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Recent evidences have shown that phenolic structures can exert many biological functions. Ellagic acid (EA), a phenolic compound, has been suggested to have cardioprotective effects. In this study we aimed to investigate the effect of EA on cardiac Ca2+ currents and contractility in rat ventricular myocytes and to elucidate the underlying mechanisms of these changes.All records measured from the freshly isolated ventricular myocytes of rat heart at 36±1 °C by using whole-cell configuration of voltage clamp. Cell shortening was measured by detecting the length of edges with video-based system at 1 Hz frequency of field stimulation. We found that EA dose dependently reduced Ca2+ currents with EC50= 23 nM. EA decreased voltage dependent L-type Ca2+ current density (ICaL) but it didn't affect the inactivation and reactivation parameters. Inhibition of adenylate cyclase (AC) with SQ-22536 (10 μM) and using probucol (antioxidant, 5 μM) had no effect on EA modulation of ICaL. Interestingly, blockage of nitric oxide synthase (NOS) with L-NAME (500 μM) and guanylate cyclase (GC) with ODQ (1 μM) abolished inhibitory effect of EA on ICaL. Moreover, EA dose dependently blunted fractional shorthening of ventricular myocytes.In conclusion, EA affects ionic and mechanical properties of ventricular myocytes starting at nanomolar concentrations. Our findings indicated that EA suppresses ICaL and exerts negative inotropic effects through activation of NOS-GC-cGMP pathway. Accordingly, EA may be useful in pathophysiological conditions whereby these effects might be favorable such as hypertension and ischemic heart diseases. [ABSTRACT FROM PUBLISHER]

Background: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are known for their benefits in conditions like cardiovascular diseases in type 2 diabetes and obesity. They also show promise for aging-related conditions with minimal side effects. However, their impact on cardiovascular risk is still debated. Notably, some long-acting GLP-1RAs cause a sustained increase in heart rate on the first day of use without a clear mechanism. To understand their short-term effects, we examined acute GLP-1R agonism on the electrical activity of elderly hearts., Methods: In this study, we utilized in vivo electrocardiography, in vitro cellular electrophysiology experiments, and biochemical measurements on heart preparations from 6-month-old (Adult) and 24-month-old (aged) BALB/c mice., Results: A single liraglutide injection (0.3 mg/kg) induced repetitive, self-sustained arrhythmogenic electrocardiograms in aged mice (24 months old) but had no effect on adults (6 months old) within the first 10 minutes. Acute application of liraglutide to isolated ventricular cardiomyocytes from aged mice significantly prolonged the late phase of action potential repolarization (APR90). This was due to suppressed K+ currents and increased persistent Na+currents (Late-INa), primarily through delayed recovery from inactivation of Na+ currents. Additionally, liraglutide increased Ca2+ spark frequency and wave formation by enhancing Ca2+ release from the sarcoplasmic reticulum, affecting both Na+ and Ca2+ regulation in aging cells. Liraglutide also induced casein kinase 2 (CK2) hyperphosphorylation in aged cardiomyocytes, which a CK2 inhibitor could reverse, normalizing APR90 by reducing Late-INa and enhancing K+ currents., Conclusion: These findings reveal that acute GLP-1R agonism can disrupt electrical signaling and induce arrhythmia in aged mice through CK2 hyperphosphorylation, providing new insights into the cardiovascular effects of GLP-1RAs in the elderly.

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca 2+ ]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca 2+ ] by releasing Ca 2+ from the sarcoplasmic reticulum (SR). Timely Ca 2+ release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca 2+ release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca 2+ release events across a range of [Ca 2+ ]. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cardiomyocyte isolation from mouse Basic Protocol 2: Preparation of cardiomyocytes for Ca 2+ imaging Basic Protocol 3: Confocal microscopy and quantitative Ca 2+ analysis using SparkMaster 2., (© 2024 Wiley Periodicals LLC.)

Doxorubicin (DOXO) induces marked cardiotoxicity, though increased oxidative stress while there are some documents related with cardioprotective effects of some antioxidants against organ-toxicity during cancer treatment. Although magnolia bark has some antioxidant-like effects, its action in DOXO-induced heart dysfunction has not be shown clearly. Therefore, here, we aimed to investigate the cardioprotective action of a magnolia bark extract with active component magnolol and honokiol complex (MAHOC; 100 mg/kg) in DOXO-treated rat hearts. One group of adult male Wistar rats was injected with DOXO (DOXO-group; a cumulative dose of 15 mg/kg in 2-week) or saline (CON-group). One group of DOXO-treated rats was administered with MAHOC before DOXO (Pre-MAHOC group; 2-week) while another group was administered with MAHOC following the 2-week DOXO (Post-MAHOC group). MAHOC administration, before or after DOXO, provided full survival of animals during 12-14 weeks, and significant recoveries in the systemic parameters of animals such as plasma levels of manganese and zinc, total oxidant and antioxidant statuses, and also systolic and diastolic blood pressures. This treatment also significantly improved heart function including recoveries in end-diastolic volume, left ventricular end-systolic volume, heart rate, cardiac output, and prolonged P-wave duration. Furthermore, the MAHOC administrations improved the structure of left ventricles such as recoveries in loss of myofibrils, degenerative nuclear changes, fragmentation of cardiomyocytes, and interstitial edema. Biochemical analysis in the heart tissues provided the important cardioprotective effect of MAHOC on the redox regulation of the heart, such as improvements in activities of glutathione peroxidase and glutathione reductase, and oxygen radical-absorbing capacity of the heart together with recoveries in other systemic parameters of animals, while all of these benefits were observed in the Pre-MAHOC treatment group, more prominently. Overall, one can point out the beneficial antioxidant effects of MAHOC in chronic heart diseases as a supporting and complementing agent to the conventional therapies., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cardiac conduction abnormalities are disorders in metabolic syndrome (MetS), however, their mechanisms are unknown. Although ventricular arrhythmia reflects the changes in QT-interval of electrocardiograms associated with the changes in cardiomyocyte action potential durations (APDs), recent studies emphasize role of intercellular crosstalk between cardiomyocytes and nonmyocytes via passive (electrotonic)-conduction. Therefore, considering the possible increase in intercellular interactions of nonmyocytes with cardiomyocytes, we hypothesized an early-cardiac-remodeling characterized by short QT-interval via contributions and modulations of changes by nonmyocytes to the ventricular APs in an early-stage MetS hearts. Following the feeding of 8-week-old rats with a high-sucrose diet (32%; MetS rats) and validation of insulin resistance, there was a significant increase in heart rate and changes in the electrical characteristics of the hearts, especially a shortening in action potential (AP) duration of the papillary muscles. The patch-clamp analysis of ventricular cardiomyocytes showed an increase in the Na + -channel currents while there were decreases in  l-type Ca 2+ -channel (LTCC) currents with unchanged K + -channel currents. There was an increase in the phosphorylated form of connexin 43 (pCx43), mostly with lateral localization on sarcolemma, while its unphosphorylated form (Cx43) exhibited a high degree of localization within intercalated discs. A high-level positively-stained α-SMA and CD68 cells were prominently localized and distributed in interfibrillar spaces of the heart, implying the possible contributions of myofibroblasts and macrophages to both shortened APDs and abnormal electrical conduction in MetS hearts. Our data propose a previously unrecognized pathway for SQT induction in the heart. This pathway includes not only the contribution of short ventricular-APDs via ionic mechanisms but also increasing contributions of the electrotonic-cardiomyocyte depolarization, spontaneous electrical activity-associated fast heterogeneous impulse conduction in the heart via increased interactions and relocations between cardiomyocytes and nonmyocytes, which may be an explanation for the development of an SQT in early-cardiac-remodeling., (© 2023 John Wiley & Sons Ltd.)

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Recent studies discuss the evidence of lesser degrees of hyperglycemia contribution to cardiovascular disease (CVD) than impaired glucose tolerance. Indeed, the biggest risk for CVD seems to shift to glucose intolerance in humans with insulin resistance. Although there is a connection between abnormal insulin signaling and heart dysfunction in diabetics, there is also a relation between cardiac insulin resistance and aging heart failure (HF). Moreover, studies have revealed that HF is associated with generalized insulin resistance. Recent clinical outcomes parallel to the experimental data undertaken with antihyperglycemic drugs have shown their beneficial effects on the cardiovascular system through a direct effect on the myocardium, beyond their ability to lower blood glucose levels and their receptor-associated actions. In this regard, several new-class drugs, such as glucagon-like peptide 1 receptor agonists (GLP-1Ra) and sodium-glucose cotransport 2 inhibitors (SGLT2i), can improve cardiac health beyond their ability to control glycemia. In recent years, great improvements have been made toward the possibility of direct heart-targeting effects including modulation of the expression of specific cardiac genes in vivo for therapeutic purposes. However, many questions remain unanswered, regarding their therapeutic effects on cardiomyocytes in heart failure, although there are various cellular levels studies with these drugs. There are also some important comparative studies on the role of SGLT2i versus GLP-1Ra in patients with and without CVD as well as with or without hyperglycemia. Here, we sought to summarize and interpret the available evidence from clinical studies focusing on the effects of either GLP-1Ra or SGLT-2i or their combinations on cardiac structure and function. Furthermore, we documented data from experimental studies, at systemic, organ, and cellular levels. Overall, one can summarize that both clinical and experimental data support that either SGLT2i or GLP-1R agonists have similar benefits as cardioprotective agents in patients with or without impaired glucose tolerance., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The correlation between long-QT and connexin 43 (Cx43) status and localization in elderly rats was determined to demonstrate a correlation between insulin resistance (I-R), ischemia-reperfusion, aging, and heart dysfunction. Male Wistar rats are grouped as 24-month-old rats (Aged-group), those with metabolic syndrome (8 months old; MetS-group), or controls (8 months old; Con-group). Both experimental groups have long-QT and low heart rate. Immunohistochemical imaging and quantification showed marked decreases in Cx43 staining of intercalated disc with less localizations in the Aged-group and MetS-group. The lateralization of Cx43 on longitudinal cell membrane was significantly high in the MetS-group than in the Con-group with no significant change in the Aged-group. Its significant cytoplasmic internalization was higher in the Aged-group than in the MetS-group. There were marked decreases in phospho-Cx43 (pCx43) staining of intercalated disc with less localizations in both groups than in the Con-group. Furthermore, lateralization of pCx43 was significantly low in the Aged-group and MetS-group, whereas there were no significant changes in the cytoplasmic internalization of both groups compared with the Con-group. Furthermore, the ratio of pCx43 to Cx43 was significantly small in both groups. We determined increases in RhoA and endothelin-1 in both groups, further supporting decreases in pCx43. Our data indicate the important role of I-R on long-QT in aging heart through alterations in both Cx43 protein level and localizations, leading to an abnormal spreading of ventricular repolarization in I-R heart.

The matrix metalloproteinases (MMPs) contribute to matrix remodeling in diabetes via tissue degradation; however, their contributions can be different depending on the pathology. For instance, MMPs are elevated in acute stress hyperglycemia, whereas they can be degraded in chronic hyperglycemia. Since studies emphasize the possible cardioprotective effect of ticagrelor (Tica) beyond its antiplatelet action, we aimed to examine whether Tica treatment can reverse the depressed heart function of metabolic syndrome (MetS) rats via affecting the expression levels of MMPs. Tica treatment of high-carbohydrate-induced MetS rats could not affect significantly the depressed contractile activity of Langendorff-perfused heart preparations. On the other hand, the Tica treatment provided a significant recovery in the reduced relaxation activity of the aortic preparations from the same animals. Histological examination of the hearts demonstrated marked damages in Mets rats, such as increases in the number of foamy cells and accumulation of collagen fiber and increases in the elastic lamellar irregularity of tunica media, while Tica treatment provided a slight improvement in the structure of left ventricle tissue. We also could not obtain a significant reverse in the high cytosolic labile Zn 2+ ([Zn 2+ ] i ) with the treatment of cardiomyocytes with Tica. Furthermore, Tica treatment of MetS rats could not significantly reverse the degraded protein levels of MMP-2 and MMP-9 in the heart, as well. Overall, we demonstrated that Tica treatment of MetS rats has no significant benefits on the depressed heart function, although provide a significant beneficial impact on vascular relaxation. This action of Tica may be through its lack of action on both MMP degradation and high [Zn 2+ ] i , which can further precipitate in cleavage of extracellular matrix in the heart., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Metabolic syndrome (MetS) became a tremendous public health burden in the last decades. Store-operated calcium entry (SOCE) is a unique mechanism that causes a calcium influx, which is triggered by calcium store depletion. MetS-induced alterations in cardiac calcium signaling, especially in SOCE are still unclear. Therefore, we aim to examine the possible role of SOCE and its components (STIM1 and Orai1) in the MetS-induced cardiac remodeling., Methods: We used male, adult (12 weeks) Wistar albino rats (n = 20). Animals were randomly divided into two groups which were: control (C) and MetS. We gave 33% sucrose solution to animals instead of water for 24 weeks to establish MetS model. In the end, papillary muscle function was evaluated, and various electrophysiological analyses were made in isolated cardiomyocytes. Additionally, STIM1 and Orai1 protein and mRNA expressions were analyzed., Results: We observed a deterioration in contractility in MetS animals and demonstrated the contribution of SOCE by applying a SOCE inhibitor (BTP2). Calcium spark frequency was increased while its amplitude was decreasing in MetS hearts, which was reversed after SOCE inhibition. The amplitude of transient calcium changes in the MetS group was decreased, and it decreased further BTP2 application. Both protein and mRNA levels of STIM1 and Orai1 were increased significantly in MetS hearts., Conclusion: Current data indicate the significant contribution of SOCE to cardiac calcium handling in the MetS model. We think MetS-induced SOCE activation is a compensation mechanism that is required for the continuum of proper cardiac functioning, although the activation can also cause cardiac hypertrophy., (© 2022. The Author(s).)

Insufficient-heart function is associated with myocardial insulin resistance in the elderly, particularly associated with long-QT, in a dependency on dysfunctional KCNQ1/KCNE1-channels. So, we aimed to examine the contribution of alterations in KCNQ1/KCNE1-current (I Ks ) to the aging-related remodeling of the heart as well as the role of insulin treatment on I Ks in the aged rats. Prolonged late-phase action potential (AP) repolarization of ventricular cardiomyocytes from insulin-resistant 24-month-old rats was significantly reversed by in vitro treatment of insulin or PKG inhibitor (in vivo, as well) via recovery in depressed I Ks . Although the protein level of either KCNQ1 or KCNE1 in cardiomyocytes was not affected with aging, PKG level was significantly increased in those cells. The inhibited I Ks in β 3 -ARs-stimulated cells could be reversed with a PKG inhibitor, indicating the correlation between PKG-activation and β 3 -ARs activation. Furthermore, in vivo treatment of aged rats, characterized by β 3 -ARs activation, with either insulin or a PKG inhibitor for 2 weeks provided significant recoveries in I Ks , prolonged late phases of APs, prolonged QT-intervals, and low heart rates without no effect on insulin resistance. In vivo insulin treatment provided also significant recovery in increased PKG and decreased PIP2 level, without the insulin effect on the KCNQ1 level in β 3 -ARs overexpressed cells. The inhibition of I Ks in aged-rat cardiomyocytes seems to be associated with activated β 3 -ARs dependent remodeling in the interaction between KCNQ1 and KCNE1. Significant recoveries in ventricular-repolarization of insulin-treated aged cardiomyocytes via recovery in I Ks strongly emphasize two important issues: (1) I Ks can be a novel target in aging-associated remodeling in the heart and insulin may be a cardioprotective agent in the maintenance of normal heart function during the aging process. (2) This study is one of the first to demonstrate insulin's benefits on long-QT in insulin-resistant aged rats by accelerating the ventricular AP repolarization through reversing the depressed I Ks via affecting the β 3 -ARs signaling pathway and particularly affecting activated PKG., (© 2021 Wiley Periodicals LLC.)

Metabolic syndrome (MetS) is associated with additional cardiovascular risk in mammalians while there are relationships between hyperglycemia-associated cardiovascular dysfunction and increased platelet P2Y12 receptor activation. Although P2Y12 receptor antagonist ticagrelor (Tica) plays roles in reduction of cardiovascular events, its beneficial mechanism remains poorly understood. Therefore, we aimed to clarify whether Tica can exert a direct protective effect in ventricular cardiomyocytes from high-carbohydrate diet-induced MetS rats, at least, through affecting sarcoplasmic reticulum (SR)-mitochondria (Mit) miscommunication. Tica treatment of MetS rats (150 mg/kg/day for 15 days) significantly reversed the altered parameters of action potentials by reversing sarcolemmal ionic currents carried by voltage-dependent Na + and K + channels, and Na + /Ca 2+ -exchanger in the cells, expressed P2Y12 receptors. The increased basal-cytosolic Ca 2+ level and depressed SR Ca 2+ load were also reversed in Tica-treated cells, at most, though recoveries in the phosphorylation levels of ryanodine receptors and phospholamban. Moreover, there were marked recoveries in Mit structure and function (including increases in both autophagosomes and fragmentations) together with recoveries in Mit proteins and the factors associated with Ca 2+ transfer between SR-Mit. There were further significant recoveries in markers of both ER stress and oxidative stress. Taken into consideration the Tica-induced prevention of ER stress and mitochondrial dysfunction, our data provided an important document on the pleiotropic effects of Tica in the electrical activity of the cardiomyocytes from MetS rats. This protective effect seems through recoveries in SR-Mit miscommunication besides modulation of different sarcolemmal ion-channel activities, independent of P2Y12 receptor antagonism., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Consumption of illicit pharmaceutical products containing sibutramine has been reported to cause cardiovascular toxicity problems. This study aimed to demonstrate the toxicity profile of sibutramine, and thereby provide important implications for the development of more effective strategies in both clinical approaches and drug design studies. Action potentials (APs) were determined from freshly isolated ventricular cardiomyocytes with whole-cell configuration of current clamp as online. The maximum amplitude of APs (MAPs), the resting membrane potential (RMP), and AP duration from the repolarization phases were calculated from original records. The voltage-dependent K + -channel currents (I K ) were recorded in the presence of external Cd 2+ and both inward and outward parts of the current were calculated, while their expression levels were determined with qPCR. The levels of intracellular free Ca 2+ and H + (pH i ) as well as reactive oxygen species (ROS) were measured using either a ratiometric micro-spectrofluorometer or confocal microscope. The mechanical activity of isolated hearts was observed with Langendorff-perfusion system. Acute sibutramine applications (10 -8 -10 -5  M) induced significant alterations in both MAPs and RMP as well as the repolarization phases of APs and I K in a concentration-dependent manner. Sibutramine (10 μM) induced Ca 2+ -release from the sarcoplasmic reticulum under either electrical or caffeine stimulation, whereas it depressed left ventricular developed pressure with a marked decrease in the end-diastolic pressure. pH i inhibition by sibutramine supports the observed negative alterations in contractility. Changes in mRNA levels of different I K subunits are consistent with the acute inhibition of the repolarizing I K , affecting AP parameters, and provoke the cardiotoxicity.

The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age-associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co-transporter gene (SGLT2) in disrupted cellular Ca 2+ -homeostasis, and mitochondrial dysfunction in age-associated cardiac dysfunction. In contrast to younger rats (6-month of age), older rats (24-month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca 2+ ([Ca 2+ ] i ) overload, indicative of disrupted cellular Ca 2+ -homeostasis. Interestingly, increased [Ca 2+ ] i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged-cardiomyocytes also displayed high Na + /Ca 2+ -exchanger (NCX) activity and blood glucose levels compared with young-controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age-associated defects in [Ca 2+ ] i -homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca 2+ -loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca 2+ ] i -homeostasis. Our studies support the notion that interventions that modulate SGLT2-activity can provide benefits in maintaining [Ca 2+ ] i and cardiac function with advanced age., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The death prevalence from cardiovascular disease is significantly high in elderly-populations, while mitochondrial-aging plays an important in abnormal function of vital organs through high mitochondrial ROS production. Mitochondria have a unique mode of action by providing ATP production and modulating the cytosolic Ca 2+ -signaling and maintain the redox status of cardiomyocytes. There is an aging-associated impairment in oxidative phosphorylation which causes a marked dysregulation of mitochondrial biogenesis. Therefore, we aimed to examine whether a mitochondria-targeting antioxidant, MitoTEMPO, can directly provide a cardioprotective effect on ventricular cardiomyocyte function under in vitro conditions. The MitoTEMPO-treatment (0.1 μM for 4-h) of aged-ventricular cardiomyocytes (from 24-mo-old rats), compared to those of the adults (from 8-mo-old rats) markedly augmented not only the depressed biochemical parameters but also the ultrastructure of mitochondria. It also provided marked protective action against increased mitochondrial superoxide formation and Bnip3 overexpression, which both markedly induce depolarized mitochondrial potential, increase reactive oxygen species, mitochondrial swelling and fission, and accelerate mitochondrial turnover via autophagy. Furthermore, it provided marked protection against spontaneous action potentials, via shortening the prolonged action potential duration, at most, through recovery in depressed K + -channel currents. Moreover, we determined significant recovery in the depressed intracellular Ca 2+ -changes under electrical stimulation in MitoTEMPO-treated the aged-cardiomyocytes. Overall, we provided important information associated with an antiarrhythmic action, thereby controlling cytosolic and mitochondrial Ca 2+ -handling, implying its possible protective role of mitochondria-targeting antioxidant-treatment during aging., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ticagrelor, a P 2 Y 12 -receptor inhibitor, and a non-thienopyridine agent are used to treat diabetic patients via its effects on off-target mechanisms. However, the exact sub-cellular mechanisms by which ticagrelor exerts those effects remains to be elucidated. Accordingly, the present study aimed to examine whether ticagrelor influences directly the cardiomyocytes function under insulin resistance through affecting mitochondria-sarco(endo)plasmic reticulum (SER) cross-talk. Therefore, we analyzed the function and ultrastructure of mitochondria and SER in insulin resistance-mimicked (50-μM palmitic acid for 24-h) H9c2 cardiomyocytes in the presence or absence of ticagrelor (1-µM for 24-h). We found that ticagrelor treatment significantly prevented depolarization of mitochondrial membrane potential and increases in reactive oxygen species with a marked increase in the ATP level in insulin-resistant H9c2 cells. Ticagrelor treatment also reversed the increases in the resting level of free Ca 2+ and mRNA level of P 2 Y 12 receptors as well as preserved ER stress and apoptosis in insulin-resistant H9c2 cells. Furthermore, we determined marked repression with ticagrelor treatment in the increased number of autophagosomes and degeneration of mitochondrion, including swelling and loss of crista besides recoveries in enlargement and irregularity seen in SER in insulin-resistant H9c2 cells. Moreover, ticagrelor treatment could prevent the altered mRNA levels of Becklin-1 and type 1 equilibrative nucleoside transporter (ENT1), which are parallel to the preservation of ultrastructural ones. Our overall data demonstrated that ticagrelor can directly affect cardiomyocytes and provide marked protection against ER stress and dramatic induction of autophagosomes, and therefore, can alleviate the ER stress-induced oxidative stress increase and cell apoptosis during insulin resistance.

Obesity-induced insulin resistance and type 2 diabetes mellitus can ultimately result in various complications, including diabetic cardiomyopathy. In this case, cardiac dysfunction is characterized by metabolic disturbances such as impaired glucose oxidation and an increased reliance on fatty acid (FA) oxidation. Mitochondrial dysfunction has often been associated with the altered metabolic function in the diabetic heart, and may result from FA-induced lipotoxicity and uncoupling of oxidative phosphorylation. In this review, we address the metabolic changes in the diabetic heart, focusing on the loss of metabolic flexibility and cardiac mitochondrial function. We consider the alterations observed in mitochondrial substrate utilization, bioenergetics and dynamics, and highlight new areas of research which may improve our understanding of the cause and effect of cardiac mitochondrial dysfunction in diabetes. Finally, we explore how lifestyle (nutrition and exercise) and pharmacological interventions can prevent and treat metabolic and mitochondrial dysfunction in diabetes., (© 2019 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Azoramide is identified as a new compound with the dual properties for the improvement of ER-folding capacity in various cells as well as for the treatment of T2DM. Although the effect of azoramide in glucose-homeostasis in mammalians is not known very well, a limited number of experimental studies showed that it could improve the insulin sensitivity in genetically obese mice. Therefore, here, we aimed to investigate the direct effect of azoramide on insulin signaling in insulin-resistant (IR) cardiomyocytes using IR-modelled ventricular cardiomyocytes. This model was established in H9c2 cells using palmitic acid incubation (50-μM for 24-h). The development of IR in cells was verified by monitoring the cellular 2-DG6P uptake assays in these treated cells. The 2-DG6P uptake was 50% less in the IR-cells compared to the control cells, while azoramide treatment (20-μM for 48-h) could prevent fully that decrease. In addition, azoramide treatment markedly preserved the IR-induced less ATP production and high-ROS production in these IR-cells. Furthermore, this treatment prevented the functional changes in mitochondria characterized by depolarized mitochondrial membrane potential and mitochondrial fusion or fusion-related protein levels as well as cellular ATP level. Moreover, this treatment provided marked protection against IR-associated changes in the insulin signaling pathway in cells, including recovery in the phosphorylation of IRS1 and Akt as well as the protein level of GLUT4 and Akt. Our present results, for the first time, demonstrated that azoramide plays an important protective role in IR-cardiomyocytes, at most, protective action on mitochondria. Therefore, one can suggest that azoramide, as a novel regulator, can provide direct cardioprotection in the IR-heart, at most, via affecting mitochondria and can be a good candidate as a new drug for the treatment of IR-associated cardiovascular disorders in mammalians with systemic IR.

Aging is an important risk factor for cardiac dysfunction. Heart during aging exhibits a depressed mechanical activity, at least, through mitochondria-originated increases in ROS. Previously, we also have shown a close relationship between increased ROS and cellular intracellular free Zn 2+ ([Zn 2+ ] i ) in cardiomyocytes under pathological conditions as well as the contribution of some re-expressed levels of Zn 2+ -transporters for redistribution of [Zn 2+ ] i among suborganelles. Therefore, we first examined the cellular (total) [Zn 2+ ] and then determined the protein expression levels of Zn 2+ -transporters in freshly isolated ventricular cardiomyocytes from 24-month rat heart compared to those of 6-month rats. The [Zn 2+ ] i in the aged-cardiomyocytes was increased, at most, due to increased ZIP7 and ZnT8 with decreased levels of ZIP8 and ZnT7. To examine redistribution of the cellular [Zn 2+ ] i among suborganelles, such as Sarco/endoplasmic reticulum, S(E)R, and mitochondria ([Zn 2+ ] SER and [Zn 2+ ] Mit ), a cell model (with galactose) to mimic the aged-cell in rat ventricular cell line H9c2 was used and demonstrated that there were significant increases in [Zn 2+ ] Mit with decreases in [Zn 2+ ] SER . In addition, the re-distribution of these Zn 2+ -transporters were markedly changed in mitochondria (increases in ZnT7 and ZnT8 with no changes in ZIP7 and ZIP8) and S(E)R (increase in ZIP7 and decrease in ZnT7 with no changes in both ZIP8 and ZnT8) both of them isolated from freshly isolated ventricular cardiomyocytes from aged-rats. Furthermore, we demonstrated that cellular levels of ROS, both total and mitochondrial lysine acetylation (K-Acetylation), and protein-thiol oxidation were significantly high in aged-cardiomyocytes from 24-month old rats. Using a mitochondrial-targeting antioxidant, MitoTEMPO (1 µM, 5-h incubation), we provided an important data associated with the role of mitochondrial-ROS production in the [Zn 2+ ] i -dyshomeostasis of the ventricular cardiomyocytes from 24-month old rats. Overall, our present data, for the first time, demonstrated that a direct mitochondria-targeting antioxidant treatment can be a new therapeutic strategy during aging in the heart through a well-controlled [Zn 2+ ] distribution among cytosol and suborganelles with altered expression levels of the Zn 2+ -transporters.

Role of β 3 -AR dysregulation, as either cardio-conserving or cardio-disrupting mediator, remains unknown yet. Therefore, we examined the molecular mechanism of β 3 -AR activation in depressed myocardial contractility using a specific agonist CL316243 or using β 3 -AR overexpressed cardiomyocytes. Since it has been previously shown a possible correlation between increased cellular free Zn 2+ ([Zn 2+ ] i ) and depressed cardiac contractility, we first demonstrated a relation between β 3 -AR activation and increased [Zn 2+ ] i , parallel to the significant depolarization in mitochondrial membrane potential in rat ventricular cardiomyocytes. Furthermore, the increased [Zn 2+ ] i induced a significant increase in messenger RNA (mRNA) level of β 3 -AR in cardiomyocytes. Either β 3 -AR activation or its overexpression could increase cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels, in line with significant changes in nitric oxide (NO)-pathway, including increases in the ratios of pNOS3/NOS3 and pGSK-3β/GSK-3β, and PKG expression level in cardiomyocytes. Although β 3 -AR activation induced depression in both Na + - and Ca 2+ -currents, the prolonged action potential (AP) seems to be associated with a marked depression in K + -currents. The β 3 -AR activation caused a negative inotropic effect on the mechanical activity of the heart, through affecting the cellular Ca 2+ -handling, including its effect on Ca 2+ -leakage from sarcoplasmic reticulum (SR). Our cellular level data with β 3 -AR agonism were supported with the data on high [Zn 2+ ] i and β 3 -AR protein-level in metabolic syndrome (MetS)-rat heart. Overall, our present data can emphasize the important deleterious effect of β 3 -AR activation in cardiac remodeling under pathological condition, at least, through a cross-link between β 3 -AR activation, NO-signaling, and [Zn 2+ ] i pathways. Moreover, it is interesting to note that the recovery in ER-stress markers with β 3 -AR agonism in hyperglycemic cardiomyocytes is favored. Therefore, how long and to which level the β 3 -AR agonism would be friend or become foe remains to be mystery, yet., (© 2019 Wiley Periodicals, Inc.)

Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed significant effects in patients with diabetes or metabolic syndrome (MetS) with high cardiovascular risk. Although the increased intracellular Zn 2+ level ([Zn 2+ ] i ), oxidative stress, and altered cardiac matrix metalloproteinases (MMPs) in diabetic cardiomyopathy can intersect with different signaling pathways, the exact mechanisms are not known yet. Since either MMPs or SGLT2 have important roles in cardiac-fibrosis under hyperglycemia, we aimed to examine the role of SGLT2 inhibitor dapagliflozin (DAP) on cardiac Zn 2+ -transporters responsible for [Zn 2+ ] i -regulation, comparison to insulin (INS), together with MMP levels and systemic oxidative stress status in MetS-rats. High-carbohydrated diet-induced MetS-rats received DAP or INS for 2 weeks. DAP but not INS in MetS-rats significantly decreased high blood-glucose levels, while both treatments exerted benefits on increased total oxidative status and decreased total antioxidant status in MetS-rat plasma as well as in heart tissue. Protein levels of Zn 2+ -transporters, responsible for Zn 2+ -influx into cytosol, ZIP7 and ZIP14 were increased with significant decrease in ZIP8 of MetS-rat cardiomyoctes, while Zn 2+ -transporters, responsible for cytosolic Zn 2+ -efflux, ZnT7 was decreased with no change in ZnT8. Both treatments induced significant beneficial effects on altered ZIP14, ZIP8, and ZnT7 levels. Furthermore, both treatments exerted benefits on depressed gelatin-zymography and protein expression levels of MMP-2 and MMP-9 in MetS-rat ventricular cardiomyocytes. The direct effect of DAP on heart was also confirmed with measurements of left ventricular developed pressure. Overall, we showed that DAP has important antioxidant-like cardio-protective effects in MetS-rats, similar to INS-effect, affecting Zn 2+ -regulation via Zn 2+ -transporters, MMPs, and oxidative stress. Therefore one can suggest that SGLT2 inhibitors can be new therapeutic agents for cardio-protection not only in hyperglycemia but also in failing heart.

Functional contribution of S(E)R-mitochondria coupling to normal cellular processes is crucial and any alteration in S(E)R-mitochondria axis may be responsible for the onset of diseases. Mitochondrial free Zn 2+ level in cardiomyocytes ([Zn 2+ ] Mit ) is lower comparison to either its cytosolic or S(E)R level under physiological condition. However, there is little information about distribution of Zn 2+ -transporters on mitochondria and role of Zn 2+ -dependent mitochondrial-function associated with [Zn 2+ ] Mit . Since we recently have shown how hyperglycemia (HG)-induced changes in ZIP7 and ZnT7 contribute to Zn 2+ -transport across S(E)R and contribute to S(E)R-stress in the heart, herein, we hypothesized that these transporters can also be localized to mitochondria and affect the S(E)R-mitochondria coupling, and thereby contribute to cellular Zn 2+ -muffling between S(E)R-mitochondria in HG-cells. Mitochondrial localizations of ZIP7 and ZnT7 were demonstrated using fluorescence technique while they were confirmed in isolated mitochondrial fractions using biochemical analysis. Markedly decreased ZIP7 and increased ZnT7 levels were measured in isolated mitochondrial fractions from either HG- or doxorubicin, DOX (as positive control)-treated cardiomyocytes. Significantly increases in [Zn 2+ ] Mit and ROS production levels and depolarized mitochondrial membrane potential were also measured in HG cells. The expression levels of some key proteins, responsible for proper S(E)R-mitochondria coupling such as Mfn-1, Fis-1, OPA1, BAP31, STIM1 and PML in either HG- or DOX-cells were supported our above hypothesis, strongly. Overall, this study provides an important description about the role of ZIP7 and ZnT7, localized to both mitochondria and S(E)R and contribute to cellular Zn 2+ -muffling between cellular-compartments in HG or hypertrophic cardiomyocytes via affecting S(E)R-mitochondria coupling. Any alteration in this axis and/or cellular [Zn 2+ ] may provide new insight for prevention/therapy of HF in diabetes and/or hypertrophy., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Background: Metabolic syndrome (MetS) is a prevalent risk factor for cardiac dysfunction. Although SGLT2-inhibitors have important cardioprotective effects in hyperglycemia, their underlying mechanisms are complex and not completely understood. Therefore, we examined mechanisms of a SGLT2-inhibitor dapagliflozin (DAPA)-related cardioprotection in overweight insulin-resistant MetS-rats comparison with insulin (INSU), behind its glucose-lowering effect., Methods: A 28-week high-carbohydrate diet-induced MetS-rats received DAPA (5 mg/kg), INSU (0.15 mg/kg) or vehicle for 2 weeks. To validate MetS-induction, we monitored all animals weekly by measuring body weight, blood glucose and HOMO-IR index, electrocardiograms, heart rate, systolic and diastolic pressures., Results: DAPA-treatment of MetS-rats significantly augmented the increased blood pressure, prolonged Q-R interval, and low heart rate with depressed left ventricular function and relaxation of the aorta. Prolonged-action potentials were preserved with DAPA-treatment, more prominently than INSU-treatment, at most, through the augmentation in depressed voltage-gated K + -channel currents. DAPA, more prominently than INSU-treatment, preserved the depolarized mitochondrial membrane potential, and altered mitochondrial protein levels such as Mfn-1, Mfn-2, and Fis-1 as well as provided significant augmentation in cytosolic Ca 2+ -homeostasis. Furthermore, DAPA also induced significant augmentation in voltage-gated Na + -currents and intracellular pH, and the cellular levels of increased oxidative stress, protein-thiol oxidation and ADP/ATP ratio in cardiomyocytes from MetS rats. Moreover, DAPA-treatment normalized the increases in the mRNA level of SGLT2 in MetS-rat heart., Conclusions: Overall, our data provided a new insight into DAPA-associated cardioprotection in MetS rats, including suppression of prolonged ventricular-repolarization through augmentation of mitochondrial function and oxidative stress followed by improvement of fusion-fission proteins, out of its glucose-lowering effect.

Aging in humans represents declining in cardio-protective systems, however its mechanisms are not known yet. We aimed to analyse how aging affects key mechanisms responsible for contractile dysfunction via comparing the improperly synchrony between electrical and mechanical activities in male aged-rats (24-month old) comparison to those of adult-rats (6-month old). We determined significantly increased systemic oxidative stress with decreased antioxidant capacity, clear insulin resistance and hypertrophy in aged-rats with normal fasting blood glucose. We also determined significantly high level of reactive oxygen species, ROS production in fluorescent dye chloromethyl-2',7'-dichlorodihydrofluoroscein diacetate (DCFDA) loaded isolated cardiomyocytes from aged-rats, confirming the increased oxidative stress in these hearts. In situ electrocardiograms, ECGs presented significant prolongations in RR- and QT-intervals in the aged-rats. Invasive hemodynamic measurements demonstrated marked increases in the heart rate and mean arterial pressure and decreases in the ejection-fraction and preload-recruitable stroke-work, together with depressed contraction and relaxation activities in aortic rings. In light and electron microscopy examinations in aged-rats, significant increases in muscle fibre radius and amount of collagen fibres were detected in the heart as well as markedly flattened and partial local splitting in elastic lamellas in the aorta, besides irregularly clustered mitochondria and lysosomes around the myofilaments in cardiomyocytes. MitoTEMPO treatment of tissue samples and cardiomyocytes from aged-rats for 1-h induced significant structural improvements. In the second part of our study, we have shown that mitochondria-targeted antioxidant MitoTEMPO antagonized all alterations in the heart samples as well as penylephrine-induced contractile and acetylcholine-induced relaxation responses of aged-rat aortic rings. Overall, the present data strongly support the important role of mitochondrial oxidative stress in the development of aged-related insufficiencies and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in aging-associated complications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Intracellular labile (free) Zn 2+ -level ([Zn 2+ ] i ) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn 2+ ] i is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn 2+ ] i on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn 2+ ] i induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of ≥ 3 min following [Zn 2+ ] i increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn 2+ ] i induced inhibitions in voltage-dependent K + -channel currents, such as transient outward K + -currents, I to , steady-state currents, I ss and inward-rectifier K + -currents, I K1 , reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both I ss and I K1 , while no effect on I to . However, the increased-[Zn 2+ ] i could induce marked activation in ATP-sensitive K + -channel currents, I KATP , depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn 2+ ] i with no change in mRNA level of Kv4.2, while the mRNA level of I KATP subunit, SUR2A was increased significantly with increased-[Zn 2+ ] i , being reversible with DTT. Overall we demonstrated that high-[Zn 2+ ] i, even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K + -currents, although activation of I KATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn 2+ ] i can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Zn 2+ -homoeostasis including free Zn 2+ ([Zn 2+ ] i ) is regulated through Zn 2+ -transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn 2+ -transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, [Zn 2+ ] i was significantly high in doxorubicin-treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKCα expression and PKCα-phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in [Zn 2+ ] i using zinc-ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn 2+ transporters. Additionally, increased [Zn 2+ ] i could induce marked activation of PKCα. Moreover, we observed marked decrease in [Zn 2+ ] i under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn 2+ transporters on an intersection pathway with increased [Zn 2+ ] i and PKCα activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well-controlled [Zn 2+ ] i via Zn 2+ transporters and PKCα can be important therapeutic approach in prevention/treatment of HF., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Clinical and experimental studies have shown an association between intracellular free Zn 2+ ([Zn 2+ ] i )-dyshomeostasis and cardiac dysfunction besides [Ca 2+ ] i -dyshomeostasis. Since [Zn 2+ ] i -homeostasis is regulated through Zn 2+ -transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn 2+ -homeostasis via alteration in Zn 2+ -transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn 2+ -influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn 2+ ] i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn 2+ ] i via decreased ER-[Zn 2+ ] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn 2+ -transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn 2+ ] i - besides [Ca 2+ ] i -dyshomeostasis.

Mechanical activity of the heart is adversely affected in metabolic syndrome (MetS) characterized by increased body mass and marked insulin resistance. Herein, we examined the effects of high carbohydrate intake on cardiac function abnormalities by evaluating in situ heart work, heart rate, and electrocardiograms (ECGs) in rats. MetS was induced in male Wistar rats by adding 32% sucrose to drinking water for 22-24 weeks and was confirmed by insulin resistance, increased body weight, increased blood glucose and serum insulin, and increased systolic and diastolic blood pressures in addition to significant loss of left ventricular integrity and increased connective tissue around myofibrils. Analysis of in situ ECG recordings showed a markedly shortened QT interval and decreased QRS amplitude with increased heart rate. We also observed increased oxidative stress and decreased antioxidant defense characterized by decreases in serum total thiol level and attenuated paraoxonase and arylesterase activities. Our data indicate that increased heart rate and a shortened QT interval concomitant with higher left ventricular developed pressure in response to β-adrenoreceptor stimulation as a result of less cyclic AMP release could be regarded as a natural compensation mechanism in overweight rats with MetS. In addition to the persistent insulin resistance and obesity associated with MetS, one should consider the decreased heart work, increased heart rate, and shortened QT interval associated with high carbohydrate intake, which may have more deleterious effects on the mammalian heart.

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca 2+ homeostasis in hypertrophic hearts. This study investigated molecular mechanism underlying ROCK inhibition-mediated cardioprotection against pressure overload-induced cardiac hypertrophy, with a focus on Ca 2+ homeostasis. Cardiac hypertrophy model was established by performing transverse aortic constriction (TAC) in 8-week-old male rats. Groups were assigned as SHAM, TAC and TAC+Fas (rats undergoing TAC and treated with fasudil). Rats in the TAC+Fas group were administered fasudil (5mg/kg/day), and rats in the SHAM and TAC groups were treated with vehicle for 10 weeks. Electrophysiological recordings were obtained from isolated left ventricular myocytes and expression levels of proteins were determined using western blotting. Rats in the TAC group showed remarkable cardiac hypertrophy, and fasudil treatment significantly reversed this alteration. TAC+Fas myocytes showed significant improvement in reduced contractility and Ca 2+ transients. Moreover, these myocytes showed restoration of slow relaxation rate and Ca 2+ reuptake. Although L-type Ca 2+ currents did not change in TAC group, there was a significant reduction in the triggered Ca 2+ transients which was reversed either by long-term fasudil treatment or incubation of TAC myocytes with fasudil. The hearts of rats in the TAC group showed a significant decrease in ROCK1, ROCK2, RyR2 protein levels and p-PLB S16/T17 /SERCA2 ratio and increase in RhoA expression and MLC phosphorylation. However, fasudil treatment largely reversed TAC-induced alterations in protein expression. Thus, our findings indicate that upregulation of the RhoA/ROCK pathway is significantly associated with cardiac hypertrophy-related Ca 2+ dysregulation and suggest that ROCK inhibition prevents hypertrophic heart failure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The objective of this study was to examine the effect of swimming exercise on aging-related Ca2+ handling alterations and structural abnormalities of female rat heart., Methods: For this purpose, 4-month and 24-month old female rats were used and divided into three following groups: sedentary young (SY), sedentary old (SO), and exercised old (Ex-O). Swimming exercise was performed for 8 weeks (60 min/day, 5 days/week). Myocyte shortening, L-type Ca2+ currents and associated Ca2+ transients were measured from ventricular myocytes at 36 ± 1°C. NOX-4 levels, aconitase activity, glutathione measurements and ultrastructural examination by electron microscopy were conducted in heart tissue., Results: Swimming exercise reversed the reduced shortening and slowed kinetics of aged cardiomyocytes. Although the current density was similar for all groups, Ca2+ transients were higher in SO and Ex-O myocytes with respect to the SY group. Caffeine-induced Ca2+ transients and the integrated NCX current were lower in cardiomyocytes of SY rats compared with other groups, suggesting an increased sarcoplasmic reticulum Ca2+ content in an aged heart. Aging led to upregulated cardiac NOX-4 along with declined aconitase activity. Although it did not reverse these oxidative parameters, swimming exercise achieved a significant increase in glutathione levels and improved structural alterations of old rats' hearts., Conclusions: We conclude that swimming exercise upregulates antioxidant defense capacity and improves structural abnormalities of senescent female rat heart, although it does not change Ca2+ handling alterations further. Thereby, it improves contractile function of aged myocardium by mitigating detrimental effects of oxidative stress.

The Zn 2+ in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO 4 (extracellular exposure) or Zn 2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn 2+ exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO 4 - or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn 2+ -exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn 2+ can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion membrane potential and changes in some cytosolic signaling proteins as well as a defect in ER-mitochondria cross talk.

Hypertension is one of the major risk factors of cardiac hypertrophy and magnesium deficiency is suggested to be a contributing factor in the progression of this complication. In this study, we aimed to investigate the relationship between intracellular free Mg(2+) levels and electrophysiological changes developed in the myocardium of L-NAME induced hypertensive rats. Hypertension was induced by administration of 40 mg/kg of L-NAME for 6 weeks, while magnesium treated rats fed with a diet supplemented with 1 g/kg of MgO for the same period. L-NAME administration for 6 weeks elicited a significant increase in blood pressure which was corrected with MgO treatment; thereby cardiac hypertrophy developing secondary to hypertension was prevented. Cytosolic free magnesium levels of ventricular myocytes were significantly decreased with hypertension and magnesium administration restored these changes. Hypertension significantly decreased the fractional shortening with slowing of shortening kinetics in left ventricular myocytes whereas magnesium treatment was capable of restoring hypertension-induced contractile dysfunction. Long-term magnesium treatment significantly restored the hypertension-induced prolongation in action potentials of ventricular myocytes and suppressed Ito and Iss currents. In contrast, hypertension dependent decrement in intracellular Mg(2+) level did not cause a significant change in L-type Ca(2+) currents, SR Ca(2+) content and NCX activity. Nevertheless, hypertension mediated increase in superoxide anion, hydrogen peroxide and protein oxidation mitigated with magnesium treatment. In conclusion, magnesium administration improves mechanical abnormalities observed in hypertensive rat ventricular myocytes due to reduced oxidative stress. It is likely that, changes in intracellular magnesium balance may contribute to the pathophysiology of chronic heart diseases.

Purpose: Due to the increasing use of wireless technology in developing countries, particularly mobile phones, the influence of electromagnetic fields (EMF) on biologic systems has become the subject of an intense debate. Therefore, in this study we investigated the effect of 2.1 GHz EMF on contractility and beta-adrenergic (β-AR) responsiveness of ventricular myocytes., Materials and Methods: Rats were randomized to the following groups: Sham rats (SHAM) and rats exposed to 2.1 GHz EMF for 2 h/day for 10 weeks (EM-10). Sarcomere shortening and Ca(2+) transients were recorded in isolated myocytes loaded with Fura2-AM and electrically stimulated at 1 Hz, while L-type Ca(2+) currents (I(CaL)) were measured using whole-cell patch clamping at 36 ± 1°C. Cardiac nitric oxide (NO) levels were measured in tissue samples using a colorimetric assay kit., Results: Fractional shortening and amplitude of the matched Ca(2+) transients were not changed in EM-10 rats. Although the isoproterenol-induced (10(-6) M) I(CaL) response was reduced in rats exposed to EMF, basal I(CaL) density in myocytes was similar between the two groups (p < 0.01). Moreover, EMF exposure led to a significant increase in nitric oxide levels in rat heart (p < 0.02)., Conclusions: Long-term exposure to 2.1 GHz EMF decreases β-AR responsiveness of ventricular myocytes through NO signaling.

Purpose: Antiplatelet therapy has been widely used for management of patients with ischaemic heart diseases or thrombotic events. Experimental studies have shown that ticlopidine and clopidogrel decreased L-type Ca(2+) currents (ICaL), altered action potential (AP) duration and thence exerted negative inotropic effects. In this study we tested if ticagrelor, a non-thienopyridine agent, has any influence on contractile and electrical properties of isolated ventricular myocytes., Methods: Cardiomyocytes were isolated from male rat hearts with an enzymatic dissociation procedure and left ventricular myocytes were used for experiments. The effects of ticagrelor (1 μM) on sarcomere shortening, ionic currents and action potentials were measured at 36 ± 1 °C., Results: Ticagrelor significantly reduced ICaL density (~18%, p < 0.01) of ventricular myocytes and this effect was reversible. In consistence, it also decreased sarcomere shortening of electrically stimulated cardiomyocytes (13%, p < 0.05), while it did not change relaxation rates. Repolarizing K(+) currents and AP duration were unaffected by 1 μM ticagrelor application., Conclusions: Ticagrelor exerts a significant influence on contractile properties and membrane currents of ventricular myocytes similarly to thienopyridine agents. The impact of ticagrelor on cardiac excitation-contraction coupling elements is important, since it is widely used for the treatment of patients with heart diseases.

There is evidence that phenolic structure may have biological functions. Ellagic acid (EA), a phenolic compound, has been suggested to have cardioprotective effects. EA effects were investigated on cardiac Ca currents and contractility in rat ventricular myocytes to elucidate the underlying mechanisms. Freshly isolated ventricular myocytes from rat hearts were used. EA dose-dependently reduced Ca currents (ICaL) with EC50 = 23 nM, whereas it did not affect the inactivation and reactivation parameters. Inhibition of adenylate cyclase by SQ-22536 (10 μM) and probucol (5 μM) had no effect on EA modulation of ICaL. Nitric oxide synthase block by L-NAME (500 μM) and of guanylate cyclase by ODQ (1 μM) abolished EA inhibitory effects on ICaL. Moreover, EA blunted ventricular myocytes' fractional shortening in a concentration-dependent manner. In conclusion, EA affects ionic and mechanical properties of rat ventricular myocytes starting at nanomolar concentrations. EA suppresses ICaL and exerts negative inotropic effects through activation of NOS-GC-cGMP pathways. Thus, EA may be useful in pathophysiological conditions such as hypertension and ischemic heart diseases.

There is a growing body of evidence that Diabetes Mellitus leads to a specific cardiomyopathy apart from vascular disease and bring about high morbidity and mortality throughout the world. Recent clinical and experimental studies have extensively demonstrated that this cardiomyopathy causes impaired cardiac performance manifested by early diastolic and late systolic dysfunction. This impaired cardiac performance most probably have emerged upon the expression and activity of regulatory proteins such as Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum Ca(2+)-ATPase, ryanodine receptor and phospholamban. Over years many therapeutic strategies have been recommended for treatment of diabetic cardiomyopathy. Lately, inorganic elements have been suggested to have anti-diabetic effects due to their suggested ability to regulate glucose homeostasis, reduce oxidative stress or suppress phosphatases. Recent findings have shown that trace elements exert many biological effects including insulin-mimetic or antioxidant activity and in this manner they have been recommended as potential candidates for treatment of diabetes-induced cardiac complications, an effect based on their modes of action. Some of these trace elements are known to play an essential role as component of enzymes and thus modulate the organ function in physiological and pathological conditions. Besides, they can also manipulate redox state of the channels via antioxidant properties and thus contribute to the regulation of [Ca(2+)]i homeostasis and cardiac ion channels. On account of little information about some trace elements, we discussed the effect of vanadium, selenium, zinc and tungstate on diabetic heart complications.

Recently, sodium tungstate was suggested to improve cardiac performance of diabetic rats in perfused hearts based on its insulinomimetic activity. In this study, we aimed to investigate the cellular and molecular mechanisms underlying this beneficial effect of sodium tungstate. Tungstate was administered (100 mg/kg/day) to diabetic and control rats intragastrically for 6 weeks. Blood glucose levels increased, whereas body weight, heart weight and plasma insulin levels decreased significantly in diabetic animals. Interestingly, none of these parameters was changed by tungstate treatment. On the other hand, fractional shortening and accompanying intracellular Ca(2+) [Ca(2+)](i) transients of isolated ventricular myocytes were measured, and sodium tungstate was found to improve the peak shortening and the amplitude of [Ca(2+)](i) transients in diabetic cardiomyocytes. Potassium and L-type Ca(2+) currents were also recorded in isolated ventricular cells. Significant restoration of suppressed I (to) and I (ss) was achieved by tungstate administration. Nevertheless, L-type calcium currents did not change either in untreated or treated diabetic rats. Tissue biochemical parameters including TBARS, protein carbonyl content, xanthine oxidase (XO) and xanthine dehydogenase (XDH) were also determined, and diabetes revealed a marked increase in TBARS and carbonyl content which were decreased significantly by tungstate treatment. Conversely, although XO and XDH activities didn't change in untreated diabetic rats, a remarkable but insignificant decrease was detected in treated animals. In conclusion, tungstate treatment improved diabetes-induced contractile abnormalities via restoration of dysregulated [Ca(2+)](i) and altered ionic currents. This beneficial effect is due to antioxidant property of sodium tungstate rather than normalization of hyperglycemia.