R J Marjoram
email: robin_marjoram@med.unc.edu

C Guilluy #

K Burridge

Using Magnets and Magnetic Beads to Dissect Signaling Pathways Activated by Mechanical Tension Applied to Cells

Keywords: Mechanotransduction, tension, VE-cadherin, endothelial cell

Cellular tension has implications in normal biology and pathology. Membrane adhesion receptors serve as conduits for mechanotransduction that lead to cellular responses. Ligand-conjugated magnetic beads are a useful tool in the study of how cells sense and respond to tension. Here we detail methods for their use in applying tension to cells and strategies for analyzing the results. We demonstrate the methods by analyzing mechanotransduction through VE-cadherin on endothelial cells using both permanent magnets and magnetic tweezers.

Introduction

Analyses of physical forces applied to or produced within tissues and their molecular responses at the cellular level (i.e. mechanotransduction) have become important due to the role force-sensing has in normal biology and disease. For instance, endothelial cells of the vasculature experience shear and pulsatile forces under normal conditions as well as environmental stiffening upon progression of atherosclerosis. Cells detect these forces through cell-cell and cell-extracellular matrix (ECM) interactions, where cell-surface adhesive receptors form the links between neighboring cells or between cells and the ECM [START_REF] Schwartz | Cell adhesion receptors in mechanotransduction[END_REF]. Adhesion receptors form the physical connection between the intracellular actin cytoskeleton and the surrounding environment, and some of these receptors sense tension differences and transduce this fluctuation into a chemical signal, such as activation of Rho GTPase signaling pathways [START_REF] Marjoram | Regulation of RhoA activity by adhesion molecules and mechanotransduction[END_REF]. How extracellular tension regulates cellular responses through cell-cell adhesion receptors is an important question in normal biology and disease.

Cadherins are a family of cell-cell adhesion receptors that are major components of adherens junctions and have been associated with tension sensing in cells [START_REF] Schwartz | Cell adhesion receptors in mechanotransduction[END_REF]. Vascular endothelialcadherin (VE-cadherin) is a classic cadherin mainly expressed on the plasma membrane of endothelial cells that line the luminal surface of blood vessels [START_REF] Harris | VE-cadherin: at the front, center, and sides of endothelial cell organization and function[END_REF]. The initial discovery of VE-cadherin showed that this cadherin was involved with the barrier function of the endothelial layer by controlling permeability [START_REF] Lampugnani | A novel endothelial-specific membrane protein is a marker of cell-cell contacts[END_REF]. VE-cadherin at cell-cell junctions becomes disorganized during leukocyte trafficking between neighboring endothelial cells [START_REF] Maschio | Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions[END_REF]. Tumor cells also induce disruption of the VE-cadherin contacts [START_REF] Lewalle | Alteration of interendothelial adherens junctions following tumor cell-endothelial cell interaction in vitro[END_REF][START_REF] Hamilla | VE-cadherin-independent cancer cell incorporation into the vascular endothelium precedes transmigration[END_REF]. Under fluid shear stress, endothelial cells respond to the force through a mechanosensory complex involving VEcadherin [START_REF] Tzima | A mechanosensory complex that mediates the endothelial cell response to fluid shear stress[END_REF]. Under disease states, such as atherosclerosis, the physical environment of the endothelial cells changes and so does their response to external forces [START_REF] Sadat | Biomechanical structural stresses of atherosclerotic plaques[END_REF]. Coon and colleagues showed that the transmembrane domain of VE-cadherin serves an important role associating with VEGFR2/3 to form a mechanosensory complex in endothelial cells [START_REF] Coon | Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex[END_REF]. These data suggest that VE-cadherin has essential functions in mechanotransduction in endothelial cells to allow for responses to external forces from the extracellular environment.

Various tools have been utilized to study the effects of external forces on cells, including atomic force microscopy (AFM), optical tweezers, flow systems, PDMS microneedle substrates, and FRET tension sensors [START_REF] Stapleton | Force measurement tools to explore cadherin mechanotransduction[END_REF][START_REF] Eyckmans | A hitchhiker's guide to mechanobiology[END_REF]. However, biochemical analysis is difficult with many of these techniques, whereas the use of magnetic beads to apply tension to a plate of cells readily facilitates biochemical assays. Magnetic beads also permit single cell assays, such as the measurement of bead displacements in response to repeated pulses of applied force using magnetic tweezers. The ability to perform both single cell assays as well as bulk biochemical assays makes magnetic beads a valuable tool in the study of mechanotransduction, since this is not possible using approaches that can only analyze single cells such as optical tweezers or AFM. Another benefit is the ability to apply the force to a targeted receptor unlike more general force analysis tools (i.e. flow or traction force systems). Lastly, magnetic tweezers provide a much larger dynamic range of force application when compared to optical tweezers and AFM; they are able to apply weak forces (5 pN) similar to optical tweezers as well as strong forces (1 nN) on the same level as AFM [START_REF] Neuman | Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy[END_REF]. In biology, cells are exposed to different types of force. Some forces are acute, some are sustained, some build slowly over time, and others are cyclical with periods of tension followed by relaxation. The different techniques all have advantages. The magnetic beads as used in assays by us mimic biological situations where force is applied relatively quickly for a short sustained period (permanent magnet) or a regimen of brief pulses of force with intervening periods of relaxation (magnetic tweezers). It should be noted that these force applications do differ slightly and can produce different cellular responses as was shown by the Fredberg group where they showed cellular reinforcement (stiffening) or fluidization (softening) was dependent on the force regime (frequency, amplitude) applied to the cell [START_REF] Krishnan | Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness[END_REF][START_REF] Trepat | Universal physical responses to stretch in the living cell[END_REF]. For these reasons, our lab utilizes magnetic beads for broad biochemical analyses using permanent magnets as well as fine tuned magnetic tweezers for pulling experiments to measure the stiffening response of cells to applied forces (Figure 1).

Several different studies have used magnetic beads to explore how cells respond to mechanical forces exerted on cell adhesion molecules. In early work, Wang et al. used RGD-coated magnetic beads to apply a twisting force to integrins on the surface of endothelial cells and observed a stiffening response that was dependent on the actin cytoskeleton [START_REF] Wang | Mechanotransduction across the cell surface and through the cytoskeleton[END_REF]. McCulloch's group used permanent magnets placed above cell cultures to pull vertically on collagen-coated magnetic beads adhering to the dorsal surface of cells. This allowed them to perform both single cell analysis, measuring for example increases in intracellular calcium in response to force, as well as bulk biochemical measurements on large populations of cells, such as analyzing protein tyrosine phosphorylation, which they showed increased in response to force [START_REF] Glogauer | Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching[END_REF][START_REF] Glogauer | Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts[END_REF]. In subsequent work the same group used this approach to show that sustained tension on integrins via magnetic beads coated with collagen activated RhoA [START_REF] Zhao | Force activates smooth muscle alphaactin promoter activity through the Rho signaling pathway[END_REF]. Ingber and his colleagues used magnetic tweezers to examine the effects of applying tension on magnetic beads coated with integrin ligands and implicated RhoA signaling pathways in the cellular response [START_REF] Matthews | Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels[END_REF]. Na et al. used the combination of FRET and magnetic twisting cytometry (MTC) to analyze rapid mechanochemical signaling in live cells and showed the prestressed cytoskeleton promoted rapid activation of Src upon force application [START_REF] Na | Rapid signal transduction in living cells is a unique feature of mechanotransduction[END_REF][START_REF] Na | Application of fluorescence resonance energy transfer and magnetic twisting cytometry to quantify mechanochemical signaling activities in a living cell[END_REF]. Using this approach Poh et al. also showed that force application through integrins activated Rac1 and was independent of Src activity in human airway smooth muscle cells [START_REF] Poh | Rapid activation of Rac GTPase in living cells by force is independent of Src[END_REF].

Following on from these studies, our lab has combined both biochemical analyses using permanent magnets with single cell experiments using magnetic tweezers to analyze the signaling pathways downstream from tension applied to integrins [START_REF] Guilluy | The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins[END_REF]. We used fibronectincoated beads to pull on fibroblast integrins and showed the activation of RhoA was mediated by two distinct pathways that activate the Rho GEFs, LARG and GEF-H1. Additionally, activation of RhoA via these GEFs contributed to the observed cellular stiffening [START_REF] Guilluy | The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins[END_REF]. Magnetic beads and magnets have been used to apply force to other cell adhesion molecules. For example, Tzima's lab have shown that tension applied to PECAM-1, an endothelial cell adhesion molecule implicated in endothelial mechanotransduction, activates RhoA in an integrin-dependent pathway via GEF-H1 and LARG [START_REF] Collins | Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway[END_REF]. We showed that tension on ICAM-1 on endothelial cells causes cell stiffening and helps mediate transendothelial migration of leukocytes [START_REF] Lessey-Morillon | The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration[END_REF]. In another study, DeMali's lab used a similar approach to exert force on E-cadherin using magnetic beads coated with the extracellular domain of Ecadherin. They discovered that the tension-induced recruitment of vinculin depended on the phosphorylation of vinculin at Y822 [START_REF] Bays | Vinculin phosphorylation differentially regulates mechanotransduction at cell-cell and cell-matrix adhesions[END_REF]. Another study by Kim et al. used E-cadherincoated magnetic beads to show a-catenin is an integral part of the force sensing apparatus at cell-cell junctions [START_REF] Kim | Dynamic Visualization of alpha-Catenin Reveals Rapid, Reversible Conformation Switching between Tension States[END_REF]. Other labs have also shown that vinculin, α-catenin, and actin are recruited to E-cadherin adhesions in response to force [START_REF] Le Duc | Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin IIdependent manner[END_REF][START_REF] Tabdili | Cadherin-dependent mechanotransduction depends on ligand identity but not affinity[END_REF][START_REF] Barry | alpha-catenin cytomechanics--role in cadherin-dependent adhesion and mechanotransduction[END_REF] The versatility of using magnetic beads to generate tension is illustrated in a study in which the response of an organelle, the nucleus, to tension was examined. In this work, tension was applied to isolated nuclei using magnetic beads coated with antibodies against the nuclear envelope protein nesprin-1. Unexpectedly, successive applications of force resulted in decreased bead displacement implying that the isolated nuclei were stiffening [START_REF] Guilluy | Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus[END_REF]. Together these studies demonstrate the usefulness of magnetic beads in mechanotranduction experiments.

Here we detail the methods for using magnetic beads to apply forces to surface proteins. We focus on force application to the cell adhesion receptor VE-cadherin on endothelial cells, and show that tension on VE-cadherin stimulates mechanotransduction via Rho GTPase signaling and alters protein tyrosine phosphorylation.

Materials and Methods

Materials

The superparamagnetic beads (particles that magnetize upon placement in a magnetic field but lose magnetization upon removal from the field) we utilize for these assays were 2.8 μm diameter tosyl-activated magnetic beads from Invitrogen (Dynabeads M-280 Tosylactivated; Cat. #142.03) or Dynabeads of 4.5 μm diameter (Cat. #140.13) if larger forces and surface contacts are needed. Common chemicals and experimental reagents were from Sigma Aldrich and Fisher Scientific. A list of some of the other materials used: We obtained pooled-donor primary human umbilical vein endothelial cells (HUVECs) from Lonza and cultured them in EGM2 medium up to passage 10. For experiments, HUVECs were grown to 80-100% confluent monolayers.

Ligand Conjugation to Magnetic Beads

Ligands for targeting cell surface adhesion receptors for mechanotransduction analyses can be covalently linked to the superparamagnetic beads of 2.8 μm or 4.5 μm diameter. Bead diameter should be restricted to 2-5 μm since smaller beads tend to more quickly undergo phagocytosis during 30-60 minute incubation and larger beads have stronger adhesion to the cell and would restrict bead displacement under the utilized magnetic field strengths [START_REF] Schroeder | Measurement of phagocytosis using fluorescent latex beads[END_REF][START_REF] Mcabee | Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells[END_REF][START_REF] Grinnell | Interaction of fibronectin-coated beads with attached and spread fibroblasts. Binding, phagocytosis, and cytoskeletal reorganization[END_REF]. Limiting bead size and incubation time with the cells will help avoid these problems. Here we use the human VE-cadherin extracellular domain fused at the C-terminus with the Fc domain of IgG 1 (hVEC-Fc) to target cellular VE-cadherin molecules for force application. The conjugated ligand does not have to be a physiological ligand like the extracellular domain of a cadherin, it could also be a monoclonal antibody targeting a cell surface protein, such as an antibody specific for a MHC class I receptor. We covalently cross-linked hVEC-Fc to 2.8 or 4.5 μm diameter magnetic beads (Figure 2A) in the following procedure: 3. Wash 82.5 μL (6 × 10 8 beads) of the 2.8 μm tosyl-activated Dynal beads in 1 mL Buffer B in 1.5 mL microcentrifuge tube and use the Dynal magnetic particle concentrator (MPC) to pellet beads and aspirate the buffer.

4.

Combine 20-25 μg hVEC-Fc (80-100 μL) with appropriate volume of Buffer B to bring the total volume to 200 μL and mix by pipetting. [For gel analysis take 10 μL aliquot for crosslinking analysis and mix with 10 μL 2X Laemmli Sample Buffer (Input); Figure 2B] NOTE: BSA or poly-lysine coated beads can be used as negative controls and produced in a similar manner.

5.

Combine hVEC-Fc with beads, transfer to 0.5 mL microcentrifuge tube, and incubate beads with hVEC-Fc for 18-24 hours at 37 °C on a rotor to allow for the reaction to occur and produce a covalent linkage of protein to the bead (Figure 2A).

6.

Pellet beads using the MPC and remove 10 μL aliquot and mix with equal volume of 2X; Laemmli Sample Buffer (Output), Figure 2B.

7.

Wash beads 3 times with 1 mL PBS using MPC to pellet the beads. Use the first 1 mL PBS to transfer the beads back to a 1.5 mL microcentrifuge tube.

8.

Resuspend beads in 1 mL PBS without Ca 2+ (removal of calcium ions blocks the homophilic interaction of VE-cadherin to prevent bead aggregation) to give a concentration of 6 × 10 8 beads/mL.

9.

Store beads at 4 °C for up to 3 months.

10. (Optional) Run a SDS-PAGE gel and stain with colloidal blue to analyze the hVEC-Fc crosslinking to the magnetic beads (Figure 2B).

11.

(Optional) Determine the force applied to a single bead by the magnet.

a. For the permanent magnet, we create a chamber slide using a standard microscope slide, coverslips, and nail polish to glue them in place (Figure 2C) and measure the distance the bead front moves through a channel 10 mm long and containing undiluted glycerol, a Newtonian fluid of known viscosity (η = 1.41 Pa-sec at 20 °C). The well where the magnetic beads are placed (5 μL of 1:10 dilution of beads in glycerol) is 10 mm from the edge of the slide and provides a fixed distance between the magnet and the well. The magnet is then brought into perpendicular contact with the slide edge mimicking the experimental distance. Measurements of how far the beads travel towards the magnet are taken at two time points, 15 and 30 minutes (At 30 min the 4.5 μm beads reached ∼6 mm from the magnet). Ideally, the 6 mm mark should be the center of the distance travelled for a more accurate force estimate, but cutting the glass coverslip evenly at smaller widths is difficult and often not practical. From these empirical measurements the average rate of the bead is obtained and can be used to calculate an approximate force exerted by the magnet at this fixed distance used in the experiments (6 mm). The Stokes law equation used is

Where F is the force generated by the magnet on the beads, r bead is the radius of the bead, η is the viscosity of the Newtonian liquid, and ν bead is the measured velocity of the bead. Using this method for our permanent magnet system, we have calculated the force to be ∼40 pN on the 2.8 μm bead and ∼200 pN on the 4.5 μm bead.

b. For the magnetic tweezers, this same equation is used to calculate the force applied by the magnetic pole tip on a 2.8 μm bead at a distance of 20-30 μm in another Newtonian fluid, Karo syrup (η = 3.4 Pa-sec at 20 °C). Using the same experimental force regimen, we measure the distance the bead moves and determine the rate. The force the pole tip applies with these parameters is about 20-40 pN.

Application of tension to cells through surface receptors using ligand-coated magnetic beads and permanent magnets for biochemical analyses (e.g. RhoGTPase activation, phosphotyrosine, and adhesion complex analyses)

A superparamagnetic bead coated with a specific ligand for a cell surface receptor is a useful tool for applying force to that specific receptor when placed within a magnetic field gradient. By placing the cells with adherent magnetic beads within a magnetic field of a permanent magnet, a defined force can be applied to the cell. After this treatment the cells can be lysed and processed for biochemical analyses (Figure 3A).

A permanent magnet (we use Neodymium magnets which are 10 times stronger than ceramic magnets) is used to generate perpendicular, tensile forces on magnetic beads adhering to the apical surface of cells. For all experiments, the pole face of the magnet is parallel with the 10 cm culture dish surface at a height of 6 mm. At this distance the force on a single 2.8 μm magnetic bead is ∼40 pN and on a single 4.5 μm magnetic bead is ∼200 pN.

A constant force of varying duration is used for all experiments. 2. Culture HUVECs on 10 cm tissue culture dishes in EGM2 medium until an 80-100% confluent monolayer of cells is formed.

3.

Aspirate the growth media and add 5 mL of warmed PBS without Ca 2+ or Mg 2+ per dish for 2-5 minutes to perform a mild calcium switch, which disassociates cadherin interactions to free up receptors for bead ligation. (For cell-cell junction receptors like cadherins that require Ca 2+ for binding, a calcium switch can be performed to temporarily dissociate cell-cell junctions and free up receptors to interact with ligand coated beads. By adding PBS (phosphate can extract Ca 2+ from weaker cell-cell junctions like endothelial cells) or 4 mM EGTA in PBS (EGTA chelates Ca 2+ and is more stringent for tighter cell-cell contacts like epithelial cells) for 5-60 minutes.

4.

Aspirate the PBS and add 3 mL of SFM per 10 cm dish.

5.

Vortex hVEC-Fc magnetic beads for 30-60 seconds and add predetermined volume to appropriate dishes and promptly rock the dish back and forth to disperse the beads evenly in the medium and across the dish which will prevent aggregation of the beads. (Brief, low-level sonication prior to addition to the cells may be used if the beads tend to aggregate.) a. For 2.8 μm beads, add 100 μL hVEC-Fc beads (60 × 10 6 beads) per dish.

b. For 4.5 μm beads, add 150 μL hVEC-Fc beads (7.5 × 10 6 beads) per dish.

NOTE: A confluent monolayer of HUVECs on a 10 cm dish has about 6 × 10 6 cells, so the bead addition is ≥ 1 bead per cell. More beads can be used if needed but bead aggregates start to form.

6.

Allow the beads to settle and adhere to the cells for 15-30 minutes under cell culture conditions (37°C and 5% CO 2 ). Bead adhesion to cells can be determined by observing the beads under a light microscope using a 20X objective.

7.

For treatments using magnets, normal dish lids are exchanged for lids containing 3 inch diameter magnets held in place by 2 smaller 5/8 inch diameter magnets (Figure 3A) and incubated for the designated time points at cell culture conditions or room temperature [START_REF] Glogauer | Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching[END_REF][START_REF] Guilluy | The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins[END_REF].

NOTE: These magnets are extremely strong and special attention needs to be taken to avoid personal injury or equipment damage. A designated area and spatial distance between individual magnets needs to be maintained.

8.

Remove magnet lids, replace original lid, and place dishes on ice. NOTE: All handling of samples from here on is done on ice at 4°C.

9.

Aspirate medium from the dishes. (Tilting dishes for 30 seconds to remove remaining medium helps to keep total lysate volumes equal.)

10. Add 500 μL of ice-cold lysis buffer per dish, scape the lysate from each dish with a cell scraper, and transfer the lysates to 1.5 mL microcentrifuge tubes.

11.

Pellet the magnetic beads using the MPC and transfer lysates to new 1.5 mL microcentrifuge tubes. (For analysis of associated adhesion complexes, wash the magnetic beads 3 times with 1 mL of lysis buffer. Add 50 μL 2X Laemmli Sample Buffer to beads, boil tubes, and store at -20°C.) NOTE: One way to normalize data from these samples since pulling can cause noticeable bead loss is to count the beads during the last washing step using a hemacytometer or cellometer. The ratios of magnet-treated sample bead number over the no-magnet control bead number can be used to normalize the volume of samples loaded onto gels for SDS-PAGE and western blot analysis.

12. Pellet the insoluble fraction in the lysates by using a microcentrifuge at 16,000 g for 5 minutes at 4°C and transfer lysates to new tubes. (The insoluble pellet can be washed with lysis buffer and resuspended in 2X Laemmli Sample Buffer for analysis of the TX;100 insoluble cell fraction).

13. Remove a 50 μL aliquot from each lysate and add to new tubes containing an equal volume of 2X Laemmli Sample Buffer, boil the tubes, and store at -20°C. (These samples can be used for total protein controls, phosphoprotein analysis, soluble versus insoluble fraction analysis, etc.)

14.

The remaining 450 μL of lysate can be used for pull down experiments such as Rho GTPase activation assays, immunoprecipitation experiments, or other biochemical assays. We utilize the lysates for Rho GTPase activity assays as described previously [START_REF] Wittchen | Analysis of low molecular weight GTPase activity in endothelial cell cultures[END_REF].

Magnetic tweezer application of pulsed forces for measuring cell stiffening

Magnetic tweezers can be used to apply pN tensional force to magnetic beads bound to cell surface receptors. When coupled to live cell imaging, this experimental system allows the measurement of local viscoelastic properties by tracking the bead displacement due to a known force generated by the magnetic field from an electromagnetic pole tip. We used the 3-dimensional force microscope (3DFM) designed by the Center for Computer Integrated Systems for Microscopy (http://cismm.cs.unc.edu) to apply tension to various cell surface receptors, including integrin [START_REF] Guilluy | The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins[END_REF], ICAM-1 [START_REF] Lessey-Morillon | The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration[END_REF] or as we show here, VE-cadherin. The 3DFM is composed of thin and flat magnetic poles mounted on an Olympus IX81-ZDC2 inverted microscope (Olympus) equipped with a 40× objective (Olympus UplanLN 40×/ 0.75) and a highspeed Rolera EM-C2 camera (QImaging) to record bead movement. Description and calibration of the 3DFM system has been detailed previously [START_REF] O'brien | Magnetic Manipulation for Force Measurements in Cell Biology[END_REF]. Here we describe how to use ligand-coated magnetic beads in this experimental system to measure endothelial cell mechanical properties in response to tension applied to VE-cadherin.

1.

Culture HUVECs in EGM2 medium on sterile glass coverslips (24 × 50 mm) coated with collagen (10 μg/ml) within a cloning ring. Grease, such as silicon vacuum grease is applied around the cloning ring to prevent medium leakage. Cells are cultured until they reach 80-100% confluence for at least 24 hours.

2.

Aspirate the growth media and add 500 μL of warmed PBS per dish for 5 minutes.

3.

Remove the PBS and add 500 μL of SFM.

4. Vortex 2.8 μm hVEC-Fc beads for 1 min (see hVEC-Fc beads preparation in 2.2) and add 10 3 beads per cloning ring. Allow the beads to adhere for 30 minutes under cell culture conditions (37°C and 5% CO 2 ). NOTE: Other bead sizes can be selected (from 1 to 4.5 μm) to generate different amounts of force. For example, 4.5 μm beads can be used to apply up to 10 nN force.

5.

Remove the medium and replace with SFM. Place the coverslip on the microscope stage, remove the cloning ring and approach the magnetic pole to the coverslip surface (at approximately 80 μm above the cell surface to avoid any damage to the cells).

6. Select a cell that has only one bead bound to its surface and position the tip 20-30 μm away from bead. NOTE: the distance between the tip and the bead can be adjusted depending on magnetic pole calibration, with our system a distance or 20-30 μm yields a 20-40 pN force (See section 2.2).

7.

Eliminate any remnant magnetization from the pole tips (activation of the degauss mode as detailed previously [START_REF] Spero | High throughput system for magnetic manipulation of cells, polymers, and biomaterials[END_REF]), start the force protocol, and record bead movement at 30 frames/second. NOTE: Typical force protocol generates 10 cycles of 4 sec extension (force on ∼20-40 pN) and 3 sec recovery (force off).

8.

Eliminate any remnant magnetization from the pole tips before moving the magnetic pole within the sample.

9.

Repeat steps 6 and 7.

Measure and analyze the bead displacement. Changes in the mechanical properties of the cells in response to force application can be determined by measuring the bead displacement for each pulse. For example, a decrease in bead displacement will indicate an increase in cellular stiffness, as described previously when forces are applied to integrin-based adhesion [START_REF] Guilluy | The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins[END_REF]. Measurement of the bead displacement (micrometer) can be performed using Video Spot Tracker (software designed by the Center for Computer Integrated Systems for Microscopy and Manipulation -http://cismm.cs.unc.edu). Relative bead displacement can be calculated by normalizing the displacement for each pulse to that observed during the first pulse. NOTE: Regarding statistical analysis, beads that show displacements of less than 10 nm (detection resolution) and loosely bound beads are excluded from analysis. Depending on the sample size (n=number of beads), parametric (ANOVA) or non-parametric (Kruskal-Wallis) statistical tests can be used to analyze bead displacement. To quantify the change in local stiffness, the spring constant (Pa) can be calculated for each pulse by fitting the bead displacement and force magnitude to a modified Kelvin-Voigt model [START_REF] Bausch | Measurement of local viscoel a sticity and forces in living cells by magnetic tweezers[END_REF] for a viscoelastic liquid.

Results and Discussion

Human VE-cadherin extracellular domain/Fc fusion protein (hVEC/Fc) crosslinking to 4.5 μm tosyl-activated magnetic beads

We covalently bonded hVEC-Fc to 4.5 μm tosyl-activated magnetic beads to provide a more stable ligand for force application to cellular VE-cadherins. In Figure 2A, the reaction between the tosyl groups of the magnetic beads and the primary amines of hVEC-Fc is depicted. The end result is a ligand covalently bonded to a magnetic bead, which is a useful tool for force application experiments since the ligand is less likely to dissociate from the bead during force application. To confirm the adsorption of hVEC-Fc onto the tosylactivated magnetic beads, we analyzed 10 μL aliquots (∼2 μg of protein) of the hVEC-Fc protein input (IN) and output (OUT) from the reaction mixture by SDS-PAGE followed by colloidal blue staining (Figure 2B). A 120 kDa hVEC-Fc protein band of about 2 μg (determined by BSA loading controls) is evident in the input lane and absent in the output lane indicating that the hVEC-Fc protein has been covalently bound to the surface of the bead. This data shows that the beads are ready for use in force application experiments.

Biochemical analyses of mechanical tension applied to VE-cadherin on HUVECs by hVEC/Fc-coated magnetic beads

We utilized the 4.5 μm, hVEC-Fc-coated magnetic beads to apply mechanical tension to VE-cadherin on the surface of HUVECs by using a permanent magnet set-up depicted in Figure 3A. This procedure was used to carry out several biochemical assays to determine how HUVECs respond to forces on VE-cadherin. Rho GTPases are master regulators of the cellular cytoskeleton, and their activities are important in mechanotransduction. For this reason, we measured activation levels of two Rho GTPases, RhoA and Rac1, after 1 or 5 minutes of force application on VE-cadherin (Figure 3B). RhoA activity increased significantly by 5 minutes, whereas Rac1 showed decreased activation by 5 minutes. This data supports that HUVECs respond to mechanical tension on VE-cadherin by activating

RhoA and inhibiting Rac1 activity potentially through crosstalk in a similar tug-of-war mechanism as shown previously [START_REF] Daneshjou | Rac1 functions as a reversible tension modulator to stabilize VE-cadherin trans-interaction[END_REF][START_REF] Guilluy | Rho protein crosstalk: another social network?[END_REF].

Given that cell-cell junctions are prominent sites of tyrosine phosphorylation, we examined whether mechanical tension on VE-cadherin affected the level of protein tyrosine phosphorylation (Figure 3C). The hVEC-Fc magnetic beads and a permanent magnet were used to apply force on HUVECs as above. Cell lysates were prepared at varying times and the levels of protein tyrosine phosphorylation (pTyr) were determined by Western blot using a phosphotyrosine-specific antibody (4G10). Compared to the control lane, there were several protein bands between 150-25 kDa that changed pTyr levels with the addition of hVEC-Fc beads as well as when tension was applied to the beads (arrows signify protein bands of interest). Simple engagement of the hVEC-Fc with VE-cadherin elevated pTyr levels. This was a surprising finding, but the recent report that VE-cadherin associates with VEGFR2/3 through the transmembrane domain might explain this observation [START_REF] Coon | Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex[END_REF]. The hVEC beads can cause clustering of these membrane proteins that would lead to increases in VEGFR signaling and tyrosine phosphorylation. Whereas tension on VE-cadherin decreased the level of pTyr to an intermediate value or to the resting level. A band of ∼25 kDa shows an increase in pTyr levels with force application relative to control beads and beads alone. Further experiments are needed to identify these pTyr proteins and whether tension is broadly activating protein tyrosine phosphatases or inhibiting protein tyrosine kinases, or both.

The last biochemical analysis we utilized was to identify components of the adhesion complex in HUVECs that are isolated with the hVEC-Fc magnetic beads (Figure 3D). Endogenous VE-cadherin was detected using an antibody that only recognizes a site in the cytoplasmic tail of VE-cadherin, which is not present in the hVEC-Fc recombinant protein.

Importantly, we observed VE-cadherin in all treatments confirming an association between cellular VE-cadherin and the hVEC-Fc magnetic beads. Interestingly, a slight increase in VE-cadherin recruitment was detected with 5 minutes of tension. Several cellular proteins are known to associate with the cytoplasmic tail of VE-cadherin to form an adhesion complex including α-catenin and actin. We detected both of these proteins within the adhesive complex isolates from all treatments. However, a small increase in α-catenin levels with 5 minutes of tension was detected that is consistent with previous studies showing that tension on VE-cadherin recruits cellular components of the VE-cadherin adhesion complex [START_REF] Huveneers | Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling[END_REF][START_REF] Yonemura | alpha-Catenin as a tension transducer that induces adherens junction development[END_REF]. Quantification of the α-catenin to VE-cadherin protein bands was done to determine if the increase in α-catenin was independent of increasing VE-cadherin (Figure 3D, graph).

The analysis shows a trend that α-catenin increases relative to the amount of VE-cadherin but it was not statistically significant (p = 0.1). The possible increase in α-catenin is likely dependent on both force and increased VE-cadherin presence.

Cellular stiffening of HUVECs pulled on by hVEC/Fc magnetic beads using magnetic tweezers

To investigate how cells adapt their mechanical properties in response to tension applied to VE-cadherin, we used 2.8 μm hVEC-Fc-coated magnetic beads bound to HUVECs. We observed typical viscoelastic displacement for each pulse of tensional force (Figure 4A). We found that progressive application of pulses of constant force induced a significant decrease in cellular strain (Figure 4B), indicating local cellular stiffening. This result signifies that tension applied to VE-cadherin triggers adhesion remodeling, which produces a local stiffening. This is consistent with work from others demonstrating that mechanical tension regulates VE-cadherin-based adhesion growth [START_REF] Liu | Mechanical tugging force regulates the size of cell-cell junctions[END_REF][START_REF] Barry | Local VE-cadherin mechanotransduction triggers long-ranged remodeling of endothelial monolayers[END_REF]. Liu et al. showed that endothelial cell-cell adhesion size is regulated by tugging forces through balancing myosin and Rac1 activities [START_REF] Liu | Mechanical tugging force regulates the size of cell-cell junctions[END_REF]. Interestingly, Barry et al. showed that externally applied tension triggers vinculin recruitment to VE-cadherin [START_REF] Barry | Local VE-cadherin mechanotransduction triggers long-ranged remodeling of endothelial monolayers[END_REF]. Vinculin is recruited by VE-cadherin to a subset of endothelial cell-cell junctions called focal adherens junctions (FAJs) that undergo remodeling upon increased cellular tension [START_REF] Huveneers | Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling[END_REF]. Tension-dependent recruitment was similarly shown for E-cadherin [START_REF] Le Duc | Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin IIdependent manner[END_REF]. Since we observed that application of tension to VEcadherin activates RhoA and stimulates the recruitment of α-catenin (Figure 3B and 3D), it will be interesting to test the involvement of these proteins using the magnetic tweezers. Identifying the molecular events, which regulate this mechanical response may help to understand how mechanical stress controls endothelial functions.

Conclusions

Ligand-coated magnetic beads are a very useful tool for measuring cellular tension since they can be used in diverse analyses. We describe here their application in tension assays using permanent magnets in biochemical readouts as well as magnetic tweezers to examine the response of single cells to pulses of force. We have illustrated these applications of magnetic beads to analyze the effects of tension on VE-cadherin on endothelial cells. We have shown that force applied to VE-cadherin causes an increase in RhoA activation with a correlative decrease in Rac1 activation, a change in protein tyrosine phosphorylation levels, as well as a stiffening response to multiple short pulses of force. These are just some of the assays the magnetic beads can be used for, but other types of biochemical analysis are possible (e.g. other signaling pathways, mass spectrometry, etc.), as well as using the beads and the permanent magnet system beneath the culture dish to apply compressive forces to cells. The reaction output lane shows a loss of the hVEC-Fc band from the solution indicating the protein was conjugated to the beads. C, Chamber slide set-up for obtaining a coarse measurement of the amount of force applied to a single magnetic bead by a permanent magnet. Using a microscope slide, two square coverslips were cemented to the slide forming a 10 mm channel. Glycerol was pipetted onto the slide to fill the channel (50 μL). A third coverslip was scored and broken in half to form the ceiling of the chamber through which the beads will run. A fourth square cover slip was used to cover the remaining channel and create a well in between which 5 μL of a 1:10 dilution of beads in glycerol were loaded and the chamber closed and allowed to equilibrate for 5 minutes before the magnet was applied perpendicularly to the side of the slide as indicated. Measurements of how far the bead front migrated at two time points (15 and 30 minutes) were taken at three points and used to calculate the mean bead velocity (ν). This variable was then used to calculate the force of the magnet. analysis of VE-cadherin adhesion complex proteins associated with the hVEC-Fc magnetic beads from treatments with hVEC-Fc beads with no exposure to the permanent magnet or 1 or 5 minutes of magnet exposure. Cell lysates from these treatments were also analyzed. VE-cadherin, α-catenin, and actin were detected. The graph shows quantification of the αcatenin and VE-cadherin protein bands pulled down by the magnetic beads and plotted as the ratio of α-catenin/VE-cadherin, mean ± SEM for n=3.

  Concentrator MPC-S (Invitrogen; Cat. #120.20) Colloidal Blue Stain (Invitrogen) Neodymium magnets, 3″ × 1/2″ disc, NdFeB -grade N52 (K&J Magnetics, Inc.) Neodymium magnets, 5/8″ × 1/4″ disc, NdFeB -grade N52 (K&J Magnetics, Inc.) 10 cm plastic cell culture dishes (Costar) VE-cadherin antibody (Santa Cruz, F-8) Phosphotyrosine antibody (Millipore, 4G10) Actin antibody (Sigma) α-catenin antibody (BD) cell scraper, 25 cm (Sarstedt) PBS, pH 7.6 without Ca 2+ or Mg 2+ (Invitrogen) Coverslips (Corning): Square; No. 1; Material: borosilicate glass; Thickness: 0.12 to 0.16mm; Size: 22 × 22mm. Rectangle; No. 1.5; Material: borosilicate glass; Size: 24 × 50mm Clear nail polish Microscope slide (Fisher Scientific) Vacuum grease (Fisher Scientific) Cloning rings (Fisher Scientific)

1 . 4 c. PBS 2 .

 142 Prepare buffers as per Invitrogen Dynabead M-280 Tosyl-activated protocol: a. Buffer B -0.1 M Sodium Phosphate Buffer, pH 7.4 b. Buffer D -0.01 M Sodium Phosphate, 0.0137 M NaCl, and 0.5% (w/v) delipidated BSA, pH 7.Resuspend lyophilized hVEC-Fc in sterile PBS to 250 μg/mL, divide into 100 μL aliquots, and store at -80 °C.

Figure 1 .

 1 Figure 1. Use of protein-coated magnetic beads in cell surface receptor tension experimentsA, Permanent magnets can be used to apply pulling forces to ligand-coated magnetic beads adhered to cell surface receptors. Cell responses to tension on the timescale of 15 seconds and greater can be analyzed by various biochemical assays using this method. B, Magnetic tweezers can utilize ligand-coated magnetic beads to probe tension responses by individual cells on shorter timescales than permanent magnets and measure real time responses using live cell imaging.

Figure 2 .

 2 Figure 2. Preparation of hVEC-Fc magnetic beadsA , Tosyl-activated magnetic beads allow for chemical crosslinking of the ligand to the beads. B, Colloidal blue staining of SDS-PAGE analysis of hVEC-Fc conjugation to Dynal magnetic beads. The reaction input lane (IN) shows a protein band of molecular weight corresponding to hVEC-Fc (∼120 kDa) and amount (∼2 μg) was present prior to conjugation. The reaction output lane shows a loss of the hVEC-Fc band from the solution indicating the protein was conjugated to the beads. C, Chamber slide set-up for obtaining a coarse measurement of the amount of force applied to a single magnetic bead by a permanent magnet. Using a microscope slide, two square coverslips were cemented to the slide forming a 10 mm channel. Glycerol was pipetted onto the slide to fill the channel (50 μL). A third coverslip was scored and broken in half to form the ceiling of the chamber through which the beads will run. A fourth square cover slip was used to cover the remaining channel and create a well in between which 5 μL of a 1:10 dilution of beads in glycerol were loaded and the chamber closed and allowed to equilibrate for 5 minutes before the magnet was applied perpendicularly to the side of the slide as indicated. Measurements of how far the bead front migrated at two time points (15 and 30 minutes) were taken at three points and used to calculate the mean bead velocity (ν). This variable was then used to calculate the force of the magnet.

Figure 3 .

 3 Figure 3. Permanent magnet force application and biochemical assays for Rho GTPase activation, protein phosphotyrosine levels, and adhesion complex analysisA, A diagram of the permanent magnet setup on the lid of 10 cm culture dish and a general flowchart of the permanent magnet assay. The position of the magnet places the face of the magnet about 6 mm from the bottom of the dish (arrow), which fixes the distance of the magnet from the cells, makes the field strength constant, and permits control of the time of exposure to the magnetic field. B, RhoA and Rac1 activity assays were done on untreated control cell lysates or lysates from cells treated with hVEC-Fc beads with no exposure to the permanent magnet or 1 or 5 minutes of magnet exposure. Western blots of RBD-pulldowns (activated RhoA) or PBD-pulldowns (activated Rac1) and cell lysates for total protein are shown. Quantification of protein bands was done by densitometry and results are displayed in the graphs (n=4; p ≤ 0.05). C, Western blot analysis for protein phosphotyrosine levels in cell lysates that were treated as above. Arrows indicate protein bands of interest that show changes in phosphotyrosine levels when compared to the control lane. D, Western blot

Figure 4 .

 4 Figure 4.Cellular stiffening of HUVECs in response to pulses of force applied to hVEC-Fc magnetic beads using magnetic tweezers. A, Typical displacement of a 2.8 μm hVEC-Fc bead bound to HUVEC during application of cycles of 4 sec extension (force on: 40 pN) and 3 sec recovery (force off). Decreasing displacement is highlighted in red, demonstrating cellular stiffening. B, Change in bead displacement during 7 force pulses applied to hVEC-Fc beads (n=14). Displacements were calculated relative to the displacement generated by the first pulse of force (error bars represent SEM).

Methods.Author manuscript; available in PMC 2017 February 01.

Acknowledgments

We would like to thank Richard Superfine for access to the 3DFM and Tim O'Brien and Luke Osborne for helpful guidance with its use. All authors contributed to writing and editing of this paper. RM was supported by a NIH postdoctoral training grant (T32-CA009156) and an American Cancer Society fellowship (PF-13-318-01). CG is supported by grants from the Agence National de la Recherche (ANR-13-JSV1-0008), from the European Union Seventh Framework Programme (Marie Curie Career Integration n°304162) and from the Institut National de la Santé et de la Recherche Médicale (INSERM). This research was funded through NIH grants HL114388, GM029860 and GM103723 to KB.


Rianne Nederlof

Simone Denis

Benjamin Lauzier

Christine Des Rosiers

Markku Laakso

Jacob Hagen

Carmen Argmann

Ronald Wanders

Riekelt H Houtkooper

Markus W Hollmann

Nederlof

Sander M Houten

Dr Coert J Zuurbier
email: c.j.zuurbier@amc.uva.nl

Nederlof Rianne

Denis Simone

Rosiers Lauzier Benjamin

Acute detachment of hexokinase II from mitochondria modestly increases oxygen consumption of the intact mouse heart

Keywords: oxygen consumption, cardiac metabolism, hexokinase II, stable isotopes

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A C C E P T E D

M A N U S C R I P T

Introduction

The glycolytic enzyme hexokinase (HK) facilitates the immediate phosphorylation of glucose upon entering the cell, thereby trapping glucose within the cell. The heart mainly contains the isoforms HK I and II, in approximately equal amounts [START_REF] Smeele | The effect of standard chow and reduced hexokinase II on growth, cardiac and skeletal muscle hexokinase and low-flow cardiac ischaemia-reperfusion injury[END_REF]. HKI is present in almost all cells, mainly localized at the outer mitochondrial membrane, and hardly regulated by hormonal or metabolic signals. In contrast, HKII is mostly present in insulin-sensitive tissue (skeletal muscle, adipose tissue, heart) with its activity, expression and cellular localization (cytosolic or mitochondrial) highly regulated by hormonal or metabolic signals, and responsive to pathological conditions such as diabetes, heart failure, cancer and ischemia-reperfusion (IR) [START_REF] Wilson | Hexokinases[END_REF][START_REF] Mathupala | Hexokinase-2 bound to mitochondria: Cancer's stygian link to the "Warburg effect" and a pivotal target for effective therapy[END_REF][START_REF] Gürel | Hexokinase cellular trafficking in ischemia-reperfusion and ischemic preconditioning is altered in type I diabetic heart[END_REF][START_REF] Nederlof | Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in the heart[END_REF][START_REF] Balestra | Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?[END_REF][START_REF] Roberts | Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy[END_REF].

It remains unclear, however, what the metabolic consequences are of decreased mitochondrial HKII (mtHKII) or decreased total cardiac HKII on energy substrate selection and oxygen consumption within the intact heart. Fueger et al [START_REF] Fueger | Hexokinase II partial knockout impairs exercise-stimulated glucose uptake in oxidative muscles of mice[END_REF][START_REF] Fueger | Phosphorylation barriers to skeletal and cardiac muscle glucose uptakes in high-fat-fed-mice[END_REF] did show a reduced cardiac glucose uptake during exercise or an insulin clamp in in vivo HKII +/-as compared to wild-type hearts, but cellular glucose metabolism was not further studied. Cellular studies have suggested increased activity of HK when bound to mitochondria, due to privileged access to mitochondrially-produced ATP [START_REF] Wilson | Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function[END_REF]. Binding of HKII to mitochondria resulted in increased glycolysis in isolated cell cultures provided with glucose-only substrate, whereas cytosolic HKII did not contribute to glycolysis [START_REF] John | Subcellular localization of hexokinase I and II directs the metabolic fate of glucose[END_REF][START_REF] Calmettes | Hexokinase and cardioprotection[END_REF]. Whether these data would directly translate to similar effects (i.e. decreased glycolysis and/or glucose oxidation with decreased mtHKII) in the intact heart with high energy turnover and supplied with several substrates remains to be examined.

In addition, it is now well established that reductions in mtHKII makes the heart sensitive to IR injury [START_REF] Nederlof | Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in the heart[END_REF][START_REF] Zuurbier | Mitochondrial hexokinase and cardioprotection of the intact heart[END_REF][START_REF] Halestrap | The role of hexokinase in cardioprotection-mechanism and potential for translation[END_REF]. Several mechanisms have been proposed to explain how diminished mtHKII in tissues or organs can increase sensitivity to IR damage [START_REF] Nederlof | Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in the heart[END_REF][START_REF] Calmettes | Hexokinase and cardioprotection[END_REF][START_REF] Halestrap | The role of hexokinase in cardioprotection-mechanism and potential for translation[END_REF]. One putative mechanism figuring prominently in IR injury, is the activity of the respiratory chain, activation of which often results in increased IR injury [START_REF] Burwell | Cardioprotection by metabolic shut-down and gradual wake-up[END_REF][START_REF] Pell | Moving forwards by blocking backflow. The Yin and Yang of MI therapy[END_REF]. No information is currently available whether decreased cardiac mtHKII,

A C C E P T E D M A N U S C R I P T

either acutely or chronically, actually results in increased respiratory activity (oxygen consumption) within the intact heart. Accordingly, herein we examine the cardiac metabolic consequences of alterations in expression and cellular localization of HKII. Cardiac oxygen consumption and energy substrate metabolism in cytosol and mitochondria were determined in isolated heart models during perfusion conditions that were previously shown to have reduced mtHKII, i.e. 1) acutely through peptide treatment disrupting mtHKII binding in the presence of glucose, lactate and pyruvate [START_REF] Smeele | Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis[END_REF], or

2) chronically through deletion of one HKII copy in the presence of glucose, lactate, pyruvate, insulin and fatty acids (FFA) [START_REF] Smeele | Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis[END_REF][START_REF] Wu | Reduction in hexokinase II levels results in decreased cardiac function and altered remodelling after ischemia-reperfusion injury[END_REF]. Cardiac metabolism of both heart models was determined using either stable isotope labeled glucose or stable isotope labeled palmitate to assess relevant metabolic fluxes using mass isotopomer distribution analysis [START_REF] Ruiz | Metabolic tracing using isotopelabeled substrates and mass in the perfused mouse heart[END_REF]. To further examine whether cardiac metabolism is sensitive to the total amount of HKII chronically present, metabolic programming at the transcriptional level was compared between HKII +/-and WT hearts using RNA sequencing.

In the present study we hypothesize that within the intact heart 1) acute decreases in mtHKII are associated with decreased glucose breakdown and increased oxygen consumption, 2) chronic decreases in total and mtHKII result in altered cardiac metabolism (glucose and fatty acid breakdown), oxygen consumption, and differential (metabolic) gene transcription.

A C C E P T E D M A N U S C R I P T

Methods

Animals

C57BL/6J HKII +/- [START_REF] Heikkinen | Hexokinase II-deficient mice: prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes[END_REF] and their wild-type (WT) littermates were obtained from the in house breeding facility, as reported before [START_REF] Smeele | Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis[END_REF]. The wild-type (WT) mice in the TAT-HKII series were all C57BL/6J and obtained from Charles River. Experiments were performed with male mice between 2-4 months old. All experiments were approved by the animal ethics committee of the Academic Medical Center, Amsterdam, The Netherlands. Although the experimenter at the day of experiment was aware of genotype and peptide treatment (this was inherent to the execution of the experiment because the experimenter had to select the animals from the breeding facility and make the peptide solution), all results assessed (metabolic, mechanical) were performed in a blinded fashion.

Heart Perfusion

Mice were heparinized (15 IU) and anesthetized with pentobarbital (80 mg kg -1 ). Following tracheotomy, the mice were mechanically ventilated and a thoracotomy performed. The hearts were cannulated in situ with perfusion started before excision of the heart. Following stabilization of LV pressure, all hearts were perfused for 30 min, starting at baseline (t=0 min). For the TAT-HKII peptide series, we used the identical substrate composition of our previous study demonstrating the TAT-HKII induced ~ 30% decrease in mitochondrial HKII [START_REF] Smeele | Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis[END_REF], i.e. 11 mM glucose, 1 mM lactate, 0.1 mM pyruvate and 0.5 mM glutamine. For the HKII +/-series with ~ 50% less cardiac HKII in the in vivo condition [START_REF] Wu | Reduction in hexokinase II levels results in decreased cardiac function and altered remodelling after ischemia-reperfusion injury[END_REF], the isolated hearts were also perfused with in vivo-like substrates: 5.5 mM glucose, 1 mM lactate, 0.1 mM pyruvate, 0.5 mM glutamine, 100 mU/L insulin, 0.05 mM L-carnitine and 0.3 mM palmitate bound to 3% albumin. Throughout perfusion, effluent samples were collected for oxygen partial pressures (at baseline and 25 min perfusion for the TAT-HKII peptide series, and at 25 min perfusion for the HKII +/-series) and lactate and pyruvate concentration (at 25 min perfusion for both series). For the TAT-HKII peptide series an estimate of glucose uptake was derived from glucose measurements in the influent and effluent samples using a bloodgas analyser (RapidPoint400, Siemens). Influent samples were collected at the end of each experiment. TAT and TATHKII peptide were dissolved in perfusate Krebs-Henseleit solution and administered at 1% of total perfusion flow through a side-arm connected to a custom-made mixing chamber. Peptide administration was started at baseline (t= 0 min) perfusion and continued to the end of the 30 min perfusion period. At the end hearts were immediately frozen in liquid nitrogen, weighted and stored at -80 °C until further analysis.

For the TATHKII peptide series we restricted ourselves to similar substrate composition for which we had been able to demonstrate previously, with the elaborate and demanding technique of immunogold labeling of HKII in hundreds of EM images (Smeele Km et al, CircRes 2011), that the used dosage of 200 nm TATHKII reduced mtHKII by about 30%. For the partial KO we were not restricted to a specific substrate composition, because we had shown previously that the difference in HKII occurs both in isolated hearts perfused with or without insulin, or in the in vivo condition with fatty acids present (Wu R, Smeele KM et al, Circ Res 2011). Therefore, we choose for the most physiological composition of the perfusate, i.e. next to glucose-lactate-pyruvate, also the addition of insulin and fatty acids. This allowed evaluation of the role of HKII for cardiac metabolism within a high metabolic contextance.

Mass isotopomer distribution analysis

The isotope distribution of lactate, pyruvate and citric acid cycle intermediates was determined in heart tissue freeze clamped after Langendorff perfusion. Heart tissue (~100 mg wet weight) was homogenized in 1 mL 8% (w/v) sulfosalicylic acid and 100 μL of internal standard (0.2 mM 2phenylbutyric acid) using an Ultra-Turrax and sonication or the Tissuelyser (Qiagen). The resulting suspension was centrifuged (10 minutes, 12000 g), after which the pH of the supernatant was adjusted to 7 by adding 30% NaOH dropwise. The sample was then split in two. In half of the extract the keto groups were directly oximated by addition of 50 μL 5M hydroxylamine hydrochloride and incubation at 60°C for 30 minutes. The other half of the extract was first reduced by 50 μL 200 mM NaBH 4 , and incubated at room temperature for 30 minutes. After inactivation and neutralization using hydrochloric acid and KOH/MOPS, respectively, the sample was further treated with citrate lyase and hydroxylamine, to convert citrate into oximated oxaloacetate (750 μL of 500 mM triethylamine pH 7.4, 100 mM MgSO 4 , 50 mM EDTA, 10 μL 5M hydroxylamine hydrochloride and 2.5U citrate lyase). The mixture was sonicated and incubated for 5 minutes at 37°C after which the sample was acidified by adding 100 μL saturated sulfosalicylic acid and centrifuged (10 minutes, 1200 g). Next, both samples were reacidified by adding 100 μL 37% HCl, and saturated with salt by adding ~0.2 g NaCl. The organic acids were extracted twice by adding 3 mL ethylacetate. Both ethylacetate extracts were pooled and dried. The residue was derivatized by addition of 75 μL N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide, and incubation at 80°C for 60 minutes. Within 24 hours, the sample (1 μL) was analyzed by gas chromatography-mass spectrometry (GC-MS). Peak intensities were integrated, corrected for natural abundance, and used for the calculation of

A C C E P T E D M A N U S C R I P T

contribution of glucose and palmitate to acetyl-CoA formation for citrate synthesis [START_REF] Ruiz | Metabolic tracing using isotopelabeled substrates and mass in the perfused mouse heart[END_REF].

Concentrations of citric acid, 2-ketoglutaric acid, malic acid, lactic acid, succinic acid, pyruvic acid and fumaric acid were determined using 0.2 mM 2-phenylbutyric acid as internal standard.

All hearts in the TAT series were perfused with [U- 13 were generated by aligning the fastq files to the mouse genome mm10 (UCSC) with STAR and then using featureCounts to count overlaps with exons. Final counts were grouped at the gene level [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF].

Outlier detection identified 2 samples (1 WT and 1 HKII +/-), which were removed from subsequent analysis. Differential expression analysis was conducted with R the packages limma (with voom transformation) [START_REF] Smyth | Limma: Linear Models for Microarray Data[END_REF] and DESeq2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNAseq data with DESeq2[END_REF](Love et al, 2014). Low count genes were removed in the limma analysis, genes were kept if they had at least one count per million in at least 5 samples. A false discovery rate (Benjamini-Hochberg) of 0.05 was chosen as the cut off.

Statistics

Values are presented as mean ± SEM. Differences between TAT and TATHKII groups for mechanical performance and oxygen consumption were analysed by a two-way ANOVA for repeated measurements followed by contrast comparison (SPSS version 23). Differences between groups were analyzed with paired or unpaired Student's t-test when applicable. Differences were considered statistically significant at P<0.05.

A C C E P T E D M A N U S C R I P T

Results

Functional effects of acute detachment of HKII using TATHKII peptide treatment in ex vivo

Langendorff hearts

The impact of 25 min TATHKII peptide treatment was compared with that of control, the TAT peptide (Figure 1). Peak systolic ventricular pressure (Fig. 1A) and RPP (Fig. 1C) showed a moderate decrease over time in the control group, probably as a result of deterioration of the preparation over time. No changes in heart rate over time were observed in the control group (Fig. 1B). TATHKII also significantly reduced RPP during 25 min peptide perfusion. However, TATHKII was without significant effect on these changes in mechanical performance when compared to vehicle. In contrast, TATHKII treatment resulted in a significant, albeit modest, increase in oxygen consumption as compared to vehicle (Figure 1D). No differences in flow or heart weight were present between groups (Table 1).

These data demonstrate that acute detachment of HKII from mitochondria in the intact heart increased oxygen consumption with no direct effect on mechanical performance.

Metabolic effects of acute detachment of HKII using TATHKII peptide treatment in ex vivo

Langendorff hearts

Next we evaluated whether acute detachment of HKII from mitochondria affected glucose metabolism. Lactate and pyruvate efflux rate (reflecting glycolysis from exogenous labeled glucose)

was low in all hearts, probably due to the lack of insulin in these conditions (Figure 2A). TATHKII treatment was without significant effect on the rate of anaerobic glycolysis, albeit the rate was reduced by 43% (p=0.28 To further explore possible mechanisms for the increased oxygen consumption and reduced glucose metabolism in the TAT-HKII treated hearts, we examined glucose uptake and 13 C intermediates in the two groups of hearts (table 2). TAT-HKII treatment was associated with a non-significant trend (p=0.131) in decreased glucose uptake, and a significant increase in tissue pyruvate concentration.

Functional effects of chronic partial HKII knockout in ex vivo Langendorff hearts

Mechanical performance and oxygen consumption for WT and HKII +/-hearts are given in Figure 3.

Peak systolic pressure, heart rate and RPP are not different between the genotypes. Flow and heart weight were also not different between the genotypes (Table 1). Cardiac oxygen consumption is also not affected by deletion of one HKII copy.

In these insulin and FFA perfused hearts (in addition to glucose, lactate, pyruvate and glutamine) mechanical performance is similar to hearts perfused without insulin and FFA (compare to Fig. 1), when comparing baseline conditions between WT and TAT groups. However, adding 100 mU/L insulin and 0.3 mM FFA show a non-significant trend (p=0.084) in lowering cardiac oxygen consumption (Fig. 1D vs Fig. 3D; 7.6 ± 0.6 vs 6.3 ± 0.4 µmol/min/g ww , respectively).

Metabolic effects of partial HKII knockout in ex vivo Langendorff hearts

Anaerobic glycolysis rates in these hearts perfused with additional insulin and FFA was much higher (0.36 ± 0.02 µmol/min; Fig. 4A) than in the hearts perfused without insulin and FFA (0.07 ± 0.03 µmol/min; Fig. 2a), likely due to the presence of the relative high insulin concentration. Partial deletion of HKII was, however, without effect on anaerobic glycolysis rate. Glucose enrichment of

A C C E P T E D M A N U S C R I P T

tissue pyruvate (table 1) was not significantly higher than in the hearts perfused without insulin and FFA, and was not affected by genotype (p=0.22). The relative contribution of glucose and palmitate to acetyl-CoA formation for citrate synthesis in TCA cycle is shown in Fig. 4B and4C, respectively.

Despite the presence of FFA (known to inhibit glucose metabolism), carbohydrate contribution to citrate synthesis was again much higher (61.4 ± 6.9%) in these insulin-perfused hearts as compared to hearts perfused without insulin (28.4 ± 4.5%; Fig 2B). However, the contribution of glucose, and lactate and pyruvate, to citrate synthesis was not affected by the partial deletion of HKII.

Correspondingly, palmitate contribution to acetyl-CoA formation for citrate synthesis was also similar between the two genotypes (Fig. 4C).

Consequences of partial HKII knockout on the cardiac transcriptome

In order to study the consequences of deletion of one HKII copy at the transcriptome level, we performed RNAseq analysis of WT and HKII +/--hearts. Analysis of differentially expressed genes revealed a small impact of the partial HKII deletion, with only the decrease in HKII expression reaching the FDR cut off of 0.05 in both limma and DESeq2 (Table 3, showing the first 5 genes with largest tendencies of being differentially expressed between the genotypes). In DESeq2, two more genes, Antxr2 and Kbtbd12, were differentially expressed with small log2 fold changes (0.24 and 0.31, respectively). Given the lack of previous association of these genes with cardiac metabolism and the overall small changes in gene expression in the HKII +/--hearts, the identification of these transcripts as differentially expressed is likely spurious.

A C C E P T E D M A N U S C R I P T

Discussion

Although it is well recognized that the degree of mitochondrion-HKII association is sensitive to many cardiac pathologies and interventions (IR, cardioprotection, insulin treatment, diabetes, hypertrophy, heart failure [START_REF] Nederlof | Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in the heart[END_REF]), the metabolic consequences for the intact heart are, surprisingly, to a large extent unknown. We now report that 1) acute disruption of HKII from mitochondria in intact hearts is associated with an increase in oxygen consumption and a nonsignificant trend of decreased glucose contribution to citrate synthase flux; 2) chronically reduced HKII (including reduced mtHKII) is not associated with alterations in cardiac oxygen consumption,metabolism, or metabolic reprogramming at the transcriptional level of the intact heart not cause.

Acute decreases in mitochondrial hexokinase II and cardiac metabolism

We have demonstrated that acute detachment of HKII from mitochondria increases cardiac oxygen consumption. We are unaware of other studies that have specifically examined the effects of mitochondria-HKII association on oxygen consumption. Our data indicates that HKII attachment to the mitochondria slows down mitochondrial respiration in a functional, working organ. This is commensurate with our previous observations that short periods of ischemia, able to induce cardioprotection and associated with increased mtHKII [START_REF] Zuurbier | Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution[END_REF][START_REF] Gürel | Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion[END_REF][START_REF] Pasdois | Extent of mitochondrial hexokinase II dissociation during ischemia correlates with mitochondrial cytochrome c release, reactive oxygen species production, and infarct size on reperfusion[END_REF], result in slower mitochondrial activation of oxygen consumption [START_REF] Zuurbier | Post-ischaemic changes in the response time of oxygen consumption to demand in the isolated rat heart are mediated partly by calcium and glycolysis[END_REF][START_REF] Zuurbier | Mitochondrial response to heart rate steps in isolated rabbit heart is slowed after myocardial stunning[END_REF]. This slowing down was only observed with glucose, and not with lactate or pyruvate substrates, suggesting a glycolytic-origin, possibly HKII, of this effect.

Since cardioprotection is associated with increased glucose metabolism [START_REF] Ji | AMPK-regulated and Akt-dependent enhancement of glucose uptake is essential in ischemic preconditioning-alleviated reperfusion injury[END_REF], it is then suggested that increased glucose metabolism results in attenuated mitochondrial function. Indeed, in the present study increased binding of HKII to mitochondria was associated with a trend for increased glucose metabolism. It is generally believed that the physical binding of HK to mitochondria increases HK activity through decreasing its K m for ATP, relieve of allosteric inhibition by G6P and glucose-1,6-

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biphosphate, and preferential access to mitochondrial produced ATP [START_REF] Wilson | Hexokinases[END_REF][START_REF] Beitner | Inhibition of mitochondrial and soluble hexokinase from various rat tissues by glucose 1,6-biphosphate[END_REF][START_REF] Lynch | Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells[END_REF]. HKII binding to mitochondria promoted glycolysis in CHO cells [START_REF] John | Subcellular localization of hexokinase I and II directs the metabolic fate of glucose[END_REF]. Increased glucose metabolism could potentially decrease mitochondrial oxygen consumption because of the higher P/O ratio for glucose relative to other substrates (less oxygen needed per ATP produced), or because of increased transport of cytosolic NADH into the mitochondria, which transport may impair oxygen consumption [START_REF] Kobayashi | Control of maximum rates of glycolysis in rat cardiac muscle[END_REF].

Alternatively, the increased oxygen consumption with similar cardiac function could also suggest a lower cardiac efficiency. Although care should be taken for over interpretation, the observed nonsignificant trend for decreased glucose uptake and increased tissue pyruvate concentration with less mtHKII, indicates that the decrease in glucose metabolism with detachment of mtHKII is controlled by impairment of glucose uptake and pyruvate oxidation within the intact heart. The increased oxygen consumption is then likely a result of a decrease in glucose metabolism, which, because no changes in mechanical work were observed, is then compensated with increased FA oxidation with its consequently higher oxygen need to provide similar ATP.

Chronic decreases in (mitochondrial) hexokinase II and cardiac metabolism

In contrast to the acute manipulation of mitochondrial HKII, we observed no changes in oxygen consumption and metabolism in the partial HKII knockout heart, which we previously showed to have 50% less total and mitochondrial HKII [START_REF] Wu | Reduction in hexokinase II levels results in decreased cardiac function and altered remodelling after ischemia-reperfusion injury[END_REF]. Since chronic reductions in proteins are notorious for inducing compensatory alterations in other genes/proteins, possibly explaining the lack of changes in cardiac metabolism and oxygen consumption with chronic reduction in HKII, we characterized the cardiac transcriptome through RNA sequenching. The absence of differential gene expression demonstrated that compensation at the level of gene expression was absent, negating a role for gene expression adaptation in this model. However, at this stage, we cannot exclude that adaptation did occur posttranscriptonally (proteins and its posttranslational modifications). That cardiac glucose metabolism was unaffected in these partial HKII knockout hearts, is in support of

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previous findings using the similar partial HKII knockout and reporting unaltered cardiac glucose uptake during sedentary conditions in these hearts [START_REF] Fueger | Hexokinase II partial knockout impairs exercise-stimulated glucose uptake in oxidative muscles of mice[END_REF][START_REF] Heikkinen | Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes[END_REF].

No insulin versus high-dose insulin and cardiac glucose metabolism

In the present study we used perfusion conditions with and without insulin, as dictated by our previous experimental conditions where effects of the HKII manipulations were already determined [START_REF] Smeele | Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis[END_REF][START_REF] Wu | Reduction in hexokinase II levels results in decreased cardiac function and altered remodelling after ischemia-reperfusion injury[END_REF]. The absence of insulin, despite glucose being present at 11 mM, resulted in a rather low rate of anaerobic glycolysis (0.07 ± 0.03 µmol/min) and glucose contribution to acetyl-CoA formation for citrate synthesis (11.2 ± 2.2%), even when compared to other heart studies using glucose-only perfusates. Total carbohydrates (glucose, lactate, pyruvate) contribution to acetyl-CoA formation for citrate synthesis amounted to near 30%, suggesting that a major part of citrate synthesis was derived from endogenous FA oxidation from stored triglycerides. It was shown before that in conditions of glucose-only perfusate myocardial triglyceride turnover contributes significantly to energy production in isolated hearts [START_REF] Saddik | Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts[END_REF]. The low rates of glucose metabolism are likely due to the presence of 1 mM lactate; physiological concentrations of lactate decrease glucose metabolism to a large extent [START_REF] Chatham | Preferential inhibition of lactate oxidation relative to glucose oxidation in the rat heart following diabetes[END_REF]. The data suggest that in perfusion conditions with glucose and lactate both being present, insulin is needed to facilitate glucose metabolism in the isolated heart. In the presence of insulin and FFA in the perfusate, carbohydrates and FFA acids contributed for 60% and 15%, respectively, to citrate synthesis in the intact hearts. These values are commensurate with values reported before in the isolated working rat heart [START_REF] Vincent | Differential modulation of citrate synthesis and release by fatty acids in perfused working hearts[END_REF], although much higher contributions of fatty acids have also commonly been observed. The relatively low fatty acid contribution could be due to the relatively high insulin concentration (100mU/L), the presence of 1 mM lactate (physiological concentration, and often neglected in isolated heart studies), knowing that lactate inhibits fatty acid oxidation [START_REF] Van Der Vusse | Interrelationship between lactate and cardiac fatty acid metabolism[END_REF], and the low, but physiologically-relevant for the fed condition [START_REF] Ruiz | MK2 deletion in mice prevents diabetes-induced perturbations in lipid metabolism and cardiac dysfunction[END_REF], fatty acid concentration (0.3 mM).

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Methodological considerations

In the current study the effects of modest changes (30-50% decrease) in HKII on cardiac metabolism were examined. These modest HKII changes probably partly explain why only modest or even no changes in cardiac metabolism were observed. Importantly, these modest changes in HKII mimic the changes in HKII that occur during pathophysiological interventions such as IR and ischemic preconditioning [24 -26], type I and type II diabetes [START_REF] Gürel | Hexokinase cellular trafficking in ischemia-reperfusion and ischemic preconditioning is altered in type I diabetic heart[END_REF], insulin treatment [START_REF] Love | Moderated estimation of fold change and dispersion for RNAseq data with DESeq2[END_REF][START_REF] Southworth | A reevaluation of the roles of hexokinase I and hexokinase II in the heart[END_REF], or cardiac remodeling during hypertrophy and heart failure [START_REF] Balestra | Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?[END_REF][START_REF] Riehle | PCG-1β deficiency accelerates the transition to heart failure in pressure overload hypertrophy[END_REF]. Were we to use a complete knockout of HKII or transgenic overexpression of HKII by more than a factor of 5, larger changes in metabolism would probably have occurred. However, we believe the imposed manipulations of HKII and their metabolic consequences in the current study are more relevant to the real life pathophysiological conditions.

In the present work effects of acutely reduced mtHKII on cardiac metabolism were examined during perfusion with a specific substrate composition of 11 mM glucose, 1 mM lactate and 0.1 mM pyruvate. It is known that this mixture results in only small contributions of glucose to ATP production [START_REF] Chatham | Preferential inhibition of lactate oxidation relative to glucose oxidation in the rat heart following diabetes[END_REF], as supported by our low rates of glycolysis and glucose contribution to citrate formation, possibly explaining that we observed only non-significant trends for TAT-HKII effects on glucose metabolism. Therefore, it would be of interest to examine glucose contribution to citrate in the absence of lactate, or in the presence of insulin in the TATHKII series. Under these metabolic substrate conditions, glycolysis and glucose contribution to citrate would be more prominent making it perhaps easier to obtain significant effects of partly decreasing mt-HKII. Further research is needed to examine these questions. Finally, myocardial oxygen consumption was increased with acute detachment of HKII from mitochondria, but not with chronic reductions in mtHKII. New research should be directed to examine whether the acute increase in oxygen consumption is only a reversible phenomenon at maintained lower levels of mtHKII, possibly explaining that in longer-term oxygen consumption normalizes again to pre-treatment levels.

In conclusion, our data suggest that an acute, but not chronic, decrease in HKII binding to mitochondria increases cardiac oxygen consumption that is associated with a trend for decreased glucose contribution towards citrate synthesis in the mitochondria.

Figure 1 :

 1 Figure 1:TATHKII peptide effects on mechanical performance and oxygen consumption of

Figure 2 :

 2 Figure 2:TATHKII peptide effects on cardiac metabolism in isolated hearts perfused with 13 C

Figure 3 :

 3 Figure 3:Mechanical performance and oxygen consumption of HKII +/-and WT isolated hearts.

Figure 4 :

 4 Figure 4:Cardiac metabolism in isolated HKII +/-and WT hearts perfused with13 C glucose or13 C palmitate, and lactate, pyruvate and insulin. Effects of HKII +/-on anaerobic glycolysis

Figure 1 A

 1 Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 A 29 Highlights

 429 Figure 4

  RNA was isolated from 6 WT and 6 HKII +/-hearts using QIAzol lysis reagent followed by cleaned up using the RNeasy kit. RNA samples were submitted to the Genomics Core Facility at the Icahn Institute and Department of Genetics and Genomic Sciences. cDNA libraries were prepared using the

	C 6 ] glucose (initial molar percent enrichment
	(MPE): 99%; Cambridge Isotope Laboratories, Andover, USA ); for the HKII +/-hearts unlabelled glucose
	or palmitate were replaced by either [U-13 C 6 ] glucose or [U-13 C 16 ] palmitate (MPE:98%; Cambridge
	Isotope Laboratories, Andover, USA), respectively, to probe both carbohydrates (CHO) and long-chain
	fatty acid metabolism (PAL). Using the stable isotopes analysis we determined: 1) anaerobic
	glycolysis from the efflux rate of [U-13 C 2.4. RNA isolation and RNA sequencing analysis
	Illumina TruSeq RNA Library Preparation kit (#RS-122-2001). Samples were run on Illumina HiSeq
	2000 at a read length of 100nt single end, and a depth of about 35 million per sample. Count files

3 ]-labeled lactate and [U-

13

C 3 ]-labeled pyruvate derived from the breakdown of exogenous [U-13 C 6 ] glucose; 2) fractional enrichment of tissue pyruvate by

13

C glucose, using the molar percent enrichment of pyruvate for all 3 C atoms of pyruvate (MPE pyruvate (M3))

[START_REF] Vincent | Differential modulation of citrate synthesis and release by fatty acids in perfused working hearts[END_REF]

; 3) fractional contribution of CHO (glucose and lactate-pyruvate) to acetyl-CoA formation for citrate synthesis (CS; from the

13

C enrichment of the acetyl and oxaloacetate moiety of citrate), this contribution is defined as pyruvate decarboxylation to citrate (PDC/CS); and 4) fractional contribution of PAL to acetyl-CoA formation for citrate synthesis (PAL/CS).

  ). From the MPE of pyruvate it can be concluded that exogenous glucose contributes 38% to endogenous pyruvate formation, the remainder probably coming from citrate synthesis is depicted in Figure2B. Carbohydrates contributed approximately 28% to the citrate synthase flux, with the contribution of lactate being 18% and that of glucose 10%. TATHKII treatment resulted in a trend towards a decrease of glucose contribution to citrate synthase flux (p=0.076).

exogenous lactate and pyruvate (table

1

). TATHKII peptide treatment was without effect on the MPE of pyruvate, i.e. the pyruvate formation from glucose. The relative contribution of carbohydrates to

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Table 1 .

 1 Physiological parameters of isolated mouse hearts and13 C enrichments of tissue pyruvate isolated from Langendorff-perfused mouse hearts perfused with [U-13 C 6 ]glucose.

		TAT	TATHKII	WT	HKII +/-
	Heart weight (mg)	165±5	178±5	198±8	210±8
	Coronary flow (ml/min/g ww )	11.6±0.8	11.8±0.9	10.3±0.5	10.5±0.9
	MPE pyruvate (M3)	38.2±2.9	34.8±1.9	44.4±5.1	37.0±3.0
	Values are means ± SE. (n=8/9 for TAT/TATHKII hearts and n=16/17 for WT/HKII+/-hearts)

Table 2 .

 2 Glucose uptake and13 C intermediates of metabolism derived from13 C enrichment

		TAT	TATHKII	P value
	Glucose uptake (µmol/min/G ww )	1.20 ± 0.59	0.27 ± 0.18	0.131
	Pyruvic acid	20 ± 2	27 ± 2	0.033*
	Lactic acid	8301 ± 1251	6208 ± 691	0.135
	Citrate	778 ± 58	843 ± 53	0.424
	2-ketoglutaric acid	5 ± 0	6 ± 0	0.087
	Malic acid	232 ± 37	183 ± 20	0.235
	Succinic acid	566 ± 35	479 ± 28	0.068
	Fumaric acid	29 ± 6	27 ± 4	0.761
	Values are means ± SE. (n=8/9 for TAT/TATHKII hearts). Metabolites given by pmol/mg ww

Table 3 .

 3 RNA sequence and differential expression gene analysis results for two different analysis approaches (LImma and DESseq2). The first five genes with the largest differences in expression between genotypes are displayed.Values are derived from five WT and five HKII +/-hearts.

		Limma analysis			DESeq2 analysis
	gene	Adjusted P	Fold Change	gene	Adjusted P	Fold Change
	HKII	0.010	0.57	HKII	1.41 10 -13	0.68
	Kbtbd12	0.657	1.77	Antxr2	0.021	1.18
	Mir22hg	0.964	0.77	Kbtbd12	0.021	1.24
	Lonrf1	0.964	1.22	Gm7120	0.123	0.84
	Slc7a1	0.964	1.32	Wee1	0.408	1.19

Funding:

This work was supported by a research grant (NHS2010B011) of the Dutch Heart Foundation.

Disclosure statement:

No relevant disclosures exist for this work

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Authors Contributions

RN, SMH and CJZ contributed to the design and conduct of the study, data collection and analysis, data interpretation and manuscript writing; BL contributed to design and conduct of the study, data interpretation and manuscript writing; SD, JH, CA contributed to data collection and analysis, data interpretation and manuscript writing; CDR, ML, RW, RHH and MWH contributed to data interpretation and manuscript writing. All authors approved the final version of the manuscript.