Cardiac myosin inhibitor, CK-586, minimally reduces systolic function and ameliorates obstruction in feline hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule sarcomere inhibitors are promising novel therapeutics for the management of obstructive HCM (oHCM) patients and have shown efficacy in left ventricular outflow tract obstruction (LVOTO) relief. The objective of this study was to explore the 6-, 24-, and 48-hour (h) pharmacodynamic effects of the cardiac myosin inhibitor, CK-586, in six purpose-bred cats with naturally occurring oHCM. A blinded, randomized, five-treatment group, crossover preclinical trial was conducted to assess the pharmacodynamic effects of CK-586 in this oHCM model. Dose assessments and select echocardiographic variables were assessed five times over a 48-h period. Treatment with oral CK-586 safely ameliorated LVOTO in oHCM cats. CK-586 treatment dose-dependently eliminated obstruction (reduced LVOTOmaxPG), increased measures of systolic chamber size (LVIDs Sx), and decreased select measures of heart function (LV FS% and LV EF%) in the absence of impact on heart rate. At all tested doses, a single oral CK-586 dose resulted in improved or resolved LVOTO with well-tolerated, dose-dependent, reductions in LV systolic function. The results from this study pave the way for the potential use of CK-586 in both the veterinary and human clinical setting.

Calculation of ATP production rates using the Seahorse XF Analyzer.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions.

Citrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.

Deuterium-Stabilized (R)-Pioglitazone (PXL065) Is Responsible for Pioglitazone Efficacy in NASH yet Exhibits Little to No PPARγ Activity.

The antidiabetic drug pioglitazone is, to date, the most efficacious oral drug recommended off-label for the treatment of nondiabetic or diabetic patients with biopsy-proven nonalcoholic steatohepatitis (NASH). However, weight gain and edema side effects have limited its use for NASH. Pioglitazone is a mixture of two stereoisomers ((R)-pioglitazone and (S)-pioglitazone) that interconvert in vitro and in vivo. We aimed to characterize their individual pharmacology to develop a safer and potentially more potent drug for NASH. We stabilized the stereoisomers of pioglitazone with deuterium at the chiral center. Preclinical studies with deuterium-stabilized (R)-pioglitazone (PXL065) and (S)-pioglitazone demonstrated that (R)-pioglitazone retains the efficacy of pioglitazone in NASH, including reduced hepatic triglycerides, free fatty acids, cholesterol, steatosis, inflammation, hepatocyte enlargement, and fibrosis. Although both stereoisomers inhibit the mitochondrial pyruvate carrier, PXL065 shows limited to no peroxisome proliferator-activated receptor gamma (PPARγ) activity, whereas (S)-pioglitazone appears responsible for the PPARγ activity and associated weight gain. Nonetheless, in preclinical models, both stereoisomers reduce plasma glucose and hepatic fibrosis to the same extent as pioglitazone, suggesting that these benefits may also be mediated by altered mitochondrial metabolism. In a phase 1a clinical study, we demonstrated safety and tolerability of single 7.5-mg, 22.5-mg, and 30-mg doses of PXL065 as well as preferential exposure to the (R)-stereoisomer in comparison to 45-mg pioglitazone. Conclusion: PXL065 at a dose lower than 22.5 mg is predicted to exhibit efficacy for NASH equal to, or greater than, 45-mg pioglitazone without the potentially detrimental weight gain and edema. The development of PXL065 for NASH represents a unique opportunity to leverage the therapeutic benefits of pioglitazone, while reducing or eliminating PPARγ-related side effects.

Perm1 promotes cardiomyocyte mitochondrial biogenesis and protects against hypoxia/reoxygenation-induced damage in mice.

Normal contractile function of the heart depends on a constant and reliable production of ATP by cardiomyocytes. Dysregulation of cardiac energy metabolism can result in immature heart development and disrupt the ability of the adult myocardium to adapt to stress, potentially leading to heart failure. Further, restoration of abnormal mitochondrial function can have beneficial effects on cardiac dysfunction. Previously, we identified a novel protein termed Perm1 (PGC-1 and estrogen-related receptor (ERR)-induced regulator, muscle 1) that is enriched in skeletal and cardiac-muscle mitochondria and transcriptionally regulated by PGC-1 (peroxisome proliferator-activated receptor gamma coactivator 1) and ERR. The role of Perm1 in the heart is poorly understood and is studied here. We utilized cell culture, mouse models, and human tissue, to study its expression and transcriptional control, as well as its role in transcription of other factors. Critically, we tested Perm1's role in cardiomyocyte mitochondrial function and its ability to protect myocytes from stress-induced damage. Our studies show that Perm1 expression increases throughout mouse cardiogenesis, demonstrate that Perm1 interacts with PGC-1α and enhances activation of PGC-1 and ERR, increases mitochondrial DNA copy number, and augments oxidative capacity in cultured neonatal mouse cardiomyocytes. Moreover, we found that Perm1 reduced cellular damage produced as a result of hypoxia and reoxygenation-induced stress and mitigated cell death of cardiomyocytes. Taken together, our results show that Perm1 promotes mitochondrial biogenesis in mouse cardiomyocytes. Future studies can assess the potential of Perm1 to be used as a novel therapeutic to restore cardiac dysfunction induced by ischemic injury.

TANK-Binding Kinase 1 Regulates the Localization of Acyl-CoA Synthetase ACSL1 to Control Hepatic Fatty Acid Oxidation.

Hepatic TANK (TRAF family member associated NFκB activator)-binding kinase 1 (TBK1) activity is increased during obesity, and administration of a TBK1 inhibitor reduces fatty liver. Surprisingly, liver-specific TBK1 knockout in mice produces fatty liver by reducing fatty acid oxidation. TBK1 functions as a scaffolding protein to localize acyl-CoA synthetase long-chain family member 1 (ACSL1) to mitochondria, which generates acyl-CoAs that are channeled for ß-oxidation. TBK1 is induced during fasting and maintained in the unphosphorylated, inactive state, enabling its high affinity binding to ACSL1 in mitochondria. In TBK1-deficient liver, ACSL1 is shifted to the endoplasmic reticulum to promote fatty acid re-esterification in lieu of oxidation in response to fasting, which accelerates hepatic lipid accumulation. The impaired fatty acid oxidation in TBK1-deficient hepatocytes is rescued by the expression of kinase-dead TBK1. Thus, TBK1 operates as a rheostat to direct the fate of fatty acids in hepatocytes, supporting oxidation when inactive during fasting and promoting re-esterification when activated during obesity.

Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3.

Catecholamines stimulate the mobilization of stored triglycerides in adipocytes to provide fatty acids (FAs) for other tissues. However, a large proportion is taken back up and either oxidized or re-esterified. What controls the disposition of these FAs in adipocytes remains unknown. Here, we report that catecholamines redirect FAs for oxidation through the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Adipocyte STAT3 is phosphorylated upon activation of ß-adrenergic receptors, and in turn suppresses FA re-esterification to promote FA oxidation. Adipocyte-specific Stat3 KO mice exhibit normal rates of lipolysis, but exhibit defective lipolysis-driven oxidative metabolism, resulting in reduced energy expenditure and increased adiposity when they are on a high-fat diet. This previously unappreciated, non-genomic role of STAT3 explains how sympathetic activation can increase both lipolysis and FA oxidation in adipocytes, revealing a new regulatory axis in metabolism.

Sub-nanowatt microfluidic single-cell calorimetry.

Non-invasive and label-free calorimetry could become a disruptive technique to study single cell metabolic heat production without altering the cell behavior, but it is currently limited by insufficient sensitivity. Here, we demonstrate microfluidic single-cell calorimetry with 0.2-nW sensitivity, representing more than ten-fold enhancement over previous record, which is enabled by (i) a low-noise thermometry platform with ultralow long-term (10-h) temperature noise (80 µK) and (ii) a microfluidic channel-in-vacuum design allowing cell flow and nutrient delivery while maintaining a low thermal conductance of 2.5 µW K-1. Using Tetrahymena thermophila as an example, we demonstrate on-chip single-cell calorimetry measurement with metabolic heat rates ranging from 1 to 4 nW, which are found to correlate well with the cell size. Finally, we perform real-time monitoring of metabolic rate stimulation by introducing a mitochondrial uncoupling agent to the microchannel, enabling determination of the spare respiratory capacity of the cells.

A novel approach to measure mitochondrial respiration in frozen biological samples.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

Itaconate modulates tricarboxylic acid and redox metabolism to mitigate reperfusion injury.

OBJECTIVES: Cerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress. METHODS: The objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatment to mitigate reperfusion injuries in in vitro and in vivo model systems. We conducted metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized the metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of the treated/untreated mice. RESULTS: We demonstrate that the "immunometabolite" itaconate slowed tricarboxylic acid (TCA) cycle metabolism and buffered redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. The addition of itaconate to reperfusion fluids after mouse cerebral IR injury increased glutathione levels and reduced reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids increased post-reperfusion injury, while administration of itaconate normalized these metabolites. In mouse cranial window models, itaconate significantly improved hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increased survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock. CONCLUSIONS: We hypothesize that itaconate transiently inhibits SDH to gradually "awaken" mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.

Do Two Mitochondrial Wrongs Help Make Cells Right?

Using an unbiased genetic screen, To et al. map genes that enhance or suppress growth defects in response to different mitochondrial inhibitors to model mitochondrial disease. The findings have novel implications for the interconnectivity of bioenergetic pathways, and suggest a provocative strategy to treat primary mitochondrial disorders.

Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function.

The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.

Knockdown of Ant2 Reduces Adipocyte Hypoxia And Improves Insulin Resistance in Obesity.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

Metformin Inhibits Progression of Head and Neck Squamous Cell Carcinoma by Acting Directly on Carcinoma-Initiating Cells.

Metformin may reduce the progression of head and neck squamous cell carcinoma (HNSCC); however, whether metformin acts by altering the host metabolism or targets cancer-initiating cells remains poorly understood. This gap in knowledge has prevented the stratification of patient populations who are most likely to benefit from metformin treatment. Here, we explored whether metformin acts directly on HNSCC cells to inhibit aberrant cell growth. To investigate the tumor cell autonomous effects of metformin, we engineered representative HPV- and HPV+ HNSCC cells harboring typical genetic alternations to express the yeast mitochondrial NADH dehydrogenase (NDI1) protein, which is insensitive to metformin. NDI1 expression rescued the inhibitory effects of metformin on mitochondrial complex I, abolished the ability of metformin to activate AMP-activated protein kinase, and inhibited mTOR signaling both in vitro and in vivo, and was sufficient to render metformin ineffective to prevent HNSCC tumor growth. This experimental system provided an opportunity to identify metformin-regulated transcriptional programs linked to cancer cell growth inhibition in the tumor microenvironment. Remarkably, computational analysis of the metformin-induced transcriptome revealed that metformin downregulated gene expression signatures associated with cancer stemness and epithelial-mesenchymal transition, concomitant with increased expression of squamous differentiation genes. These findings support that metformin may act directly on cancer-initiating cells to prevent their progression to HNSCC, which may inform the selection of patients at risk of developing HNSCC in future early-stage clinical trials. SIGNIFICANCE: Metformin's ability to directly target HNSCC-initiating cells instead of exerting cancer preventive activity based solely on its systemic effects may inform the selection of patients in future precision prevention trials.

Mitochondrial biogenesis is altered in HIV+ brains exposed to ART: Implications for therapeutic targeting of astroglia.

The neuropathogenesis of HIV associated neurocognitive disorders (HAND) involves disruption of mitochondrial homeostasis and increased neuroinflammation. However, it is unknown if alterations in mitochondrial biogenesis in the brain underlie the neuropathogenesis of HAND. In this study, neuropathological and molecular analyses of mitochondrial biogenesis and inflammatory pathways were performed in brain specimens from a well-characterized cohort of HIV+ cases that were on antiretroviral regimens. In vitro investigations using primary human astroglia and neurons were used to probe the underlying mechanisms of mitochondrial alterations. In frontal cortices from HAND brains compared to cognitive normal brains, total levels of transcription factors that regulate mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were decreased. Immunohistochemical analyses revealed that TFAM was decreased in neurons and increased in astroglia. These changes were accompanied by decreased total mitochondrial DNA per cell and increased levels of messenger RNA for the proinflammatory cytokine interleukin (IL)-1ß. To determine how IL-1ß affects astroglial bioenergetic processes and mitochondrial activity, human astroglial cultures were exposed to recombinant IL-1ß. IL-1ß induced mitochondrial activity within 30 min of treatment, altered mitochondrial related gene expression, altered mitochondrial morphology, enhanced adenoside triphosphate (ATP) utilization and increased the expression of inflammatory cytokines. WIN55,212-2 (WIN), an aminoalkylindole derivative and cannabinoid receptor agonist, blocked IL-1ß-induced bioenergetic fluctuations and inflammatory gene expression in astroglia independent of cannabinoid receptor (CB)1 and peroxisome proliferator-activated receptor (PPAR) γ. A PPARα antagonist reversed the anti-inflammatory effects of WIN in human astroglia. These results show that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HAND brains and that targeting astroglial bioenergetic processes may be a strategy to modulate neuroinflammation.

Choline Uptake and Metabolism Modulate Macrophage IL-1ß and IL-18 Production.

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1ß and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1ß-dependent inflammation.

Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice.

The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mtDNA damage and myocardial aging using a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared to WT. We found that baseline mitophagy receptor-mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

Integrated In Vivo Quantitative Proteomics and Nutrient Tracing Reveals Age-Related Metabolic Rewiring of Pancreatic ß Cell Function.

Pancreatic ß cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in ß cell function.