Electrocardiographic and histopathological characterizations of diabetic cardiomyopathy in rats

Diabetes is a clinical condition that is associated with insulin deficiency and hyperglycemia. Cardiomyopathy, retinopathy, neuropathy, and nephropathy are well known complications of the elevated blood glucose. Diabetic cardiomyopathy is a clinical disorder that is associated with systolic and diastolic dysfunction along with cardiac fibrosis, inflammation, and elevated oxidative stress. In this study, diabetes was induced by intraperitoneal injection of streptozotocin (STZ) 50 mg/kg. We determined the plasma levels of cardiac troponin-T (cTnT) and creatinine kinase MB (CK-MB) by ELISA. Diabetic rats showed abnormal cardiac architecture and increased collagen production. Significant elevation in ST-segment, prolonged QRS, and QT-intervals and increased ventricular rate were detected. Additionally, diabetic rats showed a prolongation in P wave duration and atrial tachyarrhythmia was observed. Plasma levels of cTnT and CK-MB were elevated. In conclusion, these electrocardiographic changes (elevated ST-segment, prolonged QT interval, and QRS complex, and increased heart rate) along with histopathological changes and increased collagen formation could be markers for the development of diabetic cardiomyopathy in rats.


Introduction
The defect in insulin formation and release leads to diabetes mellitus, which is characterized by an elevation in blood glucose levels. Several pathogenetic processes set behind the progressive damage of beta cells (Cerf 2013) and depletion in insulin secretion or manifestation of insulin resistance in diabetic patients. Prolonged hyperglycemia leads to macrovascular and microvascular damage leading to several complication, including retinopathy, nephropathy, neuropathy, and cardiomyopathy (Cole and Florez, 2020). Additionally, development of cardiovascular complications such as ischemic heart disease, peripheral vascular disorder, and stroke is related to microvascular complications (Panari, 2016). In diabetic cardiomyopathy, the progression of cardiac fibrosis would induce dysfunctions of the cardiac cycle that can progress to heart failure (Jia et al. 2018). Additional various metabolic disorders are accompanied with diabetic cardiomyopathy such as diminished nitric oxide levels, elevated oxidative stress, inflammation, and enhanced activity of renin-aldosterone angiotensin system (Tan et al. 2020). The occurrence of diabetic cardiomyopathy is linked to several pathophysiological pathways, including the formation of advanced glycation end-products (AGEs), disturbances in microRNA, O-linked-Nacetylglucosamine and exosome (O-GlcNAc) pathways (Ducheix et al. 2018), adenosine monophosphate (AMP)activated protein kinase (AMPK) (Sun et al. 2020), protein kinase C (PKC) (Yin et al. 2019), and nuclear factor kappa B (NF-κB) formation (Fan et al. 2018).
In diabetes, damage-associated molecular pattern molecules (DAMPs) formation triggers toll-like receptor 4 (TL4)/NF-κB signaling that promotes high yield of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) (El-Gizawy et al. 2020;Nasr et al. 2021;Saber 2018). These inflammatory cytokines derive the progress of cardiac dysfunction in diabetic heart. Moreover, the enhanced formation of NF-κB due to stimulation of TLR4 or excessive release of reactive oxygen species (ROS) could stimulate fibrotic changes and collagen formation through enhancing the activity of tissue growth factor β (TGF-β), which could lead to diabetic cardiomyopathy (Khalil et al. 2020;Saber 2018). This study is aimed to investigate the electrocardiographic and histopathological changes associated with diabetic cardiomyopathy in rats.

Animal manipulation
Male Wistar rats with an average weight (175-220 g) and 1 year age were supplied from the animal facility of the Faculty of Pharmacy, Delta University for Science and Technology, Egypt. Animal manipulation, feeding, and housing conditions were conducted according to . All experiments were performed following the approval of the Experimental Animal Ethics Committee of Suez Canal University No.: 201807PHDA1.

Chemicals and test kits
Streptozotocin (STZ), used in the induction of type-1 diabetes, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrahydrofuran (THF) was purchased from El-Gomhouria Company for Trading Chemicals and Medical Appliances (Cairo, Egypt) and were used for inhalation anesthesia. cTnT, CK-MB, TNF-α, and IL-6 were measured by ELISA using specific kits obtained from Cusabio Technology LLC (Houston, TX, USA). Primary monoclonal antibodies that were used for the immunostaining of NF-κB were bought from Affinity Biosciences Inc (Changzhou, China). The EnVision™ FLEX immunohistochemistry detection kit was purchased from Dako Inc. (Glostrup, Denmark).

Experimental approach
Freshly prepared STZ (50 mg/kg) was injected intraperitoneally for the induction of type I diabetes (Raj et al. 2012). Plasma glucose level was detected in the fourth day. Rats were considered diabetic if plasma glucose level is > 250 mg/ dL (n = 7). Additional 7 healthy normal rats served as corresponding control groups. The diabetic nor the normal groups received any treatment. Body weight was measured at the beginning of the experiment before STZ injection. It was measured every 4 days throughout the study period.

Electrocardiography monitoring
At the end of the experiment (45 days), electrocardiography (ECG) charts were recorded. Animal were anesthetized using THF inhalation and located in supine position after full anesthesia. Electrodes were fixed subcutaneously in the animal's paws and were connected to Kaden Yasen™ ECG-903 device (Zhuhai City, Guangdong, China). The Lead II ECG traces were recorded to analyze P wave anomalies, QT interval, QRS interval, ST-segment elevation, and ventricular rate.

Collection of plasma
At the end of the experiment, animals were anesthetized by THF inhalation and blood samples were collected form orbital sinus using a sterile capillary tube that were inserted into the eye inner canthus under light anesthesia. Blood samples were collected in EDTA precoated tubes then immediately centrifuged at 5000 rpm/12 min. The supernatant was separated in stored at − 80 °C.

Evaluation of heart weight changes
Animals were sacrificed, the heart were surgically isolated, and excess blood were removed and weighted. For the calculation of heart weight to body weight ratio, the following formula were applied: [heart weight (g)/body weight (g)].
Left and right ventricles were separated by the incision of the atrioventricular septum. The isolated ventricles were cut transversely into two portions. The first portion was immediately stored at − 80 °C and were used for the preparation of cardiac tissue homogenate. The second portion was fixed in 4% neutral buffered formalin solution, then embedded in paraffin for immunohistochemical and histopathological studies.

Histopathological examination
Paraffin-embedded section were cut in 4 µm slices and stained with hematoxylin and eosin (H&E). Sections were evaluated using a light microscope (Optika B-352A, BG, Italia) for the detection of histopathological anomalies at × 400 magnification . Additionally, ventricular dilatation and cardiac wall thickness were measured. The following histopathological scoring was applied: 0 (absence of histopathological anomalies); 1 (mild; cardiomyocytes degeneration with slight infiltration of inflammatory cells); 2 (moderate to extensive cardiomyocyte degeneration with diffuse inflammatory cells infiltration); and 3 (severe; necrotic tissue with massive inflammatory cells infiltration) (Joukar et al. 2010). The degree of cardiac tissue damage was determined by examining 20 randomly chosen fields for each group.

Determination of cTnT, CK-MB, IL-6, and TNF-α levels
Plasma levels of the tested markers were detected by ELISA technique following the manufacturer's protocol. Standard, control, and test samples were placed to ELISA plate precoated with monoclonal antibodies specific for the tested immunogens. The conjugates of both markers were added to each well then 1 h incubation at room temperature followed by adding the color substrate. After 30 min incubation, the enzymatic reactions yielded a blue color that was converted into yellow color after the addition of stop solution. Microplate reader was used for the measurement of optical density.

Statistical analysis
All data were analyzed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). Data were expressed as mean ± standard deviation (SD). Means of both groups were compared using unpaired t-test.

Body weight and heart weight to body weight ratio
Body weight of diabetic animals were reduced throughout the study period after injection with STZ ( Fig. 1a, b). In contrary, the body weight of normal untreated rats was continuously increasing. Additionally, the reported drop in the body weight was displayed in the calculated percentage of body weight change (P ≤ 0.0001). Furthermore, the ratio of heart weight to body weight was significantly (P ≤ 0.01) elevated in the diabetic group confirming the progression to cardiomegaly (Fig. 1c).

ECG recordings
The diabetic rats displayed significant (P ≤ 0.01) increase of ventricular rate compared to the healthy group (Fig. 2a,b). Moreover, additional ECG anomalies were detected such as increased QT interval (P ≤ 0.0001), increased corrected QT intervals (QTc) (P ≤ 0.0001), and elevation in QRS interval (P ≤ 0.0001) (Fig. 2a,c,d,e). Similarly, the duration of P wave of diabetic rats was significantly (P ≤ 0.01) prolonged ( Fig. 2a, g). Regarding the elevation of ST-segment elevation, a consistent increase in the ST-segment elevation was scored compared to (P ≤ 0.001) the normal group (Fig. 2a,f), and trial tachyarrhythmias was observed in diabetic rats (Fig. 2a).

Ventricular dilatation and ventricular wall thickness
The diabetic group showed a notable dilatation in the left ventricle compared to the normal group (P ≤ 0.0001) indicating the occurrence of diabetic cardiomyopathy (Fig. 3a,  b). Regarding the ventricular wall thickness, a significant (P ≤ 0.0001) decrease in the ventricular wall thickness was clearly observed in diabetic rats relative to control group (Fig. 3a,d). In contrary, the dilatation of the right ventricle in both groups remained unaffected (Fig. 3a,c).

Histopathological examination
Histopathological examination of left ventricles showed an abnormal architecture and disarray of cardiomyocytes (Fig. 4c,d) compared to normal rats (Fig. 4a, b). Additionally, enlarged cardiomyocytes with box-shaped nucleus were detected. The cardiac myofibers demonstrated an abundant helical appearance with myofibers arrangement around a fibrous central core (Fig. 4d). The cardiac myofibers were organized in deviant angles or perpendicular arrangement (Fig. 4c), while the myofibers' arrangement in normal healthy untreated rats was parallel pattern (Fig. 4a). The diabetic group demonstrated increased histopathological (P ≤ 0.01) scoring as an indication for these histopathological anomalies and increased infiltration of inflammatory cells (Fig. 4e). Examination of the cardiac vasculature showed an increase in the formation of perivascular fibrosis in diabetic rats (Fig. 4d) compared to the normal group (Fig. 4b).

Effect on cTnT, CK-MB, IL-6, and TNF-α
High readings of cTnT and CK-MB are indicators of cardiac damage; STZ-treated rats displayed a substantial elevation (P ≤ 0.0001) in both markers compared to untreated group (Fig. 5a,b). Similarly, the measurement of cardiac levels of IL-6 and TNF-α showed significant (P ≤ 0.0001) increase compared to the normal group (Fig. 5c,d).

Discussion
Diabetes is a major medical issue worldwide. Several complications are linked to diabetes, including nephropathy, retinopathy, neuropathy, and cardiomyopathy. In our study, induction diabetes was conducted by intraperitoneal injection of STZ. The STZ-induced diabetes is not mechanistically understood. However, since STZ is a chemical analogue of glucose, it is permeated in beta cells, inducing a Fig. 1 Changes in body weight and heart-to-body weight ratio. a Effect on body weight. b Effect on percentage change in body weight. c Effect on heart weight to body weight ratio. Data were analyzed using the unpaired t-test with Welch's correction and were expressed as mean ± SD. **** = P ≤ 0.0001, * = P ≤ 0.05 with respect to the normal group (n = 7) subsequent cytotoxic reaction that generates reactive nitrogen and oxygen species which by turn damage the beta cells that secret the insulin. Diabetes induction leads to loss of weight due to reduced lipogenesis and enhanced utilization of fats as an energy source (Beylot 1996;Garg et al. 2017).
Diabetic cardiomyopathy is usually connected with electrocardiographic, and histopathological abnormalities (Jia et al. 2016). The anatomical signs of diabetic cardiomyopathy are left ventricular dilatation that accompanied with to left ventricular dysfunction in the early diastolic filling with enhanced isovolumetric relaxation duration (Jia et al. 2016). Systolic dysfunction would be followed by symptomatic heart failure (Lee et al. 1997). The cardiac dysfunction is believed to be raised from the stiffness of cardiomyocytes, cardiac fibrosis, and cardiac hypertrophy (Lee et al. 1997). This dysfunction stimulates the sympathetic tone, which leads to an increased activity of sarcoplasmic reticulum by exaggerated stimulation of β-receptors and ryanodine receptors leading to an intensification of Ca ++ release and ventricular tachycardia (Assis et al. 2019). In Addition, cardiomyopathy can be developed due to inflammatory reactions or cardiac fibrosis (Krejci et al. 2016). The extreme liberation of Ca ++ ions from the sarcoplasmic reticulum in cardiomyocytes could cause irregular repolarization, which could explain the prolongation in QT and QTc intervals (Paavola et al. 2016). Development of diabetic cardiomyopathy will lead to the formation of left ventricular bundle block, which might result in dyssynchronous activation of the left ventricles. This dysfunction in ventricular conductivity might justify the prolongation of the QRS complex (Akgun et al. 2014). Cardiac dysfunction in the current model progressed to ischemic dilated cardiomyopathy. This could be explained by the observed atrial arrhythmias observed in our study and (Ducas and Ariyarajah, 2013). Furthermore, ischemic-dilated cardiomyopathy was ensured by an increase in ST-segment elevation. Diabetic cardiomyopathy is a cardiomyopathy that takes place in the absence of notable coronary artery disease (Jia et al. 2018). However, diabetic cardiomyopathy could be accompanied with coronary vascular abnormalities that may affect the coronary blood flow. Decreasing coronary supply and myocardial perfusion will result in impairing the ventricular function which leads to subsequent clinical outcomes (Sandesara et al. 2018). Several structural and functional abnormalities in coronary blood vessels may occur in diabetics, luminal obstruction, infiltration of inflammatory cells, Fig. 2 Effect on ECG recordings. a Differential effects on ECG parameters in diabetic rats compared to normal untreated rats. ST segment elevation (blue lines). b Ventricular rate (beat/ min). c QRS interval duration. d QT interval duration. e Corrected QT-interval duration. f ST-segment elevation. g P wave duration. Data were analyzed using the unpaired t-test with Welch's correction and were expressed as mean ± SD. **** = P ≤ 0.0001, *** = P ≤ 0.001, ** = P ≤ 0.01, and * = P ≤ 0.05 with respect to the normal group (n = 7) and vascular fibrosis as structural changes while functional changes include dysfunction endothelial cells dysfunction, impaired vasorelaxation, and vasoconstriction, diminished cardiac perfusion (Sandesara et al. 2018).
The advancement of dilated diabetic cardiomyopathy would stimulate fibrosis, which is considered a familiar marker for cardiomyopathy (Ho et al. 2010). The reduction in adenosine triphosphate (ATP) levels could lead to impaired perfusion and energetics of cardiomyocytes. This would result in reduced cardiac functionality, in addition to microvascular abnormalities (Raman et al. 2019). The modified cardiac functions encourage mutations in the genes encoding myofibers proteins, which may lead to an enhanced fibrosis (Araco et al. 2017).
Many proteins are incorporated in cardiac myofibers contractions. Troponin is one of these proteins. Three complex proteins represent troponin: troponin C, I, and T (Kraus et al. 2018). The detection of cTnT along with CK-MB in plasma is crucial for the diagnosis of cardiac dysfunctions (C.C. et al. 2010). CK enzyme is important for the conversion of creatinine with the assistance of ATP molecules to form phosphocreatine and adenosine diphosphate (ADP) (Bessman & Carpenter 1985). CK is differentiated into three isoforms that are distributed in different body tissues. CK-MB is mainly found in cardiac muscles; CK-MM is expressed in skeletal muscles and CK-BB, and the brain CK isoform (Schlattner et al. 2006). The damage of CK-rich tissue will lead to high CK levels detected in plasma (Schlattner et al. 2006). Therefore, detection of cTnT and CK-MB in plasma could be used for the diagnosis of cardiomyopathy, as was previously described by other clinical (Ali et al. 2016, Odum andYoung, 2018) and animal studies (Al-Rasheed et al. 2017).
NF-κB is believed to regulate inflammatory reactions by enhancing the production of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β (Gianello et al. 2019;Saber et al. 2019a;Zhai et al. 2020). This inflammatory response is quick, as NF-κB is previously stored inactivated form in the cytoplasm. Moreover, activated NF-κB stimulates cardiac hypertrophy and fibrosis (Nakamura and Sadoshima, 2018). Diabetes is always coupled with elevated ROS activity and stimulation of the renin-angiotensin-aldosterone system, which results in the subsequent increase in NF-κB formation and excess of pro-inflammatory cytokines (Jia et al. 2016;Saber et al. 2019b;Thomas et al. 2014). These mediators are responsible for the quick progression of diabetic cardiomyopathy (Ali et al. 2020, Yu andLiu, 2003). The elevated levels of TNF-α and IL-6 could impair the capability of cardiomyocytes to handle the Ca ++ ions through lowering the sarcoplasmic/ endoplasmic reticulum Ca ++ ATPase production (Villegas et al. 2000). This was previously shown in type I model of diabetes induced by STZ in mice, where IL-6 knock out lessened the progress of diabetic cardiomyopathy and enhanced cardiac function (Zhang et al. 2016). Moreover, NF-κB activation increased the release of ROS , which could be due to the increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Mariappan et al. 2010). Pyrrolidine dithiocarbamate derived inhibition of NF-κB restored cardiac function in diabetic animals and improved the mitochondrial structural integrity and prevented oxidative stress (Mariappan et al. 2010).

Conclusion
In conclusion, diabetic cardiomyopathy in rats could be diagnosed with several ECG anomalies, including STsegment elevation, prolongation of QRS complex and QT Fig. 4 Histopathological examination of cardiac tissue. Photomicrographic representation of heart (× 400). a Normal myofibers architecture in normal group (stained with H&E). b Perivascular fibrosis (blue arrow) in the normal group (stained with Masson's trichrome stain). c Disarray of cardiac myofibers in the diabetic group (stained with H&E) with infiltration of inflammatory cells (black arrow). d Perivascular fibrosis (blue arrow) of the diabetic group (stained with Masson's trichrome stain). It shows a helical arrangement of cardiac myofibers (yellow arrow). e Histopathological scoring. Data were analyzed using the unpaired t-test with Welch's correction and were expressed as mean ± SD and. **** = P ≤ 0.0001, *** = P ≤ 0.001, ** = P ≤ 0.01, and * = P ≤ 0.05 with respect to the normal group (n = 7) interval, and increased ventricular rate. The presence of atrial tachyarrhythmia could be due to atrial dilatation. Moreover, diabetes-induced cardiac injury leads to an elevation in cardiac specific biomarkers such as cTnT and CK-MB. Several histopathological alterations were detected in the hearts of diabetic rats. Cardiac myofibers took the arrangement of perpendicular angles and encircled a fibrotic helical core, which is a substantial anatomical hallmark of dilated cardiomyopathy. Collagen formations was increased in the perivascular area of the coronary blood vessels with enhanced infiltration of inflammatory cells. Additionally, diabetic cardiomyopathy is correlated with high IL-6 and TNF-α cardiac levels.
Author contribution Mahmoud E. Youssef contributed to conception, data acquisition and analysis, manuscript synthesis, histological examinations, image analysis, and data validation. Mona F. El-Azab, Marwa A. Abdel-Dayem, Galal Yahya, and Ibtesam S. Alanazi contributed to data analysis and discussion and manuscript synthesis. Sameh Saber handled animals, provided software, and conducted statistical analysis. All authors have read and approved the manuscript for publication.

Data availability
The data used in the current study are available from the relevant authors upon reasonable request.

Declarations
Ethics approval and consent to participate All experiments were performed following the approval of the Experimental Animal Ethics Committee of Suez Canal University No.: 201807PHDA1.

Conflicts of interest
The authors declare no competing interests. Fig. 5 cTnT, CK-MB, IL-6, and TNF-α levels. a Plasma levels of cTnT. b Plasma levels of CK-MB. c IL-6 in the cardiac levels. d TNF-α in the cardiac levels. Data were analyzed using the unpaired t-test with Welch's correction and were expressed as mean ± SD and. **** = P ≤ 0.0001, *** = P ≤ 0.001, ** = P ≤ 0.01, and * = P ≤ 0.05 with respect to the normal group (n = 7)