Effects of Naringin on Cardiomyocytes From a Rodent Model of Type 2 Diabetes

Diabetic cardiomyopathy (DCM) is a primary disease in diabetic patients characterized by diastolic dysfunction leading to heart failure and death. Unfortunately, even tight glycemic control has not been effective in its prevention. We have found aberrant diastolic Ca2+ concentrations ([Ca2+]d), decreased glucose transport, elevated production of reactive oxygen species (ROS), and increased calpain activity in cardiomyocytes from a murine model (db/db) of type 2 diabetes (T2D). Cardiomyocytes from these mice demonstrate significant cell injury, increased levels of tumor necrosis factor-alpha and interleukin-6 and expression of the transcription nuclear factor-κB (NF-κB). Furthermore, decreased cell viability, and reduced expression of Kir6.2, SUR1, and SUR2 subunits of the ATP-sensitive potassium (KATP) channels. Treatment of T2D mice with the citrus fruit flavonoid naringin for 4 weeks protected cardiomyocytes by reducing diastolic Ca2+ overload, improving glucose transport, lowering reactive oxygen species production, and suppressed myocardial inflammation. In addition, naringin reduced calpain activity, decreased cardiac injury, increased cell viability, and restored the protein expression of Kir6.2, SUR1, and SUR2 subunits of the KATP channels. Administration of the KATP channel inhibitor glibenclamide caused a further increase in [Ca2+]d in T2D cardiomyocytes and abolished the naringin effect on [Ca2+]d. Nicorandil, a KATP channel opener, and nitric oxide donor drug mimic the naringin effect on [Ca2+]d in T2D cardiomyocyte; however, it aggravated the hyperglycemia in T2D mice. These data add new insights into the mechanisms underlying the beneficial effects of naringin in T2D cardiomyopathy, thus suggesting a novel approach to treating this cardiovascular complication.


INTRODUCTION
Type 2 Diabetes mellitus (T2D) is public health threat with a significant increase in affected individuals worldwide in the last decade (Zimmet et al., 2016). Globally the associated healthcare cost of diabetes has been projected to exceed $2.1 trillion by 2030 (Bommer et al., 2018). Epidemiologic data show that the frequency of heart diseases in diabetic patients is much higher, and the prognosis is worse than in populations without diabetes (Thrainsdottir et al., 2005;Lehrke and Marx, 2017;Singh et al., 2018). One significant complication associated with type 2 diabetes is diabetic cardiomyopathy (DCM), characterized by diastolic dysfunction, ventricular hypertrophy, and interstitial fibrosis leading to heart failure in the absence of dyslipidemia, hypertension and coronary artery disease (Nicolino et al., 1995;Redfield et al., 2003;Singh et al., 2018;Dillmann, 2019). While hyperglycemia provokes noticeable effects on cardiomyocyte function, blood glucose levels are not necessarily predictive of cardiac dysfunction (Diamant et al., 2003). Management of T2D -DCM involves lifestyle modifications, including diet, regular physical activity, and pharmacological therapies for glucose management, lipid abnormalities, hypertension, and cardiac failure (Pappachan et al., 2013). Although hyperglycemia appears to drive the pathogenesis of DCM, firm glycemic control in diabetic patients has not translated into a significant reduction in either morbidity or mortality (Wang and Reusch, 2012). Unfortunately, despite decades of intense clinical research, no therapies have substantially protected DCM development in diabetic patients.
The study aimed to investigate the potential cardioprotective effect of naringin in cardiomyocytes isolated from T2D mice. Our results demonstrate that the naringin exerts cardioprotection through multiple mechanisms 1) Reducing [Ca 2+ ] d ; 2) Improving cardiomyocyte glucose uptake; 3) Decreasing production ROS; 4) Reducing the level of myocardial tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the expression of NF-κB; 5) Attenuating calpain activity, cardiac injury and improving cardiomyocyte viability; 6) Increasing the expression of Kir6.2, SUR1, and SUR2 subunits of the ATP-sensitive potassium (K ATP ) channels. In addition, our results provide new insight into the mechanisms by which naringin exercises cardioprotection in T2D cardiomyocytes.

Animals
Male homozygous C57BL/10 (WT-control) and C57BL/KsJ-db/ db (T2D) mouse colonies were established using founders obtained from the Jackson Laboratory (Bar Harbor, ME, United States). Control and T2D 12 weeks old mice were housed under a constant temperature (24°C) and 12-h light/ 12-h dark cycle, with free access to water and fed with pelletized commercial chow diet. All experimental animal procedures were carried out following the Care and Use Handbook of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committees at the Mount Sinai Medical Center, Miami Beach, FL, United States.

Diabetic Model
12-week old C57BL/KsJ-db/db mice, which spontaneously develop hyperglycemia due to a leptin receptor mutation with phenotypic features of human T2D, were used as our mouse model of T2D (Hummel et al., 1966). We used fasting blood glucose levels to corroborate the diabetic condition. Mice with a 12 h fasting glucose level >250 mg/dl for three consecutive tests were considered diabetic and used experimentally.

Mouse Blood Collection and Glucose Determinations
Blood samples (5 µL) for glucose measurements were obtained from 12 h fasting T2D and control mice from the tail vein on two occasions before beginning the treatment protocol and after the 4 weeks of treatment immediately prior the removing the heart. Blood glucose concentrations were measured on a glucometer (AlphaTRAK ® Glucose Meter, Abbott Animal Health, Abbott Park, IL United States) (Andrikopoulos et al., 2008).

Naringin Treatment
Control and T2D mice (12 weeks old) were treated with naringin 20, 40, or 60 mg/kg/day (d) dose (Sigma-Aldrich, MO United States) administered intraperitoneally (i.p.) once daily for 4 weeks. Untreated control and T2D mice received equal volumes of normal saline solution administered i. p. daily for the same period. Mice were divided randomly into eight groups. Group 1. Control mice in which no therapeutic intervention was made during the study (n 20 mice); Group 2: Control mice treated with naringin (20 mg/kg/d i. p.) (n 5 mice); Group 3: Control mice treated with naringin (40 mg/kg/d i. p.) (n 5 mice); Group 4: Control mice treated with naringin (60 mg/kg/d i. p.) (n 20 mice); Group 5. T2D mice in which no therapeutic intervention was made during the study (n 20 mice); Group 6: T2D mice treated as Group 2, (n 5 mice); Group 7: T2D mice treated as Group 3, (n 5 mice); Group 8: T2D mice treated as Group 4 (n 20 mice). At the end of the 4 weeks treatment, all mice were fasted for 12 h, heparinized (1000 U/kg, i. p.), anesthetized with ketamine (100 mg/kg), and xylazine (5 mg/kg), and the heart was removed to prepare isolated cardiomyocytes. Blood samples were drawn from the tail vein for a final glucose determination before the heart was removed.

Glibenclamide Treatment
In the second cohort of mice (12 weeks old), we examined the importance of K ATP channels on the naringin cardioprotective effects in T2D cardiomyocytes by testing the effect of the K ATP channel blocker glibenclamide. For this study, mice were divided into six groups (n 5 mice per group). Group 1. Control mice that did not receive therapeutic intervention; Group 2. Control mice treated with glibenclamide (10 μg/kg i. p./d) for 4 weeks; Group 3: Control mice treated with glibenclamide (10 μg/kg, i. p./ d) for 48 h and then with glibenclamide (10 μg/kg, i. p.) in combination with naringin (60 mg/kg i. p./d) for 4 weeks; Group 4. T2D mice that did not receive therapeutic intervention; Group 5: T2D mice were treated as Group 2; Group 6: T2D mice were treated as Group 3. Untreated control and T2D mice received equal volumes of normal saline solution administered i. p. daily for the same period. After the 4 weeks of treatment, cardiomyocytes were isolated from all experimental animals, following the protocol previously described for naringin treatment above.

Nicorandil Treatment
In the third cohort of mice (12 weeks old), we assessed whether a K ATP channel agonist could imitate the cardioprotective effect of naringin in T2D cardiomyocytes regarding the [Ca 2+ ] d and ROS production. Mice were divided into four groups (n 5 mice per group). Group 1. Control mice in which no therapeutic intervention was made during the study; Group 2. Control mice treated orally with nicorandil (10 mg/kg/d) for 4 weeks; Group 3: T2D mice in which no therapeutic intervention was made during the study; Group 4: T2D mice were treated as Group 2. After the 4 weeks of treatment, cardiomyocytes were isolated from all experimental animals, following the protocol previously described for naringin treatment above. Blood samples were obtained for glucose determination before the heart was removed.

Isolation of Cardiomyocytes and Inclusion Criteria
Hearts from anesthetized mice were rapidly removed, attached to a cannula, and mounted onto a temperature-controlled (37°C) Langendorff reverse coronary perfusion system for enzymatic dissociation of cardiomyocytes as previously described (Liao and Jain, 2007). All cardiomyocytes utilized in this study (within 5 h after isolation) were rod-shaped, had well-defined striation spacing, did not contract when perfused with Tyrode solution containing Ca 2+ , and had a resting sarcomere length of ≥1.75 µm (Wussling et al., 1987) after being electrically stimulated (1 ms square pulse, X1.5 threshold voltage, 0.5 Hz, for 3 min).

Ca 2+ -Selective Microelectrodes
The procedure for manufacturing the doubled-barreled Ca 2+ selective microelectrodes has been described previously (Eltit et al., 2013). Ca 2+ ionophore II (ETH 129) (Sigma-Aldrich, MO, United States) was used to backfill the microelectrode tip and pCa 7 (pCa −log free [Ca 2+ ]) the shanks of the Ca 2+selective barrel. 3M KCl was used to fill the tip and shank of the membrane potential barrel. Each ion-selective microelectrode was individually calibrated in solutions with known [Ca 2+ ] (pCa3 to pCa8), and only those microelectrodes with Nernstian slope (28.5 mV per pCa) between pCa3 and 7 were used experimentally (Lopez et al., 1983;Lopez et al., 2000;Eltit et al., 2013) (Figure 1E). Ca 2+ -selective microelectrodes were calibrated again after completing [Ca 2+ ] d measurements, and if the two calibrations curves showed a difference greater than 3 mV (pCa6-7), the data was discarded.

Recording of Diastolic Calcium Concentration
Isolated cardiomyocytes were impaled with double-barreled Ca 2+ -selective microelectrodes for the determination [Ca 2+ ] d , and the potentials were recorded via a high-impedance amplifier (WPI 773 electrometer, FL, United States) as described previously (Lopez et al., 2000;Lopez et al., 2017). The potential from the 3M KCl microelectrode barrel (V m ) was subtracted electronically from the potential recorded by the Ca 2+ ion-selective barrel (V CaE ) to produce a differential Ca 2+ -specific potential (V DiffCa ), which represent the cardiomyocyte [Ca 2+ ] d . V m and V DiffCa potentials were acquired at a frequency of 1,000 Hz with AxoGraph software (version 4.6; Axon Instruments, CA, United States) and stored in a computer for further analysis. The following criteria were used to accept individual cardiomyocytes [Ca 2+ ] d measurements from control and db/db mice: 1) a quick drop to a steady level of Vm; 2) a stable recording potential (V m and V DiffCa ) for no less than 60 s; 3) a quick return to baseline of Vm and V DiffCa potential on the exit of the microelectrodes from the cell. These criteria were not met in 15% of the total impalements carried out in control and 25% in db/db cardiomyocytes, and these data were rejected from our analysis.

Cardiomyocyte Glucose Uptake
Glucose uptake was assessed by measuring the change in fluorescence of 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose (2-NBDG; Invitrogen, CA, United States) in isolated cardiomyocytes (Gallo et al., 2018). Enzymatically isolated cardiomyocytes were 2-NBDG (250 µM) loaded for 30 min in the dark in glucose-free Tyrode solution. After the loading was completed, cardiomyocytes were washed with Tyrode standard solutions and then imaged using an invert fluorescence microscope (Zeiss Axiovert 200, NY, United States), equipped with a 50X/0.90 oil-immersion objective (Zeiss, NY, United States). Excitation light and emission light wavelengths were set at 475 and 535 nm, respectively, utilizing a filter set (XF100-2, Omega Optical, VT, United States). At the end of the experiment, only cardiomyocytes contracted in response to electrical stimulation (1 ms square pulse, X1.5 threshold voltage, at 37°C) were included. Glucose uptake for all groups was recorded for 3 min and normalized to the value from untreated control cardiomyocytes.

Calpain Activity Assay
Calpain activity was assessed in isolated cardiomyocytes using a fluorometric calpain activity assay kit (Abcam Inc, MA, United States). The lysates of cardiomyocytes were centrifuged, and the supernatant was used to detect calpain activity using the fluorescent substrate N-succinyl-LLVY-AMC. Fluorescence was detected using a microplate reader (Molecular Device, CA, United States) at 360 nm excitation and 460 nm emission wavelengths. Fluorescence measured in arbitrary units for all groups was normalized to values from untreated control cardiomyocytes.

Assay for Cardiac Injury and Cell Viability
Lactate dehydrogenase (LDH) release, a marker of cellular injury, was measured in the medium in naringin-treated and untreated control and T2D cardiomyocytes using an LDH assay kit (Sigma-Aldrich, MO, United States) according to the manufacturer's instructions. The activity of LDH was normalized to values from untreated control cardiomyocytes. Cardiomyocyte viability was assessed by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following the manufacturer's instructions  (Lopez et al., 1985;Eltit et al., 2013). The microelectrode shows a Nernstian slope (28.5 mV per pCa unit) (pCa −log [Ca 2+ ]) between pCa 3 and pCa 7 and still have a helpful response between pCa7 and 8. (F) Dose-response effects of naringin on [Ca 2+ ] d measured in quiescent cardiomyocytes isolated from control (green) and T2D mice (red). [Ca 2+ ] d was significantly higher in T2D than control cardiomyocytes (p < 0.001). Naringin reduced significantly [Ca 2+ ] d in a dose-dependent manner in T2D cardiomyocytes compared to untreated cardiomyocytes (p < 0.001), with no significant effect on control cardiomyocytes (p 0.99 compared to untreated). Experiments were conducted in nmice 5-20/group and ncells 10-21/group. Values are expressed as means ± S.D. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison tests, ns p > 0.05, *p < 0.05, ***p < 0.001.
Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 719268 (Abcam Inc, MA, United States). Data are expressed as a reduction in MTT concentration compared to untreated control cardiomyocytes.

Measurement of TNF-α and Interleukin-6 Concentrations and Western Blot Analyses
A different cohort of naringin-treated (60 mg/kg/d i. p. for 4 weeks) and untreated control and T2D mice (12 weeks old) were divided randomly into four groups (n 5 mice per group) for the determination of TNF-α and IL-6. Mice were anesthetized, the heart was removed and homogenized in 10% phosphate buffer to measure the inflammatory mediators TNF-α and IL-6 using ELISA kits (Abcam Inc, MA, United States). The measurements of these markers were carried out according to the manufacturer's protocols using a microplate reader (Molecular Devices, CA, United States). For Western blot analysis, naringin-treated (60 mg/kg/d i. p. for 4 weeks) and untreated control and T2D mice (12 weeks old) (nmice 3 per group) mice were anesthetized; the heart was removed, the ventricle dissected and then homogenized in radioimmunoprecipitation assay (RIPA) buffer for protein extraction. After protein transfer from the gel to the membrane, we sliced the membrane horizontally according to the protein molecular weight of interest (Uryash et al., 2015). The protein size was determined based on the protein standard marker. Separate membrane strips were incubated with the primary KATP antibody (Kir6.2, SUR1, SUR2, Abcam Inc, MA, United States) and NF-κB (Abcam Inc, MA, United States) 1:2,500 dilution. The intensity of the bands of these proteins was normalized to the intensity of the housekeeping protein ß-actin, which was used as a loading control (Actin, Abcam Inc, MA, United States).

Solutions
All solutions were made using ultrapure water supplied by a Milli-Q system (Millipore, Bedford, MA). Tyrode solution had the following composition (in mM): NaCl 130, KCl 2.68, CaCl 2 1.8, MgCl 2 1, glucose 5, NaHCO 3 20, NaH 2 PO 0.9, gassed with 95% O 2 and 5% CO 2 , pH 7.2. For the conditions where a Tyrode glucose-free solution was required, glucose was omitted from the solution. All experiments were performed at 37°C.

Statistical Analysis
Experimental results are expressed as means ± SD; n mice represents the number of mice used and n cells the number of successful measurements used for statistical analysis. Data were analyzed using one-way ANOVA for repeated measures, followed by Tukey's multiple comparison tests to determine significance. For Western blots, statistical analyses were carried out using the paired Student's independent t-test. A p-value of less than 0.05 was considered significant. GraphPad Prism 9 (GraphPad Software, CA, United States) was used for statistical analysis.

Naringin Enhanced Cardiomyocyte Glucose Transport and Reduced Hyperglycemia
The T2D heart is characterized by a decrease in glucose uptake (Jaswal et al., 2011). To further elucidate the effect of naringin, we carried out glucose uptake measurements in cardiomyocytes isolated from naringin-treated and untreated control and T2D mice using the fluorescent glucose analog 2-NBDG. Basal glucose uptake was reduced (60%, p < 0.001) in T2D cardiomyocytes compared to controls ( Figure 2). Naringin treatment (60 mg/kg/ d) induced a significant increase in glucose uptake in T2D cardiomyocytes (111%, compared to untreated T2D cardiomyocytes, p < 0.001). There was no significant difference in the control group between untreated and naringin-treated cardiomyocytes (p 0.99) (Figure 2).

Attenuation of Diabetes-Associated Oxidative Stress by Naringin
Oxidative stress plays a prominent role in the pathogenesis of diabetes (Guo et al., 2018). Therefore, cardiomyocyte oxidant levels for all groups were quantified using DCFDA fluorescence probe. Figure 3A shows representative records of the rate of dichlorofluorescein (DCF) fluorescence obtained from naringin treated and untreated control and T2D cardiomyocytes. Cardiomyocytes from T2D mice had a significantly increased DCF fluorescence signal rate compared to controls (204%) (p < 0.001) ( Figure 3B). Naringin treatment (60 mg/kg/d) significantly reduced the rate of DCF fluorescence in T2D Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 719268 cardiomyocytes by 55% (p < 0.001 compared to untreated T2D) but did not affect naringin-treated control cardiomyocytes (p 0.91 compared to the untreated control group) ( Figures 3A,B).

Naringin Suppressed Diabetes-Induced Myocardial Inflammation
Previous results have shown that an increase of inflammatory cytokines plays a critical role in developing diabetic cardiomyopathy (Palomer et al., 2013). We examined the effect of naringin on the levels of TNF-α and IL-6 in mice myocardium from naringin-treated and untreated-control and T2D mice. The content of proinflammatory cytokines TNF-α and IL-6 were significantly higher in myocardial tissue from T2D mice compared to control (191 and 145% respectively) (p < 0.001 compared to control, Figures 4A,B). Treatment with naringin (60 mg/kg/d) significantly reduced myocardial TNF-α and IL-6 levels in T2D by 42 and 32%, respectively (p < 0.001 compared to untreated T2D mice ( Figures 4A,B). Naringin treatment had no significant effect on TNF-α and IL-6 control myocardium (p 0.98 TNF-α and p 0.30 for IL-6).
Since NF-κB is an important mediator in the pathophysiology of DCM (Lorenzo et al., 2011), we studied the expression of NF-κB protein in myocardium from naringin-treated and untreatedcontrol and T2D mice. NF-κB expression was significantly upregulated in T2D tissue (360%) compared with control (p < 0.001) ( Figure 4C). Treatment with naringin (60 mg/kg/d) normalized the expression of NF-κB in T2D myocardium compared to untreated tissue (p < 0.001) ( Figure 4C), without affecting the expression in control naringin treated myocardium (p 0.94).

Reduction of Calpain Activity in T2D Cardiomyocytes by Naringin
The calpains represent a family of Ca 2+ -dependent proteases implicated in the pathophysiology of several inflammatory disorders of the cardiovascular system, including diabetic cardiomyopathy (Wan et al., 2015). Calpain activity was elevated by 228% in untreated T2D cardiomyocytes compared to control cardiomyocytes (p < 0.001) ( Figure 5A). Naringin treatment (60 mg/kg/d) significantly reduced calpain activity by 35% in T2D cardiomyocytes (p < 0.001 compared to untreated diabetic cardiomyocytes) ( Figure 5A) but did not change calpain activity in control cells (p 0.99) ( Figure 5A).
T2D cardiomyocyte viability, as assessed by MTT, was 41% lower than in control cardiomyocytes (p < 0.001) ( Figure 5C). Naringin treatment (60 mg/kg/d) increased T2D cardiomyocytes' viability by 37% compared to naringin-untreated T2D cardiomyocytes (p < 0.001) ( Figure 5C). Cardiomyocyte viability in controls was not significantly different in the naringin group compared to the naringin-untreated group (p 0.91) ( Figure 5C). FIGURE 2 | Naringin improved glucose transport and lowered hyperglycemia. Basal glucose uptake in T2D cardiomyocytes was significantly reduced compared to control (p < 0.001). Naringin treatment provoked a significant increase in glucose uptake in T2D cardiomyocytes compared to untreated T2D cardiomyocytes (p < 0.001). Naringin treatment did not modify cardiomyocytes' glucose uptake in control cardiomyocytes (p 0.99). Glucose uptake experiments were conducted in n mice 5/group; n cells 19-21/group. Insert shows the significant hypoglycemic effect of naringin in T2D mice (p < 0.001), however, no effect was observed in naringin-treated control mice (p 0.36 compared to untreated mice). Blood Glucose was measured in n mice 20/group; n determinations 15-20/group before and after naringin treatment. Values were normalized to control untreated cardiomyocytes (glucose uptake) or indicated as mg/dL (blood glucose) and expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, ***p < 0.001.

Glibenclamide Aggravated [Ca 2+ ] d Dysfunction and Blocked the Naringin Cardioprotective Effect
To test the role of K ATP channels in naringin cardioprotective effect control and T2D mice were treated with glibenclamide, a non-specific K ATP channel blocker (Findlay, 1992). Glibenclamide (10 μg/kg, i. p./d) induced an increase in [Ca 2+ ] d in T2D cardiomyocytes from 298 ± 31 nM to 448 ± 62 nM (p < 0.001 compared to untreated T2D cardiomyocytes) ( Figure 7A) and significantly enhanced ROS production by 47% in T2D cardiomyocytes (p < 0.001 compared to untreated T2D cardiomyocytes) ( Figure 7B). However, it did not modify either [Ca 2+ ] d or ROS production in control cardiomyocytes ( Figures 7A,B). Furthermore, glibenclamide prevented the previously observed beneficial effects of naringin on [Ca 2+ ] d and ROS production in T2D cardiomyocytes (for comparison, see Figures 1F, 3B).

Nicorandil Effect on [Ca 2+ ] d in T2D Cardiomyocytes
To further explore the involvement of the K ATP channel on the naringin cardioprotection, we tested whether nicorandil, a K ATP channel opener and nitric oxide (NO) donor (Uchida et al., 1978;Holzmann, 1983;Ishii et al., 2005; Figure S1). In addition, nicorandil modified glucose homeostasis in control and worsened hyperglycemia in T2D mice. In T2D mice fasting blood glucose level was elevated from 401 ± 31 mg/dl to 549 ± 62 mg/dl after nicorandil (p 0.001), while in control was raised from 99 ± 2 mg/dl to 131 ± 15 mg/dl (p < 0.01) upon completion of nicorandil treatment ( Figure 8F).

DISCUSSION
The most significant findings are as follows: FIGURE 3 | Naringin reduced intracellular ROS generation T2D cardiomyocytes. DCF fluorescence signal rate was significantly enhanced in T2D compared to control cardiomyocytes. (A) Representative records of DCF fluorescence signal from control (black), control naringin treated (green), T2D (red), and T2D naringin treated (purple) cardiomyocytes. (B) Average rate of DCF fluorescence signal from all cardiomyocytes groups. DCF fluorescence signal measurements were obtained from n mice 5/group; n cells 20-27/group. Values were normalized to control untreated cardiomyocytes and expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, ***p < 0.001.

Diabetic Cardiomyopathy
DCM is a particular form of heart disorder observed in diabetic patients due to an impairment of diastolic cardiac contractile function and occurs independently of other cardiac risk factors. The probability of developing cardiac dysfunction and heart failure is much higher in T2D patients than in non-diabetic subjects (Radovits et al., 2009;Bouthoorn et al., 2018). The pathophysiological mechanisms of DCM remain poorly understood, and multiple factors such as hyperglycemia, intracellular Ca 2+ dysfunction, oxidative stress, endoplasmic reticulum stress, inflammation, and mitochondrial alterations have been associated with this pathology (Tan Y. et al., 2020). Pharmacological treatment has substantially improved blood glucose levels in T2D patients' however, their predisposition to developing cardiovascular disease is much higher than nondiabetic subjects, independent of other comorbidities (Jia et al., 2018).
FIGURE 4 | Effects of naringin on TNF-α, IL-6, and NF-kB in T2D ventricular tissues. (A) T2D ventricular tissue had a significantly TNF-α higher concentration than control ventricular cell (p < 0.001). Naringin treatment significantly reduced TNF-α levels in T2D tissues compared to untreated T2D (p < 0.001). Naringin treatment did not affect TNF-a levels in control ventricular tissue (p 0.98 compared to untreated control). (B) Ventricular IL-6 levels were significantly increased in T2D heart tissues than untreated control (Continued ) FIGURE 4 | tissues (p < 0.001). Naringin significantly lowered tissue IL-6 in T2D ventricular cells (p < 0.001 compared to untreated ventricular cells). No effect on basal IL-6 levels was observed in control naringin-treated ventricular tissue compared to untreated control (p 0.30). (C) Top: representative Western blotting analysis of NF-kB in myocardium from naringin-treated and untreated-control and T2D mice. Bottom: Densitometric analysis of Western blot experiments. Expression of p65-NF-kB was significantly upregulated in T2D myocardium tissue compared to control tissue (p < 0.001). Naringin treatment normalized the expression p65-NF-kB in T2D cardiac tissue (p < 0.001) with no effect on control (p 0.97). Data were normalized to housekeeping protein ß-actin and expressed. TNF-α and IL-6 measurements were obtained from n mice 5 mice/group, n determinations 10/group. Western blot experiments were carried out in n mice 3 mice/group, n WB 4/group. Values are expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, ***p < 0.001.

Naringin Improved Cardiomyocytes' Basal Glucose Transport and Lowered Blood Glucose
Diabetes results from a decreased glucose uptake into insulinsensitive tissues (Defronzo, 2009). In the current study, T2D cardiomyocytes had a significantly reduced glucose uptake compared to control, and naringin improved the depressed T2D cardiomyocyte's glucose transport. Furthermore, naringin significantly lowered the blood glucose level in T2D mice. Although the downstream pathways by which impaired glucose transport contributes to diabetes are still poorly understood, it is widely accepted that a decrease in GLUT4 expression or deficient translocation plays a critical role (Stenbit et al., 1997). Thus, overexpression of GLUT4 in the db/db mice regularizes the anomalous glucose blood levels and cardiac contractile dysfunction (Belke et al., 2000). Naringin improved the depressed T2D cardiomyocyte's glucose transport, which could be related to an increase in GLUT4 expression and/or its antioxidant effects on GLUT4 translocation (Dhanya et al., 2015).

Naringin Effect on [Ca 2+ ] d in T2D Cardiomyocytes
We found that cardiomyocytes from T2D mice showed an impaired diastolic Ca 2+ regulation compared to controls. Intracellular Ca 2+ dysregulation has been implicated as an important cause of DCM (Levy et al., 1994;Sheikh et al., 2012). In healthy cardiomyocytes, diastolic [Ca 2+ ] is in the range of 100-120 nM (Marban et al., 1980;Mijares et al., 2014). It is maintained through the interplay of Ca 2+ transport mechanisms and ion channels, including sarcolemma and intracellular organelles, Ca 2+ influx-efflux, and ryanodine receptor-mediated Ca 2+ leak (Bers and Ginsburg, 2007;Bers, 2014;Freichel et al., 2017;Smith and Eisner, 2019). Although the specific mechanism underlying the diastolic Ca 2+ alterations in T2D cardiomyocytes have not been established yet, cardiac dysfunction in diabetes has been associated with detrimental expression of the sarcoplasmic reticulum Ca 2+ pump SERCA2 (Belke et al., 2004;Suarez et al., 2008) and an aberrant ryanodine receptor Ca 2+ leak (Santulli et al., 2015). This study revealed that naringin treatment significantly reduced [Ca 2+ ] d overload in T2D cardiomyocytes, which had never been investigated previously. This protective effect could be attributed to a combination of its antioxidant and anti-inflammatory effects as well as its ability to enhance K ATP channel expression, as we found in the present study. However, we discerned that the effect of naringin on [Ca 2+ ] d is not linked to its potential insulinotropic effect (Hameed, 2018) because, in isolated cardiomyocytes from T2D mice (devoid of any insulin action), we observed a dosedependent reduction of [Ca 2+ ] d when incubated with naringin (100-300 µM) (Supplementary Figure S2).

Oxidative Stress, Inflammation, Cardiac Injury, and Cell Viability
A significant finding of the present study is the increase in ROS generation in cardiomyocytes isolated from T2D mice. It is widely accepted that increased ROS plays a vital role in the pathogenesis of DCM (Evans et al., 2003). Oxidative stress appears to generate insulin resistance by altering intracellular signaling, including increased ryanodine receptor phosphorylation and downregulation of sarco-endoplasmic reticulum Ca 2+ -ATPase transcription (Evans et al., 2003). The present study revealed that naringin induced a marked reduction of ROS generation, an FIGURE 5 | Effects of naringin on calpain activity, cardiac injury, and cell viability. (A) Calpain activity was significantly increased in T2D cardiomyocytes compared to control cardiomyocytes (p < 0.001). Naringin treatment significantly reduced calpain activity compared to untreated T2D cardiomyocytes (p < 0.001). Naringin treatment did not affect basal calpain activity in control cardiomyocytes (p 0.99 compared to untreated). Calpain data was obtained from n mice 5/group; n cells 15-20/group. (B) LDH leak was significantly higher in T2D cardiomyocytes compared to control cardiomyocytes (p < 0.001). Naringin treatment significantly diminished the LDH leak compared to untreated T2D cardiomyocytes. Naringin did not modify basal LDH leak in control cardiomyocytes (p 0.81 compared to untreated). LDH measurements were obtained from n mice 5/group; n cells 17-20/group. (C) T2D cardiomyocytes' viability was significantly less than in control cardiomyocytes (p < 0.001). Naringin treatment increased T2D cardiomyocytes' viability compared to naringin-untreated cardiomyocytes (p < 0.001). Cardiomyocyte viability was not significantly different in the naringin group compared to the naringin-untreated control group (p 0.91 compared to untreated). Data for cell viability was obtained from n mice 5/group and n cells 19-30/group. All values were normalized to control untreated cardiomyocytes and expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, ***p < 0.001.
Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 719268 important hallmark in T2D. These findings concur with previous reports about the antioxidant effects of naringin in diverse tissues (Dhanya et al., 2015), which appear to be mediated by increasing activities of catalase and superoxide dismutase (Kwatra et al., 2016), suppressing proinflammatory cytokine production (Marfella et al., 2003) and thus preventing mitochondrial ROS generation, rather than enhancing cellular ROS removal systems (Facundo et al., 2006). Furthermore, recent studies have underscored the notion that Ca 2+ and ROS signaling systems are intimately integrated, such that Ca 2+dependent regulation of components of ROS homeostasis might influence intracellular redox balance and vice versa (Brookes et al., 2004). Thus, the reduction of ROS generation by naringin may be a consequence of lowering [Ca 2+ ] d and/or the activation of K ATP . Proinflammatory cytokines, like TNF-α and IL-6, and the transcriptional factor NF-κB are involved in the pathogenesis of DCM (Lorenzo et al., 2011;Palomer et al., 2013;Grubic Rotkvic et al., 2021). Our results revealed that TNF-α and IL-6, and NF-κB were elevated in the T2D myocardium, which confirms the involvement of inflammation in the pathogenesis of diabetes-induced cardiac injury (Grubic Rotkvic et al., 2021). Activation NF-κB is one of the key transcription factors involved in the expression of proinflammatory cytokines and hypertrophyrelated genes (Yang et al., 2009). Its activation in the diabetic heart causes the release of interleukins and TNF-α and amplifies oxidative stress resulting in endothelial dysfunction in T2D (Yang et al., 2009). Naringin demonstrated its cardioprotective nature by reducing the content of proinflammatory cytokines and the expression of transcriptional factor NF-κB, actively involved in developing T2D-induced cardiomyopathy (Lorenzo et al., 2011).
In addition, naringin's effects were accompanied by a decrease in the elevated calpain activity in T2D treated cardiomyocytes. Calpains are a family of Ca 2+ -dependent serine proteases, which play a vital role in physiological and pathological processes, including diabetes mellitus and ischemia (Wan et al., 2015). Calpains activation by Ca 2+ causes proteolysis-induced cardiomyocyte injury and death (Trumbeckaite et al., 2003). The elevated calpain activity agrees with previous studies, which showed calpain activation in cultured endothelial cells and vascular tissues from T1D and T2D subjects (Wang et al., 2008;Chen B. et al., 2014). The effect of naringin on calpain activity could be mediated by reducing [Ca 2+ ] d elicited by this flavonoid.
T2D cardiomyocytes showed an elevated index of cell damage evidenced by an elevated LDH leak and lower cell viability (MTT assay) compared to control cardiomyocytes. Naringin significantly lowered LDH leak (cardiomyocyte injury) and increase cell viability compared to untreated T2D cardiomyocytes. It is plausible to suggest that both of these naringin effects could be the result of the reduction of [Ca 2+ ] d since intracellular Ca 2+ dyshomeostasis can activate proapoptotic or necrosis signaling pathways, which will reduce cell viability, as has been shown in diabetic myocardium (Frustaci et al., 2000;Cai et al., 2002).

Effects of Naringin on the Expression of Cardiac K ATP Channels
Our study shows a reduction in the expression of Kir6.2, SUR1, and SUR2 subunits of K ATP channels in T2D cardiac cells. Our data partly concur with a previous report by Fancher (Fancher et al., 2013), who studied the expression of Kir6.1 and SUR1 and found a reduction of both subunits' expression in diabetic cardiomyocytes. K ATP channels play a key role in regulating cell functions under physiologic and pathological conditions (Noma, 1983;Inagaki et al., 1995). Structurally K ATP channels are composed of two types of subunits, the inwardly rectifying K + channel (Kir6.1, Kir6.2) and the sulphonylurea subunits (SUR1 and SUR2) (Inagaki et al., 1995;Shyng and Nichols, 1997). Interestingly, our study shows that treatment with naringin provoked the upregulation of Kir6.2, SUR1, and SUR2 subunits of the K ATP channels in T2D cardiomyocytes compared with untreated T2D ventricular cells. These findings suggest that the downregulation of cardiac K ATP may play an essential role in the pathophysiology of diabetic cardiomyopathy. , and SUR2 subunits in T2D show a significant downregulation compared to control tissue (64, 63, and 37%, respectively). Naringin treatment significantly increased the expression of Kir6.2 (173%), SUR1 (130%), and SUR2 (100%) subunits compared with untreated T2D ventricular homogenate. The SUR2 expression in naringin treatment-T2D was greater than control by 26% and control naringin-treated. by 22% (p < 0.001). No effect on K ATP channels subunits in control ventricular tissues was observed. Data were normalized to housekeeping protein ß-actin and expressed as means ± S.D. n mice 3, n westerns 3-4. Statistical analysis was done using paired Student's independent t-test, ns p > 0.05, **p < 0.01, ***p < 0.001.  (12%) in the presence of glibenclamide. One possibility is that naringin, through its antioxidant effect, also inhibits the sarcolemmal cation (Ca 2+ and Na + ) influx activated by oxidative stress as observed in erythrocyte (Brand et al., 2003;Shaik et al., 2012). This data support the concern about the use of sulfonylurea hypoglycemic agents (first generation) in diabetic patients due to an increased occurrence of adverse cardiovascular outcomes (Pantalone et al., 2009;Tzoulaki et al., 2009;Aronson and Edelman, 2014;Gilbert and Krum, 2015), and the higher hospital mortality after angioplasty myocardial infarction (Garratt et al., 1999;Bell and Goncalves, 2019). However, other studies have suggested that it's use in patients with T2D is not associated with increased cardiovascular risk and all-cause mortality (Rados et al., 2016). Therefore, the safety of sulfonylurea in diabetic patients remains controversial and routine utilization as second-line agents may be acceptable in the short term, in line with the American Diabetes Association Standards of Care (American Diabetes, 2020).

Nicorandil Mimics the Effects of Naringin on [Ca 2+ ] d in T2D
Nicorandil is a cardioprotective agent with two postulated mechanisms of action: an increased K + conductance of the cardiomyocytes by activation K ATP channel and a NO donor, increasing cyclic guanosine monophosphate concentration in the cardiac cell (Uchida et al., 1978;Holzmann, 1983;Tsuchida et al., 2002;Mccully and Levitsky, 2003). Our data show that nicorandil mimics the effects of naringin lowering [Ca 2+ ] d in T2D cardiomyocytes, a pharmacological action that appears to be mediated mainly by K ATP channels since glibenclamide pretreatment inhibited mostly of its effect on [Ca 2+ ] d in T2D cardiomyocytes (Supplementary Figure S2). The remained effect of nicorandil on [Ca 2+ ] d in the presence of glibenclamide (26%) could be attributed to its NO donor effect (Taira, 1989). Interestingly, in contrast to naringin, nicorandil further elevated blood glucose levels in T2D. These findings allow us to suggest that the effects of naringin and nicorandil on [Ca 2+ ] d appear to be mediated mainly by its action of K ATP channels (Mccully and Levitsky, 2003) and not by its NO donor effect.

Study Limitations
Despite the originality of our study, some limitations should be acknowledged. The cardioprotective effects of naringin were studied in young male T2D mice (12 weeks) and not in females or in aged mice (24 months), which may have more advanced cardiomyopathy. The effect of naringin on cardiomyocyte's glucose uptake was established only on basal conditions and not upon insulin stimulation. The cardioprotective action of naringin was explored at doses between 20 and 60 mg/kg/d bodyweight but not at higher doses (>60 mg/kg/d). The short-term treatment (4 weeks) did not allow us to establish whether a more extended treatment may provoke greater cardioprotection in T2D cardiac cells. Finally, the involvement of sarcolemmal K ATP or mitochondrial K ATP channels on the naringin cytoprotection was not discerned, and its effect on T2D cardiomyocyte's contractile response was not studied. In control cardiomyocytes glibenclamide did not modify [Ca 2+ ] d (p 0.06) and DCF rate (p 0.99). Experimental data were obtained from n mice 5 mice/group; n cells 15-21/group. Values are expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, **p < 0.01, ***p < 0.001.

CONCLUDING REMARKS
The present study demonstrated that the citrus fruit flavonoid naringin protects against cardiomyopathy in T2D mice. The mechanism by which naringin exerts its cardioprotective effect appears to be multifaceted. The results of this study relate its cardioprotective effects to the reduction of intracellular Ca 2+ overload, limiting the increase in ROS generation, reducing calpain activity, TNF-α and IL-6 level, decreasing NF-κB expression, and upregulating K ATP channels. These results suggest that naringin may be a novel therapeutic candidate for protection against diabetes-induced myocardial dysfunction.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ] d measurements, n mice 5/group and n cells 13-18/group. For the fasting glucose determination, n mice 5/group and n measurements 10/group. Values are expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, **p < 0.01, ***p < 0.001.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care and Use Committees at the Mount Sinai Medical Center, Miami Beach, FL, United States.

AUTHOR CONTRIBUTIONS
JL and JA contributed conception and design of the study; AU, AM, VF, and JL performed the experiments. AU, AM, VF, JA, and JL analyzed the experimental data. All authors contributed to manuscript revision and approved the submitted version.

FUNDING
This work was supported by the Florida Heart Research Foundation to JL.

ACKNOWLEDGMENTS
We thank P. D. Allen for his comments on the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.719268/ full#supplementary-material Supplemental Figure S1 | Glibenclamide partially inhibited the effect of nicorandil on [Ca 2+ ] d in T2D cardiomyocytes. To discriminate the mechanisms of action of nicorandil due to K ATP channel-opening properties from those due to its NO-donor properties [Ca 2+ ] d was measured in control and T2D isolated cardiomyocytes before and after incubation with either: (A) the K ATP channel opener nicorandil (300 µM); (B) the K ATP channel antagonist glibenclamide (10 µM); (C) glibenclamide (10 µM) for 2 h, followed by nicorandil (300 µM). Nicorandil reduced significantly [Ca 2+ ] d in T2D but not in control cardiomyocytes. Glibenclamide produced a further elevation of [Ca 2+ ] d in T2D cardiomyocytes but not in controls cardiomyocytes; glibenclamide, followed by nicorandil, significantly reduced the effect of nicorandil on [Ca 2+ ] d . n mice 3 controls and 3 T2D mice and n cells 20-22/group. Values are expressed as means ± S.D. Statistical analysis was performed as described before, ns p > 0.05, ***p < 0.001.