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ORIGINAL RESEARCH
published: 19 December 2019
doi: 10.3389/fendo.2019.00881
Frontiers in Endocrinology | www.frontiersin.org 1December 2019 | Volume 10 | Article 881
Edited by:
Debbie C. Thurmond,
Beckman Research Institute,
United States
Reviewed by:
Amira Klip,
Sick Kids Research Institute, Canada
Jonathan Bogan,
Yale University, United States
*Correspondence:
Gwyn W. Gould
gwyn.gould@strath.ac.uk
†Present address:
Peter R. T. Bowman and
Gwyn W. Gould,
Strathclyde Institute of Pharmacy and
Biomedical Sciences, Glasgow,
United Kingdom
Specialty section:
This article was submitted to
Molecular and Structural
Endocrinology,
a section of the journal
Frontiers in Endocrinology
Received: 25 September 2019
Accepted: 03 December 2019
Published: 19 December 2019
Citation:
Bowman PRT, Smith GL and
Gould GW (2019) Cardiac SNARE
Expression in Health and Disease.
Front. Endocrinol. 10:881.
doi: 10.3389/fendo.2019.00881
Cardiac SNARE Expression in Health
and Disease
Peter R. T. Bowman 1†, Godfrey L. Smith 2and Gwyn W. Gould 1
*†
1Henry Wellcome Laboratory of Cell Biology, College of Medical, Veterinary and Life Sciences, Institute of Molecular Cell and
Systems Biology, University of Glasgow, Glasgow, United Kingdom, 2College of Medical, Veterinary and Life Sciences,
Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom
SNARE proteins are integral to intracellular vesicular trafficking, which in turn is the
process underlying the regulated expression of substrate transporters such as the
glucose transporter GLUT4 at the cell surface of insulin target tissues. Impaired insulin
stimulated GLUT4 trafficking is associated with reduced cardiac function in many disease
states, most notably diabetes. Despite this, our understanding of the expression and
regulation of SNARE proteins in cardiac tissue and how these may change in diabetes is
limited. Here we characterize the array of SNARE proteins expressed in cardiac tissue,
and quantify the levels of expression of VAMP2, SNAP23, and Syntaxin4—key proteins
involved in insulin-stimulated GLUT4 translocation. We examined SNARE protein levels
in cardiac tissue from two rodent models of insulin resistance, db/db mice and high-fat
fed mice, and show alterations in patterns of expression are evident. Such changes may
have implications for cardiac function.
Keywords: diabetes, cardiomyopathy, SNARE proteins, insulin resistance, GLUT4
INTRODUCTION
Effective regulation of metabolism is essential in all cell types in order to ensure that ATP generation
requirements are met. This is particularly important within highly energetic organs such as the
heart, where the contractile action of cardiomyocytes must be continually fuelled in order to
maintain the pumping of approximately 5 liters of oxygen and nutrient rich blood into and around
the systemic circulation every minute. It is also vital that the heart exhibits metabolic flexibility in
order to adapt its contractile output in response to increased demands e.g., during exercise. Normal
cardiac metabolism is characterized by predominant use of fatty acids as a metabolic substrate, with
a relatively lower utilization of glucose (1). This is logical as fat is a more abundant and energy rich
fuel source, making it ideal for scenarios where sustained moderate levels of ATP are required.
However, several cardiac disease states are partly defined (and potentially caused) by deficits in
glucose uptake and metabolism.
The predominant type 2 diabetic phenotype is characterized by peripheral insulin resistance,
whereby insulin no longer effectively stimulates the uptake of glucose into muscle and adipose
tissue. The mechanism underlying this condition is multifactorial, but is strongly linked to
obesity (2). This physiological action of insulin is important in maintaining glycaemic control
post-prandially, and indeed diabetes is diagnosed by an elevation in fasting blood glucose (or
circulating glycated hemoglobin) above clinically defined thresholds. In a contracting working
heart preparation from a diabetic mouse model, radiolabelled substrates revealed an impairment
of insulin stimulated glycolysis and glucose oxidation and increased basal fatty acid utilization
(associated with reduced myocardial efficiency) and an impaired ability of insulin to reduce this (3).
Bowman et al. Cardiac SNARE Proteins
Additionally, increasing the rate of fatty acid delivery to control
hearts resulted in reduced glycolysis and glucose oxidation,
enhanced fatty acid utilization, and reduced cardiac performance
(3). This demonstrates the importance of insulin-stimulated
glucose uptake to fatty acid metabolism (and vice versa) and
therefore cardiac efficiency and performance and exemplifies
potential defects in these systems in diseases such as diabetes.
Cardiovascular disease has long been established to be a
leading cause of death in the diabetic population, in part
attributable to the high rate of vascular disease that occurs
on account of sustained excessive glucose (and fat) in the
bloodstream (4). However, there is also a direct pathological
effect of diabetes upon cardiac function, characterized by
initial diastolic dysfunction prior to structural remodeling and
progression to heart failure, termed diabetic cardiomyopathy (5–
7). There is strong evidence that metabolic impairments such
as the intramyocellular accumulation of lipids in the heart and
associated cardiac insulin resistance may be critical early factors
in the progression of this condition (8–10). Most notably, in a
mouse model of diabetic cardiomyopathy, overexpression of the
insulin sensitive glucose transporter GLUT4 restored aberrant
cardiac metabolic and contractile function to values observed
in controls (11,12). Additionally, insulin resistance/glycaemic
control has also been demonstrated to be of prognostic
significance in human post-myocardial infarction patients (13–
15), with an experimental rat model identifying the onset of
cardiac (not systemic) insulin resistance to be correlated with
adverse recovery/remodeling (16).
It has been established that general concepts derived from
other insulin sensitive cell types are applicable to the heart, for
example that GLUT4 is the functionally predominant glucose
transporter (17). Under basal conditions the majority of GLUT4
is not located at the cell surface but rather distributed between the
general endosomal recycling pathway and a depot of specialized
GLUT4 storage vesicles (GSVs) that can be rapidly mobilized
to the cell surface in response to activation of the insulin
receptor (18). Both the sorting of GLUT4 through different
intracellular compartments and the fusion of GSVs with the
plasma membrane (PM) requires the action of specific SNARE
proteins (19). In skeletal muscle and fat tissue, the SNARE
proteins that regulate GSV fusion with the PM are VAMP2,
SNAP23, and Syntaxin 4, whereas Syntaxin 6 and 16 mediate
sequestration of GLUT4 into pools of GSVs at the trans Golgi
network (20–25).
Numerous studies have used a variety of techniques (including
knock down or expression of dominant mutant proteins)
to disrupt the function of SNARE proteins in the insulin
sensitive cell model 3T3-L1 adipocytes and demonstrated a clear
inhibitory effect upon insulin dependent GLUT4 trafficking and
glucose uptake, with the precise defect depending on the protein
targeted (26–30). Furthermore, in vivo examination of mice over
or under-expressing Syntaxin 4 identified improved/impaired
skeletal muscle insulin stimulated glucose uptake and therefore
also whole body glucose tolerance, attributed to corresponding
effects on insulin stimulated GLUT4 translocation (31,32).
To underline this strong association between SNARE proteins,
GLUT4 trafficking, and glycaemic control, even manipulation of
the expression of ancillary proteins such as Munc18c and Doc2b
that regulate SNARE interactions has significant consequences
on GLUT4-PM integration and glucose uptake (33,34). Insulin-
stimulated GLUT4 translocation in peripheral tissues thus
exhibits a considerable degree of mechanistic overlap, with
studies emphasizing the role of Syntaxin 4 and SNAP23 in fat and
muscle cells, and transgenic mice clearly establishing that levels of
expression of these SNAREs correlate with whole-body glycaemic
control (31,32).
The involvement of SNAREs in multiple steps of GLUT4
trafficking make them an intriguing potential target in the
context of disease. There are many theories relating to insulin
resistance, such as inhibition of proximal insulin signaling via
either lipid mediated activation of protein kinase C (35,36) or
altered release of adipocytokines from expanded and inflamed
adipose tissue (37,38). However, there is also evidence from
rodent models correlating altered SNARE protein (Syntaxin4,
Syntaxin6, VAMP2, VAMP3, SNAP23, Munc18) expression or
localization with skeletal muscle and adipose insulin resistance
(31,32,39–41). Additionally, in human type 2 diabetic patients
enhanced syntaxin 8 expression in adipose tissue was significantly
associated with reduced GLUT4 expression and impaired whole
body glucose tolerance (42). While it is not clear from these
studies whether alterations in SNARE protein levels are causal
or adaptive changes, initial studies from 2 independent insulin
resistant models indicate that targeting SNARE proteins may be
a viable strategy to restore insulin stimulated GLUT4 trafficking
and improve metabolic outcomes (43–45).
Any attempt to investigate these initial observations in the
heart is constrained by very limited prior characterization of
SNARE protein expression in cardiomyocytes, let alone how
they may interact or be of functional importance. This is in
contrast to considerable investigation in adipose and skeletal
muscle. The only specific prior attempt to identify the expression
and involvement of SNAREs in cardiac GLUT4 trafficking was
performed in a mouse atrial cell line (HL-1) and restricted to
assessment of all VAMP (v-SNARE) isoforms (46). Expression
of VAMP2/3/4/5/7 (but not VAMP1 or 8) protein was detected,
and this was confirmed in lysate generated from mouse heart.
Targeted silencing of each isoform revealed a role for VAMP2
and VAMP5 in the insulin stimulated appearance of GLUT4-myc
at the PM. However, separate work with rodent cardiomyocytes
identified the expression of VAMP1/2/3/4/5/8 (but not VAMP7)
mRNA transcripts (47). Whilst this study did not investigate
the expression of all VAMPs beyond the use of RT-PCR, this
data highlights that even within this limited field contradictions
have emerged regarding the expression of VAMP1/7/8 in the
heart. It is possible that this is in part due to the use of isolated
cardiomyocytes (47). Whilst isolation of cells prior to analysis
reduces interference in the sample from other subpopulations of
cardiac cells, if maintained in culture dedifferentiation may start
to occur which could impact protein expression.
Therefore, the aim of this work was to characterize the
expression of a wide range of SNARE isoforms within primary
adult cardiac tissue. Furthermore, it was assessed if the expression
of these proteins (in addition to GLUT4) was altered in 2
different diabetic mouse models. This study is the first step
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Bowman et al. Cardiac SNARE Proteins
FIGURE 1 | Quantification of Syntaxin 4 expression in the heart. Protein lysates were generated from 20-week old male mouse cardiac tissue and subjected to
SDS-PAGE and immunoblotting alongside purified recombinant Sx4. Lysates were incubated with antibodies probing for the expression of Syntaxin 4 and GAPDH
(loading control) as shown. Protein loaded refers to 10 or 20 µg of protein for the M1-M3 mouse cardiomyocyte samples or 0.5–1.5 ng of purified Syntaxin 4 protein
(lacking the transmembrane domain and thus migrating faster than endogenous, full length Syntaxin4), as indicated. M1-3 indicates biologically independent mouse
cardiac samples, where each sample is composed of lysate generated from 2 to 3 individual hearts. The approximate positions of molecular weight markers are
indicated. Borders indicate where images have been cropped for presentation purposes only. Data from a representative immunoblot is shown, quantified in Table 1.
TABLE 1 | Quantification of SNARE protein expression in primary mouse cardiac tissue.
Sample SNAP23 Copies/mg Syntaxin 4 Copies/mg VAMP2 Copies/mg
Mouse Heart 2.07 ×1012 (±5.66 ×1011) 7.26 ×1011 (±2.67 ×1010) 3.23 ×1011 (±3.56 ×1010 )
3T3-L1 Adipocytes 5.28 ×1012 1.72 ×1012 2.28 ×1012
Densitometry was used to quantify Syntaxin 4, SNAP23, and VAMP2 expression from known amounts of primary cardiac tissue and purified recombinant protein, within immunoblots
such as that depicted in Figure 1. Values for recombinant proteins were used to generate a standard curve that allowed estimation of the absolute amount of each protein in a given
amount of total cardiac lysate. This value is expressed in copies per mg of total lysate and is the mean (±S.E.M.) of at least 3 biologically independent replicate samples. Values from
3T3–L1 adipocytes are those reported in Hickson et al. (48).
toward uncovering the role of different SNAREs in insulin
stimulated cardiac GLUT4 trafficking and is of clinical relevance
due to the association of cardiac insulin resistance with diabetic
cardiomyopathy and myocardial infarction. SNAREs are also
important in both non-insulin stimulated (e.g., contraction
mediated) GLUT4 trafficking and the trafficking of the fatty
acid transporter CD36 (46). Therefore, this work is valuable
and foundational in the context of the regulation of cardiac
metabolism in general.
RESULTS AND DISCUSSION
Quantification of VAMP2, Syntaxin 4, and
SNAP23 in Cardiac Lysates
In adipocytes and muscle, the SNARE proteins associated with
GSV-PM fusion are VAMP2, Syntaxin 4, and SNAP23 (26,27,
30). We first sought to quantify expression of these SNAREs
in rodent cardiac lysates. Recombinant SNARE proteins were
expressed and purified from bacteria and used as standards in
quantitative immunoblotting to compare to the signal obtained
from mouse cardiac samples. An example of this technical
approach is shown in Figure 1 for quantification of Syntaxin
4 expression, with quantification of all 3 SNAREs reported in
Table 1. Comparative values displayed for 3T3-L1 adipocytes
were obtained from previously published work using a similar
approach (48). This technique was also performed with a
pooled lysate generated from cardiac tissue derived from 3
individual rat hearts. The mean values obtained closely matched
those of the mouse samples (1.45 ×1012 copies per mg
SNAP23; 6.82 ×1011 copies per mg Sx4; 3.05 ×1011 copies
per mg VAMP2).
In 3T3-L1 adipocytes there are approximately 2–3 fold more
SNAP23 molecules in comparison to VAMP2 and Syntaxin 4
(48). It is acknowledged that within this study our technical
approach will be associated with a margin of error, however
this phenomenon may not be specific to adipocytes as a
similar relative expression of key SNARE proteins is recorded
here from two sources of primary cardiomyocytes. The reason
underlying this observation requires further study but most
plausibly may reflect the involvement of SNAP23 in multiple
other fusion events relevant to other biological processes.
Additionally, this data provides one possible explanation as to
why 3T3-L1 adipocytes display a greater fold insulin response
compared to primary cardiomyocytes. Despite expressing less
GLUT4 (49), 3T3-L1 adipocytes express abundantly more of
each SNARE protein thereby generating a more favorable
ratio between the availability of transporters and fusion
machinery. However, it must also be acknowledged that the
heart tends to display an elevated basal rate of glucose
uptake in order to support contractile demands (50), which
would therefore reduce any fold insulin response. These data
also confirm that, like 3T3-L1 adipocytes, Syntaxin 4 and
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Bowman et al. Cardiac SNARE Proteins
FIGURE 2 | Cardiac SNARE expression in db/db and db/m mice. Protein lysates were generated from db/db and control mouse hearts and subjected to SDS-PAGE
and immunoblotting, probing for the expression of numerous SNARE proteins and GLUT4. Representative immunoblots for selected proteins only are shown. A
representative Ponceau S staining of a membrane is displayed, which was used as a loading control. For each sample, 40 µg of protein lysate was loaded. Each
sample (1–6 on figure) is composed of lysate generated from a separate heart.
VAMP2 are expressed at broadly similar levels on a per
cell basis.
SNARE Expression in Diabetic Mouse
Models
Insulin resistance may have an important role in the development
of limitations in cardiac contractile performance in diabetic
individuals. Accordingly, given their involvement in multiple
steps of GLUT4 trafficking, SNARE proteins may be implicated
in this clinically relevant pathophysiological setting. It is
therefore necessary to characterize the array of SNARE trafficking
machinery present in primary cardiac tissue in order to probe
known functions of these proteins (in other cell types) in the
heart, and novel physiological roles or interactions specific to
cardiac function. It is challenging to obtain a significant amount
of human myocardial samples for biochemical investigation,
particularly from specific patient populations and appropriately
matched controls. In order to circumnavigate this issue, many
animal models of disease have been developed in the field of
cardiac physiology. In the context of diabetes one of the most
widely utilized and characterized models is the db/db mouse,
which has a leptin receptor mutation that results in obesity
and blood parameters mimicking the human diabetic state
(51). Importantly, this model not only displays systemic insulin
resistance, but also cardiomyocyte-specific insulin resistance
and reduced cardiac contractility (recorded at the level of the
whole heart) (52,53), making it an ideal tool for investigation
of DCM.
Within this study, cardiac segments from 6 db/db and control
(db/m) mice were lysed and probed for the expression of a
broad range of SNARE proteins and GLUT4 via immunoblotting.
A typical dataset is displayed in Figure 2. Consistent with
previous analysis of cardiac protein expression from this diabetic
mouse model (53,54), GLUT4 expression was found to be
significantly (P<0.05) reduced in db/db lysates. We observed
clear and consistent immunoblot signals for Sx2, 4, 5, 8,
and 16, SNAP23, 29 and 47, and VAMP2, 3, 4, 5, and 8
(Supplementary Table 1), but as shown for a subset in Figure 2,
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Bowman et al. Cardiac SNARE Proteins
FIGURE 3 | Cardiac SNARE expression in HFD fed and control mice. Protein lysates were generated from HFD and control mouse hearts and subjected to
SDS-PAGE and immunoblotting, probing for the expression of SNARE proteins and GLUT4. Representative immunoblots for selected proteins are shown together
with Ponceau S staining, which was used as a loading control. Each sample is composed of lysates generated from 2 to 4 individual hearts.
no significant differences between db/m and db/db mice were
observed for any SNARE proteins. In contrast to Schwenk et al.
(46) but consistent with Peters et al. (47), we were unable to
detect VAMP7 in primary cardiac tissue yet obtained a clear
signal for VAMP8. However, in support of Schwenk et al. (46)
but in opposition to Peters et al. (47), we were unable to
detect VAMP1.
The db/db mouse is considered to be a good representation
of the established human diabetic phenotype. However,
there are legitimate questions regarding the translational
relevance of this model. Whilst there is almost certainly a
genetic component in many cases of type 2 diabetes (55),
more commonly lifestyle factors—e.g., food intake and
activity levels—that determine overall net energy balance
are recognized as key factors in diabetes prevalence. In
particular, obesity greatly increases the risk of an individual
developing diabetes (2). Therefore, high fat diet (HFD) based
interventions in rodents have been developed extensively.
This produces a less severe diabetic phenotype typically
analogous to a pre-diabetic state (56), however still captures
reduced cardiac contractility in the context of insulin resistance
(57). It is likely that the mechanisms underpinning complex
phenotypes such as insulin resistance and cardiomyopathy
may be multifactorial and vary with disease progression.
Therefore, we examined SNARE protein expression in a mouse
HFD model.
As can be observed in Figure 3 and Table 2, an almost
identical range of SNARE proteins were detected in this second
independent mouse cohort as observed in Figure 2. Due to
limitations in sample availability, lysates from 2 to 4 individual
hearts were pooled in order to generate each of the 4 samples
displayed, which reduced the statistical power of subsequent
analysis. With this limitation in mind, there was no impact of
the pro-diabetic phenotype upon GLUT4 expression. However,
statistically significant elevations in VAMP5 (78% increase) and
SNAP29 (68%) expression were detected. It appeared as though
VAMP2 expression may have been reduced in at least one
experimental group, however this effect was not found to be
statistically significant.
The samples used in Figures 2,3were obtained from sections
of frozen tissue. Therefore, measurements could not be obtained
that could confirm or refute that these lysates were from insulin
resistant hearts. However, we note that the db/db mouse is
a reliable and well characterized model and our observation
of reduced GLUT4 expression supports the disease phenotype
being evident in these samples. Importantly, published data from
the same control and HFD mice cohort confirms that samples
used here were from mice that displayed significant weight gain
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Bowman et al. Cardiac SNARE Proteins
TABLE 2 | Quantification of SNARE protein expression in HFD primary cardiac lysates.
Protein Detected? Difference? Protein Detected? Difference?
SNAP23 Yes No Syntaxin 16 Yes No
SNAP29 Yes Yes, significantly (P=0.04)
increased in HFD lysates by 68%
GLUT4 Yes No
SNAP47 Yes No VAMP1 No N/A
Syntaxin 2 Yes No VAMP2 Yes 30% lower expression in HFD
lysates, difference was not
significant (P=0.33)
Syntaxin 3 Yes No VAMP3 Yes No
Syntaxin 4 Yes No VAMP4 Yes No
Syntaxin 5 Yes No VAMP5 Yes Yes, significantly (P=0.02)
increased in HFD lysates by 78%
Syntaxin 6 Yes No VAMP7 No N/A
Syntaxin 7 No N/A VAMP8 Yes No
Syntaxin 8 Yes No
Immunoblotting was used to detect the presence or absence of a range of proteins in cardiac lysates generated from HFD and CHOW control mice. Where a clear and reproducible
difference was observed between groups, densitometry was used to quantify expression relative to total protein (from Ponceau stained images) and an unpaired Student’s t-test was
used to assess statistical significance (N =2). The level of significance was set at P =0.05.
and a trend toward delayed systemic glucose clearance during
a glucose tolerance test compared to controls—indicative of
reduced insulin sensitivity (58).
We have shown a range of SNARE proteins are expressed
in primary cardiac samples. This exceeds the machinery we
may expect to be required for the regulated trafficking of
substrate transporters such as GLUT4 or CD36. Indeed, the
heart is known to secrete vasoactive natriuretic peptides in
order to regulate blood pressure and therefore cardiac loading,
and recent work has also highlighted the emerging role that
numerous cardiokines may have in modulating both cardiac
remodeling in response to sustained pathophysiological stress
through auto/paracrine signaling and also remote physiological
functions through endocrine actions (59). The secretion of
these signaling proteins will be reliant upon the action of
SNAREs. Our study will provide the platform necessary to
relate SNARE function to specific physiological actions, and
therefore ultimately investigate any potential role in disease.
Additionally, this study provides clarity where there had
previously been conflicting data regarding the expression of
VAMP1/7/8. Obtaining identical results for these proteins
from 2 independent mouse lines provides confidence in
these findings.
Interestingly, in the HFD model SNAP29 and VAMP5
expression were elevated relative to control samples. VAMP5
has previously been linked to GLUT4 trafficking, but only in a
cardiomyocyte cell line model (46). In contrast, the functional
role of SNAP29 is not clear, particularly in the heart. Given
that impaired GLUT4 trafficking underlies insulin resistance, it
is unclear why expression of a related protein would remain
elevated beyond any initial compensatory period. It is interesting
to note however that elevated SNARE protein levels were
observed in skeletal muscle of the ZDF rat model of diabetes,
which were reversed by treatment with thiazolidinediones (39).
Hence, there is a precedent for elevated SNARE expression in
insulin resistance. In contrast to the study of skeletal muscle
insulin resistance which saw elevations in Sx4 and VAMP2,
here we report elevated VAMP5 in cardiac tissue from HFD
mice. VAMP5 has also been implicated in the regulation of
contraction-mediated glucose uptake in cardiomyocytes (46)
and thus our observation may reflect a functional distinction
between skeletal muscles and cardiomyocytes. Elevated VAMP5
may change (or reflect) the contractile profile of the heart in the
early stages of diabetes prior to the onset of cardiomyopathy.
It might be speculated that enhanced contraction is used to
compensate for reduced insulin mediated glucose uptake at
certain times. Alternatively, the observed changes may relate
to the trafficking of another target, perhaps a fatty acid
transporter. Enhanced cardiac lipid content is one of several
factors in the onset of insulin resistance, but this can only
occur with increased trafficking of the relevant transporters to
the cell membrane. Further work will be required to answer
these questions and to define whether the changes observed
in cardiac SNARE proteins in disease models are causal or
adaptive responses.
CONCLUSION
This study has detailed, for the first time, the expression of a
wide range of SNARE proteins in primary cardiac samples. This
foundational work paves the way for future studies investigating
the interactions and roles of these proteins in several different
aspects of cardiac physiology, both under normal working
conditions and in disease.
MATERIALS AND METHODS
Primary Cardiac Samples
All primary cardiac tissues used during this study were kind
gifts from colleagues at the University of Glasgow; procedures
were undertaken in accordance with the United Kingdom
Animals (Scientific Procedures) Act of 1986 and conform
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Bowman et al. Cardiac SNARE Proteins
TABLE 3 | Information regarding primary cardiac samples.
Sample Key details Gifted by
Db/db and db/m
mice
13–15-week-old male mice either hetero
or homozygous for a mutation in the leptin
receptor gene
Dr. Augusto
Montezano
High Fat Diet fed
and control mice
Approximately 20-week-old male mice fed
standard CHOW diet for 8 weeks
postnatally prior to switching to a diet
whereby 60% of calories were obtained
from fat for the next 12 weeks (or
maintained on CHOW as control)
Dr. Anna
White
to the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health and
approved by the Glasgow University Ethical Review Board.
In all cases tissues were received as sections of myocardium
that had been snap frozen either at −80◦C or in liquid
nitrogen. Key details pertaining to each sample are listed
in Table 3. All of the samples gifted were from male
animals, therefore it is acknowledged that there may be
sex specific differences in the reported findings that require
further investigation.
Generation of Lysates
The procedure for generation of cardiac lysates was the
same regardless of the origin of the tissue. First of all, a
large section of myocardium was excised from each heart.
These sections were generated in the same way by the same
experimenter, without prior separation into distinct anatomical
regions. The vast majority of myocardium is ventricular tissue,
and therefore any large section of cardiac tissue will almost
certainly originate predominantly from this area. Each section
was placed within a plastic 10 cm dish on ice in RIPA buffer
at a ratio of approximately 1-part tissue to 5 parts buffer.
The tissue was then manually diced with a sterile scalpel
blade into sections as small as possible, prior to further
mechanical lysis via a Dounce style tissue grinder. Samples were
placed on ice for a further 20 min, prior to repetition of this
mechanical lysis technique. The lysates were then centrifuged at
17,500 g for 15 min at 4◦C. The supernatant was then collected,
and the pellet was discarded. Prior to immunoblotting the
protein concentration of each sample was calculated via micro
BCA assay.
Generation and Quantification of Purified
Protein
Recombinant purified protein samples were generated as
described previously by Sadler et al. (60). First of all, plasmids
encoding sequences for Syntaxin 4, SNAP23, and VAMP2
tagged with glutathione S-transferase (GST) but in the case
of Sx4 and VAMP2 lacking their transmembrane domains
to increase expression were transformed into BL-21 E.coli
and subsequently amplified in serial cultures, using ampicillin
(100 µg/mL) resistance as a selection marker. Once in the optimal
growth range and volume, recombinant protein production was
induced via Isopropyl B-D-1-thiogalactopyranoside (0.5 mM)
incubation overnight at 22◦C. The following day cells were
lysed, and target proteins were extracted through incubation
of the lysate for 2 h with glutathione beads. Thereafter,
the beads were washed with PBS and then the samples
were eluted. Prior to use within this study, the GST tags
were cleaved from the proteins. Quantification of purified
protein concentration was performed by running samples
through SDS-PAGE against known amounts of BSA and
then performing Coomassie staining in order to visualize all
protein bands, as described by Sadler et al. (60). Gels were
scanned and then densitometry was performed in order to
generate a protein standard curve with BSA signals, which
was then used to estimate the concentration of each purified
SNARE sample.
SDS-PAGE and Immunoblotting
Samples were defrosted on ice, combined 1:1 with 2x Laemmli
sample buffer, and then heated to 65◦C for 10 min. Homemade
polyacrylamide gels of the appropriate percentage were cast
and then lysates were loaded in order to achieve the desired
(often equal) amount of protein across different samples. These
were then separated via gel electrophoresis and subsequently
subjected to immunoblotting as described previously by Sadler
et al. (60). Densitometry was performed via Image Studio Lite
in order to quantify the generated images. Either GAPDH
expression or Ponceau S staining was used in order to assess total
protein loading.
Antibodies
Anti-GLUT1 (#652) and anti-GLUT4 (#654) were from AbCam
(Cambridge, United Kingdom). Anti-GAPDH (#4300) was from
Ambion (Foster city, California, USA). Anti-VAMP1 (#104002),
anti-VAMP2 (#104202), anti-VAMP3 (#43080), anti-VAMP4
(#136002), anti-VAMP5 (#176003), anti-VAMP7 (#232003),
anti-VAMP8 (#104302), anti-SNAP23 (#111202), anti-SNAP29
(#111303), anti-SNAP47 (#111403), anti-syntaxin2 (#110022),
anti-syntaxin3 (#110032), anti-syntaxin4 (#110042), anti-
syntaxin5 (#110053), anti-syntaxin7 (#110072), anti-syntaxin8
(#110083), and anti-syntaxin16 (#110162) were from Synaptic
Systems (Goettingen, Germany). Anti-syntaxin6 (#610635)
was from BD Biosciences (Franklin lakes, New Jersey, USA).
Fluorescent secondary antibodies were from LI-COR Biosciences
(Lincoln, Nebraska, USA).
Statistical Analysis
Statistical testing was performed with GraphPad Prism 7. Where
appropriate, this consisted of an unpaired t-test, assessing the
difference in protein expression between one of the experimental
groups and relevant control. The level of significance was set
at P=0.05.
DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to
the corresponding author.
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Bowman et al. Cardiac SNARE Proteins
ETHICS STATEMENT
The animal study was reviewed and approved by Glasgow
University Ethical Review Board.
AUTHOR CONTRIBUTIONS
PB designed and performed the experiments, analyzed the
data, prepared the figures, and wrote the first draft. GS
and GG conceived the study, obtained funding, designed the
study, and supervised the laboratory work. GS and GG edited
the manuscript.
FUNDING
This work was supported by a Ph.D. scholarship to PRTB
(FS/14/61/31284) and project grants (PG/18/47/33822)
from The British Heart Foundation to GG and
GS, and Diabetes UK (15/0005246 and 13/0004696
to GG).
ACKNOWLEDGMENTS
Data in this manuscript was submitted as part of PB’s Ph.D. thesis
to the University of Glasgow, 2019. We thank Dr. Anna White
and Dr. Augusto Montezano for supplying tissues and Dr. Jessica
Sadler for purified proteins.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fendo.
2019.00881/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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