Supplementation with a cocoa–carob blend, alone or in combination with metformin, attenuates diabetic cardiomyopathy, cardiac oxidative stress and inflammation in Zucker diabetic rats

  Citation: García-Díez, E.; López-Oliva, M.E.; Caro-Vadillo, A.; Pérez-Vizcaíno, F.; Pérez-Jiménez, J.; Ramos, S.; Martín, M.Á. Supplementation with a Cocoa–Carob Blend, Alone or in Combination with Metformin, Attenuates Diabetic Cardiomyopathy, Cardiac Oxidative Stress and Inflammation in Zucker Diabetic Rats. Antioxidants 2022,11, 432. https:// doi.org/10.3390/antiox11020432 Academic Editor: Stanley Omaye Received: 21 January 2022 Accepted: 17 February 2022 Published: 21 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Supplementation with a Cocoa–Carob Blend, Alone or in Combination with Metformin, Attenuates Diabetic Cardiomyopathy, Cardiac Oxidative Stress and Inflammation in Zucker Diabetic Rats Esther García-Díez 1, María Elvira López-Oliva 2, Alicia Caro-Vadillo 3, Francisco Pérez-Vizcaíno 4,5,6, Jara Pérez-Jiménez 1, Sonia Ramos 1and MaríaÁngeles Martín1,7,* 1Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC), 28040 Madrid, Spain; esther.gar[email protected] (E.G.-D.); [email protected] (J.P.-J.); [email protected] (S.R.) 2Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain; [email protected] 3 Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; [email protected] 4Departamento de Farmacología y Toxicología, Facultad de Medicina, Universidad Complutense de Madrid, 28040 Madrid, Spain; [email protected] 5Instituto de Investigación Sanitaria Gregorio Marañón (IISGM), 28007 Madrid, Spain 6CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, 28029 Madrid, Spain 7CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, 28029 Madrid, Spain *Correspondence: [email protected] Abstract: Diabetic cardiomyopathy (DCM) is one of the main causes of mortality among diabetic patients, with oxidative stress and inflammation major contributors to its development. Dietary flavonoids show strong antioxidant and anti-inflammatory activities, although their potential additive outcomes in combination with antidiabetic drugs have been scarcely explored. The present study investigates the cardioprotective effects of a cocoa–carob blend (CCB) diet, rich in flavonoids, alone or in combination with metformin, in the development of DCM. Zucker diabetic fatty rats (ZDF) were fed with a CCB rich-diet or a control diet, with or without metformin for 12 weeks. Glucose homeostasis, cardiac structure and function, and oxidative and inflammatory biomarkers were analysed. CCB improved glucose homeostasis, and mitigated cardiac dysfunction, hypertrophy, and fibrosis in ZDF rats. Mechanistically, CCB counteracted oxidative stress in diabetic hearts by down-regulating NADPH oxidases, reducing reactive oxygen species (ROS) generation and modulating the sirtuin-1 (SIRT1)/ nuclear factor E2-related factor 2 (Nrf2) signalling pathway, overall improving antioxidant defence. Moreover, CCB suppressed inflammatory and fibrotic reactions by inhibiting nuclear factor kappa B (NF κ B) and pro-inflammatory and pro-fibrotic cytokines. Noteworthy, several of these effects were further improved in combination with metformin. Our results demonstrate that CCB strongly prevents the cardiac remodelling and dysfunction observed in diabetic animals, highlighting its potential, alone or in adjuvant therapy, for treating DCM. Keywords: heart; flavonoids; diabetes; antioxidants; metformin; SIRT1; Nrf2 1. Introduction Type 2 diabetes (T2D) represents a major worldwide health problem because of its high prevalence and associated cardiovascular complications [ 1 ]. Among them, diabetic cardiomyopathy (DCM) is one of the main causes of diabetes-associated morbidity and mortality [ 2 ]. There are many glucose-lowering drugs available for the treatment of T2D. Some of them may reduce the cardiovascular mortality but the risk of cardiovascular Antioxidants 2022,11, 432. https://doi.org/10.3390/antiox11020432 https://www.mdpi.com/journal/antioxidants Antioxidants 2022,11, 432 2 of 17 death is still very high in this population. Therefore, development of novel preventive or therapeutic strategies is needed to treat diabetic cardiomyopathy. DCM is defined as the existence of abnormal myocardial structure and function in the heart of diabetic patients in the absence of other cardiac risk factors [ 3 ]. Oxidative stress is considered one of the most important contributors to the pathogenesis of DCM [ 4 ]. Thus, the overproduction of reactive oxygen species (ROS) in diabetic hearts activates several detrimental pathways, including inflammation, hypertrophy, fibrosis, and cell death that eventually induce irreversible structural remodelling and dysfunction of this organ [ 5 ]. At the molecular level, hyperglycaemia directly induces the activity of NADPH oxidases (NOX) in cardiomyocytes, resulting in an excessive generation of ROS [ 6 ]. At the same time, the expression levels of the nuclear factor-erythroid 2-related factor 2 (Nrf2), involved in the cellular antioxidant defence system, and the levels of sirtuin1 (SIRT-1), a NAD-dependent histone deacetylase that protect against ROS-mediated oxidative damage, are down-regulated in diabetes, worsening the oxidative stress situation in the heart [ 7 , 8 ]. In addition, this pro-oxidant environment in the cardiomyocytes may initiate inflammatory responses via activation of nuclear factor kappa B (NFκ B) signalling, resulting in the upregulation of several inflammatory cytokines and the induction of profibrotic factors, which contribute to myocardial fibrosis and collagen deposition [ 9 ]. Considering this, agents that target both oxidative stress and inflammation are considered very promising candidates to slow down the occurrence of DCM. In the last years, natural products such as polyphenols have received widespread attention as a preventive approach to alleviate several cardiovascular diseases without adverse side effects [ 10 – 12 ]. In addition, the combination of pharmaceutical drugs and natural health substances with synergistic interactions could provide a way to make a more effective treatment [ 13 ]. Flavonoids are one of the most abundant group of polyphenols in the human diet and they have been widely investigated for their beneficial effects in several cardiovascular pathologies [ 14 ]. More recently, the study of the potential of dietary flavonoids or flavonoid rich diets during DCM is emerging. It has been indicated that flavonoids have the potential to alleviate DCM by their anti-hyperglycaemic, antioxidant and anti-inflammatory properties [ 15 ]. In this regard, cocoa flavonoids could be considered very good candidates for DCM treatment since they possess antioxidants and antidiabetic properties [ 16 , 17 ], as well as favourable cardiovascular effects [ 18 – 20 ]. However, cocoa is characterised by a bitter taste, rejected by many people. This taste is often modified by adding sugars, thus generating products with a worse nutritional profile. In contrast, carob (Ceratonia siliqua L.) bean, a Mediterranean legume rich in polymeric flavanols and other polyphenol classes, has proven to be a good option to be combined with cocoa [ 21 ], generating mixtures with a high phytochemical content. Indeed, carob is being increasingly studied for its potential beneficial effects, particularly regarding glucose homeostasis [22–24]. In view of this, we have developed a potential functional food combining cocoa powder with carob flour (in a proportion 60:40) for obtaining a cocoa–carob blend (CCB) rich in polyphenols (particularly non-extractable proanthocyanidins) with decreased bitterness (Garcia-Diez et al., submitted). The aim of this study was to investigate whether this functional food, rich in flavonoids, may prevent the development of DCM in an animal model of T2D. The potential additive effect of the CCB supplementation in combination with metformin, the first line anti-hyperglycaemic agent for the treatment of T2D, was evaluated as well. Our results provide strong evidence for the beneficial impact of the functional CCB rich-diet, alone or in combination with metformin, against the development of cardiac remodelling and dysfunction in diabetic animals and suggest the possible mechanisms involved in this protection. 2. Materials and Methods 2.1. Materials and Chemicals Nicotine adenine dinucleotide reduced salt (NADPH), 2 0 ,7 0 -dichlorofluorescin diacetate(DCFH),reducedglutathione(GSH),glutathionereductase(GR),tert-butylhydroperoxide, Antioxidants 2022,11, 432 3 of 17 o-phthaldehyde, streptavidin-biotin conjugated horseradish peroxidase (HRP), 3, 3 0 - diaminobenzidine (DAB) and glucose were purchased from Sigma Chemical (Madrid, Spain). Anti-TFG β 1 (sc-52893), anti-NADPH oxidase (NOX)-4 (sc-30141), anti-SIRT1 ( sc-74465 ), anti-phospho-Ser536-p65-NF κ B (sc-135769) anti-TNF α (sc-52746), anti-interleukin (IL)-6 (sc-57315) and anti-monocyte chemoattractant protein-1 (MCP-1) (sc-52701) were purchased from Santa Cruz Biotechnology (Quimigen, Madrid, Spain). Antiphospho-Nrf2 (#12811) was purchased from Signalway antibody (Quimigen, Madrid, Spain). Anti-NOX2 (ab-80897) and anti-CD45 (ab-10558), were purchased from Abcam (Cambrigde, UK). Terminal Transferase recombinant, biotin-16-dUTP and proteinase K were from Roche Applied Science (Roche Diagnostic, Barcelona, Spain). Bradford reagent was from BioRad (BioRad Laboratories S.A., Madrid, Spain). 2.2. Cocoa–Carob Blend Diet Cocoa–carob blend (CCB) was prepared by mixing pure cocoa powder (a kind gift from Idilia S.L., Barcelona, Spain) with carob flour (Casa Ruiz Granel Selecto S.L., Madrid, Spain) in a proportion of 60:40. The product was characterized by a high polyphenol and dietary fibre content (16.7 and 55.7 g/100 g, respectively). Among polyphenols, most of them (12.1% in CCB) were non-extractable proanthocyanidins, i.e., high molecular weight flavanols mostly associated with dietary fibre. Regarding dietary fibre, it was mostly insoluble dietary fibre (51% in CCB). Both cocoa and carob contributed with characteristic substances, as shown by chromatography analysis: thus, CCB contained theobromine ( 23 mg/100 g ) from cocoa and D-pinitol (1.22%) derived from carob. A detailed characterization of CCB has been provided elsewhere (García-Díez et al., submitted). CCB rich-diet (10%) was produced by adding 100 g/kg of the CCB to the AIN-93G diet. The resulting CCB diet was isoenergetic and its composition is given in Table 1. Table 1. Composition of the experimental control and cocoa–carob diets. Component (g/Kg Dry Weight) Control Cocoa–Carob Blend Casein 140 140 Dextrose 155 155 Sucrose 100 92 Fat 40 40 t-BHQ (tert-butylhydroquinone) 0.008 0.008 Mineral mix. 35 35 Vitamin mix. 10 10 L-Cys 1.8 1.8 Cholin bitartrate 2.5 2.5 Cellulose 100 44.5 Starch 415.7 379 Cocoa–carob powder - 100 Energy (KJ/Kg diet) 15048 15048 2.3. Animals and Experimental Design Male Zucker diabetic fatty (ZDF, n= 32) rats and their Zucker lean controls (ZL, n= 6) were purchased from Charles River Laboratories (L’arbresle, France) at 11 weeks of age. Animals were acclimated for one week under standard controlled conditions (21 ± 1 ◦ C; 50–60% humidity 12 h day/night cycle). Afterwards, ZDF diabetic rats (12 weeks of age) were randomly sorted into four different experimental groups of eight animals: (1) ZDF diabetic rats received a standard AIN-93G diet (ZDF); (2) ZDF rats received a standard AIN93G diet and oral metformin (300 mg/kg/day) (ZDF (M)); (3) ZDF rats received the CCB diet (ZDF (CCB)) and 4) ZDF rats received the CCB diet and metformin (300 mg/kg/day) (ZDF (CCB + M)). The lean Zucker rats (ZL) received the standard AIN-93G diet. Drinking water served as the vehicle for metformin treatment of ZDF rats. During the study period Antioxidants 2022,11, 432 4 of 17 (12 weeks), all experimental groups were provided with food and water ad libitum. Food and water intakes were monitored daily, and body weight and postprandial blood glucose were followed weekly during the entire study. At 24 weeks of age, animals were fasted overnight and sacrificed. Blood samples were collected for biochemical analysis, hearts were rapidly resected, and the left ventricles (LV) excised for the different analyses. Animals were treated according to the European (2010/63/EU) and Spanish (RD 53/2013) legislation on Care and Use of Experimental Animals and the experiments were approved by the Ethics Committee from Comunidad de Madrid (PROEX 079/19). 2.4. Biochemical Determination Fasting blood samples were collected for glucose, insulin, glycosylated haemoglobin (Hb1Ac), triacylglycerols (TG), HDL, and LDL analysis. Blood glucose was determined using an Accounted Glucose Analyser (LifeScan España, Madrid, Spain). Serum insulin and Hb1Ac were analysed with an ELISA kit (Rat Insulin, Mercodia, Uppsala, Sweden; HbA1c Kit Spinreact, BioAnalitica, Madrid, Spain). Fasting glycaemia and insulinaemia were used to calculate indices of homeostatic model assessment of insulin resistance (HOMA-IR) and secretion (HOMA-B) according to the following formulas: HOMA-IR = fasting insulin (mU/mL) × fasting glucose (mM)/22.5 and HOMA-B = 20 × fasting insulin (mU/mL)/[fasting glucose (mM)-3.5], respectively. TG, T-Cho, HDL-Cho, and LDL-Cho were determined in serum by kits (BioSystems, Madrid, Spain). 2.5. Glucose Tolerance Test One week before the sacrifice, a glucose tolerance test (GTT) was performed. After overnight fasting, 35% glucose solution (2 g/kg of body weight) was administrated to rats by intraperitoneal injection and blood samples were obtained from the tail vein before the glucose load (t = 0) and at, 30, 60, 90 and 120 min after glucose administration. The measures were determined with a glucometer. Changes in glucose were calculated as the area under the curve (AUC) above the basal levels. 2.6. Echocardiographic Measurements Cardiac function was evaluated by echocardiography. Echocardiographic studies were performed in the last week of the experiments using the General Electric Vivid iq (GE Medical Systems, Changjiang, China) echocardiographic system, equipped with a 8 to 18 MHz linear probe. Images were obtained from animals lightly anesthetized with 1–2% isoflurane (IsoFlo, Abbot Laboratories, Madrid, Spain). Left ventricular (LV) end-diastolic dimensions (LVDD), LV end-systolic dimensions (LVSD), intraventricular septum diastolic diameter (IVSD), LV posterior wall diastolic diameter (LVPWD), left atrial diameters (LA), aortic dimensions (Ao), left ventricular ejection fraction (EF) and LV fractional shortening (FS) were measured following the American Society for Echocardiography’s leading-edge method [ 25 ]. All images were digitally acquired and analysed by an experienced sonographer unaware of the treatment group allocation. 2.7. Histological and Immunohistochemical Analyses Left ventricles were rinsed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at 4 ◦ C overnight, embedded in paraffin and cut into 5 µ m thick sections. For the morphometric analysis, LV sections were stained with haematoxylin and eosin (H and E) and the extent of cardiac myocyte hypertrophy was measured by cross sectional area (CSA) determination. Cardiomyocyte CSA was measured by manually outlining 120 cells per heart in HE-stained sections using ImageJ. At 400-fold magnification, seven sequential, nonoverlapping fields of view were scanned to represent the whole cross section. Ten cardiomyocytes per field, transversely sectioned, with a round shape and visible central nucleus, and located in the subendocardial layer of the LV muscle wall were selected. For the quantification of fibrosis, serial sections of LV samples were stained with Masson’s trichrome, which stains the cardiomyocytes red and the fibrosis blue. Each Antioxidants 2022,11, 432 5 of 17 section was examined in 5 randomly selected high-power fields ( × 400, × 630) and middlepower fields ( × 200) under a light microscope. Percent fibrosis area was analysed using ImageJ analysis software (Version 1.8; National Institutes of Health) and expressed as percentage of collagen area (blue) to the total area of each microscopic field. For the immunohistochemical staining, antibodies against TFG β 1, CD45, NOX2, NOX4, SIRT1, p-Nrf2, p-p65-NF κ B, TNF α , IL6 and MCP1 were used. Serial left ventricular sections were incubated with the primary antibodies overnight at 4 ◦ C, then incubated with HRP-conjugated secondary antibody and revealed with DAB substrate as chromogen and haematoxylin. TFG β 1, NOX2, NOX4, SIRT1, p-Nrf2, TNF α , IL6 and MCP1 expression was evaluated by their staining pattern: weak (1), moderate (2), diffuse (3) and intense (4). The percentages of p-p65-NF κ B positive nuclei were calculated as the number of positive nuclei × 100/total number of nuclei. The percentages of CD45 positive cells were calculated as the number of positive cell × 100/total number of cells. Six sections per group were analysed. 2.8. Determination of ROS ROS were quantified by the dichlorofluorescein (DCFH) assay based on the oxidation of dichlorofluorescein (DCF) that emits fluorescence [ 19 ]. LV homogenates were diluted with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM, KCl, 3.6 mM NaHCO 3 , 2 mM CaCl 2 , 10 mM D-glucose and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfocnic acid, pH 7.4 ) and incubated with 5 µ M DCFH for 30 min at 37 ◦ C in darkness. Fluorescence was measured in a microplate reader (Bio-Tek, Winooski, VT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Proteins in LV homogenates were measured by using the Bradford reagent. 2.9. Determination of Protein Carbonyl Content Protein oxidation of LV homogenates was measured as carbonyl levels [26]. Samples were homogenized in 0.25 M Tris (pH 7.4), 0.2 M sucrose and 5 mM 1,4-dithiothreitol (DTT) buffer and derivatized with 2,4 dinitrophenylhydrazine (DNPH). Absorbance was measured at 360 nm and carbonyl content was expressed as nmol/mg protein using an extinction coefficient of 22,000 nmol L −1 cm −1 . Protein in LV homogenates was determined by the Bradford reagent. 2.10. GSH Levels and GPx and GR Activities The concentration of GSH was evaluated by a fluorometric assay already described in [ 26 ]. The method considers the reaction of GSH with o-phthalaldehyde (OPT) at pH 8.0. The LV samples were homogenized in 50 mM phosphate buffer pH 7.0, and proteins were precipitated with 5% trichloroacetic acid and then centrifuged for 30 min at 10,000×g. The fluorescence was measure at 460 nm—emission wavelength—and at 340 nm—excitation wavelength. For determining the activity of antioxidant enzymes (GPx and GR), LV samples were homogenized in 0.25 M Tris, 0.2 M sucrose and 5 mM DTT buffer pH 7.4 and centrifuged at 3000 × gfor 15 min. GPx activity was assayed by the oxidation of GSH by GPx, where tert-butylhydroperoxide (t-BOOH) is the substrate, coupled to the disappearance of NADPH by GR [ 26 ]. GR activity was determined by following the decrease in absorbance due to the oxidation of NADPH utilized in the reduction of oxidized glutathione [26]. 2.11. Statistical Analysis Statistical analysis was performed using the GraphPad Prism 6 software package. Gaussian distribution was assessed with the Shapiro–Wilk normality test. Parametric data were tested with one-way analyses of variance (ANOVA), while the Kruskal–Wallis test was performed for non parametric data (echocardiographic parameters). Statistically significant differences between means were determined using the Tukey and the Mann–Whitney post-hoc tests. A two-tailed p< 0.05 was considered significant. All data are expressed as mean ±standard deviation. Antioxidants 2022,11, 432 6 of 17 3. Results 3.1. Effect of CCB Alone or in Combination with Metformin on Glucose Homeostasis and Lipid Profile in Zucker Diabetic Rats As observed in Table 2, initially Zucker diabetic rats exhibited a higher body weight compared to non-diabetic ZL animals, confirming their obesity. At the end of the study, the final body weight was similar between ZL and ZDF animals, except for (ZDF (M)) group that showed a slightly but significantly higher body weight; this was indicative of the ZDF rat diabetic status. The total food intake during the 12 weeks of the study was higher in all ZDF groups as compared to ZL animals; therefore, the food efficiency was significantly decreased in ZDF animals. Table 2. Initial and final body weight, food intake and food efficiency of Zucker Lean rats (ZL); Zucker Diabetic rats (ZDF); Zucker Diabetic rats treated with metformin (ZDF (M)); Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). Data represent the means ± SD of 6–8 animals. Means in a row without a common letter differ, p< 0.05. ZL ZDF ZDF (M) ZDF (CCB) ZDF (CCB + M) Initial Body weight (g) 278 ±9a340 ±11 b341 ±20 b337 ±26 b338 ±23 b Final Body weight (g) 377 ±17 a393 ±13 ab 423 ±38 b384 ±26 ab 411 ±30 ab Body weight gain (g) 98 ±10 a55 ±8b93 ±17 a63 ±5b74 ±14 c Total food intake (g in 12 weeks) 1547 ±61 a2491 ±38 b2390 ±44 b2466 ±39 b2372 ±48 b Food Efficiency (body weight gain/total food intake) (%) 6.4 ±0.6 a2.2 ±0.3 b3.9 ±0.7 c2.5 ±0.2 bd 3.1 ±0.6 d Regarding glucose homeostasis, at the end of the study, ZDF control rats evidenced a significant increase in fasting and postprandial glucose levels, as well as in the percentage of glycosylated haemoglobin (Hb1Ac) as compared to non-diabetic ZL rats, while insulinaemia was similar in both populations (Figure 1a–d). Furthermore, ZDF rats showed glucose intolerance (measured by AUC), increased insulin resistance (HOMA-IR) and decreased pancreatic function (HOMA-B) (Figure 1e,f), which further confirmed their diabetic state. Treatment with metformin (ZDF (M)) or the CCB diet (ZDF (CCB)) for 12 weeks significantly improved all these parameters. Besides, animals fed with the CCB diet and treated with metformin (ZDF (CCB + M)) achieved values of both postprandial and fasting glucose and HbAc1 similar to those found in the non-diabetic rats (ZL). On the contrary, none of the treatments could improve the increase in TG and HDL levels in diabetics, and only the metformin treatment prevented the increase in LDL levels in ZDF animals (Figure 1g). These results indicate that a CCB rich diet significantly improved glucose homeostasis in ZDF rats. Notably, combining treatment with metformin had a superior effect on glucose control in comparison to the agents alone. Antioxidants 2022,11, 432 7 of 17 Figure 1. Effect of CCB, metformin and their combination on glucose and lipid homeostasis. (a) Postprandial glucose levels. ( b ) Fasting glucose levels. ( c ) Glycosylated haemoglobin (HbA1c). (d) Serum insulin levels. ( e ) Blood glucose levels during GTT and area under the curve (AUC) calculated from the GTT data. ( f ) Homeostasis model assessment (HOMA)-IR and HOMA-B. (g) Levels of TG, HDL, and LDL in serum. Data represent the means ± SD of 6–8 animals per condition. Means without a common letter differ, p< 0.05. Zucker lean (ZL), Zucker Diabetic rats (ZDF), Zucker Diabetic rats treated with metformin (ZDF (M)), Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). 3.2. Effect of CCB, Metformin and Their Combination on Cardiac Function in Zucker Diabetic Rats At 24 weeks old, the heart weight-to-tibia length ratio (HW/TL) was similar between groups being significantly decreased only in ZDF animals fed with the CCB rich-diet (ZDF (CCB)) (Table 3). To evaluate the effect of metformin and the CCB diet on cardiac function we performed echocardiography. Data indicated that left ventricular end-diastolic dimensions (LVDD), left ventricular end-systolic dimensions (LVSD), septum diastolic diameter (IVSD) and LV posterior wall diastolic diameter (LVPWD) did not differ among groups. However, a significantly increase in the left atrium diameter (LA) and a decrease in the aorta diameter (Ao) were observed in control diabetic rats (ZDF) compared to nondiabetic animals (ZL) (Table 3and Figure 2a). Consequently, the LA/Ao ratio, which is an indicator of LA enlargement and remodelling, was significantly increased in ZDF control rats (Figure 2b). Importantly, treatment of diabetic rats with metformin and the CCB rich-diet significantly reduced this ratio with no additional effect of the combination. Likewise, LV ejection fraction (EF) and LV fractional shortening (FS) were slightly but significantly decreased in the ZDF control group, suggesting initial systolic dysfunction in these diabetic animals (Figure 2c,d). This detrimental effect was prevented in all other Antioxidants 2022,11, 432 8 of 17 animal groups receiving metformin and CCB alone or in combination. Taken together, these data suggested that both metformin and the CCB diet could alleviate cardiac dysfunction in the left ventricle of diabetic rats. Table 3. Heart weight-to-tibia length ratio (HW/TL), left ventricular end-diastolic dimensions (LVDD), left ventricular end-systolic dimensions (LVSD), septum diastolic diameter (IVSD), LV posterior wall diastolic diameter (LVPWD), left atrium diameter (LA) and aorta diameter (Ao) of Zucker Lean rats (ZL); Zucker Diabetic rats (ZDF); Zucker Diabetic rats treated with metformin (ZDF (M)); Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). Data represent the means ± SD of 6–8 animals. Means in a row without a common letter differ, p< 0.05. ZL ZDF ZDF (M) ZDF (CCB) ZDF (CCB + M) HW/TL (g/cm) 0.32 ±0.02 a0.32 ±0.01 a0.35 ±0.03 a0.26 ±0.01 b0.31 ±0.01 a LVDD (cm) 0.73 ±0.05 a0.76 ±0.05 a0.75 ±0.08 a0.65 ±0.05 a0.68 ±0.13 a LVPWD (cm) 0.22 ±0.08 a0.20 ±0.05 a0.20 ±0.05 a0.19 ±0.04 a0.21 ±0.04 a LVSD (cm) 0.40 ±0.06 a0.46 ±0.05 a0.38 ±0.07 a0.39 ±0.06 a0.39 ±0.10 a IVSD (cm) 0.13 ±0.05 a0.14 ±0.05 a0.15 ±0.05 a0.14 ±0.05 a0.15 ±0.05 a Ao diameter (cm) 0.33 ±0.05 a0.23 ±0.05 b0.32 ±0.04 ab 0.28 ±0.08 ab 0.28 ±0.04 ab LA diameter (cm) 0.37 ±0.08 a0.57 ±0.08 b0.50 ±0.05 b0.43 ±0.07 ab 0.49 ±0.04 ab Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 17 decreased in the ZDF control group, suggesting initial systolic dysfunction in these diabetic animals (Figure 2c,d). This detrimental effect was prevented in all other animal groups receiving metformin and CCB alone or in combination. Taken together, these data suggested that both metformin and the CCB diet could alleviate cardiac dysfunction in the left ventricle of diabetic rats. Table 3. Heart weight-to-tibia length ratio (HW/TL), left ventricular end-diastolic dimensions (LVDD), left ventricular end-systolic dimensions (LVSD), septum diastolic diameter (IVSD), LV posterior wall diastolic diameter (LVPWD), left atrium diameter (LA) and aorta diameter (Ao) of Zucker Lean rats (ZL); Zucker Diabetic rats (ZDF); Zucker Diabetic rats treated with metformin (ZDF (M)); Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). Data represent the means ± SD of 6–8 animals. Means in a row without a common letter differ, p < 0.05. ZL ZDF ZDF (M) ZDF (CCB) ZDF (CCB + M) HW/TL (g/cm) 0.32 ± 0.02 a 0.32 ± 0.01 a 0.35 ± 0.03 a 0.26 ± 0.01 b 0.31 ± 0.01 a LVDD (cm) 0.73 ± 0.05 a 0.76 ± 0.05 a 0.75 ± 0.08 a 0.65 ± 0.05 a 0.68 ± 0.13 a LVPWD (cm) 0.22 ± 0.08 a 0.20 ±0.05 a 0.20 ± 0.05 a 0.19 ± 0.04 a 0.21 ± 0.04 a LVSD (cm) 0.40 ± 0.06 a 0.46 ± 0.05 a 0.38 ± 0.07 a 0.39 ± 0.06 a 0.39 ± 0.10 a IVSD (cm) 0.13 ± 0.05 a 0.14 ± 0.05 a 0.15 ± 0.05 a 0.14 ± 0.05 a 0.15 ± 0.05 a Ao diameter (cm) 0.33 ± 0.05 a 0.23 ± 0.05 b 0.32 ± 0.04 ab 0.28 ± 0.08 ab 0.28 ± 0.04 ab LA diameter (cm) 0.37 ± 0.08 a 0.57 ± 0.08 b 0.50 ± 0.05 b 0.43 ± 0.07 ab 0.49 ± 0.04 ab Figure 2. Effect of CCB, metformin and their combination on cardiac function. (a) Representative images of M-mode echocardiogram for each group with the measurement points of aorta diameters (yellow lines) and left atrium diameter (green lines). (b) LA/Ao ratio. (c) LV ejection fraction (EF). (d) LV fractional shortening (FS). Means ± SD of 6–8 samples per condition. Means without a common letter differ significantly. Zucker lean (ZL), Zucker Diabetic rats (ZDF), Zucker Diabetic rats treated with metformin (ZDF (M)), Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). 3.3. Effect of CCB, Metformin and Their Combination on LV Remodelling in Zucker Diabetic Rats Since cardiac dysfunction results from alterations in the composition and structure of the heart, we then investigated structural changes and remodelling in the LV of diabetic Figure 2. Effect of CCB, metformin and their combination on cardiac function. ( a ) Representative images of M-mode echocardiogram for each group with the measurement points of aorta diameters (yellow lines) and left atrium diameter (green lines). ( b ) LA/Ao ratio. ( c ) LV ejection fraction (EF). (d) LV fractional shortening (FS). Means ± SD of 6–8 samples per condition. Means without a common letter differ significantly. Zucker lean (ZL), Zucker Diabetic rats (ZDF), Zucker Diabetic rats treated with metformin (ZDF (M)), Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). 3.3. Effect of CCB, Metformin and Their Combination on LV Remodelling in Zucker Diabetic Rats Since cardiac dysfunction results from alterations in the composition and structure of the heart, we then investigated structural changes and remodelling in the LV of diabetic animals. Results from H and E-staining of the LV tissues (Figure 3a) revealed that cardiomyocytes were arranged in neat rows with uniform size nuclei in the ZL non-diabetic group. However, in the ZDF control animals, the myocardial fibres were disordered, and the cardiomyocytes were swollen and enlarged significantly, displaying an uneven cytoplasm and eccentric nucleus. In addition, the myocardial gap was widened, demonstrating Antioxidants 2022,11, 432 9 of 17 marked perivascular and interstitial oedema. Likewise, the levels of CD45 (marker of immune cell infiltration) were significantly increased in the myocytes of ZDF diabetic control rats (Figure 3b). Administration of metformin or CCB and their combination, strongly prevented these degenerative changes with appearance of the classical healthy cardiomyocytes. Cardiomyocyte hypertrophy in diabetic rats (ZDF) was evidenced by increased cardiomyocyte cross-sectional area (CSA), while diabetic animals treated with metformin (ZDF (M)), fed with the CCB-diet (ZDF (CCB)) or both (ZDF (CCB + M)) presented a normal cell size in comparison to the diabetic group. Next, to detect fibrosis and collagen content, Masson’s trichrome staining was performed in LV tissues. Likewise, the expression of a fibrotic marker, the transforming growth factor β 1 (TGF β 1), was determined. As observed in Figure 3c,d, interstitial and perivascular collagen fractional area and the expression levels of TGF β 1 were significantly increased in the left ventricles from ZDF diabetic rats as compared to ZL animals. All these adverse structural modifications induced by diabetes were partially prevented with metformin (ZDF (M)) or the CCB diet (ZDF (CCB)), with a further significant reduction by the combination (ZDF (CCB + M)). Indeed, the CCB richdiet combined with metformin totally prevented myocardial cellular damage (hypertrophy and fibrosis) in the LV of diabetic animals. Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 17 animals. Results from H and E-staining of the LV tissues (Figure 3a) revealed that cardiomyocytes were arranged in neat rows with uniform size nuclei in the ZL non-diabetic group. However, in the ZDF control animals, the myocardial fibres were disordered, and the cardiomyocytes were swollen and enlarged significantly, displaying an uneven cytoplasm and eccentric nucleus. In addition, the myocardial gap was widened, demonstrating marked perivascular and interstitial oedema. Likewise, the levels of CD45 (marker of immune cell infiltration) were significantly increased in the myocytes of ZDF diabetic control rats (Figure 3b). Administration of metformin or CCB and their combination, strongly prevented these degenerative changes with appearance of the classical healthy cardiomyocytes. Cardiomyocyte hypertrophy in diabetic rats (ZDF) was evidenced by increased cardiomyocyte cross-sectional area (CSA), while diabetic animals treated with metformin (ZDF (M)), fed with the CCB-diet (ZDF (CCB)) or both (ZDF (CCB + M)) presented a normal cell size in comparison to the diabetic group. Next, to detect fibrosis and collagen content, Masson’s trichrome staining was performed in LV tissues. Likewise, the expression of a fibrotic marker, the transforming growth factor β1 (TGFβ1), was determined. As observed in Figure 3c,d, interstitial and perivascular collagen fractional area and the expression levels of TGFβ1 were significantly increased in the left ventricles from ZDF diabetic rats as compared to ZL animals. All these adverse structural modifications induced by diabetes were partially prevented with metformin (ZDF (M)) or the CCB diet (ZDF (CCB)), with a further significant reduction by the combination (ZDF (CCB + M)). Indeed, the CCB rich-diet combined with metformin totally prevented myocardial cellular damage (hypertrophy and fibrosis) in the LV of diabetic animals. Figure 3. Effect of CCB, metformin and their combination on cardiac remodelling. (a) Representative sections of left ventricles stained with H and E (scale bars: 10μm) and quantitative data for myocyte cross sectional area. (b) Representative photographs of immunohistochemical staining of CD45 (brown-stained) (scale bar 10 μm). CD45 is expressed as a percentage of positive cells relative to total cells. (c) Representative images of collagen fibres in interstitial and perivascular areas shown by Masson’s trichrome staining (blue-stained) (scale bars: 10μm) and quantification of collagen areas (%). (d) Representative photographs of immunohistochemical staining of TFG-β1 (brownstained) (scale bar 20 μm) and immunoreactive score. Values are expressed as means ± SD of 6–8 Figure 3. Effect of CCB, metformin and their combination on cardiac remodelling. ( a ) Representative sections of left ventricles stained with H and E (scale bars: 10 µ m) and quantitative data for myocyte cross sectional area. ( b ) Representative photographs of immunohistochemical staining of CD45 (brown-stained) (scale bar 10 µ m). CD45 is expressed as a percentage of positive cells relative to total cells. ( c ) Representative images of collagen fibres in interstitial and perivascular areas shown by Masson’s trichrome staining (blue-stained) (scale bars: 10 µ m) and quantification of collagen areas (%). ( d ) Representative photographs of immunohistochemical staining of TFGβ 1 (brown-stained) (scale bar 20 µ m) and immunoreactive score. Values are expressed as means ± SD of 6–8 animals per condition. Means without a common letter differ, p< 0.05. Zucker lean (ZL), Zucker Diabetic rats (ZDF), Zucker Diabetic rats treated with metformin (ZDF (M)), Zucker Diabetic rats fed with a CCB rich-diet (ZDF (CCB)), and Zucker Diabetic rats treated with metformin and fed with a CCB rich-diet (ZDF (CCB + M)). Antioxidants 2022,11, 432 16 of 17 15. Jubaidi, F.F.; Zainalabidin, S.; Taib, I.S.; Hamid, Z.A.; Budin, S.B. 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