Pharmacological evaluation of novel PKR inhibitor indirubin-3-hydrazone in-vitro in cardiac myocytes and in-vivo in wistar rats

Article abstract of DOI:10.1016/j.lfs.2018.07.055

Aims: Double stranded protein kinase R cellular response is associated with various stress signals such as nutrients, endoplasmic stress, cytokines and mechanical stress. Increased PKR activity has been observed under diabetic and cardiovascular disease conditions. Most of the currently available PKR inhibitors are non-specific and have other effects as well. Thus, the aim of the present study was to examine the effect of novel PKR inhibitor indirubin-3-hydrazone (IHZ) in cultured rat H9C2 cardiomyocytes and wistar rats. Materials and methods: PKR expression was determined by Q-PCR, immunofluorescence and immunoblotting. The expression of different gene markers for apoptosis was measured by RT-PCR. Apoptosis and oxidative stress were determined by flow cytometry. KEY FINDINGS: High glucose (HG) treated H9C2 cardiomyocytes and high fructose (HF) treated wistar rats developed a significant increase in PKR expression. A significant increase in apoptosis and generation of reactive oxygen species was also observed in HG treated H9C2 cells and HF treated rats. Reduced vacuole formation and prominent nuclei were also observed in high glucose treated cells. Cardiac hypertrophy and increased fibrosis were observed in HF treated rats. All these effects of HG and HF were attenuated by novel PKR inhibitor, indirubin-3-hydrazone. Significance: Our results indicate IHZ as an effective inhibitor of PKR in vitro and in-vivo, thus it may prove very useful in blocking the multiple harmful effects of PKR.

Full text of DOI:10.1016/j.lfs.2018.07.055

LifeSciences209(2018)85–96
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Pharmacological evaluation of novel PKR inhibitor indirubin-3-hydrazone
in-vitro in cardiac myocytes and in-vivo in wistar rats
Mary Priyanka Udumulaa, Audesh Bhatb, Sureshbabu Mangalia, Jaspreet Kalraa, Indu Dharc,
Dharamrajan Srirama, Arti Dhara,⁎
a
Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Hyderabad Campus, Jawahar Nagar, Shameerpet, Hyderabad, Andhra Pradesh 500078,
India.
b
Department of Molecular Biology, Central University of Jammu, India
Department of Clinical Sciences, University of Bergen, Norway
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Indirubin-3-hydrazone
High glucose
High fructose
Cardiomyocytes
Aims: Double stranded protein kinase R cellular response is associated with various stress signals such as nu-
trients, endoplasmic stress, cytokines and mechanical stress. Increased PKR activity has been observed under
diabetic and cardiovascular disease conditions. Most of the currently available PKR inhibitors are non-specific
and have other effects as well. Thus, the aim of the present study was to examine the effect of novel PKR
inhibitor indirubin-3-hydrazone (IHZ) in cultured rat H9C2 cardiomyocytes and wistar rats.
Materials and methods: PKR expression was determined by Q-PCR, immunofluorescence and immunoblotting.
The expression of different gene markers for apoptosis was measured by RT-PCR. Apoptosis and oxidative stress
were determined by flow cytometry. KEY FINDINGS: High glucose (HG) treated H9C2 cardiomyocytes and high
fructose (HF) treated wistar rats developed a significant increase in PKR expression. A significant increase in
apoptosis and generation of reactive oxygen species was also observed in HG treated H9C2 cells and HF treated
rats. Reduced vacuole formation and prominent nuclei were also observed in high glucose treated cells. Cardiac
hypertrophy and increased fibrosis were observed in HF treated rats. All these effects of HG and HF were at-
tenuated by novel PKR inhibitor, indirubin-3-hydrazone.
Significance: Our results indicate IHZ as an effective inhibitor of PKR in vitro and in-vivo, thus it may prove very
useful in blocking the multiple harmful effects of PKR.
1. Introduction
pathogens [9]. PKR plays an important role in signal transduction and
transcriptional control of several inflammatory pathways such as IkB/
Diabetes and cardiovascular disorders constitute the most sig-
nificant health burden to worldwide population [1]. Type I and type II
diabetic patients are at increased risk of developing cardiovascular
diseases [2]. Under obese and metabolic stress conditions several in-
flammatory pathways get activated which in turn lead to activation of
downstream signaling molecules such as Jun NH2-terminal kinase
(JNK) and NFKB. These pathways play a significant role in development
of insulin resistance and cardiovascular diseases by controlling the in-
flammatory responses in metabolic tissues, inhibition of insulin re-
ceptor signaling and disruption of systemic glucose and lipid home-
ostasis [3–8]. Double stranded protein kinase R (PKR) is one such
molecule that is integrated with both nutrient and pathogen response
system, is activated by DsRNA, several stress signals, nutrients and
NFKB and JNK [10–12]. Previous studies have reported that PKR gene
silencing (PKR−/−) in mice relates with improvement in insulin re-
sistance [12,13]. Increased translocation and myocardial expression of
PKR has been demonstrated in human subjects suffering from con-
gestive heart failure (CHF) [14]. Congestive heart failure (CHF) is as-
sociated with cardiomyocyte hypertrophy, apoptosis and inflammation.
PKR plays a significant role in development of CHF by intensifying
apoptosis and inflammation of cardiomyocytes [15]. Based on this re-
search pharmacologically targeting PKR may be an effective ther-
apeutic strategy for treatment of diabetes and vascular complications.
Indirubin is one of the main active ingredients of Chinese herb drug
and has been known for its anti-proliferative effect in inhibition of
cyclic cyclin dependent kinase (CDK) and glycogen synthase kinase-3ẞ
⁎
Corresponding author at: Department of Pharmacy, Birla Institute of Technology and Science Pilani Hyderabad Campus, Jawahar Nagar, Shameerpet, Hyderabad,
Andhra Pradesh, 500078, India.
E-mail address: artidhar@hyderabad.bits-pilani.ac.in (A. Dhar).
https://doi.org/10.1016/j.lfs.2018.07.055
Received 28 December 2017; Received in revised form 18 July 2018; Accepted 30 July 2018
Availableonline01August2018
0024-3205/©2018PublishedbyElsevierInc.

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LifeSciences209(2018)85–96
A
B
C
HG+IHZ-10μM
IHZ-30μM
HG
HG+IHZ-3μM
IHZ-10μM
HG+IHZ-30μM
IHZ-3μM
500
***
400
300
200
100
0
%%%
%%%
C
M
M
M
M
M
M
HG
μ
μ
10
μ
30
μ
μ
μ
3
IHZ-3
+IHZ
IHZ-10IHZ-30
+IHZ +IHZ
HG
HG HG
Fig. 1. Schematic representation for synthesis of novel PKR analogue (indirubin 3 hydrazone (IHZ)) and concentration and time dependent effect of IHZ on PKR
levels. (A) Briefly describes the scheme developed for synthesizing indirubin 3 hydrazone (IHZ). The reagents and conditions for synthesis of IHZ were (a) 2-
Bromoacetic acid, K2CO3, DMF, 4 h,110 °C; (b) Acetic anhydride, 135–140 °C, 10 h; (c) K2CO3, CH3OH (d) NH2NH2.H2O, C2H5OH, 80 °C, reflux. (B)
Immunohistochemistry was done to determine the concentration dependent effect of IHZ on PKR levels using PKR specific antibody. Briefly, cultured cells were
incubated with different treatment groups Control (C), high glucose (HG) (25 mM), and IHZ (3, 10, 30 μM) alone or co-incubated with HG. (C) Time dependent
studies for determining the PKR expression. The cultured cells were incubated with different treatment groups Control (C), high glucose (HG) (25 mM), and the most
effective dose of IHZ (10 μM) alone or co-incubated with HG. for 3, 6, 12 and 24 h. (D) Immunohistochemistry, western blotting and gene expression was studies were
carried out for comparing the efficacy of IHZ (10 μM) with standard PKR inhibitor C16 (5 μm) on PKR activity. (E) PKR expression was significantly increased in HF
treated rats, which was significantly attenuated by administration of IHZ. n = 6 for each group was taken for each study ***p < 0.001, **p < 0.01, *p < 0.5 vs.
control. %%%p < 0.001, %p < 0.5 vs. HG, %p < 0.5 vs. HF, #p < 0.5 vs. HG + C16.
86

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C
HG
C
HG+IHZ-3 H
HG+IHZ-6 H
IHZ-12 H
HG+IHZ-12 H
HG+IHZ-24 H
IHZ-3 H
IHZ-6 H
IHZ-24 H
500
***
400
300
200
100
0
%%%
%%%
C
H
H
HG
-3 H
Z-6 H
12
Z-3HZ-6H
-24 H
-
Z
Z-12 Z-24H
IH IH
Z
Z
IH IH
+IH +IH
+IH +IH
HG HG
HG HG
Fig. 1. (continued)
(GSK-3 ẞ) [16]. We have also reported recently that indirubin-3-oxime
(I3O) has beneficial effects against high glucose induced oxidative
stress and apoptosis in cultured rat cardiomyocytes. Further, we found
that I3O is also capable of attenuating high glucose induced increased
PKR expression [17]. Indirubin- 3-hydrazone (IHZ), a novel derivative
of indirubin is a reported cyclin dependent kinase (CDK) inhibitor
[16,18]. However, the effect of IHZ on PKR signaling is not yet re-
ported, so the aim of the present study was to investigate the effect of
IHZ on PKR signaling pathway and underlying molecular mechanism.
washed and filtered by column chromatography to obtain indirubin 3
hydrazone (IHZ) (6).
2.2. Cell culture
H9C2 cells (a myoblast cell line purchased NCCS, Pune, India) were
cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing
10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml
streptomycin at 37°C in a humidified atmosphere of 95% air and 5%
CO2. The cells at 3–4 passages were exposed to high glucose (25 mM)
alone or in combination with PKR inhibitor, C16 (Sigma Aldrich, MO,
USA).
2. Materials and methods
2.1. Novel PKR inhibitor
2.3. Animals
The synthesis of novel PKR inhibitor was given in Fig. 1. In the first
step of reaction we used anthranilic acid (1) with bromoacetic acid and
potassium carbonate in presence of ethyl acetate to yield corresponding
(carboxymethylamino) benzoic acid (2) which was then filtered and
purified through column chromatography to obtain 1-acetyl-1H-indol-
3-yl acetate (3). Later to this reaction indoline-2,3-dione (commercially
purchased) (4) was added and dissolved in methanol in presence of
potassium carbonate to yield indirubin (5), resultant indirubin was
dissolved in hydrazine and ethanol. Finally, resulting compound was
5–6-week-old male wistar rats with a mean body weight (b.w.) of
180–220 g were randomly divided into 4 groups of 6 animals in each
group as follows: Control (C), Diabetes Control with 20 g of fructose in
drinking water (HF), high fructose and IHZ (HF + IHZ), IHZ alone.
Animals were housed as 3 rats per polycarbonated cage in a tempera-
ture and humidity controlled room (22
1 °C, 45–60% humidity) with
a set of 12 h light-dark cycle. The rats were fed a commercially avail-
able rat pellet diet ad libitum throughout the 6 weeks experimental
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D
5
4
3
2
1
0
600
400
200
0
***
***
%%%
#
C
HG
HG+C16
%%%
%%%
%%%
HG+IHZ
C16
IHZ
2.0
1.5
1.0
0.5
0.0
1.5
***
**
%%%
1.0
0.5
0.0
E
%
%%%
PKR
C
HG HG+C16 C16
C
HG HG+IHZ IHZ
B-ACTIN
2.5
*
2.0
1.5
1.0
0.5
0.0
%
PKR
β-ACTIN
C
HF
HF+IHZ IHZ
Fig. 1. (continued)
period. Control groups were supplied with normal drinking water ad
libitum. Diabetes control group were fed with FR20 were supplied with
20% fructose solution. The animals were maintained according to the
rules and regulations of the Institutional ethical committee (Ethical
approval number: HYD/IAEC/2016/06).
formaldehyde for 10 min, permeabilised in 0.1% triton-x, blocked with
3% BSA for 1 h at room temperature, incubated with primary antibodies
for over-night at 4 °C, then incubated with secondary antibodies for 2 h
and counterstained with DAPI.
2.6. Real time quantitative PCR (RT-PCR)
2.4. Induction of hyperglycemia
Total RNA from cultured cells was isolated using RNA isolation kit
(HI Pura, Germantown, Md., USA). The real-time PCR (RT-PCR) was
performed in CFX connect apparatus associated with the Bio-rad CFX
manager software (version 3.1) using SYBR Green PCR Master Mix (Bio-
Rad Laboratories Ltd., India).
Low dose (35 mg/kg b.w.) of streptozotocin (STZ), (Sigma Aldrich,
MO, USA) was given to diabetic control and high fructose and IHZ
group while the animals in normal control group were injected with
vehicle buffer only and all biochemical parameters were measured in
plasma.
2.7. Western blotting
2.5. Immunohistochemistry
Briefly, aliquots of cell lysates (50 μg each) (n = 6 for each group)
were separated on 6–10% SDS-PAGE, electrotransferred to a poly-
vinylidene difluoride (PVDF) membrane (Bio-Rad) and blocked with
Cells were plated on a 6 well plate (n = 6 for each group). After
treatment for 24 h cells were washed with PBS and fixed with 4% para-
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LifeSciences209(2018)85–96
5% nonfat milk in TBS-Tween buffer (20 mM Tris–HCl, pH 7.4, 135 mM
NaCl, 0.1% Tween) for 1.5 h at room temperature. Next the membrane
were incubated with the appropriate primary antibodies (Santa Cruze
Biotechnology, CA, USA) respectively, followed by incubation with
horseradish peroxidase conjugated secondary antibody for 1 h. After
extensive washing, immunoreactive protein was detected with an
Enhanced Chemiluminescence Detection System (ECL; Amersham
Biosciences Corp.) Each immunoblot experiment was repeated 3 times.
3.2. IHZ inhibits HG induced PKR activity in vitro
We previously reported that HG induced PKR expression was atte-
nuated by Indirubin 3-oxime, recently from docking analysis we found
novel analogue of indirubin, indirubin hydrazone (IHZ) has inhibitory
activity on PKR. In this study we investigated the in-vitro potential of
IHZ in H9C2 cells on PKR by RT-PCR, immunohistochemistry and
western blotting. The mRNA and protein expression of PKR, which is a
key modulator involved in glucose homeostasis was significantly in-
creased in HG incubated cells and was significantly attenuated by IHZ
(10 μM) co-incubated with HG (Fig. 1D). In heart tissue also we found
increased PKR expression (Fig. 1E). This was significantly decreased by
IHZ co-incubated with HF.
2.8. Estimation of lactate dehydrogenase activity and nitrite levels
Cells were incubated with high glucose, C16 and novel PKR in-
hibitor or high glucose alone for 24 h, later the supernatant was col-
lected and incubated with working reagent for 5 min according to
manufacturer's instructions and absorbance was taken using plate
reader at 340 nm–356 nm.
3.3. Effect of IHZ on gene markers of apoptosis
We examined the effect of IHZ on gene markers associated with
apoptosis. H9C2 cells were incubated with HG alone and in combina-
tion with C16 and IHZ for 24 h and analyzed for gene expression. The
mRNA expression of NFKB (Fig. 2C), an oxidative stress marker, JNK
(Fig. 2C) and caspase-3 (Fig. 2B) an apoptotic and inflammatory tran-
scription factor was significantly increased in HG incubated H9C2 cells
which was significantly decreased by IHZ (10 μM) co-incubated with
HG (Fig. 2A). Protein expression studies using western blot was per-
formed for JNK and phospho-JNK. There was no change in protein
expression of JNK but increased expression of phospho-JNK was ob-
served when incubated with HG for 24 h compared to untreated cells
this was significantly attenuated by IHZ with HG (Fig. 2A). Immuno-
fluorescence studies for JNK and caspase-3 (Fig. 2B) were performed
and HG significantly increased the JNK expression which was atte-
nuated by IHZ co-incubated with HG (Fig. 2A).
2.9. Measurement of reactive oxygen species and apoptosis
The formation of peroxynitrite was determined by a DCFH assay.
Cells were collected after incubation for 24 h with different treatment
groups. For ROS cells were loaded with a membrane-permeable, non-
fluorescent probe 2,7′-dichlorofluorescin diacetate (CM-H2DCFDA,
5 μmol/l) for 2 h at 37 °C in FBS-free DMEM in the dark. For apoptosis
cells were incubated with annexin and propidium iodide for 15 min.
After washing with PBS 3 times, apoptosis and ROS were measured
using flow cytometry.
2.10. Labelling of H9c2 cells with rhodamine for autophagy and hand E
staining
Cells were grown on coverslips and incubated with Tetra methyl
rhodamine methyl ester (TMRM) (100 nm) and Hematoxylin (H) in
separate wells for 30 min in dark at 37 °C. H coverslips were counter-
stained with Eosin and observed under confocal microscope for au-
tophagy and cell integrity.
3.4. Effect of IHZ on HG induced apoptosis and ROS generation
Previously, we have reported that HG induces apoptosis and ROS, so
here we have examined whether IHZ can prevent HG induced apoptosis
and ROS in H9C2 cells and wistar rats or not. HG treated cells have
shown more early apoptotic cells and ROS production which was sig-
nificantly attenuated by C16 and IHZ co incubated with HG. IHZ along
with HG show significant reduction in early apoptotic percentage of
cells and ROS production compared to HG + C16 (3A). IHZ along with
HF attenuated ROS produced by HF (Fig. 3A and B).
2.11. Measurement of fibrosis and cardiac hypertrophy
Hearts were isolated and sectioned of 5 μm, weighed (cardiac hy-
pertrophy) and stained with Sirius red for 1 h and washed with 2
changes of alcohol, later observed for collagen deposition or fibrosis.
3.5. Effect of IHZ on iNOS production and LDH
2.12. Statistical analysis
Inducible nitric oxide (iNOS) production is a key mediator to pro-
mote apoptosis, LDH an important marker in cardiomyopathy so we
examined whether HG can induce iNOS and LDH. so in H9C2 cells in-
cubated with HG (25 mM) there was significant increase in iNOS and
LDH production when compared with untreated cells which was sig-
nificantly decreased by IHZ (10 μM) co-incubated with HG (Fig. 4A).
However, it was not significant when compared to HG + C16.
Data obtained from separate experiments are expressed as
mean
SEM. Statistical analysis was performed using ANOVA with
post hoc Bonferroni's test. A p value of < 0.05 was considered to be
statistically significant (n = 6 for each group).
3. Results
3.1. Effect of novel PKR inhibitor (IHZ) on HG induced PKR expression
3.6. Effect of IHZ on HG elicited autophagy in cardiomyocytes and cellular
Novel PKR inhibitor at different concentrations (3, 10, 30 μm) was
incubated with HG in-vitro in H9C2 cells (Fig. 1B). C16 was used as a
standard for comparison of PKR inhibition levels. Incubation of HG
with novel inhibitor at 3 μm for 24 h did not showed any significant
reduction, whereas incubation of HG with 10 and 30 μm showed almost
75% of the PKR inhibition (Fig. 1B). So based on this observation we
have selected 10 μm concentration and incubated for different time
points 3,6,12 and 24 h however significant reduction in PKR expression
was observed at 12 and 24 h time point (Fig. 1C).
integrity
Autophagy is followed by oxidative stress, in the present study we
have examined the reduced vacuole formation in HG treated cultured
cells which may be due to autophagy using rhodamine staining.
Prominent nuclei and irregular shaped cell structure was found in cells
treated with HG, Moreover when cells were exposed to HG along with
IHZ vacuole formation and cell structure was recovered when com-
pared to HG alone treated cells (Fig. 4B).
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LifeSciences209(2018)85–96
3.7. Effect of IHZ on HF induced changes in biochemical markers, fibrosis
and cardiac hypertrophy in heart
PKR can be very effective agents against the harmful effects of high
glucose. Although, Lancaster et al. [21] have reported that PKR deletion
does not interfere in the progression and development of high fat diet
(HFD) induced obesity. Interestingly, they found that increased PKR
levels or its activation causes inflammation of adipose tissue resulting in
impairment of glucose metabolism. However, they also suggested that
several independent studies need to be carried out to contradict the
results obtained from Nakamura et al. Thus, further detailed studies are
required to explore role of PKR in cardiovascular and metabolic com-
plications. Moreover, currently there is a lack of selective PKR in-
hibitors and most of the compounds available, such as imoxin, 2-ami-
nopurine used as PKR inhibitors are costly.
All the biochemical estimations were done in plasma samples
(Fig. 5A). For fibrosis and cardiac hypertrophy, the heart sections were
then deparaffinized with Xylene, rehydrated with alcohol and water.
The rehydrated sections were stained using Sirius red, collagen de-
position and heart size was significantly increased in HF treated rats
this was significantly attenuated by IHZ (Fig. 5B and C).
4. Discussion
Thus in the present study we designed and synthesized novel PKR
inhibitor, IHZ analogue using indirubin as a backbone (Fig. 1A). We
report for the first time that IHZ attenuated high glucose and HF in-
duced increased PKR expression in cultured rat cardiomyocytes. The
attenuation was much more significant compared to standard PKR in-
hibitor C16 (Fig. 1B, C). Recent studies have reported that PKR in-
tegrates nutrient overload, ER stress and inflammation. Inhibition of
PKR activation reduces JNK phosphorylation which is responsible for
insulin resistance. Activated PKR also induces overexpression of NFKB
Metabolic disorders including obesity, insulin resistance, impaired
glucose tolerance has limited treatment options and has become a
growing health concern across world [19,20]. PKR a serine/threonine
protein kinase is activated by cytokines, stress signals, interferon's and
viral infection and plays an important role in the nutrient/pathogen
sensing interface and acts as a key modulator of chronic metabolic in-
flammation, insulin sensitivity and glucose homeostasis [17]. PKR has
been implicated in the pathogenesis of insulin resistance as well as
congestive heart failure [14]. Thus selective and specific inhibitors of
A
600
***
400
%%%
C
HG
HG+C16
%%%
###
200
0
HG+IHZ
C16
IHZ
2.0
2.5
2.0
1.5
1.0
0.5
0.0
***
1.5
%%%
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
C HG HG+C16 C16
C
HG HG+IHZ IHZ
***
%%%
Fig. 2. IHZ attenuates HG induced expression of JNK, caspase-3 and NFKB.
Incubation of cultured cells with HG (25 mM) significantly increased expression of JNK, and caspase-3 which was significantly attenuated by IHZ (10 μM) on co-
incubation with HG (A). Effect of IHZ on JNK expression was determined by performing immunohistochemistry, and western blotting using antibodies specific for
JNK and phospho-JNK, (B) Representative results obtained from immunohistochemistry and mRNA expression studies for determining the expression of caspase-3
with specific caspase-3 antibody, (C) Demonstrates gene expression studies for NFKB and JNK. n = 6 for each group ***p < 0.001, **p < 0.01 vs. control.
%%%p < 0.001, %%p < 0.01, %p < 0.5 vs. HG treated group. #p < 0.5 vs. HG + C16.
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B
C
HG
HG+C16
IHZ
C16
HG+IHZ
15
10
5
600
400
200
0
***
***
%
%%%
%%%
%%%
0
C
6
4
2
0
8
***
***
6
4
2
0
%%
%%
%%%
%%%
Fig. 2. (continued)
causing transcription of many apoptotic markers such as caspase-3
[17,18]. So we investigated whether the novel molecule IHZ can at-
tenuate the harmful effects of HG on JNK and NFKB expression
(Fig. 2C). Here we report that IHZ attenuates HG induced JNK ex-
pression in cultured H9C2 cardiac myocytes (Fig. 2A). Moreover the
effect was much more significant than the standard PKR inhibitor C16.
Oxidative stress and ROS production has negative influence on
glucose tolerance, insulin resistance and hyperglycaemia [22]. HG is a
known inducer of oxidative stress [17]. IHZ attenuates the HG and HF
induced oxidative stress in cultured cardiomyocytes (Fig. 3A). The re-
duction in ROS production was ever more significant than standard PKR
inhibitor C16 (Fig. 3A). Apoptosis plays a crucial role in various me-
tabolic diseases such as ischemia, hypertension and diabetes [22].
Tissue injury, disease condition and stress signals ultimately lead to
caspase-3 activation which in turn will induce cell death via activation
of nucleases and proteases [23,24]. PKR mediates apoptosis through
multiple mechanisms, via interacting with death ligands such as TNF-α
and FasL [23]. IHZ attenuates HG induced early apoptosis in cultured
cardiomyocytes (Fig. 3B). Moreover, as shown the FACS analysis, the
effect was more significant compared to C16. HG induced increased
caspase-3 immunofluorescence as well as mRNA expression was also
attenuated by IHZ in cultured cardiomyocytes for 24 h (Fig. 2B). Lactate
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A
250000
200000
150000
100000
50000
0
400
300
200
100
0
***
***
%
C
HG
HG+C16
%%%
%%%
###
Z
C
C
HF
H
I
IHZ
HG
C16
IHZ
HF+
HG+IHZ
C16
IHZ
HG+C16HG+IHZ
B
80
L
E.A
L.A
N
60
40
20
0
C
HG
HG+C16
%%
**
%
#
C
HG
HG+C16HG+
IHZ
C16
IHZ
HG+IHZ
C16
IHZ
Fig. 3. IHZ attenuates HG induced increase reactive oxygen species production and apoptosis: incubation of cultured cardiomyocytes with HG and HF treated rats for
24 h induced increase in ROS generation which was significantly attenuated by IHZ when co-incubated with HG and HF which was analyzed by FACS and plate
reader (A). Annexin V/alexa fluor 488 assay was performed for apoptosis. Cells were incubated with HG for 24 h and analysis was performed using annexin V alexa
fluor 488, the results explain percentage of live, early, late apoptotic and necrosis cells falling in that particular gating (B). n = 5 for each group ***p < 0.001,
**p < 0.01 vs. control, %p < 0.5, %%p < 0.01, %%%p < 0.001 vs. HF and HG. ###p < 0.001 vs. HGC16. #p < 0.5 vs. HGC16 early apoptotic cells.
dehydrogenase (LDH), a soluble cytoplasmic enzyme is an important
marker to detect necrosis. LDH is present in almost all the cells and is
released into extracellular space when plasma membrane is gets da-
maged [25,26]. In the present study we found that in incubation of
H9C2 cardiac myocytes with HG lead to increased LDH levels, whereas
co-incubation of IHZ with HG significantly attenuated the increased
LDH levels in cultured rat cardiomyocytes (Fig. 4A). NFκB is one of the
key transcription factors involved in triggering the events that are as-
sociated with the inflammatory pathways. Further, it has also been
reported that inducible nitric oxide synthase activation is coupled/
linked with the activation of NFκB [27,28]. Therefore, we investigated
the effect of IHZ on iNOS levels on HG treated cardiomyocytes. HG
treated cells co-incubation with IHZ has significantly reduced iNOS
levels compared to HG treated cultured cardiomyocytes (Fig. 4A).
Autophagy has been depicted as cellular degradation pathway
playing an important role in cellular homeostasis. At basal levels au-
tophagy is used to maintain biological function [29]. Upregulated
autophagy is a prominent marker observed under cardiomyopathy
[30]. In the present study interest has been generated in addressing the
autophagic activity of cardiomyocytes in response to HG. Severe au-
tophagy was observed in HG treated cells which was determined with
the help of a specific tracer Tetra methyl rhodamine methyl ester
(TMRM) for autophagic vacuoles [31]. Our novel molecules attenuated
this vacuole formation when incubated with HG (Fig. 4B). Additionally,
through H and E staining we have detected numerous other morpho-
logical changes (Fig. 4B) in cultured cardiomyocytes that could have
occurred as a consequence of apoptosis [32]. We found that in HG
treated cardiomyocytes, dark eosinophilic cytoplasm with fragmented
nucleus was present, a well-known marker for apoptosis. Moreover,
improvement in morphological changes was observed on co-incubating
the HG treated cells with IHZ. Results obtained from biochemical stu-
dies done in rat plasma also confers it role in improving overall lipid
profile and glucose levels and (Fig. 5A). We also measured collagen
deposition in all rats using Sirius red staining, there was a significant
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LifeSciences209(2018)85–96
A
250
200
150
100
50
***
150
100
50
***
%
%
%%%
%%%
0
0
B
HG+IHZ
HG
HG+C16
C
C16
IHZ
C
HG+IHZ
HG
HG+C16
C16
IHZ
(caption on next page)
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LifeSciences209(2018)85–96
Fig. 4. IHZ attenuates HG induced iNOS production, lactate dehydrogenase (LDH), morphological changes and autophagy.
Incubation of cultured cells with HG (25 mM) caused significant increase in nitrite levels and induced morphological changes along with the increased activity of LDH
and autopahgy, which was significantly attenuated by IHZ (10 μM) incubated with HG, n = 8 for each group. Cells after incubating with HG has shown no vacuole
formation which was attenuated by IHZ (10 μM), n = 6 for each group. ***p < 0.001 vs. control. %p < 0.5, %%%p < 0.001 vs. HG. (A) Nitrite levels were measured
with the help of Griess reagent. LDH activity was measured by commercially available kit and autophagy was determined with the help of rhodamine staining. (B)
Cellular integrity was assessed by staining the sections with H & E and autophagy was determined with the help of rhodamine staining.
increase in fibrosis in HF treated rats, this was significantly attenuated
by IHZ on co-treatment with HF (Fig. 5B). As cardiac hypertrophy is a
marker for cardiomyopathy [15] we next measured weight of hearts
and we observed if there is any change in heart size. HF treated rat
hearts showed increase in weight and size this was attenuated by IHZ
along with HF (Fig. 5C). Therefore, the present study indicates that PKR
is involved in the development of diabetes related cardiac complica-
tions and contribute an important role in progression of cardio-meta-
bolic diseases. Hence, targeting PKR may present a novel approach in
the discovery and development of therapeutic strategies which are
aimed at correcting cardio-metabolic disorders.
A
Parameters
C
HF
HF+IHZ
IHZ
Total glucose (mg/dL)
106 9
322 71
613 10
6 0.27
209 27
421 3.01
11 0.71
90 14.68
63 5.1
23 0.91
Total cholesterol (mg/ldL) 89 0.894
High density lipoprotein
(mg/dL)
24 0.81
Low density lipoprotein
(mg/dL)
62.1 1
386 8.1
291 1
56 2.1
Triglycerides (mg/dL)
13.55 0.891 105 0.551
90 0.201
18.3 0.41
2.5
2.0
1.5
1.0
0.5
0.0
***
B
C
%%%
C
HF
HF+IHZ
IHZ
30
20
10
0
***
C
HF
%%%
HF+IHZ
IHZ
Fig. 5. IHZ attenuates HF induced biochemical changes, cardiac hypertrophy and collagen deposition in-vivo in Wistar rats.
After the experimental protocol all animals were sacrificed on same day and serum was collected and hearts were harvested. (A) Biochemical changes in lipid profile
were assessed using commercially available kits. (B) Hearts were dehydrated and weighed and changes in heart were measured. (C) Paraffin embedded tissues were
sectioned of 4–5 μm and sirius red staining was done to observe the collagen deposition. Positive staining was seen in red colour, n = 6 for each group. ***p < 0.001
vs. C, %%p < 0.01, %%%p < 0.001 vs. HF. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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5. Conclusion
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Acknowledgements
This work was supported by grant from Counsel of Scientific and
Industrial Research (CSIR) (37(1643)/15/EMR-II), and Department of
Science and Technology (DST)-SERB under young scientist scheme
(YSS/2014/000164), Govt. of India and Research Initiation grant from
BITS Pilani-Hyderabad, India to Arti Dhar.
Conflict(s) of interest/disclosure(s) statement
None.
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