pyruvate dehydrogenase in the heart, indicative of increased m Ca 2+ loading. In vivo TMEM65 OE did not alter cardiac function. Conclusions: We conclude that TMEM65 has an essential function to promote m Ca 2+ efflux, likely via positive regulation of NCLX, and is critical to limiting deleterious m Ca 2+ overload in the intact heart. Ongoing work is defining the functional interactions between TMEM65 and NCLX, and establishing the utility of targeting TMEM65 to restore m Ca 2+ homeostasis in heart failure and other excitable tissue diseases.

Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia.

Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. Here, we identify an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. We determined that Drosophila tweety (tty) is highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. We performed live-cell imaging of genetically encoded metabolite sensors to determine the impact of tty and autophagy deficiencies on glial bioenergetics. We found that tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, our findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia.

IGF-1 Stimulates Glycolytic ATP Production in MCF-7L Cells.

The Insulin-like Growth Factor (IGF) system in breast cancer progression has been a matter of interest for decades, but targeting this system did not result in a successful clinical strategy. The system's complexity and homology of its two receptors-insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R)-are possible causes. The IGF system maintains cell proliferation and also regulates metabolism, making it a pathway to explore. To understand the metabolic phenotype of breast cancer cells, we quantified their real-time ATP production rate upon acute stimulation with ligands-insulin-like growth factor 1 (1GF-1) and insulin. MCF-7L cells express both IGF-1R and IR, while tamoxifen-resistant MCF-7L (MCF-7L TamR) cells have downregulated IGF-1R with unchanged IR levels. Treating MCF-7L cells with 5 nM IGF-1 increased the glycolytic ATP production rate, while 10 nM insulin did not affect metabolism when compared with the control. Neither treatment altered ATP production in MCF-7L TamR cells. This study provides evidence of the relationship between metabolic dysfunction, cancer, and the IGF axis. In these cells, IGF-1R, and not IR, regulates ATP production.

P112: ENERGIZE AND ENERGIZE-T: TWO PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDIES OF MITAPIVAT IN ADULTS WITH NON--TRANSFUSION-DEPENDENT OR TRANSFUSION-DEPENDENT ALPHA- OR BETA-THALASSEMIA

Background: Thalassemias are characterized by imbalanced globin-chain production resulting in excess α- or β-globin precipitation, hemolytic anemia, and ineffective erythropoiesis.1,2 ATP levels are reduced in thalassemic red blood cells (RBCs), despite increased energy demands.3,4 Mitapivat is an oral activator of RBC pyruvate kinase (PKR), a glycolytic enzyme that regulates ATP production.5 In a phase 2 study of patients with α- or β-non–transfusion-dependent thalassemia (NTDT), twice-daily (BID) dosing with mitapivat increased hemoglobin (Hb) levels by ≥1.0 g/dL in 80% of patients,6 supporting the broadening of mitapivat’s development in thalassemia. Aims: To report the study designs of ENERGIZE (2021-000211-23) and ENERGIZE-T (2021-000212-34), two phase 3 trials to assess the efficacy and safety of mitapivat in adults with α- or β-NTDT or transfusion-dependent thalassemia (TDT), respectively. Methods: Both studies are phase 3, multicenter, randomized, double-blind, placebo-controlled trials (Figure). In ENERGIZE, approximately 171 eligible adults with NTDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 24 weeks. Upon completion, eligible patients can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (β-thalassemia ± α-globin mutations, Hb E β-thalassemia, or α-thalassemia [Hb H disease]), Hb concentration ≤10.0 g/dL, and NTDT defined as ≤5 RBC units during the 24-week period before randomization and no RBC transfusion ≤8 weeks prior. The primary endpoint is an Hb response defined as a ≥1.0 g/dL increase in average Hb concentration from Week 12 through 24 compared with baseline. Secondary endpoints include patient-reported outcomes, changes in Hb, markers of hemolysis and erythropoiesis, and safety. In ENERGIZE-T, approximately 240 eligible adults with TDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 48 weeks. Upon completion, eligible patients can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (same genotypes as detailed for the ENERGIZE study), and TDT defined as 6–20 RBC units transfused and no transfusion-free period ≥6 weeks during the 24 weeks before randomization. The primary endpoint is a transfusion reduction response, defined as a ≥50% reduction in transfused RBC units with a reduction of ≥2 units of transfused RBCs in any consecutive 12-week period through Week 48 compared with baseline. Secondary endpoints include additional measures of transfusion burden, changes in iron markers, and safety. Results: Not yet available. Conclusions: ENERGIZE and ENERGIZE-T are the first pivotal studies to assess a potential treatment across a broad spectrum of patients with thalassemia (ie, patients with TDT and NTDT; α- and β-thalassemias). ENERGIZE and ENERGIZE-T will evaluate the efficacy and safety of mitapivat, a novel, first-in-class oral activator of PKR. Both studies are actively recruiting.

Calcium influx through the mitochondrial calcium uniporter holocomplex, MCUcx

Ca2+ flux into the mitochondrial matrix through the MCU holocomplex (MCUcx) has recently been measured quantitatively and with milliseconds resolution for the first time under physiological conditions in both heart and skeletal muscle. Additionally, the dynamic levels of Ca2+ in the mitochondrial matrix ([Ca2+]m) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca2+ into the mitochondrial matrix through MCUcx and efflux through the mitochondrial Na+ / Ca2+ exchanger (NCLX). Under these conditions [Ca2+]m was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca2+]m continue to be reported in the literature. Here we review the new findings attributed to MCUcx function and provide a framework for understanding and investigating mitochondrial Ca2+ influx features, many of which remain controversial. The properties and functions of the MCUcx subunits that constitute the holocomplex are challenging to tease apart. Such distinct subunits include EMRE, MCUR1, MICUx (i.e. MICU1, MICU2, MICU3), and the pore-forming subunits (MCUpore). Currently, the specific set of functions of each subunit remains non-quantitative and controversial. The more contentious issues are discussed in the context of the newly measured native MCUcx Ca2+ flux from heart and skeletal muscle. These MCUcx Ca2+ flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca2+ conductances and with distinct extramitochondrial Ca2+ ([Ca2+]i) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca2+]i levels and for hypothesized gatekeeping function and are discussed in the context of model cell (e.g. HeLa, MEF, HEK293, COS7 cells) measurements. Our new findings on MCUcx dependent matrix [Ca2+]m signaling provide a quantitative basis for on-going and new investigations of the roles of MCUcx in cardiac function ranging from metabolic fuel selection, capillary blood-flow control and the pathological activation of the mitochondrial permeability transition pore (mPTP). Additionally, this review presents the use of advanced new methods that can be readily adapted by any investigator to enable them to carry out quantitative Ca2+ measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨm.

An in Situ Atlas of Mitochondrial DNA in Mammalian Tissues Reveals High Content in Stem and Proliferative Compartments

Mitochondria regulate ATP production, metabolism, and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Because most measurements of mtDNA use homogenates of complex tissues, little is known about cell-type-specific mtDNA copy number heterogeneity in normal physiology, aging, and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, an in situ hybridization approach to generate a single-cell-resolution atlas of mtDNA content in mammalian tissues was validated. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared with differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared with cells within glomeruli and collecting duct epithelial cells. In mice, decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. This study provides a novel approach for quantifying species- and cell-type-specific mtDNA copy number and dynamics in any normal or diseased tissue that can be used for monitoring the effects of interventions in animal and human studies.

CCN6 REGULATES MITOCHONDRIAL RESPIRATORY COMPLEX ASSEMBLY AND ACTIVITY

BackgroundCCN6 is a multidomain protein of mesenchymal origin. Mutations in CCN6 lead to PPRD (Progressive Pseudo Rheumatoid Dysplasia), which is associated with cartilage loss, muscle wasting and restricted skeletal development. Mechanism of PPRD pathogenesis and its connection with CCN6 is unclear. Previously we documented that CCN6 localizes to mitochondria and regulates ATP production. Here we have investigated the molecular mechanism of this regulation in context of PPRD.Objective To check whether CCN6 regulates mitochondrial respiratory complex assembly and activity. To check if engineering PPRD causing CCN6 mutations alters the function of the mitochondrial respiratory complex.MethodsCCN6 expression was depleted by siRNA transfection in C28/I2 chondrocyte cell line. CCN6 mutants were generated by CRISPR‐Cas9 technology. CCN6 association with respiratory complex was assessed by 2D BN/SDS PAGE & size‐exclusion chromatography. Mitochondrial respiratory complex activity/assembly was assessed spectrophotometrically and by BN‐PAGE in‐gel assay. Transmission electron microscopy (TEM) was used to study mitochondrial morphology.ResultsSize‐exclusion chromatography and 2D BN/SDS PAGE of mitochondria reveals that CCN6 is associated with Complex I/Super complexes of mitochondrial electron transport chain. Partial depletion of CCN6 by siRNA shows close association of RER with mitochondria and alteration of its mitochondrial distribution relative to cytosol, thereby resulting in increased Complex I activity and assembly. CCN6 mutation (truncation in exon 5, similar to PPRD linked mutation), however, results in disrupted complex I activity and assembly. CCN6 mutation also correlates with distorted mitochondria and mitophagy.ConclusionCCN6 is required for the regulation of mitochondrial respiratory complex activity/assembly in chondrocytes, which involves controlled juxtaposition of RER‐mitochondria. Mutations in CCN6 disrupt respiratory complex activity/assembly and cause mitophagy.Support or Funding InformationThis work is supported by a grant from the Department of Science and Technology, Grant No‐SB/SO/BB‐016/2014 and Department of Biotechnology, Grant No‐BT/PR24244/MED/12/764/2017, Ministry of Science and Technology, Government of India. D.K.P. is supported by the Council of Scientific and Industrial Research (CSIR) fellowship, A.S is supported by DBT‐RA programme and A.G is supported by University Grant Commission (UGC), Government of India.CCN6 migrates as a high molecular weight complex with mitochondrial respiratory complex proteins.CCN6 remains as high molecular weight complex in mitochondria along with subunits of mitochondrial electron transport chain including complex I subunit NDUFB8 as shown by 2D BN‐PAGE/SDS‐PAGE (A) and size exclusion chromatography (B) of mitochondrial lysate from C28/I2 cells (chondrocyte line).Figure 1CCN6 regulates mitochondrial respiratory complex assembly and activity.(A) Increased Complex I assembly and activity was documented by BN‐PAGE western blot with NDUFB8 (Complex I subunit) antibody and in‐gel Complex I activity assay in CCN6 siRNA transfected (CCN6 partially depleted) C‐28/I2 cells (CCN6 si) as compared to control cells (Cont.si). (B) Transmission Electron Microscopy (TEM) micrographs showing decreased RER‐Mitochondria distance in CCN6 siRNA transfected cells. (C) BN‐PAGE western blot with NDUFB8 antibody and in‐gel Complex I activity of CRISPR Cas9 mediated PPRD causing CCN6 mutants shows reduced Complex I/Super complex assembly and activity. (D) TEM micrographs shows presence of mitophagy in CCN6 mutant cells.. RER: Rough endoplasmic reticulum. M: MitochondriaFigure 2

Mitochondrial flashes regulate ATP homeostasis in the heart

The maintenance of a constant ATP level ('set-point') is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart.

Glucocorticoid receptor isoforms direct distinct mitochondrial programs to regulate ATP production.

The glucocorticoid receptor (GR), a nuclear receptor and major drug target, has a highly conserved minor splice variant, GRγ, which differs by a single arginine within the DNA binding domain. GRγ, which comprises 10% of all GR transcripts, is constitutively expressed and tightly conserved through mammalian evolution, suggesting an important non-redundant role. However, to date no specific role for GRγ has been reported. We discovered significant differences in subcellular localisation, and nuclear-cytoplasmic shuttling in response to ligand. In addition the GRγ transcriptome and protein interactome was distinct, and with a gene ontology signal for mitochondrial regulation which was confirmed using Seahorse technology. We propose that evolutionary conservation of the single additional arginine in GRγ is driven by a distinct, non-redundant functional profile, including regulation of mitochondrial function.

Stanniocalcin 1 effects on the renal gluconeogenesis pathway in rat and fish

The mammalian kidney contributes significantly to glucose homeostasis through gluconeogenesis. Considering that stanniocalcin 1 (STC1) regulates ATP production, is synthesized and acts in different cell types of the nephron, the present study hypothesized that STC1 may be implicated in the regulation of gluconeogenesis in the vertebrate kidney. Human STC1 strongly reduced gluconeogenesis from 14C-glutamine in rat renal medulla (MD) slices but not in renal cortex (CX), nor from 14C-lactic acid. Total PEPCK activity was markedly reduced by hSTC1 in MD but not in CX. Pck2 (mitochondrial PEPCK isoform) was down-regulated by hSTC1 in MD but not in CX. In fish (Dicentrarchus labrax) kidney slices, both STC1-A and -B isoforms decreased gluconeogenesis from 14C-acid lactic, while STC1-A increased gluconeogenesis from 14C-glutamine. Overall, our results demonstrate a role for STC1 in the control of glucose synthesis via renal gluconeogenesis in mammals and suggest that it may have a similar role in teleost fishes.

Abstract 663: The Effects of Hypoxia and Metabolic Inhibitors on Cellular ATP and ATP/ADP ratio in Human Coronary Artery Smooth Muscle cells

Background: Although ATP is principally considered as the body’s energy currency, it is also an important signaling molecule. As availability of oxygen is an important factor in the production of ATP, it is simply assumed that the latter is tightly regulated by the former. However, how oxygen tension over the physiological range may regulate ATP production is still unclear. Here, we address the fundamental question of whether changes in environmental oxygen tension over the physiological range result in significant changes in cellular ATP in Human Coronary Artery Smooth Muscle cells (HCASMCs). Methods: Cells were seeded 24-48 hours before cellular ATP was measured in the cell population using CellTiter-Glo ® Luminescent Cell Viability Assay according to manufacturer’s instruction (n=4-6). Percent change of cellular ATP was determined and normalized to control (untreated). For the change of ATP/ADP ratio, single cells were transient transfected with Perceval or PercevalHR, fluorescent reporters of ATP/ADP ratio. The fluorescent signal was collected in a confocal microscope (LSM510) under different experimental conditions, including hypoxia. Results: Application of metabolic inhibitors for 10 minutes decreased the ATP to 104.6%±2.0% (5 mM 2-Deoxy-D-glucose/2DG),103.1% ±2.9% (6μM Oligomycin/Oligo) and 84.4%±2.7% (2DG+ Oligo) in 10 mM Glucose physiological saline (PSS); and 55.3%±0.3% (5 mM 2DG), 92.4%±3.7% (6μM Oligo) and 36.7%±2.7% (2DG+ Oligo) in glucose free PSS. Hypoxia for 10 minutes also caused a reduction in ATP to 67.0%±1.6% (10%O 2 ), 52.7%±0.6% (5%O 2 ) and 36.2%±1.8% (1%O 2 ) in 10 mM glucose PSS; 71.3%±2.4% (10%O 2 ), 57.9%±1.1% (5%O 2 ) and 40.4%±3.2% (1%O 2 ) in glucose free PSS. Inhibiting metabolism with 2DG in single cells resulted in a decrease in Perceval signal by 50.2±14.4% (n=3). Transient hypoxia (1%O 2 ) also induced a significant reduction in Perceval signal by 19.3%±5.3% (n=3). By inhibiting the electron transport chain for 10 mins, we also observed PercevalHR signal declined by 43.6%±3.6% (1μM Rotenone, n=3), 33.0%±11.4% (1μM Antimycin, n=4), 39.5%±9% (6μM Oligo, n=3) and 16.9%±4.4% (1μM CCCP, n=3). Conclusions: Our studies suggest hypoxia causes a reduction in cellular ATP and a decrease in the ATP/ADP ratio in HCASMCs.

An introduction to the Bioscience Birthday Symposium held in honour of Ole Petersen CBE, FRS

An introduction to the Bioscience Birthday Symposium held in honour of Ole Petersen CBE, FRS

The Drosophila inner-membrane protein PMI controls cristae biogenesis and mitochondrial diameter

Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Cardiac Pacemaker Cells Uniquely Match ATP Supply to Demand

Sinoatrial node cells (SANC) within the heart's pacemaker govern the heart rhythm and contraction due to constitutively active, Ca2+-activated adenylyl-cyclases (AC) that generate a high level of cAMP/protein kinase A (PKA) dependent, localized, submembrane-compartmentalized Ca2+- and AC-cycling. This rhythmic Ca2+ cycling generates rhythmic action potentials (APs), which initiate Ca2+ release and SANC contraction. To drive the heart beat SANC must tightly regulate ATP production to supply sufficient ATP for cAMP production, Ca2+-cycling and contraction. To explore how ATP supply-demand is tightly balanced in isolated SANC we measured ATP levels: in control; during PKA-inhibition by specific inhibitor peptide PKI (15μM) or by H-89 (6μM); during intracellular Ca2+-buffering by BAPTA (25μM); during muscarinic-receptors (MR) activation by carbacol 1μM±MR-inhibition by atropine (10μM). PKA-inhibition by PKI or H-89, which blocked the major ATP-consumption processes, depleted ATP by 45±6% and 44±5%, respectively. Thus, complete inhibition of these major ATP consumption processes in SANC significantly reduces ATP levels even though ATP consumption is reduced. Buffering intracellular Ca2+ depleted ATP by 54±8%. Hence, Ca2+ not only has direct effects on the surface membrane potential to ignite APs, and to activate myofilament-displacement/force production, but also regulates ATP production. In contrast to SANC, in either stimulated (3Hz) or quiescent rabbit ventricular cells the same experimental perturbations had only minor (<6%) effects on ATP levels. These control mechanisms identified by pharmacological-perturbations are apparently utilized in nature, because MR-stimulation of SANC also depleted the ATP by 45±10% (by blocking the ATP-consumption processes). Notably, atropine, a MR-antagonist, substantially reversed the MR-stimulation effect, resulting in only 18±3% ATP depletion. These data suggest that in contrast to ventricular cells, the same signals that drive, and are derived from, the utilization of ATP in SANC also tightly couple the production of ATP to match energy demand.

Uridine-5′-triphosphate Protects Against Hepatic- Ischemic/Reperfusion Injury in Mice

Mitochondrial calcium overload triggers apoptosis and also regulates ATP production. ATP and uridine-5'-triphosphate (UTP) depletion from hepatic tissue after ischemia causes cell death. ATP and UTP binds to cell membranes of the hepatocytes through P2Y receptors. Our aim was to investigate the role of UTP on the hepatic injury induced by ischemia. Isolated mouse livers were randomly divided into five groups: (1) control group; (2) ischemic group (90 min); (3) as group 2, but with the administration of UTP; (4) as group 2, but with the administration of suramin, a P2Y antagonist; and (5) as group 3, but with the simultaneous administration of suramin and UTP. There was a postischemic significant reduction in the release of liver enzymes in the animals pretreated with UTP, the intrahepatic caspase-3 activity was significantly decreased, and the intrahepatic ATP content increased compared with group 2 (ischemic untreated). UTP prevented intracellular Ca overload after hypoxia in hepatocyte cultures. In the UTP-treated groups, significantly fewer apoptotic hepatocyte cells were noted by weaker activation of caspase-3 and by the transferase-mediated dUTP nick end labeling assay. The administration of suramin prevented the beneficial effect of endogenous ATP. UTP treatment attenuated the degradation of IkappaBalpha (nuclear factor-kappaB inhibitor) by 80% during reperfusion with no effect on c-Jun N terminal kinase phosphorylation. The administration of UTP before induction of ischemia-reperfusion can attenuate hepatic injury. UTP administration decreased cytosolic Ca overload in hypoxic conditions. UTP-mediated protective effects may be regulated through nuclear factor- kappaB inactivation. These findings have important implications for the potential use of UTP in ischemic hepatic injury.

Cyclic AMP Produced inside Mitochondria Regulates Oxidative Phosphorylation

Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signaling pathways involved. We show here that protein kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP), which activates mitochondrial PKA, does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO(2)-HCO(3)(-)-sAC-cAMP-PKA (mito-sAC) signaling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability.

ADCK3, an Ancestral Kinase, Is Mutated in a Form of Recessive Ataxia Associated with Coenzyme Q 10 Deficiency

Muscle coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency has been identified in more than 20 patients with presumed autosomal-recessive ataxia. However, mutations in genes required for CoQ(10) biosynthetic pathway have been identified only in patients with infantile-onset multisystemic diseases or isolated nephropathy. Our SNP-based genome-wide scan in a large consanguineous family revealed a locus for autosomal-recessive ataxia at chromosome 1q41. The causative mutation is a homozygous splice-site mutation in the aarF-domain-containing kinase 3 gene (ADCK3). Five additional mutations in ADCK3 were found in three patients with sporadic ataxia, including one known to have CoQ(10) deficiency in muscle. All of the patients have childhood-onset cerebellar ataxia with slow progression, and three of six have mildly elevated lactate levels. ADCK3 is a mitochondrial protein homologous to the yeast COQ8 and the bacterial UbiB proteins, which are required for CoQ biosynthesis. Three out of four patients tested showed a low endogenous pool of CoQ(10) in their fibroblasts or lymphoblasts, and two out of three patients showed impaired ubiquinone synthesis, strongly suggesting that ADCK3 is also involved in CoQ(10) biosynthesis. The deleterious nature of the three identified missense changes was confirmed by the introduction of them at the corresponding positions of the yeast COQ8 gene. Finally, a phylogenetic analysis shows that ADCK3 belongs to the family of atypical kinases, which includes phosphoinositide and choline kinases, suggesting that ADCK3 plays an indirect regulatory role in ubiquinone biosynthesis possibly as part of a feedback loop that regulates ATP production.

The Role of Ca2+ in Coupling Cardiac Metabolism with Regulation of Contraction

The heart adapts the rate of mitochondrial ATP production to energy demand without noticeable changes in the concentration of ATP, ADP and Pi, even for large transitions between different workloads. We suggest that the changes in demand modulate the cytosolic Ca2+ concentration that changes mitochondrial Ca2+ to regulate ATP production. Thus, the rate of ATP production by the mitochondria is coupled to the rate of ATP consumption by the sarcomere cross-bridges (XBs). An integrated model was developed to couple cardiac metabolism and mitochondrial ATP production with the regulation of Ca2+ transient and ATP consumption by the sarcomere. The model includes two interrelated systems that run simultaneously utilizing two different integration steps: (1) The faster system describes the control of excitation contraction coupling with fast cytosolic Ca2+ transients, twitch mechanical contractions, and associated fluctuations in the mitochondrial Ca2+. (2) A slower system simulates the metabolic system, which consists of three different compartments: blood, cytosol, and mitochondria. The basic elements of the model are dynamic mass balances in the different compartments. Cytosolic Ca2+ handling is determined by four organelles: sarcolemmal Ca2+ influx and efflux; sarcoplasmic reticulum (SR) Ca2+ release and sequestration (SR); binding and dissociation from sarcomeric regulatory troponin complexes; and mitochondrial Ca2+ flows. Mitochondrial Ca2+ flows are determined by the Ca2+ uniporter and the mitochondrial Na+Ca2+ exchanger. The cytosolic Ca2+ determines the rate of ATP consumption by the sarcomere. Ca2+ binding to troponin regulates the rate of XBs recruitment and force development. The mitochondrial Ca2+ concentration determines the pyruvate dehydrogenase activity and the rate of ATP production by the F(1)-F(0) ATPase. The workload modulates the cytosolic Ca2+ concentration through feedback loops. The preload and afterload affect the number of strong XBs. The number of strong XBs determines the affinity of troponin for Ca2+, which alters the cytosolic Ca2+ transient. Model simulations quantify the role of Ca2+ in simultaneously controlling the power of contraction and the rate of ATP production. It explains the established empirical observation that significant changes in the metabolic fluxes can occur without significant changes in the key nucleotide (ATP and ADP) concentrations. Quantitative investigations of the mechanisms underlying the cardiac control of biochemical to mechanical energy conversion may lead to novel therapeutic modalities for the ischemic and failing myocardium.

RyR2 and Calpain-10 Delineate a Novel Apoptosis Pathway in Pancreatic Islets

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.