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Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
https://doi.org/10.1007/s00424-024-02967-4
ORGAN PHYSIOLOGY
SGLT2 inhibitor asapotential therapeutic approach
inhyperthyroidism‑induced cardiopulmonary injury inrats
NermeenBastawy1 · AliaaE.M.K.El‑Mosallamy2 · SamiraH.Aljuaydi3 · HudaO.AbuBakr3,10 ·
RababAhmedRasheed4 · A.S.Sadek5,6 · R.T.Khattab5 · WaelBotrosAbualyamin7,8 ·
ShereenE.Abdelaal9 · AmyF.Boushra7
Received: 13 February 2024 / Revised: 1 March 2024 / Accepted: 17 April 2024 / Published online: 3 May 2024
© The Author(s) 2024
Abstract
Hyperthyroidism-induced cardiac disease is an evolving health, economic, and social problem affecting well-being. Sodium-
glucose cotransporter protein 2 inhibitors (SGLT2-I) have been proven to be cardio-protective when administered in cases
of heart failure. This study intended to investigate the potential therapeutic effect of SGLT2-I on hyperthyroidism-related
cardiopulmonary injury, targeting the possible underlying mechanisms. The impact of the SGLT2-I, dapagliflozin (DAPA),
(1mg/kg/day, p.o) on LT4 (0.3mg/kg/day, i.p)-induced cardiopulmonary injury was investigated in rats. The body weight,
ECG, and serum hormones were evaluated. Also, redox balance, DNA fragmentation, inflammatory cytokines, and PCR
quantification in heart and lung tissues were employed to investigate the effect of DAPA in experimentally induced hyper-
thyroid rats along with histological and immunohistochemical examination. Coadministration of DAPA with LT4 effectively
restored all serum biomarkers to nearly average levels, improved ECG findings, and reinstated the redox balance. Also,
DAPA could improve DNA fragmentation, elevate mtTFA, and lessen TNF-α and IGF-1 gene expression in both organs of
treated animals. Furthermore, DAPA markedly improved the necro-inflammatory and fibrotic cardiopulmonary histologi-
cal alterations and reduced the tissue immunohistochemical expression of TNF-α and caspase-3. Although further clinical
and deep molecular studies are required before transposing to humans, our study emphasized DAPA’s potential to relieve
hyperthyroidism-induced cardiopulmonary injury in rats through its antioxidant, anti-inflammatory, and anti-apoptotic effects,
as well as via antagonizing the sympathetic over activity.
Keywords Dapagliflozin· Hyperthyroidism· Cardiopulmonary· Toxicity· Antioxidant· TNF-α
Introduction
The primary active type of thyroid hormones, T3, is well
known for regulating growth, metabolism, and a host of other
physiological processes [66]. Elevated thyroid hormone
levels are a sign of hyperthyroidism, a clinical condition
involving thyroid malfunction. In nations where iodine is
sufficient, its prevalence ranges from 0.2 to 1.3% [15]. Most
of the effects of thyroid hormone are mediated by nuclear
receptors and affect the transcription of T3-responsive genes.
Significant evidence, however, suggests that some dysfunc-
tions brought on by hyperthyroidism are caused by tissue
oxidative stress [6]. Numerous tissues, including blood ves-
sels and myocardium, contain thyroid hormone receptors.
Therefore, variations in thyroid hormone concentrations may
impact cardiovascular functions [54].
Since the turn of the 20th, extensive research has been
done on the impact of thyroid dysfunction on the cardio-
vascular system. Clinically, thyroid hormone excess or defi-
ciency may cause or worsen cardiovascular disease (CVD)
[12]. The prevalence of hyperthyroidism-related cardiac
problems appears to be higher in older individuals with
Graves’ disease or toxic untreated hyperthyroidism [40].
Hyperthyroidism may affect functional parameters of the
left ventricle, including the ejection fraction [67]. About
6% of hyperthyroid patients appear to have heart failure
as their most common clinical presentation, with roughly
half of them having left ventricular systolic dysfunction.
Compared to euthyroid subjects, patients with symptomatic
heart failure and an ejection fraction < 35% have an approxi-
mately 85% higher relative risk of dying [46, 58]. Moreover,
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1126 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
systolic blood hypertension affects up to 68% of hyperthy-
roid patients, particularly the older age group [42].
There is a correlation between tissue oxidative injury and
increased thyroid hormone levels in the blood. Most of the
data suggests that an experimentally induced hyperthyroid
state causes oxidative damage to many tissues, including the
heart and lungs, changing the pro-oxidant-antioxidant balance
characteristics of euthyroid tissues [59]. Increases in lung cell
damage, the permeability of the alveolocapillary membrane,
the total number of phagocytic cells, and the phagocytic cells’
enhanced release of nitric oxide were all caused by hyperthy-
roidism [75]. Moreover, increased thyroid hormone levels can
boost lung responses to other substances that cause oxidant
stress, as evidenced by the increased pulmonary toxicity of
oxidant gases in hyperthyroidism [25].
Sodium-glucose cotransporter protein 2 inhibitors (SGLT2-
I) are oral anti-diabetic medications that lower blood sugar
by lessening the kidney’s proximal convoluted tubules’ renal
glucose reuptake [27]. Within the past few years, many stud-
ies researched the cardioprotective role of SGLT2 inhibitors,
such as empagliflozin, dapagliflozin, and canagliflozin, in
heart failure [53, 80]. Interestingly, the cardiovascular prof-
its of SGLT2-I result from mechanisms other than glycemic
control. Moreover, SGLT2-I cardioprotective effects were
exhibited even in non-diabetic patients with established CVD
[73]. SGLT2-I reduces oxidative stress by translocating Nrf2 to
the nucleus and triggering Nrf2/ARE signaling, which lessens
myocardial fibrosis by inhibiting collagen deposition through
the traditional TGF-β/Smad pathway [41].
With a focus on potential mechanisms by which SGLT2-I
may exert their therapeutic effects, this study points to the
potential therapeutic impact of dapagliflozin on rats’ cardio-
pulmonary injury caused by hyperthyroidism and highlights
the possible underlying mechanisms.
Materials andmethods
Drugs
Levothyroxine sodium hydrate (LT4) (powder form, pack
size: 50mg, Sigma-Aldrich, MO, USA) and dapagliflozin
(DAPA) (5mg film-coated tablet, AstraZeneca Pharmaceu-
ticals LP, Indiana, USA) were used in this study. Both drugs
were of analytical grade and were dissolved in normal saline
0.9% just prior to use.
Animals housing andethical statement
Twenty-four adult male albino rats of Wistar strain
(10–12weeks, 180–200g) were purchased and sheltered in
the Animal House of the Faculty of Medicine, Cairo Uni-
versity, Egypt. Per the NIH Guidelines for the Care and Use
of Laboratory animals and strictly adherent to the ARRIVE
guidelines (Animal Research: Reporting InVivo Experi-
ments), animals were kept in a hygienic, naturally ventilated,
and pathogen-free environment in conventional stainless-steel
cages (6 rats/cage). Rats were permitted to acclimate before
starting the study for 1week with unrestricted access to rat
chow and tap water at a temperature oscillating between
22:24°C, moderate humidity (50–60%), and alternating
12/12-h dark/light cycle. This study was accepted by The
Institutional Animal Care and Use Committee, Cairo Univer-
sity (CU-IACUC), Egypt (Code: CU/III/F/15/23).
Sample size calculation
The number of animals per group was calculated by
employing the method described by [68] before conduct-
ing the study to minimize the number of used animals. As
per previous work [60], we considered the mean difference
in serum T4 level between the study groups to be 5.65,
and the standard deviation was 1.46. Thus, the sample size
is estimated as six rats per group, with a total of 24 rats
attaining a power of 80% when p value is adjusted at 0.05
or less as a level of significance.
Experimental design
The rat model of thyrotoxicosis was built up by daily
intraperitoneal (i.p) injection of levothyroxine (LT4) at
a dose of 0.3mg/kg dissolved in saline for 4weeks [37].
Rats were equally and undiscriminatingly assigned to four
groups (n = 6). For randomization of the rats, they were
labeled from 1 to 24 and were allocated into four groups
using the Rand function in Excel (ver. 16.82). The ani-
mal groups were as follows: the control group: received
the saline vehicle (1mL/kg/day, p.o) for 4weeks; DAPA
group: received DAPA dissolved in normal saline (1mg/
kg/day, p.o) for 4weeks [52], T4 group: received LT4
(0.3mg/kg/day, i.p) for 4weeks, and T4 + DAPA group:
received LT4 solely for 2weeks, then received both LT4
and DAPA for a further 2weeks at the previously assigned
doses and routes for each drug. The timeframe, drug dos-
ages, routes, and animal grouping are shown in Fig.1.
At the end of the fourth week, rats were weighed,
fasted overnight (12h), anesthetized by ketamine injec-
tion (100mg/kg, i.p) [34], and then, prepared for ECG
monitoring, blood, and sample collection.
Measurement ofbody weight
The rats’ body weight was recorded using a digital scale
(PS 750.R1) starting from day zero before administering
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1127Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
any medications and continued weekly till the end of the
study (day 28).
ECG andheart rate recording
After the conclusion of the trial, the anesthetized rats were
put supine on a wooden sterilized board for ECG monitor-
ing. Briefly, needle electrodes were implanted subcutane-
ously in the right and left paws (front and back sides) of
each animal for five minutes. Being connected to PowerLab
8SP (model ML785), Lead II ECG signals and the heart rate
were monitored, recorded, and analyzed using ECG analysis
version 2.0 software (AD instruments) [20].
Blood collection
After ECG monitoring, retro-orbital blood samples
(3mL) were collected from each anesthetized rat into
evacuated tubes. The serum was then separated by cen-
trifugation (3000rpm for 10min at 4°C). The collected
serum was stored at − 20°C before biochemical lab
investigations.
Tissue collection
A thoracotomy incision was immediately done on the anes-
thetized rats to extract the heart and lungs, followed by
washing with cold saline. The left ventricle was dissected
into two halves. Half the left ventricle and the right lung
were sent for histopathological examination. The other half
of the left ventricle and the left lung were divided into two
sets. The first set was homogenized in cold PBS (pH 7.4)
using a Teflon homogenizer, and the homogenates were
centrifuged at 14,000 × g for 20min at 4°C. The obtained
supernatant was used to assess oxidant and antioxidant tis-
sue biomarkers. The other set of samples was kept frozen
immediately in liquid nitrogen at − 80°C until the process-
ing for RT-qPCR analysis.
Biochemical analysis
Serum levels of thyroid-stimulating hormone (TSH),
T3, and T4 were analyzed by colorimetric competitive
enzyme immunoassay employing individual ELISA kits
(Abcam, United States), according to the manufacturer’s
protocol. Also, lactate dehydrogenase (LDH) (LDHI
0108021) and creatine kinase-myocardial band (CK-MB)
(CKMB0101022-3) by using kits are supplied by Spectrum
diagnostics and measured using the NS BIOTEC Chemistry
analyzer (MODEL: SCA-1200).
Oxidant/antioxidant tissue biomarkers
Assessment ofcatalase activity
According to [3], CAT activity was tested using Biodiag-
nostic kits (Egypt). A catalase inhibitor was used to halt the
reaction of catalase with a known amount of H2O2 after
1min. The remaining H2O2 combines with 3–5-Dichloro-
2-hydroxybenzene sulfonic acid and 4-aminophenazone in
the presence of peroxidase to generate a chromophore whose
color intensity is inversely proportional to the concentration
of catalase in the sample. The UNICO-UV-2100 spectro-
photometer was used to measure the absorbance at 510nm.
Assessment ofreduced glutathione
According to [19], the reduced glutathione (GSH) level was
evaluated using Biodiagnostic kits (Egypt). The tissue homoge-
nate was combined with 0.2M phosphate buffer pH 8 and 5,
50-dithiobis 2-nitrobenzoic acid (DTNB). The reduction of
DTNB is a prerequisite for the decline of glutathione levels.
Fig. 1 The timeframe, drug dosages, routes, and animal grouping
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1128 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
Glutathione gives off a yellow color, and the UNICO-UV-2100
spectrophotometer detected its absorbance at 412nm.
Assessment oflipid peroxidation
According to [48], the concentration of malondialdehyde
(MDA) was utilized as a measure of lipid peroxidation.
MDA was measured using Biodiagnostic kits (Egypt) via
evaluating the reactive species of thiobarbituric acid [74].
MDA was established using a UNICO-UV-2100 spectropho-
tometer; the absorbance of the resulting pink product was
measured at 534nm.
Assessment ofgenotoxicity byDNA fragmentation
The DNA fragmentation percentage was calculated using
the procedure that was provided by [1]. In summary, 400
μL of hypotonic lysis buffer containing 10–20mg tissue was
mixed and centrifuged at 3000 × g for 15min at 4°C. The
supernatant was separated into two portions: one was used
for the gel electrophoresis, and the other was combined with
the pellet to measure the amount of fragmented DNA by
the diphenylamine using the UNICO-UV-2100 spectropho-
tometer at 578nm. DNA fragmentation percentage in each
sample was expressed by the formula:
%DNA fragment ation = (O.D Supernatant∕O.D Super nat ant + O.D Pellet) × 100
Quantitative real‑time polymerase chain reaction
ofmitochondrial transcription factor A(mtTFA),
tumor necrosis factor‑alpha (TNF‑α), andinsulin‑like
growth factor‑1 (IGF‑1) genes
RNA was extracted from heart and lung tissues using the
QIAmp miRNAsy mini kit (QIAGEN, Hilden, Germany) as
directed by the manufacturer. The purity and concentration
of total RNA were determined using a nanodrop ND-1000
spectrophotometer. Reverse transcriptase was utilized to
synthesize cDNA from the extracted RNA (Fermentas,
EU). Using a mixture of 1 μL cDNA, 0.5mM of each
primer (Table1), and iQ SYBR Green Premix (Bio-Rad
170–880, USA), real-time PCR (qPCR) was carried out in
a total volume of 20 μL. The MyiQ real-time PCR detec-
tion device and the Bio-Rad iCycler heat cycler were used
to accomplish PCR amplification and analysis. Every assay
comprises three duplicate samples of each cDNA under
test along with a negative control that has no template; the
expression in relation to the control is determined using
Eq. 2−ΔΔCT [2].
Histological examination
Fresh portions from heart and lung tissues were dissected,
instantly fixed in 10% neutral buffered formalin for 24h,
dehydrated in scaling grades of alcohol, cleared in xylene,
and then, dipped in molten paraffin to obtain tissue blocks.
Thin Sects.(3–4μm) were sliced by a microtome, further
stained with hematoxylin and eosin to assess the cardiopul-
monary histopathological changes and picrosirius red stain
to demonstrate the interstitial myocardial and pulmonary
fibrosis [71].
Immunohistochemical analysis
As described by [56], thin Sects.(3–4 micron) were cut
from the paraffin blocks on positively charged slides. Fol-
lowing deparaffinization, the sections were treated with
0.3% H2O2/methanol and left for ten min, at room tempera-
ture to erase the action of peroxidase. Antigen retrieval
was done in citrate buffer (vol. 10mM, 95°C, pH 6) and
left to cool (1h at room temperature). Sections were then
incubated with primary antibodies overnight at 4°C; the
product codes and the source are as specified in Table2.
Later, sections were incubated in DAB to reveal peroxi-
dase activity and left overnight in phosphate-buffered
saline (PBS) at 4°C. The procedure was finalized by add-
ing DAB, followed by counterstaining with hematoxylin.
Sections were examined for the brown cytoplasmic and
nuclear areas, indicating a positive reaction against the
blue background.
Table 1 Primer sequences of
reference, mtTFA, TNF-α,
and IGF-1 genes of Rattus
norvegicus
Target genes Accession no Sequence (5′ to 3′) Product size
GAPDH (refer-
ence gene)
NM_017008.4 F: 5′-GAG ACA GCC GCA TCT TCT TG-3′
R: 5'- TGA CTG TGC CGT TGA ACT TG -3'
224bp
mtTFA NM_031326.2 F: 5′-CAT GAC GAG TTC TGC CGT TTG-3′
R: 5′-AGT AAA GCC CGG AAG GTT CTT-3′
254bp
TNF-α NM_012675.3 F:5′-ACA CAC GAG ACG CTG AAG TA-3′
R:5′-GGA ACA GTC TGG GAA GCT CT-3′
235bp
IGF-1 XM_039078402.1 F: 5′-TCT CCT AGT CCC TGC CTC TT-3′
R: 5′-TCT GTG AAG GAA GCG GCT TA-3′
183bp
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1129Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
Image acquisition, morphometric measurements,
andimage analysis
H&E, picrosirius red, and immunohistochemically stained
sections were examined and snapped at the Pathology &
Image Analyzer Research Lab, Medical Research Centre
of Excellence (MRCE), National Research Centre, Cairo,
using the image analyzer Leica Qwin 500 (LEICA Image
Systems Ltd., Cambridge, England). It comprised a Leica
DM3000 microscope with a JVC color video camera
attached to a computer system. To avoid bias in reporting
the results, we used the “comprehensive masking method”
according to [23]. Briefly, the tissue sections from differ-
ent study groups were labeled as a, b, c, d, etc. They were
examined by two contributors (a histologist and a patholo-
gist) who were unaware of the study design or the treatment
given to rats. Also, blinding was applied while recording
ECG, biochemical measurements, and statistics. The mor-
phometric measurements, taken from ten randomly chosen
non-intersecting fields × 200, were carried out on H&E-
stained sections of the heart to measure the wall thick-
ness of the left ventricle, picrosirius red-stained sections to
detect the percentage of the red-colored fibrous tissue, and
immunohistochemically stained slides (for both Caspase-3
and TNF-α antibodies) to measure the percentage of posi-
tive cells. The area, area fraction, area fill, and area per-
centage were calculated using the automated system soft-
ware. The areas to be measured were marked by a binary
color to form a binary image (Fig.2). The area of positiv-
ity was determined as an area in micrometers squared per
field. The results appeared automatically on the monitor as
a table with the measured total, mean, standard deviation,
standard error, minimum, and maximum area.
Statistical analysis
The acquired values are given as means ± S.E of the mean.
Comparisons of weight values in relation to interval weeks
between different groups were performed by two-way analy-
sis of variance (ANOVA) using SPSS software version 24.
In other parameters, the comparisons between groups were
carried out by one-way analysis of variance (ANOVA) fol-
lowed by “Duncan’s Multiple Range” test for post hoc analy-
sis using SPSS software version 24. The significance was set
at p ≤ 0.05. GraphPad software Instat (version 2) was used
to make graphs.
Results
Effect ofDAPA onbody weight inhyperthyroid rats
As shown in Fig.3, there was a weight loss in the hyper-
thyroid rats (both untreated or treated with DAPA) start-
ing after the first week of induction of hyperthyroidism and
reaching significant values from the third week till the end of
the experiment, compared to the control and DAPA groups
(p ≤ 0.05). The concomitant treatment of the hyperthyroid
rats with DAPA significantly ameliorated the weight loss
compared to the T4 group (p ≤ 0.05). DAPA alone did not
affect the body weight compared to the control group.
Effect ofDAPA onECG inhyperthyroid rats
The results of the current study show a significant increase
in heart rate in the animals with hyperthyroidism compared
to the controls (p ≤ 0.05). Animals with hyperthyroidism
treated with DAPA showed significantly lower heart rate
levels as compared to the untreated animals (p ≤ 0.05). Addi-
tionally, the untreated hyperthyroid group showed signifi-
cant prolongation in the JT interval compared to the control
groups (p ≤ 0.05). This effect was ameliorated with DAPA
treatment, with no significant difference from control lev-
els, p = 0.7, 0.8). DAPA-treated group showed significantly
shorter JT intervals as compared with the untreated group
(p ≤ 0.05). ECG records from T4 group showed impaired
repolarization of ventricles as indicated by the prolonged
QT, p = 0.002, QTc duration, p ≤ 0.05), T wave abnor-
malities (prolonged T peak-T end, p ≤ 0.05) and T wave
amplitude (p ≤ 0.05) as compared with the control group.
Table 2 Primary antibodies
used for immunohistochemistry Antibody Caspase-3 TNF-α
Source Diagnostic BioSystems, CA, USA Elabscience, TX, USA
Catalog # PDR172 E-AB-22159
Host/type Rabbit, polyclonal Mouse, monoclonal
Optimal dilution Ready to use 1:200
Cellular localization Cytoplasmic, some nuclear Cytoplasmic
Function Detect apoptosis Detect inflammation
and macrophage
activity
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1130 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
Ventricular repolarization was significantly improved after
DAPA treatment, as indicated by the significant difference
in QT, QTc, and T peak-T end. T wave amplitude (p ≤ 0.05)
from the hyperthyroidism group and the non-significant dif-
ference from the control groups (p = 0,7. 0.1, 0.7, and 0.5,
respectively) (Table3 and Fig.4).
Effect ofDAPA onserum biochemical markers
As shown in Table4, the serum samples of the hyperthyroid
(T4) group showed significantly higher concentrations of T3,
T4, LDH, and CK-MB and significantly lower concentra-
tion of TSH (p ≤ 0.05) compared to the control and DAPA
groups. On the other hand, DAPA treatment in hyperthyroid
rats substantially returned all serum biomarkers to almost
their average levels (p ≤ 0.05) compared to the T4 group. Of
note, no significant change was noticed between the control
and DAPA groups in all measured parameters.
Effect ofDAPA onoxidant/antioxidant biomarker
findings
The concentration of MDA in both organs significantly
increased in the T4 group (p ≤ 0.05), opposite the control
and DAPA groups. However, MDA concentration in both
organs was reduced to the average value after DAPA treat-
ment (Fig.5A, B). The catalase enzyme activity in heart
tissue showed a non-significant difference between all study
groups (p ≤ 0.05). On the other hand, catalase activity in
lung tissue significantly raised in the T4 group compared to
the control and DAPA groups (p ≤ 0.05). Coadministration
of DAPA to the hyperthyroid rats resulted in a significant
decline in lung catalase activity (p ≤ 0.05) compared to the
T4 group (Fig.5C, D). Reduced glutathione concentra-
tion significantly decreased in the T4 group of both organs
(p ≤ 0.05) compared to control and DAPA groups. After
DAPA administration, GSH concentration substantially
Fig. 2 A binary image demonstrating the morphometric measurement of the area parameters (area, area fill, area fraction, and area percent) to
detect the red-stained fibrous strand in the heart tissue (Picrosirius red, × 200)
Fig. 3 Weights in grams (g) in interval weeks of different groups.
Values are mean ± SE, n = 6. *, #, and $ are significant variances from
control, DAPA, and T4 groups in order at p ≤ 0.05
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1131Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
Table 3 The mean ± SE of the
measured ECG parameters
Untreated hyperthyroidism produced significant tachycardia, decreased PR interval, increased JT duration,
T amplitude and duration, and increased QTc duration, as compared to the control group. No significant
changes in the other measured ECG parameters. *, #, and $ are significant variances from control, DAPA,
and T4 groups in order at p ≤ 0.05
Groups Control DAPA T4 T4 + DAPA
HR (bpm) 260.2 ± 10.71 267.4 ± 8.57 383.4 ± 10.03*#329.4 ± 9.06*#$
P amplitude (mv) 0.05 ± 0.01 0.04 ± 0.006 0.08 ± 0.008#0.073 ± 0.01
P duration (ms) 25.16 ± 2.78 21.49 ± 2.51 19.59 ± 0.5 20.14 ± 0.38
PR interval (ms) 40.81 ± 1.23 41.57 ± 1.79 35.12 ± 0.7#39.50 ± 1.75
R amplitude (mv) 0.47 ± 0.033 0.45 ± 0.021 0.44 ± 0.039 0.46 ± 0.028
JT interval (ms) 30.87 ± 2.84 31.45 ± 0.36 50.44 ± 2.12*#34.37 ± 3.38$
T peak T end (ms) 16.3 ± 1.24 15.52 ± 0.75 29.01 ± 0.42*#18.996 ± 3.22$
T amplitude (mv) 0.03 ± 0.004 0.03 ± 0.001 0.082 ± 0.002*#0.05 ± 0.01$
QT (ms) 55.37 ± 3.94 55.03 ± 0.77 73.31 ± 1.59*#59.35 ± 3.60$
QTc (ms) 115.54 ± 8.45 116.16 ± 3.31 188.85 ± 7.33*#139.20 ± 9.93$
Fig. 4 The ECG findings in the four studied groups, hyperthyroid-
ism (T4) group showing significant increase in heart rate, shortened
PR interval, with significant prolongation of QTc and JT intervals, as
well as increased amplitude and duration of T wave. These changes
are not detected in the recordings of hyperthyroid animals treated
with DAPA
Table 4 Blood biochemical
markers in different groups
TSH, thyroid stimulating hormone; T3, threonine; T4, thyroxine; LDH, lactate dehydrogenase; CK, cre-
atine kinase. Values are mean ± SE, n = 6. *, #, and $ are significant variances from control, DAPA, and T4
groups in order at p ≤ 0.05
Parameter Control DAPA T4 T4 + DAPA
TSH (U/L) 1.16 ± 0.08 1.2 ± 0.05 0.13 ± 0.03*#1 ± 0.05$
T3 (U/L) 0.43 ± 0.08 0.83 ± 0.06 210.6 ± 20.3*#13.3 ± 1.45$
T4 (U/L) 24.6 ± 1.2 24.5 ± 0.8 150.1 ± 3*#33.4 ± 1.8*#$
LDH (U/L) 1.7 ± 0.08 1.7 ± 0.05 15.9 ± 2*#4.3 ± 0.3$
CK-MB (U/L) 0.8 ± 0.08 0.8 ± 0.08 23 ± 0.5*#2.3 ± 0.08*#$
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1132 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
increased (p ≤ 0.05) compared to T4 group in both organs
(Fig.5E, F).
Effect ofDAPA onDNA fragmentation percentages
inhyperthyroid rats
The percentage of DNA fragmentation was consider-
ably elevated (p ≤ 0.05) in the T4-treated group in both
organs compared to the control and DAPA groups. At the
same time, the percentage of DNA fragmentation of both
organs returned to average value after DAPA administra-
tion with a significant reduction (p ≤ 0.05) compared to
T4 group (Fig.6A, B).
Effect ofDAPA ontotal RNA gene expression
inhyperthyroid rats
mtTFA gene expression was significantly regressed
(p ≤ 0.05) in heart and lung tissues in the hyperthyroid group
compared to the control and DAPA groups. After oral DAPA
administration, mtTFA expression significantly elevated
(p ≤ 0.05) to 1.6 and 8.5 folds in heart and lung opposite
the control and DAPA groups, respectively (Fig.7A, B).
The expression of the TNF-α gene significantly increased
(p ≤ 0.05) in the T4-treated groups with significant decre-
ment after medication to restore the average value in both
organs (Fig.7C, D). Gene expression of IGF-1 significantly
Fig. 5 Oxidant/antioxidant biomarkers in different groups. A Malon-
dialdehyde (MDA) mM/mg protein content in heart. B Malondial-
dehyde (MDA) mM/mg protein content in lung. C Catalase activity
U/L in heart. D Catalase activity U/L in lung. E Reduced glutathione
(GSH) mM/mg protein content in heart. F Reduced glutathione
(GSH) mM/mg protein content in lung. Values are mean ± SE, n = 6.
*, #, and $ are significant variances from control, DAPA, and T4
groups in order at p ≤ 0.05
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1133Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
Fig. 6 DNA fragmentation % in all groups. A Heart DNA fragmentation percentage. B Lung DNA fragmentation percentage. Values are
mean ± SE, n = 6. *, #, and $ are significant variances from control, DAPA, and T4 groups in order at p ≤ 0.05
Fig. 7 Quantitative RT-PCR of mtTFA, TNF-α, and IGF-1 gene
expression in different groups. A Evaluation of mtTFA gene expres-
sion in the heart. B Evaluation of mtTFA gene expression in the lung.
C Evaluation of TNF-α gene expression in the heart. D Evaluation
of TNF-α gene expression in the lung. E Evaluation of IGF-1 gene
expression in the heart. F Evaluation of IGF-1 gene expression in the
lung. Values are mean ± SE, n = 6. *, #, and $ are significant variances
from control, DAPA, and T4 groups in order at p ≤ 0.05
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1134 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
increased (p ≤ 0.05) in T4-treated groups with significant
decrement after medication to retrieve to normal value in
both organs (Fig.7E, F).
Effect ofDAPA onthemyocardial histoarchitecture
inhyperthyroid rats
H&E-stained sections from the left ventricle of the control
and DAPA groups revealed a normal myocardial archi-
tecture. The branching striated cardiomyocytes appeared
with eosinophilic sarcoplasm and central vesicular nuclei.
Endomysial fibroblasts and narrow caliber capillaries
intervened between the myocardial fibers (Fig.8A, B).
Biopsies from the hyperthyroid group showed disturbed
myocardial architecture, heavy inflammatory infiltrates,
and marked myocardial vascular congestion (Fig.8C, D).
The morphometric measurement of the myocardial fiber
diameter in the hyperthyroid group showed a significant
rise at p ≤ 0.05 compared to the control and DAPA groups.
The treatment with DAPA after the induction of hyperthy-
roidism markedly alleviated the myocardial histopatho-
logical signs with mild cellular infiltrates, less vascular
congestion (Fig.8E, F), and a substantial reduction of the
myocardial diameter at p ≤ 0.05 compared to the hyper-
thyroid group.
Effect ofDAPA onthemyocardial collagen,
caspase‑3, andTNF‑α expression inhyperthyroid
rats
Microscopic examination of picrosirius red and immu-
nostained sections from the left ventricle of the control and
DAPA groups showed delicate red collagen fibers (Fig.9A,
D, respectively) with a minimal cytoplasmic expression of
caspase-3 (Fig.9B, E, respectively) and TNF-α (Fig.9C, F,
respectively). The hyperthyroid group revealed thick, wavy
collagen bundles deposited among the myocardial fibers
(Fig.9G) and intense positive cytoplasmic expression of
caspase-3 (Fig.9H) and TNF-α (Fig.9I), which showed a
considerable increase (p ≤ 0.05) opposite the control and
DAPA groups. Treatment with DAPA resulted in a signifi-
cant regression (p ≤ 0.05) in the amount of deposited col-
lagen (Fig.9J), caspase-3 (Fig.9K), and TNF-α expression
(Fig.9L) compared to the T4 group, yet with a considerable
difference opposite the control and DAPA group.
Fig. 8 H&E-stained sections from the left ventricle of different
research groups. A, B Control and DAPA groups, respectively, show-
ing typical myocardial architecture, branching striated cardiomyo-
cytes with eosinophilic sarcoplasm (arrows), and central vesicular
nuclei (N). Endomysial fibroblasts (F) and narrow caliber capillaries
(B.V) intervene between the myocardial fibers. C, D T4 group show-
ing disturbed myocardial architecture (arrow), inflammatory cel-
lular infiltrates (circle), and marked vascular congestion (B.V). E, F
T4 + DAPA group showing markedly alleviated histopathological
signs with mild cellular infiltrates (circle) and less vascular conges-
tion (B.V). G Statistical analysis of the myocardial fibers’ thickness
in different groups. Values are mean ± SE, n = 6. *, #, and $ are sig-
nificant variances from control, DAPA, and T4 groups in order at
p ≤ 0.05 (magnification: A, B × 200; C–F × 400)
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1135Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
Effect ofDAPA onthelung histoarchitecture
inhyperthyroid rats
H&E-stained sections of the lung tissue from the control and
DAPA groups were identical, showing a typical histologic
appearance of lung parenchyma with many air-filled alveoli
and alveolar sacs. The interalveolar septa were thin and
contained small, thin-walled pulmonary capillaries. Some
patent bronchioles were spotted with folded mucosal lining
(Fig.10A, B, respectively). On the other hand, administer-
ing LT4 to induce hyperthyroidism led to remarkable histo-
pathologic changes in the hyperthyroid group in the form of
noticeably thickened interalveolar septa with striking inflam-
matory infiltrates and areas of necrosis. The pulmonary ves-
sels showed significant congestion. The bronchioles were
compressed to partial occlusion at some points (Fig.10C,
D). After using DAPA as a treatment for the hyperthyroid
rats, the lung histoarchitecture acquired a healthier appear-
ance with less vascular engorgement and fewer inflammatory
infiltrates. The interalveolar septa were still thickened
at some points, resulting in compressed alveolar spaces
(Fig.10E, F). Briefly, the subsequent administration of
DAPA after the experimental induction of hyperthyroidism
had a tremendous impact on both heart and lung tissues. It
restored—at least partially—the normal histologic pattern,
which reflects the potential ameliorative effect of DAPA on
hyperthyroid-induced cardiopulmonary injury.
Effect ofDAPA onthelung tissue collagen,
caspase‑3, andTNF‑α expression inhyperthyroid
rats
Picrosirius red and immunostained sections of the lung
parenchyma from the control and DAPA groups showed
thin red collagen fibers in the parenchyma, the adventitia
of blood vessels, and the bronchioles (Fig.11A, D, respec-
tively), in addition to a weak cytoplasmic expression of
caspase-3 (Fig.11B, E, respectively) and TNF-α (Fig.11C,
Fig. 9 Picrosirius red (A, D, G, and J) and immunostained sections
with anti-caspase-3 antibodies (B, E, H, and K) and anti-TNF-α anti-
bodies (C, F, I, and L) from the left ventricle of different research
groups. A, D Control and DAPA groups, respectively, showing del-
icate red collagen fibers (arrows). G T4 group showing thick wavy
collagen bundles deposited among the myocardial fibers (arrows).
J T4 + DAPA group showing less collagen bundles (arrows). B, E
Control and DAPA groups, respectively, showing minimal caspase-3
cytoplasmic expression. (C, F) control and DAPA groups, respec-
tively, showing slight cytoplasmic TNF-α expression. H, I T4 group
showing intense positive cytoplasmic caspase-3 and TNF-α expres-
sion, respectively (arrows). K, L T4 + DAPA group showing less
cytoplasmic caspase-3 and TNF-α immunostaining, respectively
(arrows) (magnification × 200). M–O Statistical analysis of the mor-
phometric measurements of collagen, caspase-3, and TNF-α area%,
respectively. Values are mean ± SE, n = 6. *, #, and $ are significant
variances from control, DAPA, and T4 groups in order at p ≤ 0.05
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1136 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
F, respectively). The hyperthyroid rats showed a significant
increase (p ≤ 0.05) in collagen bundles deposition in lung
parenchyma (Fig.11G) besides a strong positive cytoplasmic
expression of caspase-3 (Fig.11H) and TNF-α (Fig.11I)
compared to the control and DAPA groups. The positive
effect of administering DAPA to the hyperthyroid rats was
proved by the significant decrement (p ≤ 0.05) in the amount
of deposited collagen (Fig.11J), caspase-3 (Fig.11K), and
TNF-α expression (Fig.11L) compared to the T4 group;
nevertheless, a considerable difference opposite the control
and DAPA group is still noticed.
Discussion
The hyperthyroidism-induced cardiopulmonary injury was
biochemically confirmed in the current study by decreased
serum TSH and elevated serum T3, T4, LDH, and CK-MB.
Hyperthyroidism induced a significant increase in heart rate
and shortened PR interval, with considerable prolongation
of QTc and JT intervals, as well as increased amplitude and
duration of T wave. In addition, there was a change in the
cardiopulmonary redox status, indicating oxidant/antioxi-
dant imbalance in both organs. The analysis of genotoxicity
showed that the hyperthyroid rat model’s heart and lungs had
a higher percentage of DNA fragmentation than those of the
euthyroid animals. Furthermore, compared to euthyroid rats,
hyperthyroid rats exhibited a significant rise in the expres-
sion of the TNF-α and IGF-1 genes, along with a regres-
sion in the expression of the mtTFA gene. Morphologically,
hyperthyroidism caused histopathological alterations in the
heart and lung tissues in the form of inflammatory infil-
trates, areas of necrosis, fibrosis, and vascular congestion.
In addition, the morphometric analysis showed a significant
increase in the myocardial fiber diameter along with a highly
positive cytoplasmic expression of TNF-α and caspase-3 in
both organs compared to the control and DAPA groups.
Coadministration of DAPA with LT4 effectively restored
all serum biomarkers to nearly average levels, improved
ECG signs, and reinstated the redox balance. Also, DAPA
could improve DNA fragmentation and the expression of
mtTFA, TNF-α, and IGF-1 genes in both organs of treated
Fig. 10 H&E-stained sections
from the lung tissue of different
research groups. A, B Control
and DAPA groups, respectively,
showing many air-filled alveoli
and alveolar sacs (a), thin inter-
alveolar septa (s) containing
small thin-walled pulmonary
capillaries (c), and patent bron-
chioles with folded mucosal lin-
ing (B). C, D T4 group showing
thickened interalveolar septa (s),
striking inflammatory infiltrates
(circle) with areas of necrosis
(N), markedly congested pulmo-
nary vessels (B.V), and partially
occluded bronchioles (arrow).
E, F T4 + DAPA group show-
ing less vascular engorgement
(B.V), milder inflammatory
infiltrates (circle), and thickened
interalveolar septa (s) with com-
pressed alveolar spaces (arrows)
(magnification × 200)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1137Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
animals. Furthermore, DAPA markedly improved the car-
diopulmonary histological alterations, decreased collagen
deposition, and reduced the tissue immunoexpression of
TNF-α and caspase-3.
LT4 was used in the up-to-date study to establish the rat
model with hyperthyroidism. It has been reported that LT4
stimulates thyroid function mainly by preventing the thyroid
gland from oxidatively iodinating thyroid hormones [83].
The achievement of inducing hyperthyroidism was dem-
onstrated in our study by a notable rise in serum levels of
T3 and T4, as well as a decrease in TSH levels in T4 group;
these findings were in line with those of Gluvic and col-
leagues [24].
DAPA administration to the hyperthyroidism-mod-
eled rats in this study effectively declined T3 and T4 and
increased serum TSH levels. This effect could be explained
by the reduction of sympathetic activity that SGLT2-I pro-
vides. Because SGLT2 is involved in sympathetic nervous
system activation, inhibiting it can lower renal afferent nerv-
ous activity and suppress central reflex mechanisms that lead
to widespread sympathetic activation [61]. Sympathetic
nervous system release of norepinephrine from intrathyroi-
dal adrenergic nerve terminals can directly stimulate thyroid
hormone secretion. Additionally, it seems that the periph-
eral metabolism of thyroid hormone and the sympathetic
nervous system are related [70]. The former hypothesis was
supported by decreased TSH and increased thyroid hormone
levels in smokers because of increased sympathetic nervous
activity [8].
In coincidence with former studies, the hyperthyroid rat
model given LT4 in our study experienced a weight loss
that began after the first week of hyperthyroidism induc-
tion and reached significant values by the third week of the
experiment [38]. Compared to the T4 group, the concur-
rent SGLT2-I treatment of the hyperthyroid rats consider-
ably reduced their weight loss. Theoretically, after receiving
Fig. 11 Picrosirius red (A, D, G, and J), immunostained sections
with anti-caspase-3 (B, E, H, and K), and anti-TNF-α (C, F, I, and
L) antibodies from the lung tissue of different research groups. A, D
Control and DAPA groups, respectively, showing fine red collagen
fibers in the parenchyma, adventitia of blood vessels, and bronchi-
oles (arrows). G T4 group showing thick collagen bundles (arrows).
J T4 + DAPA group showing less collagen bundles (arrow). B, E
Control and DAPA groups, respectively, showing minimal caspase-3
cytoplasmic expression. C, F Control and DAPA groups, respectively,
showing weak cytoplasmic TNF-α expression. H, I T4 group show-
ing strong positive cytoplasmic caspase-3 and TNF-α expression,
respectively (arrows). K, L T4 + DAPA group showing less cytoplas-
mic caspase-3 and TNF-α reactions, respectively (arrows) (magnifica-
tion × 200). M–O Statistical analysis of the morphometric measure-
ments of collagen, caspase-3, and TNF-α area%, respectively. Values
are mean ± SE, n = 6. *, #, and $ are significant variances from con-
trol, DAPA, and T4 groups in order at p ≤ 0.05
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1138 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
targeted therapy to restore normal hormone levels, patients
with thyroid dysfunction should regain their pre-disorder
body weight. This is not always the case, though, as multiple
studies have demonstrated [31, 43, 57].
The results of ECG recording in the present study showed
significant tachycardia with PR shortening in hyperthyroid-
ism (T4) group. Additionally, signs of impaired repolariza-
tion were indicated by prolonged JT, QTc, and both T wave
amplitude and duration. Opposingly, the T4 + DAPA group
did not experience these ECG changes. The ECG altera-
tions linked to hyperthyroidism can be attributed to several
pathogenic mechanisms. Increased metabolic rate and sym-
pathetic activity in myocardial cells led to tachycardia and
a shorter PR interval [4]. Prolonged JT and QTc intervals
were signs of impairment in the repolarization phase of the
cardiac action potential [7]. proved that the oxidative stress
and abnormal calcium handling by cardiomyocytes result-
ing from elevated thyroid hormone level were linked to the
alteration in the repolarization phase of the cardiac action
potential, which was indicated by prolonged JT, QTc inter-
vals, as well as T wave changes. The main reason for the
improvement in these results with DAPA treatment is the
significant drop in T4 level that was seen in these animals.
DAPA dramatically reduced the oxidative and apoptotic
effects of thyroid excess at the cellular levels [65]. Accord-
ing to [82], the cardioprotective impact of SGLT2-inhibitors
was achieved via the down regulation of sodium-hydrogen
antiporter 1, which in turn decreased both intracellular
sodium and calcium overloads.
Furthermore, it has been demonstrated that SGLT2
inhibitors affect sympathetic load and lower the risk of heart
failure [62]. Remarkably, it was discovered that in a num-
ber of pathophysiological stressors, such as heart failure or
post-myocardial infarction, cardiomyocytes upregulate the
expression of SGLT associated with increased sympathetic
activity [14]. The upregulated expression of SGLT in these
conditions led to intracellular sodium accumulation and
downstream myocardial dysfunction.
Several investigations that attempted to prevent the patho-
physiological cascades leading to SGLT overactivity con-
firmed the causal relationship between SGLT overexpression
and cardiomyocyte dysfunction in heart failure. Myocyte
morphology and function were preserved as a result of
reversing this effect, which was investigated both directly by
restricting SGLT activity and indirectly by targeting AMPK
activity of cardiomyocytes [63]. The findings of the present
study can be explained by these intracellular changes and
the potential protection provided by the SGLT inhibitors.
Oxidative stress is considered one of the pathophysiologi-
cal mechanisms underlying thyrotoxicity. Hypermetabolic
state results in accumulation of free radicals both on the
systemic and the cellular levels [13]. According to [32],
oxidative stress increased the generation of reactive oxygen
species (ROS) in diabetes-induced cardiomyopathy, which
resulted in cardiac apoptosis.
Our results revealed a substantial rise in MDA level in
the heart and lung tissues of hyperthyroid rats opposite the
controls, a significant decline in the GSH profile in both
organs, and an increase in the CAT levels in the lungs alone.
This finding was in line with former studies which reported
a significant rise of CAT, SOD, and MDA levels in the liver,
kidney, brain, and myocardial tissues of hyperthyroid rats
compared to the controls [49, 60]. On the contrary, [45]
observed a marked decline in myocardial tissue catalase and
SOD in rat model of thyrotoxicosis. According to Costilla
etal., a hyperthyroid condition raises ROS, which triggers
the transcription of antioxidant enzymes. That could indicate
that the oxidative stress is not adequately compensated for
[17]. Lipid peroxidation is induced in various rat tissues,
including the heart, by LT4-induced hyperthyroidism [47].
Polyunsaturated fatty acid peroxidation results in the pro-
duction of MDA. It can bind to RNA and DNA, damaging
cells [36]. Significant oxidative reactions occur when ROS
interacts with deoxyribose and nitrogenous bases in DNA.
Mutations, apoptosis, necrosis, carcinogenesis, and heredi-
tary illnesses can result from this. Nucleosomes, which are
essential for the organization of DNA within chromosomes,
rupture, leading to DNA fragmentation. This causes issues
with the compaction and coiling of DNA within chromatin.
The control of gene transcription is significantly influenced
by chromatin [11]. The study’s hyperthyroid animals also
displayed higher percentages of DNA fragmentation in
their lung and cardiac tissues, confirming the production
of hydroxyl radicals, the main species that damage DNA
more severely [33]. Also, DNA damage is documented in
our study by the downregulation of the mtTFA gene. Nuclear
genes encode the mitochondrial protein known as mtFTA,
which is moved from the cytoplasm to the mitochondria.
Mitochondrial DNA must be maintained, expressed, and
delivered. mtTFA also controls mitochondrial DNA tran-
scription, stability, repair, and replication [69]. Oxygen
radical stress oxidatively damages purine and pyrimidine
bases and directly damages DNA, primarily through strand
excision. This process can produce a variety of products and
begins with the radical-induced abstraction of a proton from
any position on the deoxyribose [55]. Overexposure to ROS
decreased the potential of the mitochondrial membrane,
which resulted in immunotoxicity and apoptotic cell death
due to an imbalance in immune redox [9]. This is supported
in the present study by the immunohistochemical results,
which showed elevated levels of apoptotic protein caspase-3
in hyperthyroid rats’ heart and lung tissues. Similar results
were observed in previous work [60].
Our study showed a significantly increased cardio-
pulmonary gene expression of IGF-1 in the hyperthyroid
group, while a significant decline was noticed after DAPA
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1139Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
treatment. The previous finding was in line with previous
studies which reported elevated IGF-1 levels with goiter
in males who have thyroid nodules, in females who have
low TSH levels, and in patients with primary and central
hypothyroidism after T4 replacement [64, 77]. It has been
previously mentioned that hyperactivation of insulin/IGF-1
signaling in diabetic patients favors cell survival via inhibit-
ing P1-3K and SIRT1, which provide a downstream activa-
tion of the p53-p21 signaling pathway. In this regard, SGLT2
inhibitors might prevent cells’ exposure to oxidative stress
and promote cellular repair via up-regulating energy dep-
rivation sensors such as AMPK and SIRT1, as well as via
inhibition of insulin/IGF-1 signaling [28].
In the current work, treatment with dapagliflozin exerted
a cardiopulmonary antioxidant effect via ameliorating oxida-
tive stress and boosting the antioxidant capacity in heart and
lung tissues. Dapagliflozin exerted its antioxidant properties
by reducing oxidative stress and boosting cardiomyocytes’
antioxidant potential [50]. There is minimal evidence of
SGLT2 expression, while the cardiomyocytes express the
SGLT1 isoform [29]. Therefore, dapagliflozin’s antioxidant
effect was attributed by Xing and colleagues using invitro
experiments, to its direct inhibition of ROS production [81].
In the present work, hyperthyroidism-induced cardio-
pulmonary injury was evidenced biochemically by elevated
serum levels of LDH and CK-MB. In many diseases, changes
in isoenzymes and plasma or serum enzymes can serve as
helpful markers of tissue damage. Increases in enzymes are
typically associated with their leakage from injured cells.
Elevated LDH level is a nonspecific finding and is observed
in various hematological and neoplastic disorders, as well
as in diseases of the heart, liver, lungs, skeletal muscles, and
kidneys [39]. Elevated levels of LDH and CK-MB are posi-
tively correlated with ischemic myocardial injury in clini-
cal practice. Both markers are released into the bloodstream
during myocardial injury [16].
Furthermore, the hyperthyroidism-related myocardial
injury was proven histologically in our study by disturbed
histoarchitecture, increased collagen deposition, severe
inflammation, vascular congestion, and increased diameter
of the myocardial fibers. These results were the same as
those of [35, 60]. On the pulmonary side, and in line with
[30, 78], hyperthyroidism led to remarkable histopathologic
changes in the lung tissues in the form of thickened inter-
alveolar septa with striking inflammatory infiltrates, areas
of necrosis, congested pulmonary vessels, and compressed
bronchioles that were partially blocked at some points.
Interestingly, a previous study reported the same pul-
monary histopathological changes in a rat model of hypo-
thyroidism [26], which could be attributed to the evoked
proinflammatory markers (mainly TNF-α and IL-6) and
the deranged redox status accompanying the disturbed
euthyroid state.
Moreover, cardiac hypertrophy development is influenced
by hyperthyroidism. Two important pathways in cardiac
hypertrophy are thought to be endoplasmic reticulum stress
and transient receptor potential canonical channels [10]. Fur-
thermore, β-adrenergic receptors, phospholamban, myosin
heavy chains, and other proteins in the cardiac myocyte are
all regulated by thyroid hormones, which can cause car-
diac hypertrophy [76]. The reversibility of cardiomyopathy
resulting from hyperthyroidism is contingent upon the tim-
ing and the proper management of the hyperthyroid condi-
tion. Patients with hyperthyroidism may, therefore, benefit
from the administration of cardioprotective agents [51]. In
our study, the substantial reduction in CK-MB and LDH
levels, as well as the attenuation of cardiopulmonary histo-
pathological changes, demonstrated the protective action of
SGLT2-I. Empagliflozin has been shown by Andreadou and
colleagues [5] to have a cardioprotective potential against
myocardial ischemia injury in animals given a Western diet.
Furthermore, in type 2 diabetic mice, DAPA decreased car-
diomyopathy [72].
Increased tissue and plasma concentrations of inflamma-
tory cytokines are linked to inflammation [21]. IL-6, IL-8,
and TNF-α are among the cytokines that are elevated in
hyperthyroidism; this increment might be the consequence
of the long-term effects of elevated thyroid hormones [22].
Earlier research indicated that LT4-induced hyperthyroid-
ism was linked to an elevation in TNF-α [44, 60], which
aligns with our immunohistochemical results that showed
a robust positive cytoplasmic expression of TNF-α in the
hyperthyroid rats compared to the euthyroid controls. A rise
in inflammatory cytokines like TNF-α brought on by NF-кB
activation resulted in cardiac complications [79]. SGLT2-I
coadministration to hyperthyroid rats significantly decreased
TNF-α levels. Per our results, Eldesoqui etal. reported that
TNF-α-antagonism was linked to a reduction in myocardial
inflammation and fibrosis and that it improved diabetic car-
diomyopathy that was experimentally induced [18].
Conclusion
Our present study demonstrated that the deranged oxidant/
antioxidant balance accompanied by the evoked proinflam-
matory and proapoptotic markers is the chief cause of hyper-
thyroidism-induced cardiopulmonary injury. Combining
DAPA with LT4 could effectively regulate inflammation and
apoptosis by downregulating TNF-α and caspase-3, combat-
ing genotoxicity, and reinstating the redox balance in heart
and lung tissues. Further, DAPA exhibited a sympatholytic
effect, documented by the improved hyperthyroid-related
ECG changes, reflecting better cardiac function. Hence, our
study introduces DAPA as a putative therapeutic candidate
with cardiopulmonary protective potential in patients with
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1140 Pflügers Archiv - European Journal of Physiology (2024) 476:1125–1143
hyperthyroidism. Further clinical and deep molecular studies
are required before transposing to humans.
Limitations ofthestudy
Our study could be improved by further molecular studies
encompassing the effect of DAPA on autophagy in myocar-
dial cells and pulmonary tissue and electron microscopic
studies to locate the cellular ultrastructural targets that open
the door for future research. In addition, we suggest per-
forming serial ECG recordings throughout the experiment
to track early changes in cardiac function by the effect of
levothyroxine and the putative protective role of DAPA.
Author contribution All authors contributed to the study conception
and design. Material preparation, data collection, and analysis were
performed by Nermeen Bastawy, Amy F. Boushra, Aliaa El-Sayed El-
Mosallamy, Samira H. Aljuaydi, Huda O. AbuBakr, Rabab Ahmed
Rasheed, and Shereen E. Abdelaal. The first draft of the manuscript
was written by A.S. Sadek, R.T. Khattab, and Wael Botros Abualyamin,
and all authors commented on previous versions of the manuscript. All
authors read and approved the final manuscript.
Funding Open access funding provided by The Science, Technology &
Innovation Funding Authority (STDF) in cooperation with The Egyp-
tian Knowledge Bank (EKB).
Data availability No datasets were generated or analyzed during the
current study.
Declarations
Ethical approval This study was conducted per the NIH Guidelines
for the Care and Use of Laboratory animals strictly adherent to the
ARRIVE guidelines and accepted by The Institutional Animal Care
and Use Committee, Cairo University (CU-IACUC), Egypt (Code: CU/
III/F/15/23).
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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Authors and Aliations
NermeenBastawy1 · AliaaE.M.K.El‑Mosallamy2 · SamiraH.Aljuaydi3 · HudaO.AbuBakr3,10 ·
RababAhmedRasheed4 · A.S.Sadek5,6 · R.T.Khattab5 · WaelBotrosAbualyamin7,8 ·
ShereenE.Abdelaal9 · AmyF.Boushra7
* Rabab Ahmed Rasheed
rabab.rasheed@ksiu.edu.eg
Nermeen Bastawy
nortymdht@cu.edu.eg
Aliaa E. M. K. El-Mosallamy
aliaamoneer@hotmail.com
Samira H. Aljuaydi
Samira_2008@cu.edu.eg
Huda O. AbuBakr
huda.omar@cu.edu.eg
A. S. Sadek
dr_ahmedsamir@med.asu.edu.eg
R. T. Khattab
dr_rehabkhattab@med.asu.edu.eg
Wael Botros Abualyamin
amyaadel@yahoo.com
Shereen E. Abdelaal
Ss.abdel-aal@nrc.sci.eg
Amy F. Boushra
wba00@fayoum.edu.eg
1 Department ofMedical Physiology, Faculty ofMedicine,
Cairo University, Cairo, Egypt
2 Department ofPharmacology, Medical Research andClinical
Studies Institute, National Research Centre, Giza, Egypt
3 Department ofBiochemistry andMolecular Biology, Faculty
ofVeterinary Medicine, Cairo University, Giza12211, Egypt
4 Department ofMedical Histology andCell Biology,
Faculty ofMedicine, King Salman International University,
ElTor46511, SouthSinai, Egypt
5 Department ofAnatomy andEmbryology, Faculty
ofMedicine, Ain Shams University, Cairo11566, Egypt
6 Department ofAnatomy andEmbryology, Faculty
ofMedicine, King Salman International University,
ElTor46511, SouthSinai, Egypt
7 Department ofMedical Physiology, Faculty ofMedicine,
Fayoum University, Fayoum, Egypt
8 Department ofNatural andPhysical Sciences, Blinn College,
Brenham, TX, USA
9 Department ofPathology, Medical Research andClinical
Studies Institute, National Research Centre, Giza, Egypt
10 Department ofBiochemistry, Faculty ofVeterinary
Medicine, Egyptian Chinese University, Cairo, Egypt
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1.
2.
3.
4.
5.
6.
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