Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of ...

27 Pages·2015·5.51 MB·English

JBC Papers in Press. Published on August 5, 2015 as Manuscript M115.643544
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.643544
Running title: Ca 1.1, CD36 and Mitochondria  
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Ca2+ Binding/Permeation via Calcium Channel, Ca 1.1, Regulates the Intracellular
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Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism

Dimitra K. Georgiou1*, Adan Dagnino-Acosta1*, Chang Seok Lee1*, Deric M. Griffin1, Hui Wang1, William R.
Lagor1, Robia G. Pautler1, Robert T. Dirksen2 and Susan L. Hamilton1#

1Department of Molecular Physiology and Biophysics
Baylor College of Medicine
One Baylor Plaza, Houston, Texas 77030
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2Department of Pharmacology and Physiology d fro
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University of Rochester Medical Center ttp
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601 Elmwood Avenue, Rochester, NY 14642 w
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#Running title: Ca 1.1, CD36 and Mitochondria gu
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§To whom correspondence should be addressed: Susan Hamilton, Department of Molecular Physiology & M
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Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA, Tel:[phone]; Fax: 9
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[phone]; E-mail: [email protected]
*The first 3 authors contributed equally to this study.

Keywords: Ca2+ channel, Ca 1.1, skeletal muscle, CaM kinase II, metabolic rate, nitric oxide synthase, CD36
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Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Running title: Ca 1.1, CD36 and Mitochondria  
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Background: Ca2+ binding and/or permeation via Ca 1.1 Ca 1.1, the skeletal muscle voltage sensor, is also
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in skeletal muscle activates CaMKII (1). a voltage dependent Ca2+ channel which allows a small
Results: Mice with a Ca2+ binding /permeation defect in Ca2+ influx into the mammalian skeletal muscle fibers.
Ca 1.1 have increased body fat, reduced fatty acid However, Ca2+ influx through this channel is not required
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metabolism and altered CD36 distribution. for E-C coupling (14). Consistent with this, in skeletal
Conclusion: Ca 1.1 regulates CD36 distribution and fatty muscle of teleost fish (15), Ca2+ influx through the Ca 1.1
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acid metabolism. channel is absent. One of the differences between teleost
Significance: New therapeutic targets are identified to Ca 1.1 and mammalian Ca 1.1 is the sequence within the
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increase skeletal muscle energy expenditure. pore. The teleost pore prevents Ca2+ permeation due to the
presence of a negatively charged aspartate in repeat II
ABSTRACT above Ca2+ binding glutamate (15). This negative charge is
Ca2+ permeation and/or binding to the skeletal likely to increase the affinity of the pore for Ca2+
muscle L-type Ca2+ channel (Ca 1.1) facilitates preventing Ca2+ from moving through the channel. Based
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activation of Ca2+/calmodulin kinase type II (CaMKII) on these findings, Schredelseker et al (15) suggested that
and Ca2+ store refilling to reduce muscle fatigue and the possibility that Ca2+ influx into mammalian skeletal
atrophy (1). Mice with a mutation (E1014K) in the muscle fibers might be vestigial. We created a mouse
Cacna1s (α subunit of Ca 1.1) gene that abolishes Ca2+ model in which glutamate in the repeat III of the pore was D
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binding within the Ca 1.1 pore, gain more body weight mutated to a positively charged lysine designed to decrease n
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and fat on a chow diet than control mice, without or abolish Ca2+ binding within the pore. These mice ad
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cChaan1g.1e-sm iend iafoteodd CinatMakKe IIo ra caticvtaivtiitoyn, ismugpgaecsttsi nmg utshcalet dofe siCganMateKdI IE, 1d0e1c4reKa soerd ECKa 2d+i sipnlfalyuexd, rdeedcurecaesde dr eafciltliivnagt ioonf d from
eneVrgy expenditure. We delineate a pathway (Cav1.1→ sarcoplasmic reticulum Ca2+ stores, and decreased protein http
CaMKII→ nitric oxide synthase (NOS) in normal synthesis. The net result of activation of this CaV1.1 ://w
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skeletal muscle that regulates the intracellular mediated pathway is increased fatigue and decreased w
distribution of the fatty acid transport protein, CD36, muscle fiber size (1). A possible interpretation of these .jbc
altering fatty acid metabolism. The consequences of findings is that Ca2+ binding within the pore or transition .org
blocking this pathway are decreased mitochondrial β- through the pore alters Ca 1.1 conformation to modulate by/
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oxidation and decreased energy expenditure. This intracellular signal transduction events. ue
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study delineates a previously uncharacterized Ca 1.1- Skeletal muscle plays a major role in regulating t o
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mediated pathway that regulates energy utilization in metabolic rate in adult humans (8). We now demonstrate M
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skeletal muscle. that the EK mice display altered metabolic function. They rch
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exhibit increased body fat, decreased energy expenditure, 9
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Excess body fat, in particular, abdominal/visceral and impaired glucose tolerance. In the current study, we 0
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obesity, increases the risk of a wide variety of human elucidate a novel role for Ca2+ permeation/binding to the
diseases, including type 2 diabetes (2), cancer (3,4), Ca 1.1 pore in the regulation of whole body energy
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dementia (5), and cardiovascular disease (6). Decreased expenditure via effects on muscle mitochondrial function.
metabolism of fat relative to carbohydrate, fatty acid
overload and/or incomplete fatty acid metabolism in EXPERIMENTAL PROCEDURES
skeletal muscle are risk factors for insulin resistance, Animals- Ca 1.1 E1014K knock-in mice (EK)
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metabolic syndrome, and obesity (7,8). Altered muscle were generated as described previously (1). In this study,
metabolism contributes to the development of insulin 6-25 week old EK and wild type (WT) male mice were
resistance and type II diabetes (9,10). The underlying used, unless otherwise indicated. All mice were housed at
mechanisms regulating skeletal muscle fuel selection and room temperature with a 12:12 hour light-dark cycle and
metabolic rate include differences in mitochondrial provided with food and water ad libitum. Mice were fed a
biogenesis, fatty acid uptake into muscle and chow diet consisting of 24% protein, 16% fat and 60%
mitochondria, and both carnitine palmitoyltransferase 1 carbohydrates. All procedures were approved by the
(CPT1) (11,12) and Sirt3-mediated pyruvate Animal Care Committee at Baylor College of Medicine
dehydrogenase activities (13). (BCM).
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Running title: Ca 1.1, CD36 and Mitochondria  
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Materials- Autocamtide 2-related inhibitory within Paravision was then used to calculate the T
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peptide (AIP), L-NG-nitroarginine methyl ester (L- relaxation time of each ROI. The T relaxation times were
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NAME), and L-carnitine, sodium palmitate, coenzyme A, averaged to give the overall T relaxation time of the
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carbonylcyanide p-trifluoromethoxyphenylhydrazone tissue. Signal intensities were normalized to a water
(FCCP), antimycin A, oligomycin A, KN 92, KN-93, phantom.
nifedipine, BayK8644 and sodium pyruvate were Body composition- Scans were performed in the
purchased from Sigma-Aldrich. N-benzyl-p-toluene mouse phenotyping core at Baylor College of Medicine
sulfonamide (BTS) was purchased from Tocris with a Lunar PIXI densitometer and were analyzed with
Biosciences. Tetramethylrhodamine, ethyl ester (TMRE) the Lunar PIXImus 2.10 software. Mice were anesthetized
and the nitric oxide sensor 4-amino-5-methylamino-2,7- with 2% isoflurane.
difluorescein (DAF-FM) were purchased from Invitrogen. Indirect calorimetry (IDC) - To obtain the
Ultra fatty acid free bovine serum albumin (BSA) was respiratory exchange ratio (VCO /VO ), 24 h of IDC was
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purchased from Roche. performed in the mouse phenotyping core at BCM with a
Magnetic Resonance Imaging (MRI) - For live VO /VCO Oxymax system (Columbus Instruments). For
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imaging, animals were anesthetized with 5% isoflurane in the cold challenge, mice were placed in the
O and maintained with 2% isoflurane in O . Animals were Comprehensive Laboratory Animal Monitoring System
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placed headfirst in a prone position on a custom-built (CLAM System, Columbus Instruments) and exposed to D
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holder while in the magnet. Respiration was monitored 4C for up to 12 h. Mouse body temperature was recorded n
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using a pressure pad placed beneath the animal and via telemetry probes implanted subcutaneously in the ad
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temperature was monitored using a rectal probe (SA abdominal area. The VO2 and VCO2 were recorded for fro
Instruments). Temperature was maintained at 37°C using metabolic rate evaluation. m
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an air heating system (SA Instruments). The Model 1025 Insulin tolerance test (ITT), glucose tolerance tests ttp
Small Animal Monitoring and Gating System software (GTT), and insulin levels- Measurements of insulin, and ://w
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was used to monitor respiratory rates and temperature (SA glucose were performed in the mouse metabolism core at w
Instruments). All images were acquired on a 9.4 T, Bruker BCM. For the ITT, mice were given an intraperitoneal .jbc
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Avance Biospec Spectrometer, 21-cm bore horizontal (I.P.) injection of insulin (0.75 U/kg BW) after 4 h fasting rg
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scanner with 35 mm volume resonator using Paravision and for the GTT mice were given an I.P injection of y
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5.0 software (Bruker Biospin). Initial pilot scans were glucose (1.5 mg/g BW) after 6 h fasting (16). ue
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performed to correctly position the mice before scanning Activity monitoring and food intake- Food intake t o
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for subcutaneous, visceral, epididymal and muscle fat. For and activity were assessed at 25°C with a CLAM System M
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subcutaneous, visceral and epididymal fat, a modified in the mouse phenotyping core at BCM. For food intake rch
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multi-slice multi-echo scan protocol was used with the monitoring, the chow diet was ground to fine powder. 9
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following parameters: Repetition time (TR)= 800 ms, echo Mice, acclimated in the cages for 24 h before the 01
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time (TE)= 10.21 ms, field of view (FOV)= 3.00 cm, slice experiment, were monitored for a period of 10 days, with a
thickness= 2.00 mm (axial scans) or 1.5-2.0 mm (sagittal less than 24 h break after the first 5 days. Activity
scans), matrix= 128x128, number of excitations (NEX)= 2, measurements included horizontal, vertical (Z), and
number of cycles= 1, and acquisition time= 3 min 24 s 800 grooming movements.
ms. A rapid acquisition with refocused echoes and variable Co-immunoprecipitation (CoIP) - Muscles
repetition time (RAREVTR) scan protocol was used to (gastrocnemius) were homogenized and lysed in IP buffer
acquire spin lattice (T ) weighted images of the liver or (10 mM Tris pH 7.4, 1% NP-40, 0.5% sodium
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muscle and had the following parameters: TR= 7500 ms, deoxycholate containing protease and phosphatase
TE= 6.57 ms, FOV= 3.00 cm, slice thickness= 1.0 mm, inhibitors). 600 g of muscle lysates with a final volume
matrix= 128x128, NEX= 1, number of cycles= 1, and of 400 l were then incubated with 4 g of either eNOS or
acquisition time= 10 min 28 s 405 ms. Analyses for all Ca 1.1 antibody at 4C overnight followed by 90 min
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liver and muscle RAREVTR images were performed with incubation with protein G agarose beads (Roche). As a
Paravision 5.0. For liver and muscle, seven or five regions negative control, the same amount of protein was
of interests (ROIs) were chosen at random, respectively, incubated with non-immunized IgG as above. After
within RAREVTR slice. The image sequence analysis tool incubation, beads were washed 3 times with IP buffer and
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Running title: Ca 1.1, CD36 and Mitochondria  
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the final immune complex was eluted by boiling in the phosphate buffer (pH 7.2), 0.5 mg/ml DAB and 5 mM
SDS sample buffer. cytochrome c.
S-nitrosylation- Proteins from diaphragm muscles Subcellular fractionation- Diaphragms excised
of WT and EK mice were assessed using iodoTMTTM from WT and EK mice were used for fractionation of
reagent for labeling (PierceTM S-Nitrosylation Western cytoplasm, nuclei, and plasma membrane using a
Blot Kit, Thermo Fisher Scientific). Total protein was MinuteTM Plasma Membrane Protein Isolation Kit (Invent
stained with the swift membrane stain (Geno Technology). Biotechnologies). Mitochondria were purified following
Western blotting, isolation of flexor digitorum the user protocol in the mitochondria extraction kit (Boster
brevis (FDB) fibers, single fiber immunocytochemistry Biological Technology).
(ICC), and proximity ligation assay (PLA)- These mRNA levels of mitochondrial proteins. RNA was
procedures were performed as described previously (1). isolated from fresh, snap frozen muscles with the
Additional antibodies used are shown in Table 1. To allow RNAzol®RT (SIGMA) according to manufacturer’s
the use of data from multiple Western blots, the intensity protocol. Diaphragm and soleus muscles were used.
of each band within a single Western blot was first Reverse transcription was done with random primers
normalized to GAPDH as a loading control and then (Invitrogen) and Superscript II reverse transcriptase
calculated as %WT average from that specific Western. (Invitrogen). The RT-PCR was performed in triplicates) in
Data were then pooled and calculated as %WT ± SEM. an AB ViiA7 RTPCR system with Syber Green (BioRad). D
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For PLA, FDB fibers were treated with either 50 μM L- Primers sequences are from Primer Bank (validated). n
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NAME, 1 μM KN93, or 1 μM AIP. The culture medium of http://pga.mgh.harvard.edu/primerbank/. Relative ad
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FDB muscle fibers was then changed to Tyrode’s solution expressions were calculated according to Livak method. d fro
with 50 μM L-NAME or 1 μM AIP in the presence of 20 GAPDH was uses as a control. Data are presented m
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μM of BTS for an additional hour in 95% O2 - 5% CO2 as%WT. ttp
incubator at 37°C for 30 min. The fibers were electrically Ubiquitination of mitochondrial proteins- ://w
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stimulated with 20 trains (100 Hz, 250 ms, 0.17 duty Mitochondrial fraction isolated as described above was w
cycle) using an RC-37FS chamber, with a constant flow of lysed in RIPA buffer. Same amount (60 µg) of .jbc
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1 ml/min of Tyrode’s solution during the stimulation. mitochondrial lysates from EK and WT hindlimb muscle rg
Fibers were then fixed with 2% paraformaldehyde in 0.1 were loaded on 5-15% SDS-PAGE gel (Bio-Rad), by/
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M phosphate buffer and incubated with the primary separated and transferred to PVDF membrane. This blot ue
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antibodies for PLA. was treated with anti-ubiquitin antibody (Santa Cruz). t o
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Analysis of Complex I-IV activity. Mitochondrial Western blot were performed as described previously (1). M
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OXPHOS complex activity assay was performed Imaging of TMRE loaded FDB fibers- Images rch
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according to Satomi et al (17). Briefly, isolated were acquired with a Plan-Apochromat 63 x 1.4 Oil DIC 9
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mitochondria were solubilized with Native PAGE Sample lens and a Zeiss LSM 510 META Confocal Microscope. 0
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buffer (Invitrogen) containing 0.3% n-dodecyl-b-D- Images (xy) were acquired setting the optical slice in the
maltoside (Sigma-Aldrich). Fifty micrograms of protein “z” axis to 1µm using the 543 nm excitation laser and the
were run on 3–12% Native PAGE Bis–Tris gel BP 560-615 emission filter. After starting the perfusion
(Invitrogen) in the Native-PAGE buffer system, anode with 25 nM of TMRE, images were acquired for up to 5
buffer as Native-PAGE buffer and cathode buffer minutes.
containing 0.02% n-dodecyl-b-D-maltoside and 0.05% TMRE fluorescence to indirectly measure fatty
deoxycolate. For in-gel catalytic activity assays of acid β-oxidation by changes in mitochondrial membrane
OXPHOS complex, gels were incubated in the following potential- The effect of BSA-conjugated palmitate on
solutions: Complex I, 5 mM Tris–HCl pH 7.4, 140 mM relative changes in mitochondrial membrane potential was
NADH and 3 mM nitro tetrazolium blue (NTB); Complex monitored with TMRE in non-quench mode (18).
II, 5 mM Tris–HCl pH 7.4, 20 mM succinate, 3 mM NTB Palmitate was conjugated to BSA at 6:1 molar ratio. To
and 200 mM phenazine methosulfate; Complex III, 50 mM monitor changes in mitochondrial membrane potential,
sodium phosphate buffer pH 7.2 and 0.5 mg/ml pre-plated FDB fibers were perfused (1 ml/min) with 5 ml
diaminobenzidine (DAB); Complex IV, 50 mM sodium of Tyrode’s (includes 5.5 mM glucose) containing 25 nM
TMRE in the presence of 0.5 mM carnitine and either 100
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Running title: Ca 1.1, CD36 and Mitochondria  
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µM of BSA conjugated palmitate or BSA control. When 21BDW, Warner Instruments) were incubated for 1 h with
indicated, 1 µM AIP was included in the solution 15 min 10 µM DAF-FM at room temperature, followed by
before starting the perfusion and then maintained in the washout with fresh DMEM. Nitric oxide production was
presence of the drug throughout the experiment. During stimulated with 3 cycles of electrical field stimulation
FCCP (2 M) incubation, to avoid variations in voltage, (EFS) using two platinum wires placed approximately 100
antimycin A (1 M) was used to block proton pumping by µm apart on both sides of the fiber. Each cycle of EFS was
complex III and oligomycin (1 M) was used to inhibit composed of 2 min of continuous application of trains
complex V from depleting ATP pools. Additionally, the every 1.5 s (100 Hz, 250 ms duration, 0.17 duty cycle)
effects of 25 mM glucose and 10 mM sodium pyruvate followed by 1 min of rest between cycles. Nitric oxide
were monitored with the 5.5 mM glucose in Tyrode’s as production was evaluated before and after the EFS
control. Fibers permeabilized with saponin as described protocol. The reactive nitrogen species sensor DAF-FM
previously (19), contained in “internal” solution (in mM): loaded into FDB fibers was excited at 0.5 Hz with the 500
140 KCl, 10 HEPES, 0.5 EGTA, 5 phosphocreatine (di- nm filter placed in a DG4 system used as light source.
Tris), 3 Mg-ATP and 0.114 CaCl , pH 7.0 with KOH. Emitted florescence was collected through a Nikon S.
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Briefly, preplated FDB fibers were exposed to 50 µg/mL Fluor objective (20x, 0.75 NA) coupled to an inverted
saponin for 30 s followed by complete washout with microscope (eclipse TE-200, Nikon) and digitized using a
internal solution. Permeabilized cells were perfused as CCD Rolera MGi-Plus camera (QImaging) with 510x256 Do
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indicated in the previous section in the presence of 0.5 mM pixels field size using 6x6 binning. Data were collected nlo
carnitine and 100 nM palmitate-CoA or 1 mM palmitate- and stored using Metafluor software (version 6.2) for ad
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carnitine as indicated (20). Fluorescence data were further analysis. The drugs tested (10 M BayK8644, 10 fro
captured through a Nikon S. Fluor objective (×20, 0.75 M nifedipine, 50 M L-NAME, 1 M KN92 or KN93) m
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numerical aperture) coupled to an inverted microscope were kept in the solution throughout the experiment. ttp
(Nikon eclipse TE-200) and digitized using a CCD Rolera Statistical analyses- Statistical analyses between ://w
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MGi-Plus camera with 510 × 252 pixels field size (using 6 two groups were conducted using Student's t-test or w
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binning). Data were collected and stored using the ANOVA. P < 0.05 was considered statistically significant. c
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Metafluor software (version 6.2) for further analysis. The effect of two independent variables was analyzed with rg
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Nitric oxide production stimulated with electrical two-way ANOVA. Analysis of covariance (ANCOVA) y g
field stimulation (EFS)- Single isolated FDB fibers plated was performed on metabolic rate with body weight or lean ue
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on coverslips and mounted in a recording chamber (RC- muscle as covariance (21). t on
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Running title: Ca 1.1, CD36 and Mitochondria  
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RESULTS mice under both fed (Fig. 2 C) and fasting (Fig. 2 D)
Effects of the EK mutation in Ca 1.1 on energy conditions. Taken together, these findings support a role
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expenditure. We previously demonstrated that a for Ca 1.1 in the control of basal metabolic rate and heat
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permeation defect in Ca 1.1 (E1014K, EK) leads to generation during cold challenge.
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increased fatigue and decreased Ca2+ store refilling, protein Effect of EK mutation on mitochondrial protein
synthesis, muscle fiber diameter, and type IIb/x fiber and mRNA levels. We previously demonstrated that the
content (1). Since skeletal muscle represents ~40% of total soleus and diaphragm muscles of the EK mice are more
body mass in mammals and contributes about 30% of the affected than the EDL muscle (1) and we, therefore,
resting metabolic rate in adult humans (22), we concentrated on soleus and diaphragm in the current study.
hypothesized that changes in Ca2+ signaling in the muscle We first assessed mitochondrial to nuclear DNA levels and
of EK mice would lead to altered muscle energy found no differences between EK and WT soleus or
utilization. We found that EK mice gained more weight diaphragm muscles (Fig. 3A.). We next assessed the
than age matched WT mice (Fig. 1 A) and had a higher protein and mRNA levels of a number of mitochondrial
percent of body weight due to fat (Fig. 1 B). Food intake proteins in diaphragm (Fig. 3 B-E), and soleus muscles
and cage activity did not differ between EK and WTmice (Fig. 3 B,C, F and G). Protein levels of COXIV, VDAC,

(Fig. 1 C and D). Fat in EK mice accumulated in SIRT3, MnSOD were reduced in both soleus and
epididymal, abdominal, visceral, and subcutaneous areas diaphragm muscles of EK compared to WT mice. D
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(Fig. 1 E and F) with no detectable changes in either However, mRNA levels for these mitochondrial proteins n
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hepatic or intramuscular fat compared to WT mice as were not decreased in the muscles of EK mice (Fig. 3E and ad
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assessed from spin-lattice (T1) relaxation time constants G). Thus, muscles of EK mice display reduced levels of d fro
using MRI (Fig. 1 G). VO was reduced (Fig. 1 H) and the some, but not all mitochondria proteins in the absence of m
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respiratory exchange ratio (RER) was increased during decreases in mRNA’s for these proteins, suggesting that ttp
both day and night in EK mice compared to WT mice, decreases in mitochondrial proteins are due to increased ://w
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indicating a preference for carbohydrate over lipid degradation. Since some mitochondrial membrane proteins w
metabolism (Fig. 1 I). Although young EK mice (6-8 are degraded by a ubiquitin dependent pathway (24,25), .jbc
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weeks) did not differ from WT mice in glucose or insulin we purified mitochondria from diaphragms of EK and WT rg
tolerance (data not shown), by 16-18 weeks of age, EK mice and assessed ubiquitination. We found a significant by/
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mice developed glucose intolerance and insulin resistance increase in the ubiquitination of mitochondrial proteins ue
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(Fig. 1 J and K) relative to WT. Consistent with the from the muscle of EK compared to WT mice (Fig. 4 A t o
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insulin resistance of the older EK mice, plasma insulin and B). The decrease in COXIV protein levels suggests M
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levels increased with age in the EK mice and were that OXPHOS may be altered in EK muscle. We assayed rch
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significantly higher than that of the WT mice at 1 year of mitochondrial OXPHOS activity as described by Satomi et 9
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age (Fig. 1L). al (17) and found a small, but significant decrease in 0
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Using indirect calorimetry we assessed heat complex IV, but not Complex I-III activity (Fig. 4 C and
production to evaluate differences in energy expenditure D).
between WT and EK mice (21). A small decrease in Alterations in mitochondrial β-oxidation in
metabolic rate was observed in the EK mice (Fig. 2 A). response to fatty acids. We assessed mitochondrial
Skeletal muscle and futile Ca2+ cycling by SERCA function in FDB fibers indirectly by measuring changes in
contribute to nonshivering thermogenesis and defects in mitochondrial membrane potential using TMRE in non-
this process are associated with obesity in mice (23). We quenching mode. TMRE fluorescence is frequently used to
assessed cold tolerance in the EK and WT mice by assess changes in mitochondrial membrane potential (26),
measuring body temperature with implanted telemetric but caution is required to avoid artifacts (18). Fig. 5 A
probes and VCO /VO with IDC upon exposure to 4C for shows that, under the conditions used in this study, TMRE
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up to 12 hours. Despite a trend toward lower body labels mitochondria (both subsarcolemmal and
temperature in the EK mice, these differences did not interfibrillar) in isolated intact FDB fibers while
reach significance (Fig. 2 B). However, heat production sarcolemmal membrane partitioning is minimal. To
(measured during the 3rd to 8th hours of cold exposure) evaluate perturbations in mitochondrial membrane
was significantly reduced in EK mice compared to WT potential driven by exogenously added fatty acids, we
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compared the effects of carnitine alone with BSA- and palmitoyl-carnitine increased the initial rate of TMRE
conjugated palmitate plus carnitine. We found linear fluorescence (i.e. β-oxidation) above that of carnitine alone
increases in TMRE fluorescence as a function of time, to a similar degree in both WT and EK fibers. These
reflecting a change in mitochondrial membrane potential, results suggest that the step that contributes to the
in the presence of carnitine alone (in 5.5 mM glucose) in decreased mitochondrial response to fatty acids in fibers
FDB fibers from both WT and EK mice (Fig. 5 B). The from EK mice lies at or upstream of ACS.
initial rate of change in TMRE fluorescence was The small decreases detected in COXIV activity in
significantly increased by the addition of BSA-conjugated the EK muscle could be contributing to the change in the
palmitate only in WT fibers (Fig. 5 C). AIP, an inhibitor of response of the mitochondrial membrane potential to fatty
CaMKII, and L-NAME an inhibitor of nitric oxide acids. To determine if the difference in the rate of change
synthase (NOS), blocked the palmitate-induced increase in the mitochondrial membrane potential upon addition of
the rate of change in TMRE fluorescence in the WT fibers palmitate in EK compared to WT fibers reflected the
(relative to carnitine alone) (Fig. 5 D and E). Both mitochondrial damage, we used different substrates to
inhibitors slowed the rate of change of TMRE fluorescence drive oxidative phosphorylation (5.5 mM glucose, 25 mM
in EK fibers treated with both carnitine and BSA- glucose, or 10 mM pyruvate). We did not detect
conjugated palmitate, but to a lesser extent (Fig. 5 F). To differences in the rate of change in TMRE fluorescence
demonstrate that changes in TMRE fluorescence were due between EK and WT fibers (Fig. 5I) with these substrates. D
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to alterations in mitochondrial membrane potential, we We conclude that the fibers from EK display a selective n
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used a mitochondrial uncoupler, FCCP, combined with decrease in fatty acid β-oxidation which can be ad
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oligomycin, to block complex V dependent ATP depletion, phenocopied in WT fibers by inhibition of either CaMKII d fro
and antimycin A, to block proton pumping by complex III. or nitric oxide synthase.  m
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The rate of change in the TMRE fluorescence was A role for nitric oxide synthase (NOS). S- ttp
significantly less in both WT and EK fibers than in the nitrosylation of proteins has been shown to either slow ://w
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presence of carnitine alone (Fig. 5 F). The initial rates of (27) or enhance (28,29) their ubiquitin-proteasome- w
change under all these conditions in intact FDB are mediated breakdown. In addition, the finding that L- .jbc
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summarized in Fig. 5 F. These data suggest that the NAME prevents the fatty acid-driven increases in TMRE rg
changes in mitochondrial function are downstream of fluorescence in WT fibers (Fig. 5 E) suggests that NOS by/
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alterations in CaV1.1, CaMKII, and NOS. activity is contributing to the pathway by which CaV1.1 ues
To determine if the reduction in mitochondrial β- regulates mitochondrial function. NOS enzymes are t o
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oxidation in response to palmitate in the EK fibers was due regulated by muscle activity (30,31), Ca2+/calmodulin (32) M
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to alterations in the rate of fatty acid uptake into the and CaMKII (33,34). Reactive nitrogen species (RNS) rch
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muscle fiber, we assessed the rate of change in TMRE generated by the NOS enzymes regulate both 9
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fluorescence in fibers permeabilized with saponin. mitochondrial biogenesis and function (35). We evaluated 0
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Permeabilized WT fibers still responded to the addition of the role of the Ca 1.1/CaMKII pathway in activity-
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BSA-conjugated palmitate in the presence of carnitine dependent RNS production in FDB fibers using 4, 5-
with an increase in the rate of change in TMRE diaminofluorescein diacetate (DAF-FM). Activity
fluorescence (Fig. 5 G and H), but the rate change in dependent changes in DAF fluorescence were significantly
TMRE fluorescence in permeabilized EK fibers was decreased in FDB fibers from EK compared to WT mice
similar for carnitine alone and carnitine plus BSA- (Fig. 6A). In WT fibers, RNS levels were enhanced by
conjugated palmitate (Fig. 5 G). Thus, differences in the BayK8644 (an activator of Ca 1.1), and inhibited by
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rate of fatty acid uptake across the sarcolemma or t-tubules nifedipine (a Ca 1.1 inhibitor), KN93 (CaMKII inhibitor),
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do not fully account for the differences in β-oxidation- and L-NAME (NOS inhibitor) (Fig. 6 A). BayK8644,
driven changes in mitochondrial membrane potential nifedipine, and KN93 did not significantly altered RNS
between EK and WT fibers. To identify additional steps in levels in FDB fibers from EK mice. On the other hand, L-
β-oxidation altered by the EK mutation, we replaced the NAME reduced RNS levels in fibers from both WT and
BSA-conjugated palmitate with either palmitoyl-CoA to EK mice, suggesting that the Ca 1.1./CaMKII pathway
V
bypass the fatty acyl-CoA synthetase (ACS) or palmitoyl- enhances, but is not absolutely required for activity
carnitine to bypass CPT1 (Fig. 5 H). Both palmitoyl-CoA dependent RNS production in muscle fibers.
7
Running title: Ca 1.1, CD36 and Mitochondria  
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CaMKII phosphorylates eNOS at S1177 (33) and decreased in EK muscle. These findings suggest CD36
nNOS at S1416 (34). Using phospho-specific antibodies, interacts with eNOS near the t-tubule membrane and that
we found decreased p-eNOS/eNOS and p-nNOS/nNOS this interaction is modulated by the Ca 1.1/CaMKII
V
levels in EK muscle (Fig. 6 B-D). Using iodoTMT and pathway.
swift stain for total protein transferred to a PVDF Whether CD36 directly binds to mitochondria is
membrane, we assessed S-nitrosylation of membrane (Fig. controversial (42,43). We examined the proximity (<40Å)
6 E and G) and mitochondrial proteins (Fig. 6 F and H). S- of CD36 to two additional proteins, CPT1, (an outer
nitrosylation was decreased in both membrane and mitochondrial membrane protein) (44) and to long-chain-
mitochondrial fractions in muscle from EK compared to fatty-acid-CoA ligase 1 (ACSL1). ACSL1 is found in
WT mice (Fig. 6 G and H). These data indicate that Ca2+ plasma membranes, peroxisomes, and the outer
binding and/or permeation through Ca 1.1 and CaMKII mitochondrial membrane. The interactions (or proximity)
V
activation increase NOS activity and this increase is of CD36 with CPT1 (Fig. 9 A and B) and CD36 with
prevented by the EK mutation in Ca 1.1. Upstream and ACSL1 (Fig. 9 C and D) were markedly decreased in EK
V
downstream pathways of NOS activity that are modulated fibers. AIP and L-NAME decreased the interactions of
by Ca2+ permeation or binding to Ca 1.1 are summarized CD36 with CPT1 and ACSL1. These findings again
V
in Fig. 6I. support a decreased interaction of CD36 with transverse
We previously showed that CaMKII was in close tubules, mitochondria and possibly other intracellular D
o
w
proximity to Ca 1.1 in the t-tubule membrane (1). To membranes and support a regulation of CD36 subcellular n
V lo
determine if NOS was in close proximity (<40Å) to location by CaMKII and NOS regulated pathways. ad
e
CaV1.1, we used both immunocytochemistry and Another potential mechanism whereby altered d fro
proximity ligation assays (PLA). As shown in Fig. 7, CD36 subcellular localization could alter muscle m
h
CaV1.1 and eNOS mostly co-localize in t-tubules of metabolism is via its effects on AMPK. Recently, ttp
skeletal muscle fibers. Samovski et al (45) suggested that CD36 suppresses ://w
w
A role for CD36. To further explore the AMPK activation (assessed as p-AMPK/AMPK) but fatty w
mechanism by which the CaV1.1/CaMKII/eNOS regulates acids relieve this inhibition. We assessed AMPK activation .jbc.o
mitochondrial function, we evaluated a potential role for in muscle of EK and WT mice and found that AMPK rg
the fatty acid (FA) transport protein CD36 (36) which activity is reduced in EK muscle (Fig. 10A and B). AMPK by/
g
regulates both FA uptake into muscle and the delivery of phosphorylates acetyl CoA carboxylase (ACC) at S79 (46) ue
s
FAs to peroxisomes and mitochondria (37). Muscle , decreasing ACC activity. We found a decrease in the t o
n
activity promotes CD36 translocation between the t- ratio of S79 phosphorylated to total ACC in EDL and M
a
tubules and mitochondria. In the t-tubules, CD36 increases diaphragm (Figure 10, C and D) suggesting increased rch
2
FA uptake and, in the mitochondria, CD36 increases the ACC activity. ACC produces malonyl CoA, which inhibits 9
, 2
rate of FA β-oxidation (38-41). Total CD36 levels were FA β-oxidation by inhibiting CPT1 (47). Hence an 0
1
9
not different between muscle from EK and WT mice (Fig. additional mechanism whereby changes in CD36 within
8 A and B). We assessed the subcellular distribution of the muscle are regulating FA β-oxidation is by altering
CD36, eNOS, Cav1.1, RyR1 and VDAC in Western blots AMPK activation.
of subcellular fractions (cytosolic, nuclear, purified
mitochondria and membranes) from EK and WT muscle. DISCUSSION
CD36 was increased in the cytosolic fractions and Ca2+ influx through the voltage dependent Ca2+ channel is
decreased in the mitochondrial and membrane fractions not required for excitation-contraction coupling, but Ca2+
from EK compared to WT muscle (Fig. 8 C and D). does bind to and permeate the pore of this channel, raising
Consistent with partial localization to the the question of the role, if any, of Ca2+ binding/permeation
membrane fraction, some CD36 colocalized with eNOS through this channel to skeletal muscle function. We
(PLA) as shown in Fig. 8 E and F. The proximity of CD36 previously demonstrated that a mutation in the Ca 1.1 pore
V
to eNOS was decreased in EK muscle and was decreased which blocked Ca2+ binding and permeation, decreased
in WT muscle by an inhibitor of CaMKII, AIP. CD36 also CaMKII activity, inhibited refilling of SR Ca2+ stores, and
immunoprecipitated with antibodies to eNOS (Fig. 8 G reduced protein synthesis. (1). Collectively these changes
and H) and the amount of the CD36 in the IPs was resulted in increased fatigue and decreased muscle fiber
8
Running title: Ca 1.1, CD36 and Mitochondria  
V

cross-sectional area. We now show that these mice (EK) both at rest and during exercise. Smith et al (36) found that
also gain weight on a chow diet without increased food CD36 deficiency decreased palmitate-supported
intake or activity, suggesting decreased energy mitochondrial respiration by 34%, but did not alter
expenditure. palmitoyl-CoA supported respiration, demonstrating that
We demonstrate an accumulation of CD36 functions upstream of acyl-CoA synthetase. Mice
abdominal/visceral and epididymal fat in the EK mice and lacking CD36 have similarities to the EK mice, including
show that these mice generate less heat when exposed to reduced FA oxidation in muscle and increased RER (47).
colder temperatures. The EK mice also exhibit insulin and We propose that one possible explanation of the activity
glucose intolerance by the time they reach 16 weeks of dependence of CD36 function is the regulation of its
age. These animals display decreased energy expenditure intracellular distribution by the Ca 1.1/CaMKII/NOS
V
(heat production), and lower respiratory quotient (RER), pathway. Even a transient contact between CD36 and
strongly supporting a significant defect in fatty acid CPT-1 could be an important mechanism for fatty acid
utilization. shuttling upon electrical stimulation and muscle
We also found that a pathway that requires Ca2+ contraction. Our data, however, cannot discriminate
binding to the pore of Ca 1.1, and activation of both between a true mitochondrial localization for CD36 versus
V
CaMKII and NOS is required for normal mitochondrial β- a transient contact with the mitochondria via nearby
oxidation (assessed as a BSA-conjugated palmitate endosomes or plasma membrane. D
o
w
induced alteration in the mitochondrial membrane Recently CD36 was shown to negatively regulate n
lo
potential). The TMRE response to palmitate is reduced in AMPK (45) which phosphorylates and decreases ACC ad
e
EK compared to WT fibers. This suggests that the activity. Decreased AMPK and p-ACC raises the d fro
mitochondria in the EK mice are not functioning possibility that malonyl CoA, produced by ACC. plays a m
h
optimally. Our data to support this include: a) decreased role in decreased FA oxidation in the muscle of EK mice. ttp
mitochondrial proteins, b) increased ubiquitination of In summary, our data suggest that Ca2+ ://w
w
mitochondrial proteins and c) a decreased mitochondrial binding/permeation of Ca 1.1 sequentially activates w
membrane potential response to palmitate (but not glucose CaMKII, and eNOS to regulaVte CD36 distribution thereby .jbc
.o
or pyruvate) in EK muscle. regulating FA β-oxidation. These effects on FA β- rg
We also found that the Ca 1.1/CaMKII/NOS oxidation could arise from S-nitrosylation of mitochondrial by/
V g
pathway regulates the intracellular distribution of CD36. In proteins, increased interaction of CD36 with mitochondria, ue
s
skeletal muscle CD36 has been shown to bind to the outer or decreased AMPK activity (Fig. 11). The relative t o
n
mitochondrial membrane (36). Continuous cycling of contributions of these pathways remain to be determined. M
a
CD36 between cell surface and intracellular compartments Our studies identify CaV1.1, CaMKII and eNOS as rch 2
has been reported (48). Continuous cycling of CD36 potential new molecular targets for therapeutic 9
, 2
between the plasma membrane, endosomes, and other interventions in metabolic syndrome. These findings also 0
1
9
intracellular organelles (i.e. mitochondria) has been have important implications for understanding the
reported (43,49,50). Yoshida et al (12) showed that CD36 mechanisms that underlie metabolic changes in obesity
deficiency decreases β-oxidation in the absence of changes and adaptations to exercise.
in mitochondrial number and enzyme content and
proposed that CD36 is a key component in fuel selection

Acknowledgements: This work is supported by grants from NIH (AR053349 to SLH and RTD; AR041802 to SLH)
Other services were provided by BCM core facilities (P30 DK079638).
Conflict of Interest. Authors declare that they have no conflicts of interest with the contents of this article.
Author contributions. DKG conceived, performed and analyzed the metabolic data in Figures 1 and 2, analyzed the PLA
data and helped to write the manuscript. ADA performed all of the TMRE studies in Figure 5 and the DAF experiments in
Figure 6 and helped to write the manuscript. CSL performed and analyzed the western blotting experiments in Figures 3
9
Running title: Ca 1.1, CD36 and Mitochondria  
V

and 6, the experiments in Figures 4, 7, 8, 9 and 10, and helped write the manuscript. DMG performed the MRI
experiments in Figure 1. HW performed all of the qRTPCR experiments and helped to write the manuscript. WRL helped
to supervise and interpret the metabolic experiments and helped write the manuscript. RGP supervised and analyzed the
MRI studies and helped with manuscript preparation. RTD helped in the supervision and interpretation of the TMRE and
DAF studies and helped in preparing the manuscript. SLH helped plan and supervise all experiments and wrote the
manuscript.
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