Modulation of N-methyl-d-aspartate receptors in isolated rat heart
ABSTRACT
This study addresses the scarcity of detailed data concerning the precise role of N-methyl-D-aspartate receptors (NMDA-R) within the cardiovascular system, particularly in the direct regulation of cardiac function. The primary objective of this investigation was to meticulously examine the acute effects of NMDA, the canonical agonist for these receptors, and DL-homocysteine thiolactone (DL-Hcy TLHC), a compound implicated in NMDA-R activation under pathological conditions. These substances were applied both individually and in specific combinations with modulators known to interact with NMDA-R: glycine, a co-agonist essential for receptor activation; memantine, a non-competitive NMDA-R antagonist; and ifenprodil, an allosteric modulator selective for GluN2B-containing NMDA-R. All experiments were conducted on the isolated rat heart, a well-established model for studying intrinsic cardiac function in a controlled environment.
The experimental setup utilized hearts from Wistar albino rats, which were retrogradely perfused through the aorta according to the standardized Langendorff technique. This method ensures a constant perfusion pressure, maintaining physiological conditions for the myocardium. The experimental protocol for all groups was designed with three distinct phases: an initial stabilization period to ensure baseline cardiac function; followed by the controlled application of the estimated substance for a duration of 5 minutes to observe acute effects; and finally, a subsequent wash-out period lasting 10 minutes to assess reversibility of effects. To comprehensively monitor myocardial performance, a high-sensitivity sensor was carefully positioned within the left ventricle. This allowed for continuous, real-time registration of several key cardiodynamic parameters, including the maximum rate of pressure development (dp/dt max), the minimum rate of pressure development (dp/dt min), systolic left ventricular pressure, diastolic left ventricular pressure, and heart rate. In parallel, coronary flow (CF), a critical measure of myocardial perfusion, was precisely determined using flowmetric techniques. Beyond functional parameters, the study also sought to investigate biochemical markers of oxidative stress. To this end, samples of the coronary venous effluent were collected and spectrophotometrically analyzed for the presence and concentration of following oxidative stress biomarkers: thiobarbituric acid reactive substances (TBARS), which indicate lipid peroxidation; nitrite (NO2-), a stable metabolite of nitric oxide; and superoxide anion (O2-) and hydrogen peroxide (H2O2), both prominent reactive oxygen species.
The findings revealed nuanced effects of the tested compounds on cardiac function and oxidative stress. Interestingly, NMDA administered alone, despite being the canonical agonist, did not induce any discernible change in any of the observed cardiodynamic or oxidative stress parameters in the isolated heart model under these acute conditions. In stark contrast, DL-Hcy TLHC, when applied alone, elicited a significant reduction across most of the assessed cardiodynamic parameters, indicating a depressant effect on myocardial performance. Similarly, the combined application of NMDA and DL-Hcy TLHC with glycine, which is essential for full NMDA-R activation, also resulted in a marked reduction of most cardiodynamic parameters, suggesting that full activation of NMDA-R, particularly in the presence of DL-Hcy TLHC, leads to cardiac depression. Furthermore, the NMDA-R antagonists, memantine and ifenprodil, independently induced a reduction in cardiodynamic parameters and coronary flow. Notably, these antagonists also influenced some of the estimated oxidative stress biomarkers, suggesting a complex interplay between NMDA-R modulation, cardiac mechanics, and redox balance.
Key words: N-methyl-D-aspartate receptors, homocysteine, memantine, ifenprodil, oxidative stress, cardiodynamics, isolated rat heart
Introduction
The N-methyl-D-aspartate receptors (NMDA-R) constitute a critical and highly complex subset within the broader family of ionotropic glutamate receptors. This family also encompasses the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-R), kainate receptors, and delta (δ) receptors, all of which play crucial roles in excitatory neurotransmission. NMDA-R are structurally defined as heterotetrameric protein complexes, meaning they are composed of four distinct protein subunits that assemble to form a functional ion channel. Specifically, each functional NMDA-R is typically formed by two obligatory GluN1 subunits, which are responsible for binding the co-agonist glycine, and two additional subunits that can be either GluN2 subunits (GluN2A-D) or, in some cases, one GluN2 subunit combined with one GluN3 subunit (GluN3A-B). The precise composition of these subunits dictates the specific pharmacological and biophysical properties of the individual receptor. These subunits are meticulously arranged to construct a central pore that traverses the cell membrane, effectively functioning as a calcium-permeable ion channel.
The unique specificities that distinguish NMDA-R from other members of the glutamate receptor family are multifaceted and physiologically significant. Foremost among these is the absolute requirement for the simultaneous presence of two distinct co-agonists for receptor activation: glutamate, the primary excitatory neurotransmitter, and glycine, an indispensable co-agonist that binds to the GluN1 subunit. Without both ligands present, the receptor remains largely inactive. Secondly, a hallmark feature of NMDA-R activation is the subsequent entry of substantial quantities of calcium ions into the cell’s interior. This significant calcium influx serves as a critical second messenger, initiating a myriad of intracellular signaling cascades that are vital for synaptic plasticity, learning, and memory, but can also be detrimental under conditions of overstimulation. Thirdly, NMDA-R function is subject to a dual layer of control: it is regulated by the binding of its specific ligands (glutamate and glycine), but also profoundly influenced by changes in the membrane potential of the cell. This voltage-dependent block by magnesium ions (Mg2+), which occludes the channel pore at resting membrane potentials, is a key mechanism that allows NMDA-R to act as “coincidence detectors,” only becoming fully active when both presynaptic glutamate release and postsynaptic depolarization occur simultaneously. The diverse array of NMDA-R characteristics, including their calcium permeability, single-channel conductance, opening rates, and the degree of voltage-dependent magnesium block, can vary significantly depending on the specific subtypes of GluN2 or GluN3 subunits that comprise the heterotetramer. This subunit diversity provides a rich pharmacological landscape for selective drug development.
N-methyl-D-aspartate (NMDA) is a synthetic compound that specifically binds to the glutamate binding site on the NMDA-R and, upon binding, exerts an effect functionally identical to that of endogenous glutamate, thus serving as a potent agonist. Homocysteine (Hcy), an amino acid intermediate in methionine metabolism, is also known to bind to the glutamate binding site of NMDA-R. Crucially, it is widely theorized that the detrimental effects associated with hyperhomocysteinemia (HHcy), a condition characterized by abnormally elevated plasma levels of Hcy (typically above 15 µmol/L), in various pathological states, particularly cardiovascular and neurological disorders, arise significantly from the excessive and pathological activation of NMDA receptors. The therapeutic use of memantine, a non-competitive NMDA-R antagonist, in the context of Alzheimer’s disease exemplifies the neuroprotective potential of modulating these receptors. Memantine exerts its beneficial neuroprotective effects by mitigating the negative consequences of excessive NMDA-R stimulation, particularly bearing in mind the fact that amyloid-beta, a key pathological protein in Alzheimer’s, is known to exert its toxic effects, in part, by increasing the concentration of glutamate in the extrasynaptic space, leading to excitotoxicity.
Beyond traditional competitive and non-competitive antagonists, substances categorized as allosteric modulators of NMDA-R are increasingly garnering significant attention within pharmacological research. This heightened interest stems from their unique capacity for fine-tuned regulation of receptor function and the potential for achieving higher selectivity based on the specific subunit composition of the receptor. For instance, ifenprodil is a well-characterized compound that causes noncompetitive inhibition of NMDA-R that specifically contain the GluN2B subunit. This subunit-selective action is particularly appealing, as it offers the possibility of modulating specific neuronal circuits with reduced off-target effects. Indeed, ifenprodil has demonstrated promising neuroprotective effects during conditions such as cerebral ischemia, underscoring the therapeutic potential of selectively targeting specific NMDA-R subtypes.
While the physiological and pathological roles of NMDA-R are relatively well-established and extensively studied within the central nervous system (CNS), there is a growing body of data indicating the widespread localization of NMDA-R in other organs and peripheral tissues, extending beyond their traditional CNS domain. This includes their presence within the intricate structures of the cardiovascular system. Early investigations involving the time and spatial distribution of radiolabeled antagonists of NMDA-R provided compelling evidence, demonstrating a widespread acceptance and binding of these receptors in a variety of peripheral organs and tissues, including the heart itself. More detailed cellular and molecular studies have since confirmed that NMDA-Rs are indeed found in diverse cell types that comprise the cardiovascular system. Specifically, they have been identified in endothelial cells, which form the inner lining of blood vessels; in vascular smooth muscle cells, which regulate vascular tone; and notably, in cardiomyocytes, the contractile cells of the heart.
The activation of NMDA-Rs is fundamentally associated with the influx of calcium ions into the cytoplasm of the cell. Consequently, an overstimulation or pathological activation of these receptors can lead to an excessive increase in intracellular calcium content. This calcium overload, in turn, can profoundly disrupt cellular homeostasis and critically imbalance the delicate equilibrium between the production and elimination of free radicals, ultimately culminating in the deleterious condition of oxidative stress. Experimental evidence supports this link: it has been shown that the inactivation of NMDA-Rs, achieved through the targeted deletion of the GluN1 subunit, resulted in a significant decrease in the production of reactive oxygen species (ROS) in the heart during conditions of hyperhomocysteinemia (HHcy). Furthermore, this inactivation also led to reduced content of nitric oxide (NO) and matrix metalloproteinase 9 (MMP9) in cardiomyocyte mitochondria, highlighting the role of NMDA-R in regulating cellular redox state and protein degradation pathways within the heart.
Despite the growing evidence of NMDA-R presence in the cardiovascular system, many fundamental questions and considerable doubts persist regarding their precise physiological function in this context, as well as their specific role in various pathophysiological conditions affecting the heart and vasculature. Accordingly, the overarching aim of this study was to systematically investigate the acute effects of N-methyl-D-aspartate (NMDA), DL-homocysteine thiolactone (DL-Hcy TLHC), and their specific combinations with glycine, memantine, and ifenprodil, on key parameters of cardiac function, coronary blood flow, and the induction of oxidative stress in the isolated rat heart. In essence, this research sought to comprehensively assess the immediate consequences of acute modulation of NMDA-R activity in the heart by these aforementioned substances and to elucidate the possible involvement and contribution of oxidative stress in mediating these observed effects.
Material and Methods
Isolated rat heart preparation
The study meticulously included a total of seventy-two animals, systematically distributed into twelve animals per experimental group to ensure statistical robustness and reproducibility of the results. All experiments were conducted exclusively on male Wistar albino rats, which were approximately 8 weeks old and possessed a body mass ranging from 180 to 200 grams, ensuring homogeneity in physiological parameters within the study cohort. These animals were sourced from the Military Medical Academy, located in Belgrade, Serbia, ensuring a consistent genetic background. Prior to any experimental manipulation, anesthesia was meticulously induced using a combination of ketamine (10 mg/kg) and xylazine (5 mg/kg), ensuring a humane and pain-free state for the animals. Following successful anesthesia, the animals were humanely euthanized via cervical dislocation, a method approved under Schedule 1 of the Animals/Scientific Procedures Act 1986, UK, for its rapid and consistent termination of life.
Immediately following euthanasia, a prompt thoracotomy was performed to quickly access the thoracic cavity. Rapid cardiac arrest was subsequently induced by carefully superfusing the heart with ice-cold isotonic saline. This swift cooling and perfusion stops myocardial activity and preserves tissue integrity. The heart was then quickly and carefully excised from the thoracic cavity and promptly attached to a specialized Langendorff apparatus (Experimetria Ltd, 1062 Budapest, Hungary). This attachment was achieved via aortic cannulation, allowing for retrograde perfusion of the coronary arteries. To facilitate subsequent measurements, the left auricle was carefully removed, and a small incision was made in the left atrium. Through this incision, a high-sensitivity sensor (transducer BS4 73-0184, Experimetria Ltd, Budapest, Hungary) was meticulously positioned within the left ventricle, enabling continuous and real-time measurement of crucial cardiac function parameters. The isolated hearts were then retrogradely perfused under a constant perfusion pressure (CPP) of 70 cmH2O. The perfusate used was a complex Krebs-Henseleit solution, a physiologically balanced buffer designed to mimic extracellular fluid. This solution was composed of the following concentrations (in mmol/L): NaCl 118, KCl 4.7, CaCl2•2H2O 2.5, MgSO4•7H2O 1.7, NaHCO3 25, KH2PO4 1.2, glucose 11 (as an energy source), and pyruvate 2. Crucially, the solution was continuously equilibrated with a gas mixture of 95% O2 plus 5% CO2 to maintain optimal oxygenation and pH buffering at a physiological temperature of 37 ºC (pH 7.4).
Experimental protocol
The experimental protocol for all isolated hearts across the various experimental groups commenced with an essential 25-minute stabilization period. During this critical initial phase, each heart was rigorously subjected to a standardized test of coronary vascular reactivity. This test involved a brief, transient occlusion (20 seconds) of the coronary flow, immediately followed by simultaneous bolus injections of 5 mmol/L adenosine (60 µL delivered at a flow rate of 10 ml/min). Adenosine is a potent vasodilator and was used to elicit maximal coronary flow, thereby assessing the heart’s intrinsic vascular responsiveness. A strict criterion was applied at this stage: if the coronary flow (CF) did not increase by at least 100% compared with its control, baseline values during this test, the heart was deemed unsuitable for the study and was disposed of, ensuring only viable and reactive preparations were used. Coronary flow was precisely determined using flowmetric techniques, providing a continuous quantitative measure of myocardial perfusion. Once the coronary flow had completely stabilized, confirmed by three repeated measurements yielding the same value, samples of coronary effluent were collected to establish a control baseline (denoted as C). Immediately following this, the specific experimental protocol for substance application was initiated. Hearts were systematically perfused with one of the following precisely prepared solutions:
1. 100 µmol/L N-methyl-D-aspartate (NMDA)
2. 100 µmol/L N-methyl-D-aspartate (NMDA) + 100 µmol/L glycine
3. 10 µmol/L DL-homocysteine thiolactone (DL-Hcy TLHC)
4. 10 µmol/L DL-homocysteine thiolactone (DL-Hcy TLHC) + 100 µmol/L glycine
5. 100 µmol/L memantine
6. 1 µmol/L ifenprodil
Each of the applied substances was administered for a fixed duration of 5 minutes, allowing sufficient time for acute effects to manifest. This application phase was consistently followed by a wash-out period lasting for 10 minutes, designed to assess the reversibility of the observed effects and to return the heart to a near-baseline state before any subsequent interventions, if applicable. During the last minute of the substance application phase (to capture the maximal effect, denoted as E) and in the final minute of the wash-out period (to assess recovery, denoted as W), samples of the coronary venous effluent were meticulously collected. These samples were crucial for subsequent biochemical analyses of oxidative stress biomarkers. Concurrently, using the sensor carefully positioned within the left ventricle, the following critical parameters of myocardial function were continuously determined and recorded:
1. The maximum rate of pressure development in the left ventricle (dp/dt max), a primary indicator of myocardial contractility.
2. The minimum rate of pressure development in the left ventricle (dp/dt min), reflecting myocardial relaxation dynamics.
3. The systolic left ventricular pressure (SLVP), representing the peak pressure generated during ventricular contraction.
4. The diastolic left ventricular pressure (DLVP), indicating the pressure within the ventricle during relaxation and filling.
5. The heart rate (HR), a fundamental measure of cardiac chronotropy.
All research procedures, encompassing animal handling, surgical preparation, and experimental execution, were scrupulously carried out in strict accordance with the European Directive for the welfare of laboratory animals, specifically No 86/609/EEC, and adhered to the fundamental principles of Good Laboratory Practice (GLP). The entire experimental protocol received formal approval from the Ethical Committee of the Faculty of Medical Sciences, University of Kragujevac, Serbia, ensuring all ethical standards were met and animal welfare was prioritized throughout the study.
Biochemical Assays
To comprehensively assess the potential for oxidative stress induced by the various experimental treatments, a series of specific biochemical parameters were meticulously determined from the collected samples of the coronary venous effluent. These analyses were performed spectrophotometrically using a Shimadzu UV 1800 instrument, a widely recognized tool for precise quantification based on light absorption. The following key oxidative stress biomarkers were targeted for measurement:
1. The index of lipid peroxidation, which serves as a crucial indicator of oxidative damage to cellular membranes, was quantified by measuring thiobarbituric acid reactive substances (TBARS).
2. The level of nitrite (NO2-), a stable end-product of nitric oxide (NO) metabolism, used as an indirect measure of NO production.
3. The level of the superoxide anion radical (O2-), a highly reactive oxygen species that is a major contributor to oxidative stress.
4. The level of hydrogen peroxide (H2O2), another critical reactive oxygen species and a precursor to more damaging free radicals.
TBARS determination (index of lipid peroxidation)
The degree of lipid peroxidation within the heart, as reflected in the coronary venous effluent, was precisely estimated by quantifying thiobarbituric acid reactive substances (TBARS). This method involves a specific chemical reaction: a 1% solution of thiobarbituric acid in 0.05 M sodium hydroxide (NaOH) was meticulously incubated with the collected coronary effluent samples. This incubation was carried out at a high temperature of 100°C for a duration of 15 minutes, a process that facilitates the formation of a colored adduct with malondialdehyde, a common end-product of lipid peroxidation. The resulting chromophore was then measured spectrophotometrically at a wavelength of 530 nm. To ensure accurate background subtraction and account for any non-specific absorbance, Krebs–Henseleit solution, the perfusate itself, was consistently used as a blank probe, providing a baseline measurement. This methodology aligns with the established technique described by Ohkawa and colleagues in 1979.
Determination of the Nitrite Level
Nitric oxide (NO), a crucial signaling molecule in the cardiovascular system, is inherently unstable and rapidly decomposes *in vivo* to form more stable nitrite (NO2-) and nitrate (NO3-) products. Therefore, the measurement of nitrite levels was employed as a reliable indirect index of nitric oxide (NO) production within the isolated heart. The quantification of nitrite was performed using Griess’s reagent, a well-established spectrophotometric method. A precise volume of 0.5 mL of the perfusate sample was first precipitated with 200 µL of a 30% sulpho-salicylic acid solution. This mixture was then vigorously vortexed for 30 minutes to ensure complete precipitation of proteins, which could interfere with the assay. Following precipitation, the sample was centrifuged at 3000 x g to separate the precipitated proteins from the soluble supernatant containing the nitrite. Equal volumes of the supernatant and Griess’s reagent were then combined. Griess’s reagent itself is composed of 1% sulphanilamide in 5% phosphoric acid and 0.1% naphthalene ethylenediamine-dihydrochloride. This combined mixture was then incubated for 10 minutes in the dark, allowing the colorimetric reaction to fully develop. The resulting absorbance, directly proportional to the nitrite concentration, was measured spectrophotometrically at a wavelength of 543 nm. To ensure accurate quantification and allow for concentration determination, the nitrite levels in the samples were calculated by comparison to a standard curve generated using known concentrations of sodium nitrite. This methodology is based on the technique described by Green and colleagues in 1982.
Determination of the Level of the Superoxide Anion Radical
The level of the superoxide anion radical (O2-), a highly reactive and damaging species, was quantitatively measured via a nitro blue tetrazolium (NBT) reaction. This assay relies on the reduction of NBT by superoxide, which produces a colored formazan product. The reaction was performed in Tris buffer, a commonly used biological buffer, with the collected coronary venous effluent samples. The resulting absorbance, indicative of superoxide levels, was measured spectrophotometrically at a wavelength of 530 nm. As with other assays, Krebs–Henseleit solution, the perfusate, was consistently employed as a blank probe to account for any background absorbance and ensure the specificity of the measurement. This method is based on the principles outlined by Auclair and Voisin in 1985.
Determination of the Hydrogen Peroxide Level
The measurement of the level of hydrogen peroxide (H2O2), another significant reactive oxygen species, was based on a well-established enzymatic assay involving the oxidation of phenol red. This reaction is specifically catalyzed by horseradish peroxidase (HRPO). In this procedure, two hundred microliters of the perfusate sample were initially combined with 800 mL of a freshly prepared phenol red solution. Subsequently, 10 µL of a 1:20 dilution of HRPO (prepared *ex tempore*, meaning immediately before use, to ensure enzyme activity) was added to initiate the catalytic reaction. For the blank probe, an adequate volume of Krebs–Henseleit solution was substituted for the coronary venous effluent, providing a baseline measurement in the absence of biological samples. The final absorbance, which correlates with the concentration of H2O2, was measured spectrophotometrically at a wavelength of 610 nm. This methodology is based on the technique originally described by Pick and Keisari in 1980.
Drugs
All pharmacological agents utilized in this meticulously designed experimental protocol were exclusively provided by Sigma-Aldrich, a globally recognized and reputable supplier of high-quality chemicals and biochemicals, ensuring consistency and purity of the test substances.
Statistical Analysis
All quantitative values obtained from the experimental measurements are uniformly expressed as the mean ± standard error (SE), providing a clear representation of the central tendency and the variability within each experimental group. For statistical comparison and determination of significance, the paired t-test was employed in the statistical analysis. This choice of test is appropriate for comparing two related sets of measurements, such as before and after substance application within the same isolated heart preparation. A p-value of less than 0.05 (p<0.05) was adopted as the threshold for statistical significance, indicating that observed differences were considered unlikely to have occurred by random chance, thereby supporting the validity of the experimental findings.
RESULTS
The Effects of N-methyl-D-aspartate on the Cardiodynamic Parameters and Coronary Flow in Isolated Rat Heart
The direct application of N-methyl-D-aspartate (NMDA), the canonical agonist for NMDA-R, to the isolated rat heart did not induce any statistically significant change in any of the meticulously observed cardiodynamic parameters. These parameters included dp/dt max, dp/dt min, systolic left ventricular pressure (SLVP), diastolic left ventricular pressure (DLVP), and heart rate (HR). Furthermore, NMDA application also failed to elicit any significant alteration in coronary flow (CF). This finding is particularly notable given NMDA's well-established role as an excitatory agonist in other systems, suggesting a nuanced or limited direct effect on isolated cardiac mechanics under these experimental conditions.
The Effects of Combined Application of N-methyl-D-aspartate and Glycine on the Cardiodynamic Parameters and Coronary Flow in Isolated Rat Heart
In contrast to the solitary application of NMDA, the simultaneous administration of N-methyl-D-aspartate (NMDA) and glycine, which is known to be an essential co-agonist for full NMDA-R activation, induced significant and widespread changes in cardiac function. This combined treatment resulted in a significant decrease in the maximum rate of pressure development (dp/dt max), indicating reduced myocardial contractility, and a significant decrease in the minimum rate of pressure development (dp/dt min), suggesting impaired relaxation. Furthermore, significant reductions were observed in systolic left ventricular pressure (SLVP), heart rate (HR), and coronary flow (CF). These findings collectively point to a substantial depressant effect on overall cardiac performance and perfusion when NMDA-R are fully activated in the presence of their co-agonist. Notably, during the subsequent wash-out period, the values of all mentioned parameters showed a significant increase, indicating a substantial degree of recovery. At the end of the wash-out phase, these recovered values were largely insignificantly different compared to the initial control values, suggesting that the observed cardiodynamic depression was largely reversible upon removal of the agonists.
The Effects of DL-homocysteine thiolactone on the Cardiodynamic Parameters and Coronary Flow in Isolated Rat Heart
The application of DL-homocysteine thiolactone (DL-Hcy TLHC), a compound implicated in NMDA-R activation under pathological conditions, directly induced significant alterations in cardiac function. Specifically, DL-Hcy TLHC caused a significant decrease in dp/dt max, indicating a reduction in myocardial contractile force, and a significant decrease in systolic left ventricular pressure (SLVP). Additionally, a significant reduction in heart rate (HR) and coronary flow (CF) was observed, pointing to both chronotropic and perfusional depression. While the diastolic left ventricular pressure (DLVP) initially showed an insignificant decrease during DL-Hcy TLHC application, this decrease unfortunately continued and even became more pronounced during the wash-out period, such that the control value and the value after the wash-out period differed significantly. This suggests a more persistent detrimental effect on diastolic function. Following the wash-out period, the values of dp/dt max and CF did show an insignificant increase, such that their recovered values were not significantly different from control. However, the values of SLVP and HR after the wash-out period remained significantly lower compared to their respective control baselines, indicating incomplete recovery of these crucial parameters.
The Effects of Combined Application of DL-homocysteine thiolactone and Glycine on the Cardiodynamic Parameters and Coronary Flow in Isolated Rat Heart
The combined administration of DL-homocysteine thiolactone (DL-Hcy TLHC) and glycine, which serves as a necessary co-agonist, resulted in a profound and statistically significant decrease across all observed cardiodynamic parameters. This included dp/dt max (contractility), dp/dt min (relaxation), systolic left ventricular pressure (SLVP), diastolic left ventricular pressure (DLVP), and heart rate (HR). Furthermore, coronary flow (CF) was also significantly reduced. This comprehensive depressant effect on myocardial function and perfusion suggests that full activation of NMDA-R by DL-Hcy TLHC, facilitated by glycine, has a widespread negative impact on isolated heart performance. Importantly, during the subsequent wash-out period, the values of all mentioned parameters showed a significant increase, indicating a substantial degree of reversibility. Most of these parameters eventually reached values statistically similar to their respective control baselines, with the notable exception of heart rate (HR), which remained significantly lower even after the wash-out period, suggesting a more persistent chronotropic effect.
The Effects of Memantine on the Cardiodynamic Parameters and Coronary Flow in Isolated Rat Heart
The administration of memantine, a well-known non-competitive NMDA-R antagonist, exerted distinct effects on the isolated rat heart. Memantine induced a significant decrease in the maximum rate of pressure development (dp/dt max), the minimum rate of pressure development (dp/dt min), systolic left ventricular pressure (SLVP), heart rate (HR), and coronary flow (CF). These findings indicate that memantine, despite being an antagonist, causes a general reduction in myocardial contractility, relaxation, pressure generation, chronotropy, and perfusion under these acute conditions. However, during the subsequent wash-out period, all the aforementioned parameters showed a significant increase, demonstrating a good degree of reversibility. Importantly, all these parameters, with the exception of heart rate (HR), successfully reached their initial control values by the end of the wash-out period, suggesting that memantine's cardiodepressant effects were largely transient. The value of diastolic left ventricular pressure (DLVP) was not significantly changed at any point during the experiment, indicating that memantine did not acutely impact this specific parameter of diastolic function.
The Effects of Ifenprodil on the Cardiodynamic Parameters and Coronary Flow in Isolated Rat Heart
The application of ifenprodil, an allosteric modulator selective for GluN2B-containing NMDA-R, induced specific and statistically significant changes in the isolated rat heart. Ifenprodil caused a significant decrease in both coronary flow (CF) and heart rate (HR) when compared with control conditions, suggesting an impact on both myocardial perfusion and chronotropy. Following the wash-out period, both of these affected parameters showed a significant increase, indicating reversibility, and ultimately reached values that were approximate to their initial control baselines. Furthermore, the maximum rate of pressure development (dp/dt max) was also observed to be significantly increased during the wash-out period, suggesting a potential rebound effect or a delayed influence on contractility. Interestingly, other cardiodynamic parameters, including dp/dt min, systolic left ventricular pressure (SLVP), and diastolic left ventricular pressure (DLVP), did not exhibit any statistically significant changes during the experiment with ifenprodil, highlighting a more selective effect profile compared to memantine or combined agonist treatments.
The Effects of N-methyl-D-aspartate on the Biomarkers of Oxidative Stress in Isolated Rat Heart
The administration of N-methyl-D-aspartate (NMDA) alone, at the tested concentration, did not induce any statistically significant change in any of the observed biomarkers of oxidative stress in the coronary venous effluent. These biomarkers included TBARS (lipid peroxidation), nitrites, superoxide anion radical, and hydrogen peroxide. This finding suggests that, in isolation, and under these acute conditions in the isolated heart model, direct NMDA application does not acutely trigger measurable oxidative stress.
The Effects of Combined Application of N-methyl-D-aspartate and Glycine on the Biomarkers of Oxidative Stress in Isolated Rat Heart
In contrast to NMDA alone, the combined administration of N-methyl-D-aspartate (NMDA) and glycine, crucial for full NMDA-R activation, induced a significant increase across several key biomarkers of oxidative stress. Specifically, significant elevations were observed in TBARS (indicating increased lipid peroxidation), nitrites (suggesting altered nitric oxide metabolism), and the superoxide anion radical. These findings indicate that full activation of NMDA-R, when both agonists are present, leads to a measurable increase in oxidative stress within the isolated heart. Importantly, the levels of all these elevated biomarkers decreased significantly following the wash-out period, returning to values that were statistically similar to control baselines, indicating the reversibility of the oxidative stress response.
The Effects of DL-homocysteine thiolactone on the Biomarkers of Oxidative Stress in Isolated Rat Heart
The application of DL-homocysteine thiolactone (DL-Hcy TLHC) alone, at the tested concentration, did not induce any statistically significant change in any of the observed biomarkers of oxidative stress in the coronary venous effluent. This finding suggests that, in isolation, and under these acute conditions in the isolated heart model, DL-Hcy TLHC, similar to NMDA alone, does not acutely trigger measurable oxidative stress. This contrasts with its cardiodepressant effects, implying that its acute mechanical impact might not be directly mediated by an immediate surge in these measured oxidative stress markers.
The Effects of Combined Application of DL-homocysteine thiolactone and Glycine on the Biomarkers of Oxidative Stress in Isolated Rat Heart
The combined administration of DL-homocysteine thiolactone (DL-Hcy TLHC) and glycine resulted in a significant increase in specific biomarkers of oxidative stress. This combination induced a notable increase in TBARS (indicating enhanced lipid peroxidation) and the superoxide anion radical. These findings suggest that the full activation of NMDA-R, mediated by DL-Hcy TLHC in the presence of glycine, leads to a measurable increase in specific oxidative stress markers within the isolated heart. During the subsequent wash-out period, the levels of both TBARS and the superoxide anion radical decreased significantly, eventually returning to values that were statistically similar to control baselines, indicating the reversibility of this oxidative stress response.
The Effects of Memantine on the Biomarkers of Oxidative Stress in Isolated Rat Heart
The administration of memantine, an NMDA-R antagonist, induced a statistically significant reduction in the production of the superoxide anion radical. This suggests a potential antioxidant or protective effect against superoxide generation by blocking NMDA-R. However, a more complex picture emerged during the wash-out period. Following the removal of memantine, this initial reduction in superoxide anion radical levels became even more pronounced, indicating a sustained or delayed antioxidant effect. Counterintuitively, and on the other hand, the values of TBARS and hydrogen peroxide were significantly increased during the wash-out period compared to their levels during the active memantine application. This suggests a possible rebound effect or an unmasking of pro-oxidant pathways once the NMDA-R antagonism is removed, highlighting a complex dynamic in redox balance.
The Effects of Ifenprodil on the Biomarkers of Oxidative Stress in Isolated Rat Heart
The application of ifenprodil, a selective NMDA-R allosteric modulator, induced a significant decrease in hydrogen peroxide levels. This suggests that inhibition of GluN2B-containing NMDA-R may specifically reduce H2O2 production. However, similar to memantine, a complex pattern emerged during the wash-out period: the production of nitrites was observed to increase significantly during the wash-out period. This increase in nitrites during wash-out, in contrast to the initial H2O2 reduction, points to a nuanced and potentially differential modulation of nitric oxide pathways or a compensatory response in redox signaling once ifenprodil's effects diminish.
DISCUSSION
The central objective of the present study was to meticulously evaluate the acute effects of a range of compounds known to modulate N-methyl-D-aspartate receptors (NMDA-R) on key aspects of cardiac function, coronary blood flow, and the status of oxidative stress in an isolated rat heart model. Specifically, we investigated the impacts of NMDA (a canonical agonist), the combination of NMDA and glycine (a necessary co-agonist for full receptor activation), DL-homocysteine thiolactone (DL-Hcy TLHC, a compound implicated in NMDA-R activation under pathological conditions), the combination of DL-Hcy TLHC and glycine, memantine (a non-competitive NMDA-R antagonist), and ifenprodil (an allosteric modulator selective for GluN2B-containing NMDA-R). The overarching aim was to precisely assess the immediate consequences of acute NMDA-R modulation in the heart by these substances and to determine the potential role of oxidative stress in mediating any observed changes.
Our experimental findings demonstrated a significant nuance in the activation requirements of cardiac NMDA-R. The direct application of NMDA alone, even at a concentration of 100 µmol/L, did not induce any statistically significant change in any of the meticulously observed cardiodynamic parameters or coronary flow. This result aligns precisely with our previous research, where application of glutamate alone at the same concentration also failed to cause changes in cardiodynamic parameters or coronary flow. This consistency across studies strongly suggests that, in this specific isolated heart experimental model, the presence of both NMDA-R co-agonists, namely NMDA (or glutamate) and glycine, is an absolute prerequisite for effective receptor activation and the subsequent elicitation of functional changes in cardiac performance. Only the combined application of both agonists, NMDA and glycine, led to significant alterations in myocardial function, including changes in dp/dt max (maximum rate of pressure development), dp/dt min (minimum rate of pressure development), systolic left ventricular pressure (SLVP), heart rate (HR), and coronary flow (CF). This necessity for dual co-agonist presence for NMDA-R activation in the heart is a critical observation, aligning with the well-established biophysical properties of these receptors as "coincidence detectors."
In contrast to NMDA, the application of DL-Hcy TLHC alone, at a concentration of 10 µmol/L, exerted a significant depressant effect on most of the observed cardiodynamic parameters and coronary flow, with the exception of dp/dt min. Furthermore, the simultaneous administration of DL-Hcy TLHC and glycine resulted in a significant reduction across all observed cardiodynamic parameters, suggesting a comprehensive negative impact on myocardial function when NMDA-R are fully engaged by this compound. These results resonate with previous investigations, such as that by Moshal and co-authors, who demonstrated that increased levels of homocysteine (HHcy) in mice led to a decrease in parameters of cardiac contractility. Their research further indicated that the use of inhibitors of NMDA-R, matrix metalloproteinase (MMP) activity, and mitochondrial permeability transition (MPT) could mitigate these changes in myocardial contractility. Based on these findings, Moshal and colleagues concluded that homocysteine, acting via NMDA-R, activates MMP and induces MPT, processes that collectively contribute to the observed decrease in myocardial contractility.
Our study further explored the impact of DL-Hcy TLHC on left ventricular pressures. DL-Hcy TLHC alone, as well as its combination with glycine, induced a significant reduction in systolic left ventricular pressure (SLVP) and an insignificant initial decrease in diastolic left ventricular pressure (DLVP). A noteworthy observation was that during the subsequent wash-out period, the value of DLVP in both of these experimental groups continued to decrease, ultimately resulting in a significantly lower value for this parameter after the wash-out period compared to the control baseline. This persistent effect on diastolic function is intriguing. Previous research by Takemoto, for instance, examined the effects of microinjection of L-homocysteine (L-Hcy) into specific brain regions, the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM), which are involved in cardiovascular regulation. Takemoto found that L-Hcy injection into RVLM induced pressor effects, while injection into CVLM induced depressor effects, both of which were abolished by prior microinjection of MK-801, an NMDA-R antagonist. However, other studies, such as those by Walker and co-workers, showed that hyperhomocysteinemia primarily induces early disturbances in myocardial *structure*, without significant changes in *function*. Joseph and colleagues reached similar conclusions. The discrepancies observed between the results of these previous studies and our current research could be attributed to fundamental differences in the experimental protocols, particularly the distinction between chronic *in vivo* models and the acute isolated heart preparation used here. The reductions in SLVP and DLVP observed in our study represent a direct effect of DL-Hcy TLHC on the heart, as the complex effects of homocysteine on the heart mediated via the autonomic nervous system and the long-term effects of chronic hyperhomocysteinemia are largely excluded in this isolated heart model. Possible underlying mechanisms mediating the direct effects of DL-Hcy TLHC could involve disturbances in calcium currents or alterations in nitric oxide (NO) production. The differing effects of NMDA alone versus DL-Hcy TLHC alone might be a result of varying affinities of NMDA-R for these respective compounds. Alternatively, there might exist another distinct mechanism through which homocysteine could exert its effects on the cardiovascular system, beyond direct NMDA-R activation. Despite these differences in individual action, the combinations of NMDA and glycine, and DL-Hcy TLHC and glycine, exhibited similar effects on SLVP, reinforcing the role of NMDA-R activation.
Regarding heart rate (HR), DL-Hcy TLHC alone, as well as its combined applications with DL-Hcy TLHC and NMDA, both in the presence of glycine, consistently induced a significant decrease in HR. This bradycardic effect is a key observation. While Muntzel and co-authors hypothesized that homocysteine might cause increases in sympathetic nerve activity, which could contribute to cardiovascular damage in hyperhomocysteinemia, their direct infusion studies found no effects on heart rate or blood pressure. Our previous laboratory results, however, showed a similar bradycardic effect of DL-Hcy TLHC on HR. Conversely, Resstel and co-workers observed an increase in HR in rats with induced mild hyperhomocysteinemia. The differing effects of homocysteine on HR across these studies may be due to the involvement of different physiological structures in regulating heart rate. In chronic and *in vivo* experiments, the primary influence on HR might stem from homocysteine's effects on NMDA-R (and potentially other glutamate receptors) within specific central nervous system structures. In contrast, the experimental protocol employed in the present study was specifically designed to focus on the direct, acute effects on the heart and its coronary circulation, thereby minimizing confounding central nervous system influences.
Similarly to heart rate, the application of DL-Hcy TLHC alone, and its combined application with both DL-Hcy TLHC and NMDA alongside glycine, consistently induced a significant decrease in coronary flow (CF). In the groups treated with the combinations of DL-Hcy TLHC and NMDA with glycine, the values of CF were significantly increased during the wash-out period, indicating a notable degree of recovery. However, in the group treated solely with DL-Hcy TLHC, the value of this parameter only slightly increased during the wash-out period, such that the control values of CF and the values after the wash-out period did not differ significantly. This observed decrease in coronary flow is consistent with findings from other studies, which most commonly propose that the effects of homocysteine on nitric oxide synthase (NOS) and subsequent disturbances in NO production are the primary mechanisms underlying this action. For instance, Abahji and colleagues assessed the effects of hyperhomocysteinemia, induced by oral methionine supplementation, on endothelial function in healthy human subjects. They found that hyperhomocysteinemia induced a significant decrease in flow-mediated vasodilatation of the brachial artery, a recognized parameter that directly reflects endothelial function and NO synthesis.
Memantine, functioning as a noncompetitive NMDA-R antagonist, induced a consistent decrease across all observed cardiodynamic parameters and coronary flow, with the notable exception of diastolic left ventricular pressure (DLVP). Importantly, all these affected parameters returned to values approximate to their control baselines during the wash-out period, except for heart rate (HR), which remained somewhat depressed. This pattern suggests a largely reversible cardiodepressant effect. A study by Makhro and co-workers provided corroborating evidence, showing that intracoronary application of memantine, along with other NMDA-R antagonists (such as eliprodil, Ro25-6981, ketamine, and MK-801), exerted negative inotropic (contractility-reducing) and chronotropic (heart rate-reducing) effects on autonomous heart function. Seeber and co-authors highlighted a complex formation between the NMDA-R subunit GluN2B and the ryanodine receptor 2 (RyR2) in neonatal rat myocardium, proposing that the negative inotropic effect induced by memantine likely occurs due to changes in intracellular calcium (Ca2+) concentrations. This aligns with the effects observed for other NMDA receptor antagonists, and it is not excluded that similar complexes may exist in older age.
Memantine is widely used clinically for the treatment of Alzheimer's disease, and consequently, most of its observed effects on the cardiovascular system have been documented as side effects during the treatment of these patients. Building on studies investigating the systemic effects of memantine as a local anesthetic, Chen and colleagues found that memantine induces a decrease in mean arterial pressure and heart rate. Beyond its bradycardic effect, NMDA-R antagonists, including memantine, also exhibit antiarrhythmic properties. Both of these actions could potentially be associated with the prolongation of the QT interval induced by memantine, a phenomenon also reported in clinical contexts. Since memantine's primary therapeutic use is in Alzheimer's disease, its effects on cerebral blood vessels and brain blood flow have been extensively examined. Intravenous administration of memantine in anesthetized rats induced a measurable decrease of cerebral blood flow, averaging 15% within 10 minutes, with further reductions reaching up to 53% over time. In a human population of patients suffering from Parkinson's disease, memantine was observed to cause a decrease in blood flow in basal ganglia and several frontal cortical areas, underscoring its cerebrovascular effects.
Ifenprodil, applied at a concentration of 1 µmol/L, induced a significant reduction in both heart rate (HR) and coronary flow (CF) values. These parameters subsequently returned to values similar to their initial baselines after the wash-out period, indicating reversibility. Furthermore, the value of dp/dt max was significantly increased during the wash-out period, suggesting a potential rebound or delayed effect on contractility. Interestingly, other cardiodynamic parameters, including dp/dt min, systolic, and diastolic pressures, did not significantly change with ifenprodil application. This absence of effect on contractility and pressures could potentially be attributed to the relatively modest dose applied, or it might reflect ifenprodil's more specific mode of action as a GluN2B-selective antagonist, which may not broadly impact all cardiodynamic parameters as seen with broader antagonists. It is important to note that there are limited data specifically addressing the effects of ifenprodil on the cardiovascular system. Monassier and co-authors assessed the effects of ifenprodil on the baroreceptor heart rate reflex in rats and reported that it did not significantly alter the basal values of hemodynamic parameters. These authors highlighted the rather complex pharmacological action of ifenprodil, considering its potential interactions with adrenergic, serotonergic, and sigma receptors. Furthermore, research data have indicated ifenprodil's action on other ion channels, including G protein-activated inwardly rectifying K+ channels, tetrodotoxin-resistant Na+ channels, N and P-type voltage-dependent Ca2+ channels, and the Na+/Ca2+ exchanger, demonstrating its multifaceted molecular targets. Despite its established neuroprotective properties, studies by Başkaya and co-workers confirmed its neuroprotective effects without significantly affecting cerebral blood flow. The reduction in HR and CF induced by ifenprodil in this research is considered a direct effect of this substance on the myocardium, as its central nervous system effects are excluded in the isolated heart model. The observed reduction in coronary flow also correlates with the concentration of nitrites, which serves as an indicator of NO production.
The subsequent part of the experimental protocol systematically addressed the dynamics of oxidative stress biomarkers during the administration of the tested substances.
Neither NMDA nor DL-Hcy TLHC, when applied alone, caused significant changes in the values of any of the observed biomarkers of oxidative stress. This suggests that individual application of these agonists, without the co-agonist glycine, does not acutely trigger measurable oxidative stress under these experimental conditions in the isolated heart. However, in stark contrast, the combined administration of NMDA and glycine induced a significant increase in TBARS values (indicating elevated lipid peroxidation), as well as increased production of nitrites and the superoxide anion radical (O2-). Similarly, the concomitant application of DL-Hcy TLHC and glycine also caused a significant increase in TBARS and O2-. Critically, the values of all these elevated parameters decreased significantly during the subsequent wash-out period, returning to levels similar to control, confirming the transient and reversible nature of this oxidative stress. These findings are consistent with previous research; for instance, McGee and Abdel-Rahman investigated the effects of ethanol on peripheral NMDA-R and showed that a bolus of NMDA significantly increased vascular NOx and reactive oxygen species (ROS). It is widely accepted that increased activity of NMDA-Rs leads to an increased content of intracellular Ca2+, which in turn can lead to an increment in nitric oxide synthase (NOS) activity and NO production, with subsequent generation of ROS. A similar study by the same research group indicated that the activation of neuronal NOS (nNOS) and the consequent increased production of NO play a crucial role in the heightened ROS production observed due to NMDA-R activation in the vasculature. Likewise, numerous authors indicate that homocysteine also causes an increase in the production of ROS and oxidative stress in the tissues of the cardiovascular system. Tyagi and colleagues, for example, highlighted the role of NMDA-R and increased production of ROS in the deleterious effects of homocysteine on the cardiovascular system, demonstrating that the increased production of ROS by homocysteine was abolished by MK-801, an NMDA-R antagonist. The absence of effects of NMDA and DL-Hcy TLHC alone on ROS production in this research, contrasted with the strong effects when combined with glycine, further supports the view that full activation of the NMDA receptors, requiring both co-agonists, is necessary to induce these oxidative stress responses in the isolated heart model.
Memantine, an NMDA-R antagonist, induced a statistically significant decrease in the production of the superoxide anion radical (O2-), suggesting a direct protective effect against this specific reactive oxygen species. However, a more complex and perhaps paradoxical dynamic emerged during the wash-out period. Following the removal of memantine, the values of TBARS and hydrogen peroxide (H2O2) were found to be significantly increased compared to their levels during the active memantine application. This phenomenon suggests a potential rebound effect or an unmasking of pro-oxidant pathways once the NMDA-R blockade is terminated, possibly due to a temporary increase in intracellular Ca2+ upon antagonist removal, sufficient to transiently enhance ROS production. Liu and co-authors, in their study, demonstrated a protective role of memantine against neuronal changes induced by methylmercury, concluding that the underlying mechanisms involve NMDA-R blockade, maintenance of Ca2+ homeostasis, and an indirect antioxidative action. Methylmercury exhibits its deleterious effects on the nervous system by over-activating NMDA-Rs, disrupting intracellular Ca2+ concentration, and increasing ROS production. Memantine has also been shown to reduce the effects of diabetes on kidneys, suggesting a role for NMDA-R in the development of diabetic nephropathy, where the antioxidative effect of memantine played a pivotal role, considering the involvement of ROS and oxidative stress in the pathogenesis of this disorder.
In a study conducted by Di Maio and others, ifenprodil significantly reduced the oxidation of thiols induced by pilocarpine in an experimental model of temporal lobe epilepsy. Based on their results, these authors concluded that ifenprodil could prevent glutamate-induced aberrant calcium influx and the consequent over-activation of NMDA-R. It is highly probable that a similar mechanism, specifically involving the impact of memantine and ifenprodil on intracellular Ca2+ flux and homeostasis, mediates the observed effects of these compounds in the cardiovascular system, offering a plausible explanation for their modulatory actions on cardiac function and oxidative stress.
CONCLUSIONS
The comprehensive findings from this investigation provide crucial insights into the acute modulation of N-methyl-D-aspartate receptors (NMDA-R) within the isolated rat heart. The observed absence of any significant effects when N-methyl-D-aspartate (NMDA) was administered alone, despite its classification as a canonical agonist, is highly informative. This lack of response is most plausibly attributed to the absence of a co-agonist for the other essential subunit of the NMDA-R in the perfusate. This interpretation is strongly supported by previous research conducted using this same experimental model, which has unequivocally demonstrated that synergistic action of both co-agonists, glutamate (or its analogue NMDA) and glycine, is an absolute prerequisite for the effective and full activation of NMDA-Rs. The results obtained from the combined application of NMDA and glycine, which indeed elicited significant changes in cardiac function, further reinforce this perspective, highlighting the "coincidence detector" property of NMDA-R even in cardiac tissues.
When considering the overall effects of DL-homocysteine thiolactone (DL-Hcy TLHC), both when applied alone and in combination with glycine, as well as drawing upon findings from other related studies conducted within our laboratory, a significant possibility emerges: that homocysteine may not exert its diverse cardiovascular effects exclusively through the direct activation of NMDA-Rs. This suggests the potential involvement of additional, yet-to-be-fully-elucidated mechanisms or molecular targets through which homocysteine contributes to cardiovascular dysfunction. Further research is warranted to comprehensively map these alternative pathways.
The observed effects of memantine and ifenprodil, both acting as antagonists of NMDA-Rs, provide pivotal insights into the physiological role of these receptors in the heart. Their administration resulted in a measurable decrease in various cardiodynamic parameters and, importantly, also modulated specific parameters of oxidative stress. This consistent finding strongly indicates that the blockade of NMDA-Rs directly influences intrinsic cardiac function and redox balance, even under physiological conditions. This emphasizes the significant and potentially continuous importance of NMDA-Rs in the precise regulation of the cardiovascular system’s function, even in a healthy, isolated state.
Based on the synthesis of all these findings, a robust conclusion can be drawn: the acute modulation of NMDA-Rs, regardless of whether it involves their activation (in the presence of both co-agonists) or their blockade by specific antagonists, profoundly and significantly affects the overall function of the cardiovascular system. This study underscores the critical yet complex role of cardiac NMDA-Rs, which extends beyond their well-known functions in the central nervous system. Therefore, their precise physiological functions and their contributions to various pathophysiological conditions warrant extensive and detailed clarification in future research endeavors. Such future studies should ideally broaden their scope to include investigations into the different and distinct structures of the cardiovascular system, beyond the isolated myocardium, to fully unravel the systemic impact of NMDA-R modulation.
ACKNOWLEDGMENTS
This research project was made possible and significantly supported by Grant no. 175043, generously provided by the Ministry of Science and Technical Development of the Republic of Serbia. Furthermore, additional crucial support was received from the Junior Project 04/2011, an initiative of the Faculty of Medical Sciences at the University of Kragujevac, Serbia. The authors express their sincere appreciation for this vital financial assistance, which was instrumental in the successful execution of this study.
CONFLICT OF INTERESTS
The authors of the present study wish to formally and unequivocally declare that they have no actual or potential conflicts of interest whatsoever to disclose. This declaration encompasses any financial, personal, or other relationships with specific persons or organizations that could potentially influence, or be perceived to influence, the objectivity, integrity, or interpretation of the research findings presented herein.s