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Sari et al. | Journal of Clinical and Translational Research 2024; 10(4): 246-255 247
of developing heart failure than people without DM (×2.4 vs. ×5, 2.2. Materials
respectively), regardless of other risk factors (age, heart disease, ®
coronary artery disease, and hypertension) [2]. Cardiomyocyte Empagliflozin (EMPA) (Jardiance ) 25 mg tablets were
hypertrophy and cardiac fibrosis are the earliest structural purchased from Anugerah Pharmindo Lestari, Indonesia.
abnormalities in DM, preceding diastolic cardiac dysfunction in Each tablet was crushed and dissolved in sterile water and
heart failure related to DM [3,4]. The expression of cytoskeletal administered at a dose of 10 mg/kg body weight (BW)/day. Liquid
contractile proteins (β-myosin heavy chain [β-MHC]) cholecalciferol (Vitamin D3) with a concentration of 400 IU/mL
(Kid-D ) was purchased from Adiguna Pharmacy, Indonesia,
®
increases in response to pathogenic stimuli, facilitating the and administered at a dose of 225 IU/day. Streptozotocin (STZ)
preservation of cardiac contractile function during periods of and all other chemicals and solvents were of analytical grade
energy depletion [5]. Apoptosis resulting from hyperglycemia and procured from Gamma Scientific Biolab, Indonesia.
further induces viable cardiomyocytes to undergo pathological
hypertrophy as a compensatory mechanism for maintaining 2.3. Experimental groups
cardiac contractile function [4].
Cardiac fibrosis is a process of pathological remodeling and The diabetic rats were divided into four groups (n = 8
excessive deposition of the extracellular matrix, which causes per group): untreated diabetic group (high-fat/high-glucose
abnormalities in the composition and quality of the extracellular [HF/HG]/STZ), diabetic group treated with EMPA 10 mg/
matrix. The expression of cardiac transforming growth factor kg BW (HF/HG/STZ+EMPA), diabetic group treated with
beta (TGF-β) has been associated with collagen deposition, Vitamin D 225 IU/day (HF/HG/STZ+VitD), and diabetic group
myocardial stiffness, and diastolic dysfunction in diabetic treated with a combination of EMPA 10 mg/kg BW and Vitamin
®
rats [6]. Diastolic dysfunction was ameliorated by inhibition D 225 IU/day (HF/HG/STZ+EMPA+VitD). EMPA (Jardiance )
of TGF-β in experimental Type 2 DM (T2DM), suggesting a 25 mg tablets were crushed and dissolved in sterile water and
central role of TGF-β signaling in the pathogenesis of heart given at a dose of 10 mg/kg BW/day. Liquid cholecalciferol
®
failure related to DM [7]. (Vitamin D3) with a concentration of 400 IU/mL (Kid-D ) was
Cardiac fibrosis and hypertrophy in T2DM are potentially given at a dose of 225 IU/day. All treatments were given by oral
reversible conditions. The sodium-glucose transport protein gavage once daily for 8 weeks.
2 inhibitor (SGLT-2i) is an antidiabetic drug that is clinically 2.4. Experimental procedure
proven to have cardiovascular benefits, though the underlying
pathomechanism is still being studied [8]. Several mechanisms Wistar rats were given a HF/HG diet for 3 weeks, containing
linked to its cardiovascular benefits include the improvement 80% normal rat chow, 15% refining lard, and 5% yolk, along
of left ventricular mass and cardiac fibrosis [9]. Similarly, with 20% HG drinking water to induce T2DM. Rat chow was
lower Vitamin D levels were identified in the DM population supplemented with Vitamin D (800 IU/kg rat chow) to ensure that
compared to those without DM and were associated with rats receive Vitamin D according to the recommended daily intake.
increased HbA1c levels [10,11]. Previous studies indicated After 3 weeks of dietary modification, animals were injected
that the renin-angiotensin-aldosterone system (RAAS) induces with low-dose STZ (35 mg/kg BW) intraperitoneally, prepared
cardiac hypertrophy in Vitamin D receptor (VDR)-knockout by dissolving STZ in a 0.01 M citrate buffer with a pH of 4.5.
mice [12], whereas Vitamin D supplementation reduced renin Fasting blood glucose (FBG) levels were measured 72 h after STZ
expression and left ventricular hypertrophy in a hypertensive injection. Increased FBG ≥200 mg/dL was used in experimental
rat model [13]. Therefore, we aim to determine the possible studies as the standard in establishing a diabetic rat model [14],
protective effects of SGLT-2i and Vitamin D administration on and rats with FBG ≥200 mg/dL were included in the study. FBG
®
cardiac hypertrophy and fibrosis in Type 2 diabetic rats. was measured using Glucometer 4 Accu-Chek (Roche Diabetes
Care, Switzerland). STZ injection can be repeated once with half
2. Materials and Methods the initial dose if the blood glucose level has not reached the
desired level. Thereafter, the animals were fed an HF/HG diet for
2.1. Animals an additional 8 weeks. At week 13, the rats were euthanized with
This is an experimental study with a post-test-only ketamine (50 mg/kg BW) and xylazine (10 mg/kg BW), followed
control group design. Thirty-two male Wistar rats (Rattus by neck dislocation. Subsequently, surgical procedures were
norvegicus; 10 – 12 weeks; 150 – 200 g) were purchased performed to extract the heart. The rats were properly buried in
from the Animal Laboratory of Pharmacology Department, accordance with local customs, similar to the burial of a human
Faculty of Medicine, Udayana University, Indonesia. Each body. The animal experimental scheme is depicted in Figure 1.
rat was housed in a cage with a lid made of aluminum wire, 2.5. Histological analysis
located indoors with sufficient lighting. Animal studies were
conducted according to the regulation by the Institute of A left ventricular biopsy was performed at week 13. The heart
Animal Studies Ethics Committee approved by the Faculty tissue samples were fixed with 10% formaldehyde phosphate-
of Medicine, Udayana University (ethical clearance #2377/ buffered solution for 24 h. The fixed tissue was dehydrated,
UN14.2.2.VII.14/LT/2022), with all possible measures taken infiltrated, and embedded in liquid paraffin to solidify. The
to minimize suffering. paraffin blocks were sectioned using microtome at a thickness

DOI: http://doi.org/10.36922/jctr.24.00010