jgp
S u p p le m ental m aterial
THE JOURNAL OF GENERAL PHYSIOLOGY
Calmettes et al.
Figure S1. Principle of spatial variance–based images analysis. Spatial variance mapping is commonly used in edge detection algorithms
to detect discrete objects (Russ, 2011). The variance value is small in uniform regions of the image and becomes large whenever a step of
intensity or line is present. Here we computed spatial variance images from cells expressing HKI-YFP or HKII-YFP to assess heterogeneity
in the cytoplasm and extract information about the localization of HKs. (A) In our algorithm, the variance map command computes a
map of the input image, where the intensity of a pixel in the output map represents the variance within a pixels window centered at the
corresponding pixel of the input image. This operation is applied to all the pixels of the original image through a local neighborhood
operation (sliding window). (B) Because mitochondria are discrete organelles, the spatial variance is large when HK fluorescence arises
predominantly from mitochondria, as shown by images of NRVM expressing HKI-YFP. Moreover, because HKI do not dissociate from
mitochondria under glucose removal, spatial variance does not change over time. (C) However, in NRVM expressing HKII-YFP and
submitted to IAA perfusion, intensity of the variance image decreases as HKII is released and diffuses evenly through the cytoplasm.
Bars, 25 µm.
Figure S2. G-6-P measurement in NRVM. This graph shows data
obtained from four different experiments where NRVM were exposed to 100 µM IAA for up to 25 min. Each point is a mean of
2–3 measures. These data indicate that intracellular G-6-P began
to increase 2 min after addition of IAA and plateaued at 25 min.
These data compare with the time course of IAA-induced HKII
displacement and support our hypothesis whereby G-6-P facilitates HKII translocation. We have also performed G-6-P measurements in NRVM after glucose removal (not depicted) and the
data shows transient increase in G-6-P similar to those obtained
previously in CHO cells (John et al., 2011).
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Figure S3. Western blot analysis of HKI and HKII overexpression
in NRVM. Quantification of the level of overexpression of HKI-YFP
(left) and HKII-YFP (right) in NRVM infected with the adenoviral
vector. The right lane in both blots shows two bands corresponding to the WT HK and the one linked to YFP (higher molecular
weight). A two-to-fivefold increase in total HK content (endogenous HK + YFP-tagged HK) was observed after infection of the cells
with our HKI-YFP and HKII-YFP adenoviral constructs.
Figure S4. Difference in expression of GLUT and HK isoforms
in neonatal and adult hearts. Total RNA was isolated from mouse
adult and neonatal hearts using an RNeasy Plus Mini kit (QIAGEN). Gel electrophoresis was used to confirm successful isolation by the presence of 18 and 26 S RNA bands. The total RNA was
then used as a template with oligo dT primers in cDNA synthesis
(SuperScript III First Strand; Invitrogen). Primers to the various
genes were constructed to generate similar sized PCR products.
Each primer pair was screened against the nr nucleotide database
(Blast) for both specific gene identity and nonspecific matches.
Preliminary PCR experiments identified unique band amplification. To control for variable amounts of starting material, two
controls of housekeeping genes (GAPDH,  actin) were included
in the RT-PCR experiments performed using the IQ Sybr Green
Supermix and an RT-PCR IQ machine (both from Bio-Rad Laboratories). Results are shown as fold differences after correction
for different levels of starting material.
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Hexokinase localization and glycolysis in cardiac myocytes
Figure S5. IAA-induced inhibition of glycolytic activity in NRVM.
(A) Typical trace obtained in NRVM with the FRET-based glucose
nanosensor Flipglu-600 µM, showing the effects of the glycolytic inhibitor IAA addition (shaded gray area). (B) Addition of
IAA (100 µM), in the presence of Cyto B to block glucose efflux
caused a dramatic decrease in the rate of glucose utilization. Panels in B show a threefold increase in the half-time of the FRET
ratio decrease associated with glucose utilization, measured in the
presence of Cyto B, before (1) and after (2) IAA addition.
Figure S6. Intracellular pH measurements in NRVMs with BCECF. To measure
changes in intracellular pH in response to
glucose removal and IAA addition, NRVM
were loaded with 1 µM of the permeable
analogue BCECF-AM for 30 min. (A and
B) Removal of glucose has almost no effect on cytosolic pH (A), whereas addition
of IAA (B) caused a small and transient
acidification (<0.05 U). At the end of each
experiment, consecutive 10 mM NH4Cl
and 80 mM Na acetate were used to verify
that BCECF correctly reported induced
intracellular alkalinization and acidification, respectively. Note that the resting pH
in the cytosol was close to 7.3. (C) HKII
displacement by cytosolic acidification in
NRVM. Intracellular acidification with 80
mM Na acetate caused partial detachment
of HKII-YFP from mitochondria within
15 min. This translocation caused by a
change in pH from 7.3 to 7.0 was also accompanied by swelling of mitochondria.
Such an effect is unlikely to occur after
removal of glucose or addition of IAA, as
the changes in intracellular pH are almost
negligible. Bar, 20 µm.
Figure S7. Calibration of the FLIPglu-600 µM FRET probe
in permeabilized NRVMs. Using the approach of Bittner et
al. (2010) in neurons and astrocytes, we were able, after
fitting the slope of glucose utilization in the absence of glucose efflux (presence of Cyto B), to calculate the rate of
glucose utilization under various experimental conditions.
We find that on average the rate of glucose utilization is
150 times higher in NRVM than ARVM. This difference
is greater than that reported by others. Many factors may
account for this difference. One is a difference in structure
between NRVM and ARVM. The ratio of mitochondria,
where HKs bind, to the cell volume, where glycolysis occurs, is greatly different, due in part to the fact that one
third of the volume of ARVM is occupied by mitochondria.
This would not affect the enzymatic assay using isolated
enzyme, but would be an important factor in our case. Another difference may be the level of expression of HKs in
ARVM after 2–3 d in culture versus freshly isolated cells.
Our data show that increasing the level of HKI by twofold reduces the difference in rate of glucose clearance between ARVM and NRVM
by sixfold. In this case our values approach those reported by others. Also, in our hands NRVM are still beating and thus require high glycolytic activity, whereas ARVM are quiescent in culture, resulting perhaps in down-regulation of glycolysis. Lastly, after calibration of the
FRET probe we express glucose utilization in terms of micromoles per second per cell, whereas activity of isolated enzyme is expressed
in micrograms per second per milligram of protein. Because of these differences it is not possible to compare the difference that we
observe in vivo, using the FRET ratios, with the difference measured in isolated enzyme preparation.
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R eferen c es
Bittner, C.X., A. Loaiza, I. Ruminot, V. Larenas, T. SoteloHitschfeld, R. Gutiérrez, A. Córdova, R. Valdebenito, W.B.
Frommer, and L.F. Barros. 2010. High resolution measurement
of the glycolytic rate. Front. Neuroenergetics. 2:26. http://dx.doi.
org/10.3389/fnene.2010.00026
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Hexokinase localization and glycolysis in cardiac myocytes
John, S., J.N. Weiss, and B. Ribalet. 2011. Subcellular localization of hexokinases I and II directs the metabolic fate of glucose. PLoS ONE. 6:e17674. http://dx.doi.org/10.1371/journal.
pone.0017674
Russ, J.C. 2011. The Image Processing Handbook, Sixth Edition.
CRC Press, Inc., Boca Raton, FL. 885 pp.