SGLT2 Inhibitor Empagliflozin Modulates Ion Channels in Adult Zebrafish Heart.

Empagliflozin, an inhibitor of sodium-glucose co-transporter 2 (iSGLT2), improves cardiovascular outcomes in patients with and without diabetes and possesses an antiarrhythmic activity. However, the mechanisms of these protective effects have not been fully elucidated. This study aimed to explore the impact of empagliflozin on ion channel activity and electrophysiological characteristics in the ventricular myocardium. The main cardiac ionic currents (INa, ICaL, ICaT, IKr, IKs) and action potentials (APs) were studied in zebrafish. Whole-cell currents were measured using the patch clamp method in the isolated ventricular cardiomyocytes. The conventional sharp glass microelectrode technique was applied for the recording of APs from the ventricular myocardium of the excised heart. Empagliflozin pretreatment compared to the control group enhanced potassium IKr step current density in the range of testing potentials from 0 to +30 mV, IKr tail current density in the range of testing potentials from +10 to +70 mV, and IKs current density in the range of testing potentials from -10 to +20 mV. Moreover, in the ventricular myocardium, empagliflozin pretreatment shortened AP duration APD as shown by reduced APD50 and APD90. Empagliflozin had no influence on sodium (INa) and L- and T-type calcium currents (ICaL and ICaT) in zebrafish ventricular cardiomyocytes. Thus, we conclude that empagliflozin increases the rapid and slow components of delayed rectifier K+ current (IKr and IKs). This mechanism could be favorable for cardiac protection.

Low-Dose Empagliflozin Improves Systolic Heart Function after Myocardial Infarction in Rats: Regulation of MMP9, NHE1, and SERCA2a.

The effects of the selective sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin in low dose on cardiac function were investigated in normoglycemic rats. Cardiac parameters were measured by intracardiac catheterization 30 min after intravenous application of empagliflozin to healthy animals. Empagliflozin increased the ventricular systolic pressure, mean pressure, and the max dP/dt (p < 0.05). Similarly, treatment with empagliflozin (1 mg/kg, p.o.) for one week increased the cardiac output, stroke volume, and fractional shortening (p < 0.05). Myocardial infarction (MI) was induced by ligation of the left coronary artery. On day 7 post MI, empagliflozin (1 mg/kg, p.o.) improved the systolic heart function as shown by the global longitudinal strain (-21.0 ± 1.1% vs. -16.6 ± 0.7% in vehicle; p < 0.05). In peri-infarct tissues, empagliflozin decreased the protein expression of matrix metalloproteinase 9 (MMP9) and favorably regulated the cardiac transporters sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) and sodium hydrogen exchanger 1 (NHE1). In H9c2 cardiac cells, empagliflozin decreased the MMP2,9 activity and prevented apoptosis. Empagliflozin did not alter the arterial stiffness, blood pressure, markers of fibrosis, and necroptosis. Altogether, short-term treatment with low-dose empagliflozin increased the cardiac contractility in normoglycemic rats and improved the systolic heart function in the early phase after MI. These effects are attributed to a down-regulation of MMP9 and NHE1, and an up-regulation of SERCA2a. This study is of clinical importance because it suggests that a low-dose treatment option with empagliflozin may improve cardiovascular outcomes post-MI. Down-regulation of MMPs could be relevant to many remodeling processes including cancer disease.

Model of Chronic Thromboembolic Pulmonary Hypertension in Rats Caused by Repeated Intravenous Administration of Partially Biodegradable Sodium Alginate Microspheres.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and life-threatening complication of pulmonary embolism. As existing animal models of CTEPH do not fully recapitulate complex disease pathophysiology, we report a new rat model for CTEPH evoked by repetitive embolization of the distal pulmonary artery branches with partially biodegradable alginate microspheres (MSs). MSs (180 ± 28 µm) were intravenously administered eight times at 4-day intervals; control animals received saline. The validity of the model was confirmed using transthoracic echocardiography, exercise testing, catheterization of the right ventricle, and histological examination of the lung and heart. The animals in the CTEPH group demonstrated a stable increase in right ventricular systolic pressure (RVSP) and decreased exercise tolerance. Histopathological examination revealed advanced medial hypertrophy in the small pulmonary arteries associated with fibrosis. The diameter of the main pulmonary artery was significantly larger in the CTEPH group than in the control group. Marinobufagenin and endothelin-1 serum levels were significantly elevated in rats with CTEPH. In conclusion, repetitive administration of alginate MSs in rats resulted in CTEPH development characterized by specific lung vasculature remodeling, reduced exercise tolerance, and a persistent rise in RVSP. The developed model can be used for pre-clinical testing of promising drug candidates.

Non-inferiority of microencapsulated mesenchymal stem cells to free cells in cardiac repair after myocardial infarction: A rationale for using paracrine factor(s) instead of cells.

A major translational barrier to the use of stem cell (SC)-based therapy in patients with myocardial infarction (MI) is the lack of a clear understanding of the mechanism(s) underlying the cardioprotective effect of SCs. Numerous paracrine factors from SCs may account for reduction in infarct size, but myocardial salvage associated with transdifferentiation of SCs into vascular cells as well as cardiomyocyte-like cells may be involved too. In this study, bone marrow-derived rat mesenchymal SC (MSCs) were microencapsulated in alginate preventing viable cell release while supporting their secretory phenotype. The hypothesis on the key role of paracrine factors from MSCs in their cardioprotective activity was tested by comparison of the effect of encapsulated vs free MSCs in the rat model of MI. Intramyocardial administration of both free and encapsulated MSCs after MI caused reduction in scar size (12.1 ± 6.83 and 14.7 ± 4.26%, respectively, vs 21.7 ± 6.88% in controls, P = 0.015 and P = 0.03 respectively). Scar size was not different in animals treated with free and encapsulated MSC (P = 0.637). These data provide evidence that MSC-derived growth factors and cytokines are crucial for cardioprotection elicited by MSC. Administration of either free or encapsulated MSCs was not arrhythmogenic in non-infarcted rats. The consistency of our data with the results of other studies on the major role of MSC secretome components in cardiac protection further support the theory that the use of live, though encapsulated, cells for MI therapy may be replaced with heart-targeted-sustained delivery of growth factors/cytokines.

Nicotinamide riboside has protective effects in a rat model of mesenteric ischaemia-reperfusion.

Acute mesenteric ischaemia is a syndrome caused by inadequate blood flow through the mesenteric vessels, resulting in ischaemia and eventual gangrene of the bowel wall. Although relatively rare, it is a potentially life-threatening condition. The maintenance of haemodynamic stability, along with adequate oxygen saturation, and the correction of any electrolyte imbalance, are of the utmost importance. However, nicotinamide adenine dinucleotide (NAD) biosynthesis modulation by precursor introduction can also be a powerful tool for preventing injury. Nicotinamide riboside is a pyridine-nucleoside form of vitamin B3 that functions as a precursor to NAD+ . The present study investigated nicotinamide riboside's effect on endothelium functional state, microcirculation and intestinal morphology in acute mesenteric ischaemia and reperfusion. Mesenteric ischaemia was simulated after the adaptation period (15 minutes) by occluding the superior mesenteric artery for 60 minutes, followed by a reperfusion period of 30 minutes. The functional state of intestinal microcirculation was evaluated by laser Doppler flowmetry. Endothelial functional activity was studied by using wire myography. Intestinal samples were stained with haematoxylin and eosin for histological analysis. The results revealed that nicotinamide riboside protects the intestinal wall from ischaemia-reperfusion injury, as well as improving the relaxation function of mesenteric vessels. Nicotinamide riboside's protective effect in small intestine ischaemia-reperfusion can be used to reduce ischaemia-reperfusion injury, as well as to preserve intestinal grafts until transplant.

Sympathetic Denervation and Pharmacological Stimulation of Parasympathetic Nervous System Prevent Pulmonary Vascular Bed Remodeling in Rat Model of Chronic Thromboembolic Pulmonary Hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) develops in 1.5-2.0% of patients experiencing pulmonary embolism (PE) and is characterized by stable pulmonary artery obstruction, heart failure, and poor prognosis. Little is known about involvement of autonomic nervous system (ANS) in the mechanisms of CTEPH. This study was aimed at evaluation of the effect of vagal and sympathetic denervation, as well as stimulation of the parasympathetic nervous system, on the outcomes of CTEPH in rats. CTEPH was induced by multiple intravenous injections of alginate microspheres. Sympathetic and vagal denervation was performed using unilateral surgical ablation of the stellate ganglion and vagotomy, respectively. Stimulation of the parasympathetic nervous system was carried out by administering pyridostigmine. The effect of neuromodulatory effects was assessed in terms of hemodynamics, histology, and gene expression. The results demonstrated the key role of ANS in the development of CTEPH. Sympathetic denervation as well as parasympathetic stimulation resulted in attenuated pulmonary vascular remodeling. These salutary changes were associated with altered MMP2 and TIMP1 expression in the lung and decreased FGFb level in the blood. Unilateral vagotomy had no effect on physiological and morphological outcomes of the study. The data obtained contribute to the identification of new therapeutic targets for CTEPH treatment.

The Hypoglycemic and Hypocholesterolemic Activity of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus Cell Culture Biomass in Rats with High-Fat Diet-Induced Obesity.

Obesity, and its consequences for human health, is a huge and complicated problem that has no simple solution. The constant search for natural and safe compounds with systemic action that can be used for obesity prophylactics and treatment is hampered by the limited availability and variable quality of biomass of wild medicinal plants. Plant cell biotechnology is an alternative approach for the sustainable production of vegetative biomass or individual phytochemicals with high therapeutic potential. In this study, the suspension cell biomass of the medicinal plants, Dioscorea deltoidea Wall., Tribulus terrestris L., and Panax japonicus (T. Nees) C.A. Mey, produced in 20 L and 630 L bioreactors, were tested for therapeutic effects in rat models with alimentary-induced obesity. Three-month intake of water infusions of dry cell biomass (100 mg/g body weight) against the background of a hypercaloric diet reduced weight gain and the proportion of fat mass in the obese animals. In addition, cell biomass preparation reduced the intracellular dehydration and balanced the amounts of intra- and extracellular fluids in the body as determined by bioimpedance spectroscopy. A significant decrease in the glucose and cholesterol levels in the blood was also observed as a result of cell biomass administration for all species. Hypocholesterolemic activity reduced in the line P. japonicus > D. deltoidea > T. terrestris/liraglutide > intact group > control group. By the sum of parameters tested, the cell culture of D. deltoidea was considered the most effective in mitigating diet-induced obesity, with positive effects sometimes exceeding those of the reference drug liraglutide. A safety assessment of D. deltoidea cell phytopreparation showed no toxic effect on the reproductive function of the animals and their offspring. These results support the potential application of the biotechnologically produced cell biomass of medicinal plant species as safe and effective natural remedies for the treatment of obesity and related complications, particularly for the long-term treatment and during pregnancy and lactation periods when conventional treatment is often contraindicated.

Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells.

Recovery of the contractile function of the heart and the regeneration of the myocardium after ischemic injury are contemporary issues in regenerative medicine and cell biology. This study aimed to analyze early transcriptional events in cardiac tissue after infarction and to explore the cell population that can be isolated from myocardial tissue. We induced myocardial infarction in Wistar rats by permanent ligation of the left coronary artery and showed a change in the expression pattern of Notch-associated genes and Bmp2/Runx2 in post-MI tissues using RNA sequencing and RT-PCR. We obtained primary cardiac mesenchymal cell (CMC) cultures from postinfarction myocardium by enzymatic dissociation of tissues, which retained part of the activation stimulus and had a pronounced proliferative potential, assessed using a "xCELLigence" real-time system. Hypoxia in vitro also causes healthy CMCs to overexpress Notch-associated genes and Bmp2/Runx2. Exogenous activation of the Notch signaling pathway by lentiviral transduction of healthy CMCs resulted in a dose-dependent activation of the Runx2 transcription factor but did not affect the activity of the Bmp2 factor. Thus, the results of this study showed that acute hypoxic stress could cause short-term activation of the embryonic signaling pathways Notch and Bmp in CMCs, and this interaction is closely related to the processes of early myocardial remodeling after a heart attack. The ability to correctly modulate and control the corresponding signals in the heart can help increase the regenerative capacity of the myocardium before the formation of fibrotic conditions.

Effect of Phytopreparations Based on Bioreactor-Grown Cell Biomass of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus on Carbohydrate and Lipid Metabolism in Type 2 Diabetes Mellitus.

In the present study, we explored the therapeutic potential of bioreactor-grown cell cultures of the medicinal plant species Dioscorea deltoidea, Tribulus terrestris and Panax japonicus to treat carbohydrate metabolism disorders (CMDs) in laboratory rats. In the adrenaline model of hyperglycemia, aqueous suspensions of cell biomass pre-administered at a dose of 100 mg dry biomass/kg significantly reduced glucose level in animal blood 1-2.5 h (D. deltoidea and T. terrestris) or 1 h (P. japonicus) after adrenaline hydrochloride administration. In a streptozotocin-induced model of type 2 diabetes mellitus, the cell biomass of D. deltoidea and T. terrestris acted towards normalization of carbohydrate and lipid metabolism, as evidenced by a significant reduction of daily diuresis (by 39-57%), blood-glucose level (by 46-51%), blood content in urine (by 78-80%) and total cholesterol (25-36%) compared to animals without treatment. Bioactive secondary metabolites identified in the cell cultures and potentially responsible for their actions were deltoside, 25(S)-protodioscin and protodioscin in D. deltoidea; furostanol-type steroidal glycosides and quinic acid derivatives in T. terrestris; and ginsenosides and malonyl-ginsenosides in P. japonicus. These results evidenced for high potential of bioreactor-grown cell suspensions of these species for prevention and treatment of CMD, which requires further investigation.

Comparative efficacy of empagliflozin and drugs of baseline therapy in post-infarct heart failure in normoglycemic rats.

The study aimed to investigate the effects of the sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin on chronic heart failure (HF) in normoglycemic rats. The effects of empagliflozin were compared with the standard medications for HF, e.g., angiotensin-converting enzyme (ACE) inhibitor fosinopril, beta-blocker bisoprolol, and aldosterone antagonist spironolactone. Myocardial infarction (MI) was induced in male Wistar rats via permanent ligation of the left descending coronary artery. One-month post MI, 50 animals were randomized into 5 groups (n = 10): vehicle-treated, empagliflozin (1.0 mg/kg), fosinopril (10 mg/kg), bisoprolol (10 mg/kg), and spironolactone (20 mg/kg). All medications except empagliflozin were titrated within a month and administered per os daily for 3 months. Echocardiography, 24-hour urine volume test, and treadmill exercise tests were performed at the beginning and at the end of the study. Treatment with empagliflozin slowed the progression of left ventricular dysfunction: LV sizes and ejection fraction were not changed and the minute volume was significantly increased (from 52.0 ± 15.5 to 61.2 ± 21.2 ml/min) as compared with baseline. No deaths occurred in empagliflozin group. The 24-hour urine volume tends to be higher in empagliflozin and spironolactone groups than in vehicle and fosinopril group. Moreover, empagliflozin exhibited maximal physical exercise tolerance in comparison with all investigated groups (289 ± 27 s versus 183 ± 61 s in fosinopril group, 197 ± 95 s in bisoprolol group, and 47 ± 46 s in spironolactone group, p = 0.0035 for multiple comparisons). Sodium-glucose co-transporter 2 inhibitor empagliflozin reduced progression of left ventricular dysfunction and improved tolerance of physical exercise in normoglycemic rats with HF. Empagliflozin treatment was superior with respect to physical tolerance compared with fosinopril, bisoprolol, and spironolactone.

Antihypoxic Action of Panax Japonicus, Tribulus Terrestris and Dioscorea Deltoidea Cell Cultures: In Silico and Animal Studies.

Chemical diversity of secondary metabolites provides a considerable variety of pharmacological actions with a significant extension due to their combinations in plant extracts. Production of plant-derived medicinal products in cell cultures has advantages because of the efficient use of different biotic and abiotic elicitors and better control of the developmental processes. Using PASS software, we predicted biological activity spectra for phytoconstituents identified in cell cultures of Panax japonicus (12 molecules), Tribulus terrestris (4 molecules), and Dioscorea deltoidea (3 molecules). Mechanisms of action associated with the antihypoxic effect were predicted for the majority of molecules. PharmaExpert software allowed analyzing possible synergistic or additive effects of the combinations of phytoconstituents associated with the antihypoxic action. Experimental studies of the antihypoxic effect of the plants' extracts in water and ethanol have been performed in 3 animal models: Acute asphyctic hypoxia (AAH), Acute haemic hypoxia (AHeH), and Acute histotoxic hypoxia (AHtH). Effects of Panax japonicus and Tribulus terrestris preparations exceeded the activity of the reference drug Mexidol in the AHtH model. In the AHeH model, all preparations demonstrated moderate activity; the most potent has been observed for Dioscorea deltoidea. Thus, we found that experimental studies in animal models have confirmed the in silico prediction.